Dissertations / Theses: 'Molecular Targeted Therapy'

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Mckiver, Bryan D. "SND1-Targeted Gene Therapy for Hepatocellular Carcinoma." VCU Scholars Compass, 2018. https://scholarscompass.vcu.edu/etd/5676.

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Staphylococcal nuclease and tudor-domain containing 1 (SND1) is an oncogene for a wide variety of cancers, including hepatocellular carcinoma (HCC). SND1 is a multifunctional protein regulating gene expression of proto-oncogenes and tumor suppressor genes, making SND1 a prime target for developing cancer therapeutics. This notion is especially attributed to HCC as most patients are diagnosed in advanced stages and the therapeutic options available for these patients are severely limited. In this study, we evaluated the therapeutic potential of a replication-defective adenovirus vector delivering SND1 shRNA (Ad.SND1sh) to human HCC cell lines, HepG3, HuH-7, and Hep3B. Adenovirus infection in HCC cells was confirmed by Western blotting and immunofluorescence. The efficacy of Ad.SND1sh to knockdown SND1 expression was confirmed via Western blot, qRT-PCR, and immunofluorescence. Ad.SND1sh did not significantly affect proliferation of the three human HCC cells but significantly inhibited their invasive and migratory capacities, as determined by wound healing and Matrigel invasion assays, respectively. As a corollary, Ad.SND1sh treatment resulted in a decrease in mesenchymal markers, such as N-cadherin, Twist, Snail, and Slug, without affecting levels of epithelial marker E-Cadherin, indicating that SND1 knockdown induces mesenchymal conversion in HCC cells. Additionally, reductions in liver cancer stem cell marker CD133 and HCC marker α-fetoprotein (AFP) were observed with SND1 knockdown. HCC cells with aberrant expression of these markers are associated with tumor initiation, recurrence, and multi-drug resistance. Our findings indicate that Ad.SND1sh may potentially be an effective therapy for advanced HCC and needs to be studied further for its clinical application.

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2

Honarvar, Hadis. "Development of Affibody molecules for radionuclide molecular imaging and therapy of cancer." Doctoral thesis, Uppsala universitet, Medicinsk strålningsvetenskap, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-298740.

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Affibody molecules are a promising class of scaffold-based targeting proteins for radionuclide-based imaging and therapy of cancer. This thesis work is based on 5 original research articles (papers I-V), which focus on optimization of molecular design of HER2-binding Affibody variants for high contrast imaging of this predictive biomarker as well as development of Affibody molecules suitable for radionuclide-based targeted therapies. Papers I and II were dedicated to evaluation of the influence of the macrocyclic chelator DOTA positioning at N-terminus, in the middle of helix-3 and at C terminus of a synthetic Affibody molecule, ZHER2:S1. These synthetic variants were labelled with different radionuclides i.e. 111In and 68Ga to study also the effect of different labels on their biodistribution properties. In paper III a 2-helix variant, Z342min, was developed using native ligation cyclization to cross-link helices one and two resulting in a stable 2-helix scaffold and characterized in vivo. This study was performed with the aim to obtain structure-properties relationship for development of smaller Affibody molecules. Papers IV and V were devoted to development of therapeutic strategies. In paper IV, a series of peptide based chelators was investigated for labelling of Affibody molecules with 188Re to provide low renal retention. In paper V, a pretargeting approach using peptide nucleic acid was investigated. These studies were performed with the aim to overcome the high renal retention of Affibody molecules when labelled with residualizing therapeutic radionuclides. Otherwise, the particle emitting radiometals could damage the kidneys more than the tumours. The results obtained for anti-HER2 Affibody molecules summarized in this thesis might be of importance for the development of other scaffold protein based targeting agents.

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3

Jokinen, E. (Elina). "Targeted therapy sensitivity and resistance in solid malignancies." Doctoral thesis, Oulun yliopisto, 2014. http://urn.fi/urn:isbn:9789526205755.

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Abstract Cancer is a major global killer and a challenge for the healthcare worldwide. Earlier cancer has been treated with surgery, radiation, chemotherapy and hormonal therapy. Unfortunately the efficiency of these therapies has shown to be limited and this has raised an enthusiasm for development of new, targeted cancer therapies that are based on activated oncogenes. The challenge of the targeted therapies is therapy resistance, de novo, adaptive and acquired. This work investigated targeted therapy sensitivity and resistance in lung cancer, breast cancer, colorectal cancer, and melanoma cell lines. The results of this study indicate that in some non-small cell lung cancer cell lines, dual PI3K and MEK inhibition is a more efficacious treatment than inhibition of either solely. It was also showed that the maximal effect of the dual inhibition can be achieved with alternative dosing schedules that are potentially more tolerable in clinical use. Furthermore, by combining ABT-263, entinostat or dasatinib to the dual PI3K and MEK inhibition, the efficiency of the therapy can be increased. Bcl-xl downregulation is a major determinant of the apoptotic response to the triple inhibitor treatment. The current work showed that cancer stem cells can mediate resistance to targeted therapies. Since these cells follow the stochastic model, concurrent therapy with a targeted agent and a stem cell targeting drug might be needed for maximal therapeutic efficiency. This study also showed that Gö6976 acts as a potent inhibitor of mutant EGFR despite the presence of T790M, the most important mechanism of acquired resistance for EGFR tyrosine kinase inhibitors in lung cancer, both in vitro and in vivo
Tiivistelmä Syöpä on yksi johtavia kuolemanaiheuttajia ja tauti on maailmanlaajuinen haaste terveydenhuollolle. Perinteiset syöpähoidot käsittävät kirurgian, sädehoidon, kemoterapian ja hormonaalisen hoidon, mutta näiden rinnalle on noussut uusia, aktivoituneiden onkogeenien signaalien estoon perustuvia hoitoja. Tämä työ tutki kohdennettuja syöpähoitoja ja näihin hoitoihin liittyvää resistenssiä keuhko-, rinta- ja paksusuolen syövän sekä melanooman solulinjoissa. Tulokset osoittavat, että joissakin ei-pienisoluisen keuhkosyövän solulinjoissa yhdistetty PI3K- ja MEK-esto aiheuttaa tehokkaamman vasteen kuin kummankaan signaalireitin esto yksistään. Tässä työssä näytettiin myös, että maksimaalinen vaste yhdistetylle PI3K- ja MEK-estolle voidaan saavuttaa vaihtoehtoisilla annostelutavoilla, jotka ovat voisivat olla paremmin siedettyjä kliinisessä käytössä kuin kahden lääkkeen jatkuva annostelu. Tämä tutkimus osoitti lisäksi, että kaksoiseston tehokkuutta voidaan lisätä yhdistämällä hoitoon kolmas lääkeaine, ABT-263, entinostaatti tai dasatinibi. Bcl-xl proteiinilla on keskeinen rooli apoptoottisen vasteen määrittäjänä näille kolmen lääkkeen käsittelyille. Tämä työ osoitti, että syövän kantasolut voivat välittää resistenssiä kohdennetuille syöpähoidoille. Nämä solut noudattavat niin kutsuttua stokastista mallia, joten parhaan vasteen saaminen saattaa edellyttää että hoito kohdentuu sekä erilaistuneisiin että kantasolutyyppisiin syöpäsoluihin. Tässä tutkimuksessa osoitettin lisäksi, että Gö6976 toimii mutatoituneen EGFR:n estäjänä, huolimatta kehittyvää keuhkosyövissä resistenssiä välittävästä T90M mutaatiosta, sekä in vitro -että in vivo -malleissa

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MAUCERI, MATTEO. "New Targeted Molecules for the Therapy of Ovarian Cancer." Doctoral thesis, Università degli Studi di Trieste, 2022. http://hdl.handle.net/11368/3031106.

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Patients with high-grade serous ovarian cancer (HGSOC), the most aggressive epithelial ovarian cancer (EOC) subtype, have a 5-year survival rate of about 93% when diagnosed at an early stage, but it drops to 30-40% when diagnosed in the advanced stage. HGSOC aggressiveness is mainly caused by the late diagnosis (51% stage III, 29% stage IV) when the tumor has already spread in the peritoneal cavity. PIN1 is a unique peptidyl-prolyl isomerase that targets the phosphorylated Ser/Thr(Pro) motifs to regulate several key proteins in different signaling pathways. Pin1 is overexpressed in several cancer types and it regulates more than 40 oncogenes and 20 tumor suppressors. Many functions are modulated through PIN1-mediated isomerization such as cell cycle progression, cellular proliferation, invasion, migration, and apoptosis. Downregulation of Pin1 decreases tumor progression. Recently, Pin1 was shown to be overexpressed in ovarian cancer (OC) which, together with the high number of interactions with other proteins, makes Pin1 a promising target for HGSOC. The aim of this work is to investigate the effects of the PIN1 inhibitor VS10 on cancer cell lines and to find the molecular signaling pathways in which Pin1 is involved. Migration, mesothelial clearance assay, and the effects on spheroid formation and preformed spheroids were studied to better understand the effects on the metastatic process. Furthermore, in order to clarify the molecular mechanism that triggers the cytotoxicity induced by Pin1 inhibition in several OC cell lines, silencing Pin1 has been demonstrated to be associated with Ser473pAkt dephosphorylation by Western Blot (WB) analysis. Additionally, cell viability and colony-forming assays showed that Akt overexpression rescued the lethal phenotype due to Pin1 knockdown in OVCAR3 and KURAMOCHI OC cell lines. Among PIN1 inhibitors, All-trans retinoic acid (ATRA), a drug in clinic for the treatment of acute promyelocytic leukemia, has been demonstrated to be active on PIN1. Our group developed many PIN1 inhibitors including VS10, a non-covalent and selective molecule, which is active in killing cancer cells. ATRA and VS10 have been combined with first- and second-line chemotherapy drugs to treat SKOV3 cell line whether these drug combinations could work synergistically to improve current therapy. This drug combination screening showed that Doxorubicin and Caelyx act in synergy with both VS10 and ATRA. This drug combination was studied in 5 sensible and 2 OC cell lines resistant to cisplatin treatment. These results candidate Pin1 as a promising new molecular target for HGSOC patients' therapy.
Patients with high-grade serous ovarian cancer (HGSOC), the most aggressive epithelial ovarian cancer (EOC) subtype, have a 5-year survival rate of about 93% when diagnosed at an early stage, but it drops to 30-40% when diagnosed in the advanced stage. HGSOC aggressiveness is mainly caused by the late diagnosis (51% stage III, 29% stage IV) when the tumor has already spread in the peritoneal cavity. PIN1 is a unique peptidyl-prolyl isomerase that targets the phosphorylated Ser/Thr(Pro) motifs to regulate several key proteins in different signaling pathways. Pin1 is overexpressed in several cancer types and it regulates more than 40 oncogenes and 20 tumor suppressors. Many functions are modulated through PIN1-mediated isomerization such as cell cycle progression, cellular proliferation, invasion, migration, and apoptosis. Downregulation of Pin1 decreases tumor progression. Recently, Pin1 was shown to be overexpressed in ovarian cancer (OC) which, together with the high number of interactions with other proteins, makes Pin1 a promising target for HGSOC. The aim of this work is to investigate the effects of the PIN1 inhibitor VS10 on cancer cell lines and to find the molecular signaling pathways in which Pin1 is involved. Migration, mesothelial clearance assay, and the effects on spheroid formation and preformed spheroids were studied to better understand the effects on the metastatic process. Furthermore, in order to clarify the molecular mechanism that triggers the cytotoxicity induced by Pin1 inhibition in several OC cell lines, silencing Pin1 has been demonstrated to be associated with Ser473pAkt dephosphorylation by Western Blot (WB) analysis. Additionally, cell viability and colony-forming assays showed that Akt overexpression rescued the lethal phenotype due to Pin1 knockdown in OVCAR3 and KURAMOCHI OC cell lines. Among PIN1 inhibitors, All-trans retinoic acid (ATRA), a drug in clinic for the treatment of acute promyelocytic leukemia, has been demonstrated to be active on PIN1. Our group developed many PIN1 inhibitors including VS10, a non-covalent and selective molecule, which is active in killing cancer cells. ATRA and VS10 have been combined with first- and second-line chemotherapy drugs to treat SKOV3 cell line whether these drug combinations could work synergistically to improve current therapy. This drug combination screening showed that Doxorubicin and Caelyx act in synergy with both VS10 and ATRA. This drug combination was studied in 5 sensible and 2 OC cell lines resistant to cisplatin treatment. These results candidate Pin1 as a promising new molecular target for HGSOC patients' therapy.

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5

Almqvist, Ylva. "Targeted Therapy of Colorectal Cancer : Preclinical Evaluation of a Radiolabelled Antibody." Doctoral thesis, Uppsala University, Radiology, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8657.

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Targeted radiotherapy (TRT) of cancer is a promising approach that enables selective treatment of tumour cells, while sparing normal tissue. The humanized monoclonal antibody A33 (huA33) is a potential targeting agent for TRT of colorectal cancer, since its antigen is expressed in more than 95 % of all colorectal carcinomas. The aim of this thesis was to evaluate the therapeutic potential of the two huA33-based TRT-conjugates, ¹⁷⁷Lu-huA33, and ²¹¹At-huA33.

The conjugates ¹⁷⁷Lu-huA33, and ²¹¹At-huA33, bound specifically to colorectal cancer cells, both in vitro and in vivo. A dose dependent cytotoxic effect of ²¹¹At-huA33 was also demonstrated in vitro. From a therapeutic perspective, both conjugates had a favourable biodistribution in tumour-bearing nude mice, with high tumour uptake and a low uptake in normal organs (with the exception of an expected thyroid uptake of ²¹¹At). After injection of ²¹¹At-huA33, the blood absorbed a slightly higher dose than the tumour, but for ¹⁷⁷Lu-huA33, the tumour received a 12 times higher dose than blood. Two days after intravenous injection of ¹⁷⁷Lu-huA33 in tumour-bearing mice, the tumours could be clearly visualised by gamma camera imaging, with very low interference from normal tissue radioactivity. In an experimental therapy study, also performed in tumour-bearing mice, there was an excellent therapeutic effect of ¹⁷⁷Lu-huA33. About 50 % of the treated animals were tumour free 140 days after injection of ¹⁷⁷Lu-huA33, while none of the non-radioactive controls survived beyond 20 days after injection of treatment substances.

In conclusion, this thesis demonstrates that the therapeutic conjugates ¹⁷⁷Lu-huA33, and ²¹¹At-huA33, are promising targeting agents that might help improve therapy of colorectal cancer.

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Pak, Ekaterina. "Resistance to Targeted Therapy in Sonic Hedgehog Subgroup Medulloblastoma: Mechanisms and Treatment Strategies." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493334.

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Aberrant activation of the Sonic Hedgehog (Shh) signaling network is implicated in many human cancers, including the most common cancer, basal cell carcinoma (BCC) and the brain tumor medulloblastoma. Suppressing Shh signaling is thus a promising strategy in oncology. The largest and most clinically advanced group of Shh signaling inhibitors comprises selective antagonists of the pathway component Smoothened (Smo). In 2012, the Food and Drug Administration approved the first Smo antagonist, and others are now approved or in clinical trials. However, there is already evidence that some patients can have primary or develop secondary resistance to therapy. Accordingly, a more comprehensive understanding of resistance mechanisms and alternative treatment approaches are needed. Here, we describe a genome-wide transposon mutagenesis screen to identify candidate resistance genes for Smo antagonists using an in vitro model of Shh-dependent medulloblastoma. Top hits from the screen include Suppressor of fused (Sufu), a known resistance gene and negative regulator in Shh signaling, and Oral-facial-digital syndrome 1 (Ofd1), a gene associated with an X-linked developmental syndrome. Independent gain- and loss-of-function experiments confirm Ofd1 as a bona fide resistance gene. Ofd1 mutant cells have reduced numbers of primary cilia, which are necessary for transducing canonical Shh signaling. Reduction of Kif3a and Ift88, two other cilia genes, also causes resistance. Strikingly, resistant cilia mutants are still dependent on active Shh signaling downstream of Smo. These mutants lack the truncated repressor form of the Shh transcription factor Gli2, but maintain full-length Gli2 levels and therefore shift the overall balance of transcriptional activators and repressors toward pathway reactivation. Importantly, we present evidence that resistance by loss of primary cilia may have clinical relevance. Subcutaneous medulloblastoma tumors in mice that acquire de novo resistance to Smo inhibition exhibit decreased numbers of primary cilia compared to tumors that remain sensitive. Additionally, resistant BCCs from patients treated with Smo antagonists have significantly more cilia gene mutations compared to untreated BCCs. Recognizing the need for more options to treat resistant tumors, we carried out a high-throughput small molecule screen in Shh-dependent medulloblastoma cells. From a set of over 900 small molecules, we identify histone deacetylase (HDAC) inhibitors as a class of promising therapeutics. While not all HDAC inhibitors are effective, we present some with similar chemical structures that work consistently within the nanomolar range across cell lines that are both sensitive and resistant to Smo inhibitors. Preliminary results indicate that inhibition of HDACs works within the Shh signaling axis and that specific HDACs may play a role in resistant human tumors. Together, these studies reveal new resistance mechanisms and explore the development of next-generation treatments in Shh-dependent tumors.
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Fröhner, Michael, Oliver W. Hakenberg, and Manfred P. Wirth. "Molecular Therapy in Urologic Oncology." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-133789.

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During recent years, significant advances have been made in the field of molecular therapy in urologic oncology, mainly for advanced renal cell carcinoma. In this hitherto largely treatment-refractory disease, several agents have been developed targeting the von Hippel-Lindau metabolic pathway which is involved in carcinogenesis and progression of the majority of renal cell carcinomas. Although cure may not be expected, new drugs, such as the multikinase inhibitors sorafenib and sunitinib and the mammalian target of rapamycine inhibitor temsirolimus, frequently stabilize the disease course and may improve survival. Fewer data are available supporting molecular therapies in prostate, bladder, and testicular cancers. Preliminary data suggest a potential role of high-dose calcitriol and thalidomide in hormone-refractory prostate cancer, whereas targeted therapies in bladder and testicular cancers are still more or less limited to single-case experiences. The great theoretical potential and the multitude of possible targets and drug combinations, however, support further research into this exciting field of medical treatment of urologic malignancies
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

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Fröhner, Michael, Oliver W. Hakenberg, and Manfred P. Wirth. "Molecular Therapy in Urologic Oncology." Karger, 2007. https://tud.qucosa.de/id/qucosa%3A27535.

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During recent years, significant advances have been made in the field of molecular therapy in urologic oncology, mainly for advanced renal cell carcinoma. In this hitherto largely treatment-refractory disease, several agents have been developed targeting the von Hippel-Lindau metabolic pathway which is involved in carcinogenesis and progression of the majority of renal cell carcinomas. Although cure may not be expected, new drugs, such as the multikinase inhibitors sorafenib and sunitinib and the mammalian target of rapamycine inhibitor temsirolimus, frequently stabilize the disease course and may improve survival. Fewer data are available supporting molecular therapies in prostate, bladder, and testicular cancers. Preliminary data suggest a potential role of high-dose calcitriol and thalidomide in hormone-refractory prostate cancer, whereas targeted therapies in bladder and testicular cancers are still more or less limited to single-case experiences. The great theoretical potential and the multitude of possible targets and drug combinations, however, support further research into this exciting field of medical treatment of urologic malignancies.
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

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9

Trisolini, Elena. "Targeted molecular characterization of adult midline and circumscribed gliomas for the identification of new potential targets for personalized therapy." Doctoral thesis, Università del Piemonte Orientale, 2020. http://hdl.handle.net/11579/114872.

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Diffuse midline gliomas (MLG) are primary brain tumours arising from thalamus, hypothalamus, brainstem, cerebellum or spinal cord, mainly occurring in children. In adults, less than 10% of diffuse gliomas arises in midline structures and recent works suggested that this subset of tumours may present with phenotypic and molecular characteristics differing from both pediatric MLG and adult supratentorial gliomas. Circumscribed gliomas (CG) are low-grade tumours but may progress to anaplasia. They have lower genetic complexity than diffuse gliomas and could be better candidate for targeted therapies, when complete surgical resection is not feasible. Unravelling the genomic landscape of MLG and CG will better define the prognostic value of molecular biomarkers and identify new therapeutic strategies that could improve patient care. Adult patients with diagnosis of MLG and CG were retrospectively identified from «Maggiore della Carità» Hospital and GH Pitié-Salpêtrière (Paris). Mutation analysis was performed by Sanger sequencing of the major hot-spots: IDH1, IDH2, H3F3A, HIST1H3B, FGFR1, TERT promoter. FISH analyses of NTRK1-2-3 rearrangements were performed by break-apart probes on tissue microarray of MLG cases. We identified 116 (French) and 47 (Italian) patients. The two cohorts showed a lower percentage of H3F3A mutations (20% vs 33%), the mutation was not associated to a worse prognosis. FGFR1 mutations were identified in 18% of cases and are restricted to MLG. NTRKs analysis in the Italian cohort showed NTRK1 translocations in 15% of cases. We reported a high rate of FGFR1 mutations in optic nerve pilocytic astrocitomas and the presence of alternative BRAF activating mutations (Thr599_Val600insThr and Val600_Lys601>Glu). Our finding of frequent and potentially targetable FGFR1 and BRAF mutations and NTRK1 translocations have important therapeutical implications in the current context of clinical trials, and further reinforces the need for molecular analyses.

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Zhang, Zhenfeng. "Study of Molecular Mechanisms of Sensitivity and Resistance to EGFR-Targeted Therapy in Lung Cancer." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1278615774.

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McCourt, Clare Margaret. "Novel strategies to enhance androgen receptor-targeted therapy in prostate cancer : a molecular and pharmacological appraoach." Thesis, Queen's University Belfast, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.579736.

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Prostate cancer is the most common cancer in men in the U.K. and the second leading cause of cancer-related death in men in the Western world. Androgen deprivation therapy (ADT), for example the anti-androgen bicalutamide, is the cornerstone treatment for prostate cancer, including advanced disease. Resistance to anti-androgens is a major impediment to the effective treatment of prostate cancer and a key contributor to the development of a castrate-resistant phenotype. Elucidating the underlying aberrant signalling pathways leading to bicalutamide resistance are necessary to design and develop novel treatment strategies that enhance the efficacy of bicalutamide and delay the emergence of castrate-resistant prostate cancer (CRPC). The chemokine IL-8 IS over-expressed in prostate cancer and has been implicated in the tumourigensis, angiogenesis and metastasis of disease. Ligand- independent activation of the AR by growth factors and chemokines is a key pathway by which prostate cancer cells can survive and flourish following ADT. We have previously reported that IL-8 can activate the AR, independent of androgen binding, leading to the downstream transcription of AR-related pro-survival and anti- apoptotic genes. In the current study, we have shown that IL-8 can induce the expression of an anti-apoptotic AR-regulated gene c-FLIP; in so doing, the regulation of c-FLIP may maintain the survival of prostate cancer cells and progression of disease. Increased expression and activity of AR co-activators is another principle pathway leading to the development of CRPC and we have shown that IL-8 can up- regulate the expression of the AR co-activator β-catenin, upstream of c-FLIP. Our data suggests that co-operation between the AR and β-catenin, following IL-8 stimulation, enhances the expression of c-FLIP. Over-expression of c-FLIP in human prostate cancer is responsible for survival and anti-apoptosis. In the current study, we have shown that molecular and pharmacological targeting of c-FLIP can enhance the efficacy of bicalutamide and is potentially an effective treatment modality for prostate cancer to delay the onset of castrate resistance. In an initial series of experiments, siRNA-mediated silencing of c-FLIP, not only resulted in apoptosis but sensitised prostate cancer cells to bicalutamide- induced apoptosis. Due to the limitations of RNAi-based therapies in the clinic; we exploited the use of several small molecules that decrease c-FLIP expression. The . HDACi droxinostat and SAHA, down-regulated c-FLIP and induced apoptosis in both the androgen-dependent cell lines tested. Additionally, pre-treatment with either HDACi sensitised cells to bicalutamide and further decreased c-FLIP expression. We extended our studies using SAHA as this HDACi has received regulatory approval for the treatment of cutaneous T cell lymphoma and clinically relevant doses were able to induce apoptosis in prostate cancer cells. The combination of SAHA and bicalutamide resulted in significant apoptosis through the down-regulation of several anti-apoptotic and pro-survival regulators. In further experiments, our findings suggest cell-specific regulation of c-FLIP in drug response relating to the relative basal activities and status of the AR and NF-KB transcription factors between 22RVl and LNCaP cells. We propose that SAHA and bicalutamide is a clinically relevant treatment strategy for the regression of 22RVl- and LNCaP- like human prostate tumours as a means to circumvent bicalutamide resistance and delay the onset of advanced CRPC. To determine the potential clinical benefit of treating prostate cancer patients with SAHA and bicalutamide, we broadened our investigations using the VCaP androgen-sensitive prostate cancer cell line, representative of castrate-resistant metastatic tumour cells that possess a hyper-activated AR. We identified that VCaP cells are intrinsically resistant to SAHA-induced apoptosis and that this may be associated with increased basal expression of c-FLIP and BCL-2 in this cell line (relative to 22RVl and LNCaP cells) and the inability of SAHA to down-regulate the expression of these anti-apoptotic proteins. Additionally, no sensitisation to bicalutamide was observed in VCaP cells and SAHA induced the transcriptional activity of the AR and NF-KB transcription factors. In summary, our findings generated using VCaP cells suggest that the combination of SAHA and bicalutamide would not be a valuable treatment modality to use in metastatic castrate-resistant patients and may cause disease progression as a result of SAHA-induced activation of transcription factors. We propose that SAHA and bicalutamide is a potentially effective treatment post-radiation or post-radical prostatectomy to delay the development of an advanced castrate-resistant phenotype.

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Toro-Gonzalez, Miguel. "LANTHANIDE-BASED CORE-SHELL NANOPARTICLES AS MULTIFUNCTIONAL PLATFORMS FOR TARGETED RADIONUCLIDE THERAPY AND MULTIMODAL MOLECULAR IMAGING." VCU Scholars Compass, 2018. https://scholarscompass.vcu.edu/etd/5647.

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Lanthanide phosphate (LnPO4) and lanthanide vanadate (LnVO4) nanoparticles (NPs) are promising platforms for theranostic applications because of their chemical stability, low solubility, low toxicity, and unique luminescence and magnetic properties. Motivated by the high radiation resistance and ability to host actinides of naturally occurring lanthanide-based compounds, LnPO4 and LnVO4 NPs were studied as radionuclide carriers for targeted radionuclide therapy using in vivoα-generators, 223Ra, 225Ac, and 227Th. The implementation of these radionuclides has shown potential for the treatment of micrometastases and solid tumors as well as challenges in the retention of decay daughters at the target site to minimize unwanted radiotoxicity. LnPO4 and LnVO4 core-shell NPs doped with either 156Eu, a “cocktail” of 85, 89Sr and 156Eu, or in vivo α-generators 223Ra, 225Ac, and 227Th were synthesized in aqueous media. In vitro retention of radionuclides was investigated by dialyzing the radionuclide-doped NPs suspensions against deionized water and quantifying the activity in dialysate aliquots over time. The crystal structure, morphology, physical stability, luminescence and magnetic properties were evaluated. Partial retention of 156Eu (~70–95%) and 85, 89Sr (>80%) was evidenced in LnPO4 core NPs, while 227Th and decay daughters were quantitatively retained in LaPO4 core + 2 shells NPs (>99%). Gd0.8Eu0.2VO4 and GdVO4 core-shell NPs showed partial retention of 223Ra (~75%), 225Ac (75–95%), 227Th (>96%), and decay daughters. Radionuclide retention was influenced by the lanthanide concentration, crystal structure, and number of shells. The partial retention of radionuclides in both LnPO4 and LnVO4 core-shell NPs may enhance the treatment efficacy while minimizing unwanted toxicity. LnVO4 core and core-shell NPs have potential as carriers of short-lived radionuclides for both diagnostic and therapeutic applications. Emission intensities were higher for LnVO4 with respect to LnPO4 NPs, whereas no significant difference was observed in the magnetic susceptibilities. GdVO4 core NPs displayed enhancement of the signal intensity in T1-weighted images. This work evidences the promising application of both LnPO4 and LnVO4 NPs as platforms for targeted radionuclide therapy and multimodal molecular imaging.

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Kashima(Yamashita), Yoriko. "Studies for maximizing value of antibody drugs against tumors." Kyoto University, 2014. http://hdl.handle.net/2433/193551.

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Altai, Mohamed. "Tumour Targeting using Radiolabelled Affibody Molecules : Influence of Labelling Chemistry." Doctoral thesis, Uppsala universitet, Enheten för biomedicinsk strålningsvetenskap, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-229090.

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Affibody molecules are promising candidates for targeted radionuclide-based imaging and therapy applications. Optimisation of targeting properties would permit the in vivo visualization of cancer-specific surface receptors with high contrast. In therapy, this may increase the ratio of radioactivity uptake between tumour and normal tissues. This thesis work is based on 5 original research articles (papers I-V) and focuses on optimisation of targeting properties of anti-HER2 affibody molecules by optimising the labelling chemistry. Paper I and II report the comparative evaluation of the anti-HER2 ZHER2:2395 affibody molecule site specifically labelled with 111In (suitable for SPECT imaging) and 68Ga (suitable for PET imaging) using the thiol reactive derivatives of DOTA and NODAGA as chelators. The incorporation of different macrocyclic chelators and labelling with different radionuclides modified the biodistribution properties of affibody molecules. This indicates that the labelling strategy may have a profound effect on the targeting properties of radiotracers and must be carefully optimized. Paper III reports the study of the mechanism of renal reabsorption of anti-HER2 ZHER2:2395 affibody molecule. An unknown receptor (not HER2) is suspected to be responsible for the high reabsorption of ZHER2:2395 molecules in the kidneys. Paper IV reports the optimization and development of in vivo targeting properties of 188Re-labelled anti-HER2 affibody molecules. By using an array of peptide based chelators, it was found that substitution of one amino acid by another or changing its position can have a dramatic effect on the biodistribution properties of 188Re-labelled affibody molecules. This permitted the selection of –GGGC chelator whichdemonstrated the lowest retention of radioactivity in kidneys compared to other variants and showed excellent tumour targeting properties. Paper V reports the preclinical evaluation of 188Re-ZHER2:V2 as a potential candidate for targeted radionuclide therapy of HER2-expressing tumours. In vivo experiments in mice along with dosimetry assessment in both murine and human models revealed that future human radiotherapy studies using 188Re-ZHER2:V2 may be feasible. It would be reasonable to believe that the results of optimisation of anti-HER2 affibody molecules summarized in this thesis can be of importance for the development of other scaffold protein-based targeting agents.

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Moretti, Luigi. "Molecular targeting for tumor radiosensitization: implications of apoptosis and autophagy signaling in combined anticancer therapy." Doctoral thesis, Universite Libre de Bruxelles, 2015. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/218736.

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The central hypothesis supporting the present work is that the effectiveness of radiation therapy for cancer is often limited due to defects in key apoptosis regulators, such as Bcl-2 family members, that contribute to cancer ability to evade apoptosis. One way to bypass this resistance to radiotherapy is to target cell death pathways, aiming to sensitize tumours to radiation and enhance the therapeutic ratio in cancer. To test this central hypothesis, we took a dual approach: one targeted apoptosis and the other targeted autophagy.
First, we focused on the apoptotic signaling. The Bcl-2 family comprises antiapoptotic members, such as Bcl-2, Mcl-1, and Bcl-XL, and proapoptotic members, such as Bax, Bak, and Bid. The Bcl-2 family controls the integrity of the outer mitochondrial membrane and is critical in determining the susceptibility of cells to apoptosis induced by the intrinsic pathway. The balance between cell survival and cell death is modulated by the ratios and interactions of antiapoptotic and proapoptotic Bcl-2 family proteins. Overexpression of Bcl-2 or Bcl-XL is observed in several cancers, including lung, colorectal, prostate, and breast cancers, and has been shown to confer resistance to various anticancer agents, including radiotherapy. In cancer cells, alterations in the amounts of these antiapoptotic Bcl-2 proteins promote cell survival, among others by contributing to their evasion from treatment-induced apoptosis. We made the observation that lung cancer cells have different radiosensitivity. On the basis of their relative response to radiotherapy, we stratified lung cancer cells into two groups (higher or lower sensitivity), and selected a representative cell line of each group for more in-depth study: A549 (resistant) and HCC2429 (sensitive). We found that the expression levels of Bcl-XL expression, which is antiapoptotic, was dramatically higher in A549, whereas almost not detected in HCC2429. We then hypothesized that AT-101, a pan-Bcl-2 inhibitor, had the potential to radiosensitize lung cancer by restoring radiation-induced apoptosis. When administered alone, AT-101 resulted in increased apoptosis in a concentration-dependent manner in both groups, with enhanced activity in HCC2429 even at lower concentration. Furthermore, AT-101 promoted radiosensitivity of A549 and HCC2429 cells (p < 0.005). A549 cells required increased AT-101 dose to achieve the same level of cytotoxicity than HCC2429 cells. These investigations suggest that the Bcl-2 family members may serve as effective therapeutic targets in lung cancer. However, the potential of AT-101 as an agent that enhances the therapeutic ratio of radiotherapy varies depending on the lung cancer clone.
Next, we turned to a different approach, focusing on the inhibition of apoptosis instead of its promotion. This work hypothesis was based on previous observations looking at the role of radiation-induced apoptosis by knockdown of Bak and Bax. The radiosensitivity of breast and lung cancer in vitro was increased through autophagy, an alternate type of programmed cell death. Consistently, radiation-induced apoptosis accounts for a minor portion of cell death in irradiated solid tumors. The hypothesis of our work was that apoptosis inhibition would increase radiation-induced autophagy and tumor sensitivity to radiation. To block apoptosis, we used Z-VAD, a broad-spectrum caspase inhibitor, and examined its in vitro and in vivo effects on breast and lung cancer models. Z-VAD markedly radiosensitized breast and lung cancer cells in vitro, with a radiation dose enhancement ratio of 1.31 (P < 0.003). The enhanced tumor cytotoxicity was associated with induction of autophagy. In both breast and lung cancer mice xenograft models, the administration of Z-VAD concurrent with radiation produced a significant tumor growth delay compared with radiation alone and was well tolerated. Interestingly, Z-VAD also had a dramatic antiangiogenic effect when combined with radiation both in vitro and in vivo. Thus, Z-VAD represents an attractive anticancer therapeutic strategy. We further explored the potential of apoptosis inhibition as a way to sensitize cancer to radiation using a more selective chemical, M867, which is a reversible caspase-3 inhibitor. In an in vivo mouse hind limb lung cancer model, the administration of M867 with ionizing radiation was well tolerated, and produced a significant tumor growth delay compared with radiation alone. A dramatic decrease in tumor vasculature and tumor cell proliferation was observed with M867 despite the reduced levels of apoptosis. The radiosensitizing effect of M867 through the inhibition of caspases was validated using a caspase-3/-7 double-knockout (DKO) mouse embryonic fibroblasts (MEF) cell model. Consistent with our previous results, autophagy contributed to the mechanism of increased cell death, following inhibition of apoptosis. Finally, we investigated the mechanism by which radiation triggers autophagy in caspase-3/7-deficient cells, and found the involvement of endoplasmic reticulum (ER) stress. The ER activates a survival pathway, the unfolded protein response, which involves ER-localized transmembrane proteins such as protein kinase-like ER kinase (PERK), inositol-requiring enzyme-1, and activating transcription factor-6. In this study, we found that PERK is essential for radiation-induced autophagy and radiosensitivity in caspase-3/7 double-knockout cells. Irradiation of these cells increased expression of phosphorylated-eIF2a. Similar results were seen after administration of tunicamycin (TM), a well-known ER stress inducer. We found that the administration of TM with radiation in MCF-7 breast cancer cells, which are lacking functional caspase-3 and are relatively resistant to many anticancer agents, enhances radiation sensitivity. Our findings revealed ER stress as a novel potential mechanism of radiation-induced autophagy in caspase-3/7-deficient cells and as a potential strategy to maximize efficiency of radiation therapy in breast cancer. Our data suggested that caspase-3 has a critical role in modulating the PERK/eIF2a pathway after radiation.
Many cancers exhibit multiple deregulations in cell death pathways, allowing for the subsequent promotion of tumor cell survival, and contributing to a relatively low response rate to therapies based on the use of pro-apoptotic strategies. As we have showed, there is a potential for novel anticancer strategies to overcome resistant cancer cells with defective apoptosis machinery in order to improve overall therapeutic outcomes. Such novel approach is to drive cancer cells towards autophagy, as demonstrated by our experiments that studied the effect of radiation on the induction of autophagy in caspase-deficient models.
Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)
info:eu-repo/semantics/nonPublished

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Senatore, Marcela Andrea Duran Haun 1974. "Meta-análise : estudos da efetividade de terapias com fármacos alvo moleculares para o tratamento do tumor renal metastático." [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/313127.

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Orientadores: Wagner Eduardo Matheus, Ubirajara Ferreira
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
Made available in DSpace on 2018-08-24T12:07:43Z (GMT). No. of bitstreams: 1 Senatore_MarcelaAndreaDuranHaun_D.pdf: 1829482 bytes, checksum: bc20dee71d7524f229d7b76160f9e38b (MD5) Previous issue date: 2014
Resumo: Atualmente existem diferentes agentes para o tratamento do câncer renal avançado. O objetivo principal desse trabalho foi realizar revisão sistemática com meta-análise dos estudos clínicos randomizados que compararam: sorafenibe, sunitinibe, bevacizumabe, temsirolimus e everolimus ao interferon-?. Para isto foi realizada revisão sistemática da literatura em diferentes bancos de dados: EMBASE, LILACS e PUBMED, identificando estudos clínicos randomizados que compararam as terapias alvo moleculares (TAM) disponíveis versus interferon-alfa para tratamento de pacientes com câncer renal avançado. Para o tratamento de 1a linha foram encontrados 10 estudos que avaliaram as terapias com sunitinibe, sorafenibe, bevacizumabe e temsirolimus; e três estudos que avaliaram o sorafenibe e everolimus como tratamento de 2a linha. O Risco Relativo (RR) da sobrevida livre de progressão (SLP) dos estudos de 1a linha foi de 0.83, intervalo de confiança (IC) [0.78-0.87] com I2= 94% e p<0.00001. Os melhores resultados foram: o estudo do sunitinibe, 0.38, IC [0.25-0.58], do bevacizumabe com RR de 0.62, IC [0.47-0.83] e do temsirolimus, 0.78, IC [0.70-0.87]. A meta-análise não demonstrou benefício quanto à sobrevida global (SG), no tratamento de 1a linha com sunitinibe e temsirolimus. Os tratamentos de 1ª linha com sorafenibe e bevacizumabe não associaram benefícios clínicos significativos. Não foi possível realizar meta-análise nos estudos do tratamento de 2a linha, pois, as populações eram diferentes. Logo, para o tratamento de 1a linha, sunitinibe e temsirolimus foram a terapias mais efetivas, quanto a SLP. No tratamento de 2a linha, o sorafenibe e everolimus foram relacionados à melhora da SLP. Em todos os estudos de 1a linha os pacientes não apresentaram melhora de SG e a qualidade metodológica não foram adequadas, portanto esses resultados devem ser analisados com cautela
Abstract: Currently, there are different agents for the treatment of advanced kidney cancer. The main aim of this study was to perform a systematic review and meta-analysis of randomized clinical trials that compared: sorafenib, sunitinib, bevacizumab, temsirolimus and everolimus. It was performed a systematic review of the literature in different databases: EMBASE, LILACS and PubMed, identifying randomized clinical trials that compared the available therapies target cells versus alpha-interferon for the patient treatments with advanced kidney cancer. For the treatment of first-line were found 10 studies that evaluated the therapy with sunitinib, sorafenib, bevacizumab and temsirolimus and three studies evaluating sorafenib and everolimus as a treatment second-line. The relative risk of progression free survival of first line studies was 0.83, confidence interval (CI) [0.78-0.87] with I2 = 94% and p <0.00001. The best results were: the study of sunitinib, 0.38, CI [0:25 to 0:58], bevacizumab with RR of 0.62, CI [0.47-0.83] and temsirolimus, 0.78, CI [0.70-0.87]. The meta-analysis showed no benefit on overall survival in first-line treatment with sunitinib and temsirolimus. The first-line treatment with sorafenib and bevacizumab not associated significant clinical benefits. Unable to perform meta-analysis on studies of second-line treatment, because the cohorts were different between them. For the treatment of first-line, sunitinib and temsirolimus were more effective therapies, as the progression free survival (PFS). In the second line treatment, sorafenib and everolimus was associated with improved PFS. In these studies, patients showed no improvement in overall survival (OS) and methodological quality were not adequate, so these results should be analyzed with caution
Doutorado
Fisiopatologia Cirúrgica
Doutora em Ciências

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17

Westerberg, Cornelia. "Development of an Affibody-based Prodrug Against HER2 for Cancer Therapy." Thesis, KTH, Proteinvetenskap, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-302213.

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Affinity proteins constitute an important category of cancer therapeutics. Owing to properties such as high target affinity and selectivity, therapeutic proteins offer more targeted therapy than small molecule drugs. The target molecules are typically proteins that are overexpressed on the surface of tumour cells, such as membrane-bound receptors. However, these surface proteins are usually expressed in normal tissues as well, resulting in on-target off-tumour toxicity. Proteins with a higher tissue selectivity are thus needed. Here, this has been addressed by developing prodrug proteins dependent on cancer-specific proteases for activation. The prodrugs were composed of a target-binding affibody (active domain) connected to a masking affibody (masking domain) by a peptide linker including a protease substrate. The target of the prodrugs developed in this project was the HER2 receptor, which is overexpressed in several cancer types. Three prodrug candidates were developed, produced and characterised based on their ability to be activated by their respective protease. The hypothesis that the prodrugs could be activated and thus bind to HER2 in cancer cells was tested using biosensor assays, as well as preliminary cancer cell assays. One of the three candidates showed strong potential to be used as a targeted therapy for cancer treatment in the future.
Affinitetsproteiner utgör en viktig kategori av cancerläkemedel. Jämfört med småmolekylära läkemedel är affinitetsproteiner mer riktade, då de har högre affinitet och selektivitet än små molekyler. Oftast utgörs det molekylära målet av ett protein som överuttrycks på ytan av cancerceller, så som membranbundna receptorer. Dessvärre uttrycks de flesta cancerspecifika proteiner i mindre mängd även i normal vävnad. Detta leder till oönskade effekter som kan ge upphov till biverkningar. I syfte att utveckla mer vävnadsspecifika läkemedel har här affibody-baserade “prodrugs”, beroende av cancerspecifika proteaser för aktivering, tagits fram. Prodrug-proteinerna i detta projekt är riktade mot HER2-receptorn, som är överuttryckt i flera typer av cancer. Tre kandidater togs fram och utvärderades med avseende på deras förmåga att aktiveras av sina respektive proteaser. För att testa hypotesen att kandidaterna kunde binda till HER2 på cancerceller efter proteasaktivering användes biosensoranalys samt experiment med cancerceller. En av kandidaterna visade stark potential att kunna användas som ett riktat läkemedel mot cancer i framtiden.

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Calvagno, Giuseppe Stefano. "Nuovi targets molecolari nel trattamento dell'epatocarcinoma. Review della letteratura e nostra esperienza." Doctoral thesis, Università di Catania, 2015. http://hdl.handle.net/10761/3772.

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BACKGROUND: Il carcinoma epatocellulare è uno dei tumori maligni più comuni e letali nel mondo . Negli ultimi 15 anni, l'incidenza di carcinoma epatocellulare è più che raddoppiata . A causa della diagnosi tardiva e / o dell avanzato sottostante quadro di cirrosi epatica, sono disponibili solo opzioni di trattamento limitate con beneficio clinico marginale per il 70% dei pazienti . Nel corso degli ultimi decenni , nessuna terapia sistemica citotossica convenzionale era efficace, contribuendo alla prognosi infausta dei pazienti con HCC . Una migliore conoscenza dei fenomeni di epatocarcinogenesi molecolare offre oggi l'opportunità di terapie mirate. MATERIALI E METODI: Una ricerca della letteratura è stata effettuata utilizzando la letteratura cancro , la PubMed , Scopus e Web of Science ( WOS ) database per le seguenti parole chiave: "carcinoma epatocellulare" "epatocarcinogenesi molecolare", "terapia mirata" e "immunoterapia". DISCUSSIONE E CONCLUSIONI: Le decisioni terapeutiche sono complesse e dipendono dalla stadiazione del tumore, dalla presenza di ipertensione portale, e dal grado della disfunzione epatica di base. La conoscenza dei meccanismi molecolari di epatocarcinogenesi ha ampliato l'orizzonte per i pazienti con carcinoma epatocellulare avanzato. Negli ultimi anni, diversi agenti con preciso target molecolare sono stati valutati in studi clinici di carcinoma epatocellulare avanzato. In futuro, nuove opzioni terapeutiche saranno rappresentate da una miscela di vaccini immunoterapia-simili e modulatori delle cellule T, integrate da inibitori mirati di vie di signaling tumorale.

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Aristizabal, Prada Elke Tatjana [Verfasser], and Christoph [Akademischer Betreuer] Auernhammer. "Preclinical in vitro investigation to evaluate novel molecular targeted therapy options for neuroendocrine neoplasms / Elke Tatjana Aristizabal Prada ; Betreuer: Christoph Auernhammer." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2018. http://d-nb.info/1166559599/34.

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Calais, Jérémie. "PSMA-targeted theranostics : from research to standard-of-care." Electronic Thesis or Diss., université Paris-Saclay, 2023. http://www.theses.fr/2023UPAST192.

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L'objectif de ce manuscrit de thèse Thèse de doctorat par Validation des Acquis de l'Expérience (VAE) est de mettre en évidence les principales études de recherche menées à UCLA qui ont conduit à l'implémentation clinique des technique théranostique ciblant le PSMA aux États-Unis. Le manuscrit est divisé en 2 parties : l'imagerie et la thérapie.Dans la première partie sont fournis les essais pivots d'efficacité diagnostique utilisés pour l'autorisation par la FDA du 68Ga-PSMA-11 (Article #1: Performance diagnostique de la TEP-PSMA pour la localisation de la récidive du cancer de la prostate: un essai clinique d'imagerie prospective multicentrique de phase 3 (n=635), PMID 30920593; Article #2: Performance diagnostique de la TEP-PSMA pour la détection des métastases ganglionnaires pelviennes avant prostatectomie radicale et curage ganglionnaire pelvien: un essai clinique d'imagerie prospective multicentrique de phase 3 (n=764), PMID 34529005), des études comparatives comparant la TEP-PSMA aux techniques d'imagerie standard (Article #3: TEP/TDM a la 18F-Fluciclovine et au 68Ga-PSMA-11 chez les patients présentant une récidive biochimique après prostatectomie avec un taux de PSA ≤2,0 ng/ml: un essai d'imagerie comparative prospectif monocentrique (n=50), PMID 31375469; Article #4: Comparaison de la TEP/TDM-PSMA et de l'IRMmp avec référence par histopathologie dans la détection, la localisation intra-prostatique et la détermination de l'extension locale du cancer primitif de la prostate: résultats dans une cohorte monocentrique d'un essai prospectif (n=74), PMID 34649942) et une étude montrant un impact significatif sur la prise en charge (Article #5: Cartographie par TEP/TDM-PSMA de la récidive biochimique du cancer de la prostate après prostatectomie radicale chez 270 patients avec un taux de PSA<1,0 ng/ml : Impact sur la planification de la radiothérapie de rattrapage (n=270), PMID 29123013) qui a conduit à un essai randomisé visant à montrer une amélioration des résultats cliniques grâce à la TEP-PSMA (Article #6: Essai randomisé prospectif de phase 3 de radiothérapie de rattrapage du cancer de la prostate guidée par TEP-PSMA [PSMA-SRT] (n=193), PMID 30616601; Article #7: Mise à jour de l'essai PSMA-SRT NCT03582774: un Essai randomisé prospectif de phase 3 de radiothérapie de rattrapage du cancer de la prostate guidée par TEP-PSMA (n=193), PMID 33386288).Dans la deuxième partie sont présentés les résultats du premier essai de phase 2 de la thérapie au 177Lu-PSMA aux USA qui a précédé l'essai VISION (Article #8 : Essai prospectif de phase 2 de radiothérapie moléculaire au 177Lu-PSMA-617 pour cancer de la prostate métastatique résistant à la castration (RESIST-PC): Résultats d'efficacité de la cohorte UCLA (n=43), PMID 34016732; Article #9: Tolérance et sécurité de la radiothérapie moléculaire au 177Lu-PSMA-617: résultats de l'essai prospectif multicentrique de phase 2 RESIST-PC NCT03042312 (n=64), PMID 34272322), des études rétrospectives multicentriques visant à affiner les critères de sélection TEP-PSMA (Article #10: Suivi des patients négatifs au PSMA par critères TEP-PSMA de l'essai VISION traités par 177Lu-PSMA: une analyse rétrospective multicentrique (n=301), PMID 35273096; Article #11: Ratio tumeur/glande salivaire par TEP-PSMA pour prédire la réponse à la thérapie par 177Lu-PSMA: une étude rétrospective multicentrique internationale (n=237), PMID 36997329; Article #12: Nomogrammes pour prédire les résultats après traitement au 177Lu-PSMA chez les patients atteints d'un cancer de la prostate métastatique résistant à la castration: une étude rétrospective internationale multicentrique (n=270), PMID 34246328) et une revue narrative des mécanismes de résistance à la thérapie au 177Lu-PSMA (Article #13: Prédicteurs de réponse et utilisation dans le monde réel de la radiothérapie moléculaire ciblée du cancer de la prostate: PSMA et au-delà. PMID 35609224)
The aim of this manuscript for PhD by Accreditation of Prior Learning is to highlight the key research studies conducted at UCLA which lead to the clinical implementation of PSMA-theranostics in the US. The manuscript is divided in 2 parts: imaging and Therapy.In the first part are provided the pivotal trials of diagnostic efficacy used for the FDA approval of 68Ga-PSMA-11 (Article #1: Assessment of 68Ga-PSMA-11 PET Accuracy in Localizing Recurrent Prostate Cancer: A Prospective Multicenter Single-Arm Phase 3 Clinical Trial (n=635), WP Fendler at al. JAMA Oncol 2019 PMID 30920593; Article #2: Diagnostic Accuracy of 68Ga-PSMA-11 PET for Pelvic Nodal Metastasis Detection Prior to Radical Prostatectomy and Pelvic Lymph Node Dissection: A Multicenter Prospective Phase 3 Imaging Trial (n=764), TA Hope et al. JAMA Oncol 2021 PMID 34529005), head-to-head comparison trials comparing PSMA PET to standard of care imaging techniques (Article #3: 18F-Fluciclovine and 68Ga-PSMA-11 PET/CT in patients with biochemical recurrence after prostatectomy at PSA levels of ≤2.0ng/ml: a prospective single-center single-arm comparative imaging trial (n=50), J Calais et al. Lancet Oncol 2019 PMID 31375469; Article #4: Head-to-head comparison of 68Ga-PSMA-11 PET/CT and mpMRI with histopathology gold-standard in the detection, intra-prostatic localization and local extension of primary prostate cancer: results from a prospective single-center imaging trial (n=74), I Sonni et al. J Nucl Med 2022 PMID 34649942) and a study showing significant impact on management (Article #5: 68Ga-PSMA-11 PET/CT mapping of prostate cancer biochemical recurrence following radical prostatectomy in 270 patients with PSA<1.0ng/ml: Impact on Salvage Radiotherapy Planning (n=270), J Calais et al. J Nucl Med 2018 PMID 29123013) that lead to a randomized imaging trial powered for clinical outcome (Article #6: Randomized prospective phase 3 trial of 68Ga-PSMA-11 PET/CT molecular imaging for prostate cancer salvage radiotherapy planning [PSMA-SRT] (n=193), J Calais et al. BMC cancer 2019 PMID 30616601; Article #7: Update from PSMA-SRT Trial NCT03582774: A Randomized Phase 3 Imaging Trial of Prostate-specific Membrane Antigen Positron Emission Tomography for Salvage Radiation Therapy for Prostate Cancer Recurrence Powered for Clinical Outcome (n=193), J Calais et al. Eur Urol Focus PMID 33386288).In the second part are provided the results of the first phase 2 trial of 177Lu-PSMA therapy in the US that preceded the VISION trial (Article #8: Prospective phase 2 trial of PSMA-targeted molecular RadiothErapy with 177Lu-PSMA-617 for metastatic Castration-reSISTant Prostate Cancer (RESIST-PC): Efficacy results of the UCLA cohort (n=43), J Calais et al. J Nucl Med 2021 PMID 34016732; Article #9: Safety of PSMA-targeted molecular radioligand therapy with 177Lu-PSMA-617: results from the prospective multicenter phase 2 trial RESIST-PC NCT03042312 (n=64), J Calais et al. J Nucl Med 2021 PMID 34272322), multicenter retrospective studies aiming at refining the PSMA-PET selection criteria (Article #10: Outcome of patients with PSMA-PET/CT screen failure by VISION criteria and treated with 177Lu-PSMA therapy: a multicenter retrospective analysis (n=301), M Hotta et al. J Nucl Med 2022 PMID 35273096; Article #11: PSMA PET Tumor-to-Salivary Gland Ratio to Predict Response to 177Lu-PSMA Radioligand Therapy: An International Multicenter Retrospective Study (n=237), M Hotta et al. J Nucl Med 2023 PMID 36997329; Article #12: Nomograms to predict outcomes after 177Lu-PSMA therapy in men with metastatic castration-resistant prostate cancer: an international, multicenter, retrospective study (n=270), A Gafita et al. Lancet Oncol 2021 PMID 34246328) and a narrative review of the mechanisms of resistance to 177Lu-PSMA therapy (Article #13: Predictors and Real-World Use of Prostate-Specific Radioligand Therapy: PSMA and Beyond. A Gafita et al. Am Soc Clin Oncol Educ Book 2022 PMID 35609224)

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Heinen, Tiago Elias. "Potencial terepêutico de inibidores de TRK no tratamento de sarcoma de Ewing : um estudo celular e molecular." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2015. http://hdl.handle.net/10183/150631.

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O sarcoma de Ewing (SE) é um dos mais agressivos tipos de câncer pediátrico. Apesar dos significativos avanços no tratamento dessa doença, ainda há uma grande necessidade no aumento das taxas de cura, redução da toxicidade quimioterápica e redução da resistência ao tratamento. Tem sido proposto que SE provém de precursores neuronais, podendo ter sua fisiologia afetada, pois, por neurotrofinas (NTs). Examinamos a influência de receptores de NTs (Trks) em SE. Foram avaliadas a expressão proteica de NTs (NGF e BDNF) e seus receptores (TrkA e TrkB, respectivamente) em amostras de tumores de pacientes com SE, e a expressão de mRNA nas linhagens celulares RD-ES e SK-ES-1. O tratamento das linhagens com o pan-inibidor de Trks (K252a) modificou a morfologia celular e diminuiu a expressão de mRNA de NGF, TrkA, BDNF e TrkB. Ainda, a inibição de Trks diminuiu drasticamente a proliferação e capacidade clonogênica celular. Efeitos sinérgicos foram observados quando as células foram tratadas em conjunto com baixas doses de quimioterápicos, tanto em células selvagens de SE, quanto nas quais induzimos quimiorresistência. Esse estudo sugere, pela primeira vez, que a inibição de Trks reduz a proliferação e sobrevivência celular em SE, além de aumentar a sensibilidade ao tratamento quimioterápico.
Ewing's sarcoma (ES) is one of the most aggressive types of pediatric cancer. Despite significant advances in the treatment of this disease, there is still a great need in increasing cure rates, reducing chemotherapy toxicity and treatment resistance. It has been proposed that ES might derive from neuronal precursors and may be influenced, therefore, by neurotrophins (NTs). We have examined the influence of Trk neurotrophin receptors in ES. Protein expression of NTs (NGF and BDNF) and their receptors (TrkA, and TrkB, respectively) was detected in tumor samples from patients with ES, and mRNA expression was analyzed in the RD-ES, SK-ES-1 cell lines. Treating cells with a Trk Pan-inhibitor (K252a) altered cell morphology and decreased the mRNA expression of NGF, TrkA, BDNF, and TrkB. In addition, Trk inhibition dramatically decreased cell proliferation and clonogenic capacity. Synergistic effects were observed when cells were treated in combination with low doses of cytotoxic chemotherapeutics, both in normal ES cells and cells in which chemoresistance was induced. The results suggest for the first time that Trk inhibition can reduce the proliferation and survival of ES cells and sensitize them to cytotoxic chemotherapy.

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Persson, Mikael. "Antibody Mediated Radionuclide Targeting of HER-2 for Cancer Diagnostics and Therapy : Preclinical Studies." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6798.

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Liao, Rachel Grace. "Functional Studies of Candidate Oncogenes in Non-Small Cell Lung Cancer." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11173.

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Cancer is a set of complex genetic diseases driven by diverse genomic alterations. The genomic study of cancer has enabled the discovery of novel, targetable events in almost all cancer types and in turn, has led to the development of new, targeted cancer therapies benefiting patients; however, the recent explosion of genomic datasets has also resulted in huge lists of new oncogenic factors of unknown biological relevance, and uncertainty over how best to use the data appropriately to influence patient care. Some of the most pressing questions surround the use of statistical methods to identify actionable genomic alterations in cancer and the identification of driving oncogenes in the context of the genomic evolution of cancer cells, undergone before, during, and after prolonged treatment regimens.

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Oliveira, Mónica Catarina Castro. "Proteasome-proteins: are these putative targets for basal-like breast cancer therapy with AAV-vectors?" Master's thesis, Universidade de Aveiro, 2016. http://hdl.handle.net/10773/18553.

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Mestrado em Biologia Molecular e Celular
O cancro da mama do tipo basal (BLBC) é um grupo de tumores muito agressivo associado a um mau prognóstico. De momento, não existe nenhum tratamento eficaz para o BLBC, uma vez que rapidamente adquirem resistência às terapias normalmente usadas. Assim, é urgente encontrar novas abordagens para tratar esta doença. Com base em dados anteriores, o objetivo geral deste estudo foi avaliar se o PSMA2, uma proteína do proteassoma, seria um alvo putativo para a inibição para terapia em BLBC. Desta forma, o primeiro objetivo específico foi avaliar o efeito anti-tumorigénico de vírus adeno-associados (AAV) capazes de entregar short hairpin RNAs (shRNA), anteriormente validados, capazes de inibir a expressão do PSMA2 em xenotransplantes de células BLBC em ratinho. Para atingir esse objetivo, foram testados in vivo, vetores AAV2 com shRNAs para os genes PLK1 e PSMA2 para diferentes concentrações de partículas virais (2x1010, 2x109, 2x108 partículas virais/tumor), em que células MDA-MB-468 BLBC foram injetadas na mama de ratinhos nude. Após cerca de um mês, foram realizadas injeções intratumorais com AAVs duas vezes por semana. A administração de AAV2-shPSMA2 resultou numa diminuição no crescimento do tumor sem toxicidade evidente, e este efeito foi mais significativo na concentração de 2x109 partículas virais/tumor. O segundo objetivo específico foi analisar a expressão de PSMA2 em amostras humanas de cancro da mama, o que indica que há também uma importância clínica na inibição deste gene, uma vez que se mostrou estar associado a características menos favoráveis relacionadas com tumores da mama do tipo basal. Em conclusão, embora ainda preliminar, os resultados obtidos abrem a possibilidade de direcionar uma terapia genética em BLBC usando vetores AAV recombinantes que entregam shRNAs para silenciar especificamente a expressão do gene PSMA2.
Basal-like breast cancer (BLBC) is an aggressive group of tumours associated to poor patient prognosis. Currently, there is no effective treatment for BLBC once they rapidly acquire resistance to standard therapies. For this reason, novel approaches to treat this disease are urgently needed. Based on previous data, the general goal of this study was to evaluate if PSMA2, a proteasome protein, was a putative target for inhibition in BLBC therapy. In this way, the first specific aim was to evaluate the anti-tumorigenic effect of adeno-associated virus (AAV)-based vectors, that were able to deliver validated short hairpin RNAs (shRNAs) that inhibit the expression of PSMA2 in BLBC mouse xenografts. To achieve that aim, we have tested, in vivo, AAV2 vectors with shRNAs for the genes PLK1 and PSMA2 for different concentrations of viral particles (2x1010, 2x109, 2x108 VP/tumour), MDA-MB-468 BLBC cells were injected into the mammary fat pad of nude mice and, after nearly one month, intratumoral injections with AAVs were performed twice a week. The delivery of AAV2-shPSMA2 resulted in a decrease in tumour growth with no obvious toxicity, and this effect was more significant at the concentration of 2x109 VP/mouse. The second specific aim was to analyse the expression of PSMA2 in human breast cancer samples, which indicated that there is also a clinical importance in inhibiting this gene, once it showed to be associated with less favourable features that are linked to basal-like breast tumours. In conclusion, although still preliminary, the results obtained open a possibility to direct a gene-based therapy in BLBC using recombinant AAVs that deliver shRNAs that specifically silence PSMA2 gene expression.

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Lee, Min-Hyung. "The Function of SUV39H Histone Methyltransferase in Alveolar Rhabdomyosarcoma." Case Western Reserve University School of Graduate Studies / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=case1283373657.

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Nishiya, Adriana Tomoko. "Administração intratumoral de uma toxina engenheirada ativada por uroquinase (UPA) e metaloproteinase (MMP) para o tratamento do melanoma oral canino: estudo piloto." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/5/5155/tde-06042018-132036/.

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Os melanomas malignos em cães são uma das mais frequentes neoplasias diagnosticadas na cavidade oral. Infiltração local, recidiva (15-41%) e o alto potencial para metástases em linfonodos regionais (18-53%) e pulmões (23-27%) nos animais acometidos, conferem uma menor sobrevida (131-818 dias), ressaltando a necessidade e importância do estudo de novas terapias para o tratamento efetivo da doença. As uroquinases (UPA) e metaloproteinases (MMPs) são proteases superexpressas em uma variedade de células tumorais e raramente estão presentes em células fisiologicamente normais. A toxina do Bacillus anthracis é composta por três proteínas chamadas: fator letal (LF), fator de edema (EF) e antígeno protetor (PA). A toxina foi reengenheirada para a formação de dois tipos de PAs chamadas PAU2-R200A e PAL1-I207R, ativadas por UPA e MMPs da superficie das células tumorais, respectivamente, formando um complexo semelhante a um poro celular para permitir a internalização da LF. A citotoxicidade dessa associação reengenheirada PAU2-R200A, PAL1-I207R e LF ocorre quando a LF atinge o meio intracelular e causa a morte celular por interrupção da via de sinalização celular MAPkinase. O objetivo deste estudo é avaliar o potencial terapêutico da toxina reengenheirada do Bacillus anthracis, PAU2-R200A, PAL1-I207R e LF, dependentes de UPA e MMP, em melanomas orais de cães. Três etapas foram propostas para este estudo: o estudo in vitro da citotoxicidade de 5 linhagens de melanomas caninos submetidas à toxina reengenheirada, a avaliação da expressão de UPA e MMP em amostras parafinadas de melanoma oral canino e o tratamento intratumoral com a toxina modificada em cães com melanomas orais espontâneos. A linhagem GMGD2 foi a única que demonstrou sensibilidade à toxina estudada, apesar da concentração inibitória de 50% das células ter sido alta (IC50=4.964,16 mg/dl) em relação a linhagem controle HT29-RJ (IC50=179,47). As demais linhagens não demostraram redução da viabilidade celular com o aumento da concentração da toxina reengenheirada e não atingiram a IC50. Dentre as amostras de melanomas submetidos a imuno-histoquimica, 76,6% expressavam tanto uroquinases quanto metaloproteinases. Melanomas orais espontâneos de cães variando de 231,8 a 18601,6 mm3 em volume, sem evidências de metástases, foram tratados com as aplicações da toxina modificada por via intratumoral, previamente à excisão, realizada nos dias 07 ou 14 do tratamento. Dentre os animais estudados, todos apresentaram evolução favorável classificada como doença estável e resposta parcial. Somente um animal apresentou reação local. Nenhum dos pacientes apresentou efeito colateral sistêmico importante. Os resultados sugerem que existe potencial terapêutico da toxina reengenheirada do Bacillus anthracis sobre os melanomas bucais caninos e futuros ensaios clínicos são possíveis em cães e de extrema importância para o estudo mais aprofundado da toxina como nova terapia antineoplásica
Malignant melanomas in dogs are one of the most frequent malignancies diagnosed in the oral cavity. Local infiltration, recurrence (15-41%) and the high potential for regional lymph nodes metastases (18-53%) and lungs (23-27%) in the affected animals, confer a lower survival (131-818 days), emphasizing the necessity and importance of the study of new therapies for the effective treatment of the disease. Urokinase (UPA) and metalloproteinases (MMPs) are overexpressed proteases in a variety of tumor cells and are rarely present in normal physiological cells. Bacillus anthracis toxin is composed of three proteins called lethal factor (LF), edema factor (EF) and protective antigen (PA). The toxin was re-engineered for the formation of two types of PAs called PAU2-R200A and PAL1-I207R, activated by UPA and MMPs from the surface of tumor cells, respectively, forming a cell-like complex to allow the internalization of the LF. The cytotoxicity of this association PAU2-R200A, PAL1-I207R and LF occurs when LF reaches the intracellular environment and causes cell death by disruption of the MAPkinase cell signaling pathway. The objective of this study is to evaluate the therapeutic potential of UPA and MMP-dependent Bacillus anthracis toxin (PAU2- R200A, PAL1-I207R and LF) to treat oral melanomas in dogs. Three steps were proposed: cytotoxicity assay of 5 lineages of canine melanomas submitted to the reengineered toxin, immunohistochemistry study for UPA and MMP expression in paraffin samples of canine oral melanoma and intratumoral treatment with toxin in dogs with spontaneous oral melanomas. The lineage GMGD2 was the only one that showed sensitivity to the toxin studied, although 50% inhibitory concentration of the cells was high (IC50 = 4,964.16 mg / dl) in relation to the HT29-RJ control lineage (IC 50 = 179.47). Among the samples of melanomas submitted to immunohistochemistry, 76.6% expressed both urokinase and metalloproteinases. Spontaneous oral melanomas of dogs ranging volume from 231.8 to 18601.6 mm3 with no evidence of distant metastases, were treated with the applications of intratumoral re-engineered toxin prior to surgical excision. All of them has presented favorable evolution classified as stable disease and partial response. Only one animal had a local allergic reaction. None of the patients had a significant systemic side effects. The results suggest that there is a potential therapeutic effect of re-engineered anthrax toxin on canine melanomas and future clinical trials are possible in dogs and extremely important for further studies on the role of the B. anthracis toxin as a new antineoplastic agent

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Sengal, Asmerom Tesfamariam. "Prognostic, predictive, and therapeutic role of FGFR2 isoforms and cognate FGF ligands in endometrial cancer." Thesis, Queensland University of Technology, 2020. https://eprints.qut.edu.au/205851/1/Asmerom%20Tesfamariam_Sengal_Thesis.pdf.

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This project investigated the role of FGFR2 isoforms (FGFR2b/FGFR2c) and their cognate FGF ligands in endometrial cancer development, prognosis, and treatment response via designing and validating an innovative BaseScope RNA in-situ hybridization assay and generating patient tumour-derived organoids. FGFR2c and high FGF18 expression were significantly associated with aggressive tumour characteristics and poor survival outcome. It was also noted FGFR2c expression is associated with progestin treatment failure in atypical hyperplasia and well-differentiated endometrial cancers. Overall, FGFR2c and FGF18 are independent prognostic biomarkers that could improve our ability to predict patient prognosis and predict response to FGFR inhibitor treatment in endometrial cancer.

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Thomas, Sean Casey. "A Developed and Characterized Orthotopic Rat Glioblastoma Multiforme Model." Thesis, Virginia Tech, 2020. http://hdl.handle.net/10919/100772.

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This thesis project serves to fill experimental gaps needed to advance the goal of performing pre-clinical trials using an orthotopic rat glioblastoma model to evaluate the efficacy of high-frequency electroporation (H-FIRE) and QUAD-CTX tumor receptor-targeted cytotoxic conjugate therapies, individually and in combination, in selectively and thoroughly treating glioblastoma multiforme. In order to achieve this, an appropriate model must be developed and characterized. I have transduced F98 rat glioma cells to express red-shifted firefly luciferase, which will facilitate longitudinal tumor monitoring in vivo through bioluminescent imaging. I have characterized their response to H-FIRE relative to DI TNC1 rat astrocytes. I have demonstrated the presence of the molecular targets of QUAD in F98 cells. The in vitro characterization of this model has enabled preclinical studies of this promising glioblastoma therapy in an immunocompetent rat model, an important step before advancing ultimately to clinical human trials.
Master of Science
Treating glioblastoma multiforme (GBM), a form of cancer found in the brain, has not been very successful; patients rarely live two years following diagnosis, and there have been no major breakthrough advances in treatment to improve this outlook for decades. We have been working on two treatments which we hope to combine. The first is high-frequency electroporation (H-FIRE), which uses electrical pulses to kill GBM cells while leaving healthy cells alive and blood vessels intact. The second is QUAD-CTX, which combines a toxin with two types of protein that attach to other proteins that are more common on the surface of GBM cells than healthy cells. We have shown these to be effective at disproportionately killing human GBM cells growing in a lab setting. Before H-FIRE and QUAD-CTX may be tested on humans, we need to show them to be effective in an animal model, specifically rats. I have chosen rat glioma cells that will behave similarly to human GBM and a rat species that will not have an immune response to them. I have made these cells bioluminescent so that we may monitor the tumors as they grow and respond to our treatments. I have also shown that QUAD-CTX kills these rat glioma cells, as does H-FIRE. Because of this work, we are ready to begin testing these two treatments in rats.

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Hendricks, Gabriel L. "Modulating Influenza and Heparin Binding Viruses’ Pathogenesis with Extrinsic Receptor Decoy Liposomes: A Dissertation." eScholarship@UMMS, 2013. http://escholarship.umassmed.edu/gsbs_diss/674.

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Influenza is a severe disease in humans and animals, causing upwards of 40,000 deaths every year in America alone. Influenza A virus (IAV) also causes periodic pandemics every 10 to 50 years, killing millions of people. Despite this, very few effective therapies are available. All strains of IAV are prone to developing resistance to antibodies due to the high mutation rate in the viral genome. Because of this mutation rate, a yearly vaccine must be generated before every flu season, and efficacy varies year to year. IAV has also mutated to escape several of the clinically-approved small molecule inhibitors. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of IAV. IAV attachment is mediated by many individually weak hemagglutinin–sialic acid interactions that all together make a strong attachment to a host cell. Polymerized sialic acid analogs can recreate these interactions and block infection. However, they are not ideal therapeutics due to solubility issues and in vivo toxicity. We used liposomes as a novel means for delivery of the sialic acid-containing glycan, sialylneolacto-N-tetraose c (LSTc). LSTcbearing decoy liposomes form multivalent, polymer-like interactions with IAV. Decoy liposomes competitively bind IAV in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. LSTc decoy liposomes co-localize with IAV, while control liposomes do not. Inhibition is specific, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind IAV or inhibit infectivity. LSTc decoy liposomes prevent the spread of IAV during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high-avidity interactions with IAV hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging strains.

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Yang, Ya-Ting. "Molecularly targeted therapy for ovarian cancer." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1149015359.

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Trigila, Carlotta. "Development of a portable gamma imaging system for absorbed radiation dose control in molecular radiotherapy." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS285.

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La thérapie interne par radionucléides est encore aujourd’hui un domaine peu exploité parmi les différentes modalités de traitement contre le cancer. Son spectre d’applications est toutefois en pleine évolution grâce notamment à l'apparition de nouveaux radiopharmaceutiques émetteurs beta ou alpha (peptides, alpha-thérapie ²²³ Ra, alpha-immunothérapie ²²¹ As,...) (Ersahin 2011). Dans ce contexte, la grande hétérogénéité des doses délivrées et des effets observés, à la fois en terme de toxicité et de réponse, démontrent qu'une dosimétrie personnalisée est essentielle pour optimiser le traitement (Strigari 2011). En pratique clinique, la dosimétrie de la tumeur et des organes à risque (foie, rein, ...) repose sur l’image de la biodistribution et de la cinétique précise du radiopharmaceutique chez chaque patient. Ces images peuvent être réalisées avec un traceur pré-thérapeutique pour planifier le traitement ou après celui-ci, afin de corréler directement les effets observés aux doses délivrées de manière à optimiser le protocole (activité maximum à injecter, intervalle entre les injections). Les contraintes de détection imposées par les protocoles de traitement sont très différentes de celles associées à un examen diagnostique (Flux 2011, Konijnenberg 2011). Les gamma-caméras conventionnelles ne sont ainsi pas adaptées à la détection de fortes activités de rayonnements gamma d'énergies inférieures à 100 keV (²²³ Ra) ou supérieures à 300 keV (¹³¹I, ⁹⁰Y). D’autre part, les fortes activités des traceurs injectés exigent généralement que le patient reste isolé, ce qui le rend donc plus difficilement accessible par les techniques d’imagerie standard. Enfin, la disponibilité de ces systèmes est incompatible avec un échantillonnage temporel précis de la cinétique du traceur, qui joue un rôle très important dans la quantification des doses absorbées. L'objectif de ma thèse était de proposer de nouvelles approches instrumentales visant à renforcer le contrôle de la dose délivrée aux patients lors d'un traitement de radiothérapie moléculaire. Ceci est réalisé en réduisant les incertitudes associées à la quantification de l'activité (et donc au calcul de la dose absorbée) grâce à l'utilisation d'un système d'imagerie compact et hautement optimisé. Il consistait à mettre au point et à optimiser une gamma-caméra mobile miniaturisée à haute résolution spécialement conçue pour améliorer l'évaluation quantitative individuelle de la distribution hétérogène et de la bio-cinétique du radiotraceur avant et après administration du traitement. L'étude était axée sur le traitement des maladies bénignes et malignes de la thyroïde à l'aide de l'¹³¹ I. Le premier prototype de la caméra, avec un champ de vue de 5x5 cm² , consiste en un collimateur à trous parallèles à haute énergie, réalisé en impression 3D et optimisé par simulations Monte Carlo, couplé à un scintillateur inorganique continu, lu par une technologie récente basée sur des matrices de photomultiplicateurs au silicium (SiPM). Ses propriétés intrinsèques, en termes d'énergie et de réponse spatiale, ont été testées avec des sources ponctuelles de ⁵⁷ Co et ¹³³ Ba. Le premier prototype de la caméra a été calibré avec de l'¹³¹ I. La calibration du système conduit à une résolution spatiale globale de (3.14±0.03) mm et à une sensibilité moyenne de (1.23±0.01) cps/MBq, le deux à 5 cm de distance. Nous avons effectué les premières études précliniques avec l'utilisation de différents fantômes thyroïdiens imprimés en 3D, avec et sans nodules, remplis de ¹³¹ I. Des résultats très prometteurs ont été atteints (valeurs de RC proches de l’unité), qui mettent en évidence ses performances adaptées à une quantification précise dans un contexte clinique assez réaliste
Targeted radionuclide therapy is still a developing area among the different treatment modalities against cancer. However, its range of applications is rapidly expanding thanks to the emergence of new radiopharmaceuticals labeled with beta or alpha emitters (peptides, ²²³ Ra alpha-therapy, ²²¹ As alpha- immunotherapy, ...) (Ersahin 2011). In that context, the large heterogeneity of absorbed doses and the range of effects observed, both in terms of toxicity and response, demonstrate that individualized patient dosimetry is essential to optimize this therapy (Strigari 2011). In clinical practice, patient-specific dosimetry of tumors and organs-at-risk (liver, kidney, ...) is image-based and rely on the quantification of radio- pharmaceutical uptake as a function of time. These images can be obtained from either a pre-therapy tracer study or from a previous therapy procedure. The detection constraints imposed by the treatment protocols are very different from those associated with diagnostic imaging. (Flux 2011 Konijnenberg 2011). Thus, conventional gamma cameras are not suited for detecting high activity of gamma emitters with energy below 100 keV (²²³ Ra) or greater than 300 keV (¹³¹ I, ⁹⁰Y ). Moreover, high activities of the injected tracer typically require isolation of the patient, making the use of standard imaging devices difficult. Finally, the availability of these devices is incompatible with an accurate temporal sampling of the kinetics of the tracer, which is a key parameter for the quantification of the absorbed doses. The objective of my thesis was precisely to propose new instrumental and methodological approaches aiming to strengthen the control of the dose released to patients during molecular radiotherapy. This is achieved by reducing the uncertainties associated to activity quantification (and therefore to the absorbed dose calculation) through the use of a compact and highly optimized imaging system. Specifically, the work consisted in the development and optimization of a miniaturized, high-resolution mobile gamma camera specifically designed to improve the individual quantitative assessment of the heterogeneous distribution and biokinetics of the radiotracer before and after treatment administration. The study was focused on the treatment of benign and malign thyroid disease with ¹³¹ I. The first prototype of the mobile camera, with a field of view of 5x5 cm², consists of a high-energy parallel- hole collimator, optimized with Monte Carlo simulation and made with 3D printing, coupled to a 6 mm thick continuous CeBr3 scintillator readout by a recent and well-suited technology based on arrays of Silicon Pho- tomultiplier (SiPMs) detectors. Its intrinsic properties, in term of energy and spatial response, have been tested with collimated point source of ⁵⁷Co and ¹³³Ba. The first feasibility prototype has been then calibrated with a line and five cylindrical sources filled with ¹³¹ I. The system calibration leads to an overall spatial resolution of (3.14±0.03) mm at a distance of 5 cm and a sensitivity that decreases with distance and slightly changes with source size. An average sensitivity of (1.23±0.01) cps/MBq has been found at 5 cm. In order to test the quantification capability of the camera, the first preclinical planar studies involved the use of different 3D-printed thyroid phantoms filled with ¹³¹ I, with and without nodules. Although corresponding to a relatively ideal, but realistic, clinical situation (no superimposition of background activity), the optimized imaging features of the camera leads to very promising results, with activity recovery factors that deviate of around 2% from the unity

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Culp, W. David. "Identifying molecular targets for cancer therapy /." Stockholm, 2007. http://diss.kib.ki.se/2007/978-91-7357-188-3/.

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Couture, Frédéric. "Étude des implications biochimiques et moléculaires sous-jacentes à la pharmacothérapie ciblée contre la proprotéine convertase PACE4 dans le cancer de la prostate." Thèse, Université de Sherbrooke, 2018. http://hdl.handle.net/11143/11782.

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Le cancer de la prostate est le cancer le plus fréquent chez les hommes et la capacité des tumeurs à développer une résistance face aux thérapies anti-androgéniques vient souvent compromettre le pronostic des patients. Le développement de nouvelles approches thérapeutiques afin de circonvenir à la progression de ces tumeurs représente un besoin important la gestion de ce type de cancer. Plusieurs démonstrations récentes établissent l’implication de la famille des proprotéines convertases dans la progression tumorale. Ces enzymes ont pour fonctions biologiques de cliver une variété de précurseurs protéiques jouant des rôles importants dans la tumorigénèse. Dans le cancer de la prostate, la proprotéine convertase PACE4 est fortement surexprimée dans les cellules cancéreuses et joue un rôle dans la prolifération et la capacité à former des tumeurs, ce qui en fait une cible thérapeutique d’intérêt. En ce sens, des inhibiteurs peptidomimétiques ont été développés dans l’optique de la thérapie ciblée contre la PACE4. Toutefois, dans le but de développer une approche thérapeutique optimale, il convient néanmoins de comprendre le niveau de redondance fonctionnelle entre les différents membres de la famille des convertases, qui sont connus pour partager plusieurs de leurs substrats, ainsi que les mécanismes moléculaires régissant l’activité de la PACE4 et de ses substrats sous-jacents. L’utilisation d’une approche de répression génique stable envers les différentes convertases a permis de mettre en lumière les fonctions uniques de la PACE4 dans la progression tumorale. De plus, grâce à une approche de protéomique comparative, le premier substrat de la PACE4 dans le cancer de la prostate; le growth differenciation factor 15, a été découvert. Ce substrat permet de commencer à dresser l’implication de PACE4 dans le paysage moléculaire du cancer de la prostate. Grâce à des modalités d’imagerie moléculaire, l’emploi de versions radiomarquées des inhibiteurs peptidiques a également permis de démontrer que les composés s’accumulent dans les cellules cancéreuses en fonction des niveaux de PACE4 présents, et ce, tant in cellulo qu’in vivo. Ces données suggèrent un potentiel pour le développement d’un examen théranostique pour prédire la réponse tumorale à la pharmacothérapie anti-PACE4. Finalement, l’analyse de l’épissage alternatif de l’ARNm de PACE4 a permis l’élucidation des caractéristiques biochimiques et des fonctions spécifiques d’une nouvelle isoforme; la PACE4-altCT, qui est exprimée chez les cellules cancéreuses de la prostate, mais aussi d’autres types de cancer. Cette découverte a permis de redéfinir le modèle de travail en intégrant le concept de la rétention intracellulaire de cette isoforme qui semble médier la plupart de l’activité pro-proliférative reliée à l’activité PACE4, ce qui en fait la cible pharmacologique principale des inhibiteurs peptidiques dans le cancer de la prostate, mais aussi un biomarqueur potentiel.
Abstract: Prostate cancer is the most common cancer among men. The capabilities of tumors to adapt and overcome antiandrogenic therapy is persistently worsening patient’s prognostic and the development of novel therapeutic approaches to circumvent tumor progression therefore represents an unmet need. Many reports now demonstrate the implication of the enzymes from the proprotein convertase family in the progression of tumor from many cancer types. These enzymes are responsible for the processing of various protein precursors playing important roles in tumorigenesis. In prostate cancer, the proprotein convertase PACE4 is strongly overexpressed in cancer cells and plays a role in cell proliferation and tumor formation thus making a strong case for its use as a pharmacological target. For this reason, PACE4 peptidomimetic inhibitors were generated to develop PACE4-targeted therapies. However, to develop an optimal therapeutic approach regarding the inhibition of this enzyme, a complete understanding of the level of functional redundancy between the different convertases in prostate cancer is needed. Moreover, understanding the molecular mechanisms both upstream and downstream of PACE4 in prostate cancer cells would allow a better understanding of the considerations underneath such a therapeutic strategy. Using a stable gene silencing approach to knockdown all co-expressed member of the convertase family in prostate cancer cells, the roles of PACE4 in tumor progression were found to be unique and non-redundant among the other family member. Through a comparative proteomic approach, the first PACE4-specific substrate in prostate cancer; growth and differentiation factor 15, was identified. With this substrate growth factor, it is now possible to initiate the dissection of PACE4 biochemical functions in the prostate cancer molecular landscape. Using a radiolabelled version of the PACE4 peptide inhibitors, it was possible to demonstrate using molecular imaging that when applied in cellulo and in vivo, the compound is uptaken by cancer cells as well as by tissues according to their PACE4 expression levels. These data suggest that such PACE4 molecular imaging with pharmacological inhibitor could be developed as a theranostic assay to predict which tumor could be treated by PACE4-targetted therapy. Lastly, PACE4 mRNA alternative splicing analysis permitted the discovery of a new PACE4 isoform; named PACE4-altCT, which is strongly overexpressed by prostate cancer cells as well as other cancer types. As this isoform displays specific biochemical features and functions, notably being intracellularly retained and mediating most of the PACE4-associated cell growth capabilities, this discovery further redefined our working model, pointing to PACE4-altCT as the pharmacological target of inhibitory peptides in prostate cancer as well as a potential biomarker.

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EL, BOUSTANI MAGUIE. "AP3M2, NEW MOLECULAR TARGET IN COLORECTAL CANCER THERAPY." Doctoral thesis, Università degli Studi di Trieste, 2021. http://hdl.handle.net/11368/2987980.

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Annually, around 1 million cases of CRC are diagnosed, with 50% death rate, representing 8% of cancer-related deaths worldwide. The implementation of early diagnosis screening programs contributed to the reduction of the incidence and the mortality rates. The combination of chemotherapeutic regimens (5-FU, oxaliplatin and irinotecan) and targeted drugs (EGFR and VEGF inhibitors) have improved patients' prognosis. However, the combination of some types of chemical and biological therapies failed in the fourth line treatment and resistance to targeted therapies is the major limit in CRC. Under these circumstances, we have conducted an RNAi screening aiming to identify new targetable oncogenes. Starting from the studies conducted by the Cancer Genome Atlas that identified a list of amplified genes in CRC, we analyzed putative genes that their Knockdown could negatively influence cell viability in different CRC cell lines, indicating the possible involvement in CRC. After a careful bioinformatic analysis I found that one gene called AP3M2 could be a successful candidate. AP3M2 (adaptor related protein complex 3 subunit mu 2) encodes for the neuronal Mu subunit of the heterotetrameric adaptor-related protein complex 3 (AP-3), which recognizes tyrosine-based sorting signals within the cytoplasmic domains of transmembrane cargo proteins and is involved in the biogenesis of lysosome-related organelles. Deletion of this gene in mice was associated with susceptibility to drug-induced seizures and fewer synaptic vesicles. In the other hand, AP3M2 amplicons were observed in the primary tumor and maintained in at least two passages of breast cancer xenograft. AP3M2 is also in the list of genes in recurrent amplicons associated with reduced survival in breast cancer. The new "druggable" gene was found to be altered in 6% of CRC patients and was associated with reduced survival rate according to the TGCA data. Our experiments confirmed it to be overexpressed in colon cancer patient tissues and cancer cell lines and proved to be a potent oncogene by promoting the colorectal cancer cell lines viability, adhesion and colony formation. Interestingly, AP3M2 levels affects the expression of other CRC associated protein such as P62, ATG7and ARF6 involved in autophagy and influenced ROS levels in colorectal cancer cell lines.

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Bapat, Aditi Ajit. "Inhibition of Ape1's DNA repair activity as a target in cancer identification of novel small molecules that have translational potential for molecularly targeted cancer therapy /." Connect to resource online, 2009. http://hdl.handle.net/1805/2062.

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Thesis (Ph.D.)--Indiana University, 2009.
Title from screen (viewed on February 2, 2010). Department of Biochemistry and Molecular Biology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Mark R. Kelley, Millie M. Georgiadis, John J. Turchi, Martin L. Smith. Includes vitae. Includes bibliographical references (leaves 114-133).

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Vergani, E. "BRAF AND MAPK PATHWAY MOLECULES FOR TARGETED THERAPY OF MALIGNANT MELANOMA." Doctoral thesis, Università degli Studi di Milano, 2012. http://hdl.handle.net/2434/173422.

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The clinical activity of the BRAF inhibitor PLX4032 (vemurafenib) in patients with BRAFV600E mutant melanoma is limited primarily by the development of resistance leading to tumor progression. Strategies to overcome primary and acquired resistance are required. In a panel of 27 genetically characterized patient-derived melanoma cell lines the sensitivity to PLX4032 was dependent on BRAFV600E and independent from other gene alterations that commonly occur in melanoma, such as CDKN2A, and mutations of TP53, PTEN loss, and BRAF and MITF gene amplification. To investigate the molecular basis underlying acquired resistance to BRAF inhibitor, PLX4032-resistant cells were derived from a high sensitive BRAFV600E melanoma cell line, and used as a model. The resistant variant line showed increased AKT and ERK phosphorylation and enhanced IGF-1R/PI3K signaling. Combined treatment with PLX4032 plus PI3K inhibitors resulted in significant cell growth inhibition by decreasing pAKT and pERK signaling. To explore molecular mechanisms underlying primary resistance two melanoma cell lines lacking sensitivity to PLX4032 were used as models. Resistance to PLX4032 was maintained after CRAF down-regulation by siRNA, indicating that CRAF is not involved in the activation of ERK in the resistant cell lines. Treatment with the MEK inhibitor UO126 inhibited cell growth and decreased ERK phosphorylation indicating alternative activation of MEK-ERK signaling. Genetic characterization by MLPA and analysis of pTyr signaling by MALDI-TOF mass spectrometry revealed the activation of MET and SRC signaling, associated with the amplification of MET and of CTNNB1 and CCND1 genes, respectively. Testing of co-inhibition of the MET, SRC and MAPK signaling pathways by the combined treatment with the MET inhibitor, SU11274 or the SRC inhibitor, BMS-354825 plus PLX4032 resulted in a significant inhibitory effect on melanoma cell proliferation, survival, migration and invasive capacity. These results support combinatorial approaches targeting MAPK pathway at different nodes and intercepting parallel signal transduction pathways as a strategy to override resistance to BRAF inhibitors.

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Grilli, Giada <1992&gt. "Molecular target therapy and immunotherapy of rare lung tumors." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2022. http://amsdottorato.unibo.it/10156/3/Tesi_GrilliGiada_.pdf.

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Lung cancer is an heterogeneous disease, with 1-2% of rare histology. New molecular profiling technologies, such as next generation sequencing (NGS), haverevolutionized the assessment of molecular alteration in clinical practice. We analyzed a cohort of 1408 NSCLC-A patients treated at the Sant'Orsola- Malpighi University Hospital from 2019 to 2021. This analysis was performed using the oncomine focus thermo fischer panel. Of them, 410 (29%) had rare alteration (RET 3%, NTRK 0,2%,FGFR1 2%, MET exon14 skipping 3%, BRAF V600 4%, ALK fusion EGFR exon 20 2%) and 36 (2%)had a uncommon mutation. We enrolled 7 RET- rearranged patients in CRETA and J2G-MC-JZJC clinical trials assessing respectively unselective and selective RET-inhibitors , another 7 patients tested positive for the BRAF V6006 mutation and have been enrolled in the Array clinical trial assessing a novel combination of anti-BRAF and anti-mek agents . Other molecular alterations found are KRAS (Gly12Cys), FGFR1-4 mutation, MET skipping ex14 mutations, respectively eligible for other ongoing open studies such as Amgen 20190009 comparing efficacy of sotorasib vs docetaxel, Fight-207 assessing activity of pemigatinib and CINC280J12201 assessing activity of the novel met inhibitor capmatinib. In 2018 we joined the CHANCE clinical trial,a multicenter study evaluating the efficacy and safety of atezolizumab in patients withrare lung cancer histologies where and 14 patients have been so far enrolled in the Bologna site. Our studies underline the need of tailored approach to NSCLC patients and our results showed that precision medicine is feasible and is an effective approach to cancer treatment.

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38

Pan, Jie. "Molecularly targeted therapy on a new preclinical mouse model for gastric neuroendocrine tumors." Diss., Ludwig-Maximilians-Universität München, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-159343.

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Neuroendocrine tumors are a heterogeneous group of malignancies with an increasing prevalence. Since there is not much progress in therapy, model systems are urgently needed. We have a CEA424-SV40 TAg transgenic mouse model which develops spontaneous tumors in the antral region of the stomach. In addition, several cell lines derived from the tumor were established. Gene expression analysis of the tumor tissue as well as cell lines revealed neuroendocrine markers. Therefore we further characterized this model with special emphasis on the cells of origin and used it for testing new targeted treatment protocols. To analyze CEA424-SV40 TAg mouse model in more detail, tumor tissue as well as the cell lines derived from the primary tumor were investigated by immunohistochemistry, immunofluorescence, western blot, and ELISA. Antibodies used were directed at SV40 TAg, Ki-67, chromogranin A, chromogranin B, secretin, H+-K+-ATPase, glucagon, and transcription factors NeuroD1 and Nkx2.2. Plasma hormone levels of serotonin and secretin were measured by ELISA. Immunostainings of SV40 TAg and Ki-67 revealed highly proliferative tumors cells. The tumors stained intensively for the neuroendocrine markers chromogranin A, chromogranin B, secretin and glucagon. The tumor tissue as well as the cell lines expressed transcription factors NeuroD and Nkx2.2, which are involved in the differentiation of the neuroendocrine lineage. Hormone levels of serotonin and secretin in the plasma of the transgenic mice were dramatically elevated when compared with normal littermates, thus supporting the neuroendocrine phenotype. As the neuroendocrine phenotype of CEA424-SV40 TAg transgenic mouse was confirmed, molecularly targeted therapies were tested in this model system both in vitro and in vivo. Cell lines were tested for drug sensitivity with mTOR inhibitors (RAD001, NVP-BEZ235), paclitaxel, E2F inhibitor, HSP90 inhibitor, and p53 stabilizer Nutlin-3a. All the drugs tested in vitro could efficiently inhibit cell proliferation in a dose dependent manner. From these drugs the mTOR inhibitor RAD001 was chosen for the in vivo experiment. Daily feeding of 10 mg/kg RAD001 inhibited the tumor development and prolonged the survival time of the CEA424-SV40 TAg transgenic mice dramatically. The effects of the RAD001 treatment on tumor cells were achieved mainly through inactivating mTOR-p70S6K and mTOR-4EBP1 signaling as proven by western blot and immunohistochemistry. Still, some cells must develop escape mechanisms, since the tumor tend to grow. To gain a better understanding of the T antigen transforming mechanisms as well as the possible escape mechanisms, some efforts were made on the tumor originating cells in the CEA424-SV40 Tag transgenic mouse model. Possible candidates for these tumor originating cells in the stomach are the newly described epithelial as well as mesenchymal stem cells. In a first attempt, the expression feature of epithelial and mesenchymal stem cell markers were analyzed. Established cell lines as well as tumor tissue from the tumor bearing mice were investigated by reverse transcription PCR (RT-PCR), immunohistochemistry, immunofluorescence, western blot, and microarray analysis. From several markers analyzed, the tumor cell lines showed a high expression level of the potential epithelial stem cell marker Bmi1 in RT-PCR and cDNA expression array. This could be further substantiated by western-blotting and immunostaining. Consequently, Bmi1 message could also be found in the growing tumors both in mRNA and protein levels. Experiments using siRNA to knock down the SV40-TAg expression showed that the Bmi1 expression went down in the cell lines thus showing the interrelationship. On the other hand, the mesenchymal stem cell marker Etv1 was also found to be expressed in the tumor tissue and cell lines derived from the tumor. More interestingly, Etv1 expression level was up-regulated over the time course of the tumor development. From these, an Etv1 positive mesenchymal cell could be a possible candidate for transformation. Since the CEA-promoter used for the generation of the T-antigen transgenic animals contains Etv1 binding sites, it is tempting to speculate, that this may drive the transcription of the T antigen. In conclusion, our data provide convincing evidence that CEA424-SV40 TAg mice are a clinically relevant model for neuroendocrine tumor. Testing of molecularly targeted therapies both in vitro and in vivo offered promising candidates for further clinical evaluation. Thus, this new model system could be of great value not only for studies on the mechanisms of how SV40 TAg induces neuroendocrine tumors but also for exploring novel targeted therapy in a preclinical setting.
Neuroendokrine Tumore stellen heterogene Tumore mit ansteigender Prävalenz dar. Da es nur geringe Verbesserungen in der Therapie gibt, besteht ein Bedarf an neuen Modellsystemen. In unserer Arbeitsgruppe wurde das CEA424-SV40-T-Antigen transgene Mausmodell entwickelt, in welchem sich spontan Tumore im Bereich des Magenantrums bilden. Gleichzeitig wurden Zelllinien aus diesem Tumor etabliert. Die Analyse der Genexpression im Tumor und in den Zelllinien ergaben Hinweise auf einen neuroendokrinen Tumortyp. Deshalb wurde der Phänotyp des Tumors auch mit Blick auf den Ursprung der Tumore analysiert und für die Testung von Therapieoptionen eingesetzt. Die Analyse der CEA-424-SV40-TAg-Mäuse sowie der Tumorlinien wurden mittels Immunhistochemie, Immunfluoreszenz, Westernblot und ELISA untersucht. Dazu wurden Antikörper gegen das SV40-T-Antigen, den Proliferationsmarker Ki-67, Chromogranin A, Chromogranin B, Sekretin, die H-K-ATPase, Glukagon und die Transkriptionsfaktoren NeuroD und Nkx2.2 eingesetzt. Die Plasma Hormonkonzentrationen von Serotonin und Sekretin wurden mittels ELISA bestimmt. Die Färbung für das SV40-T-Antigen und Ki-67 zeigte einen hoch proliferierenden Tumor. Dieser war hoch positiv für die neuroendokrinen Marker Chromogranin A, Chromogranin B, Sekretin und Glukagon. Sowohl der Tumor als auch die Tumorzelllinien exprimierten die Transkriptionsfaktoren NeuroD und Nkx2.2, welche an der Differenzierung von neuroendokrinen Zellen beteiligt sind. Die Hormonkonzentrationen von Serotonin und Sekretin waren im Plasma der transgenen Mäuse deutlich erhöht. Dies ergab das Gesamtbild eines neuroendokrinen Karzinoms. In diesem Modell wurden nun verschiedene molekular begründete Therapien in vitro und in vivo getestet. So wurde an den Zelllinien die Empfindlichkeit von mTOR Inhibitoren (RAD001, NVP-BEZ235), Paclitaxel, E2F -Inhibitor, Hsp90-Inhibitor und dem p53 Inhibitor Nutlin3 getestet. Alle verwendeten Substanzen konnten die Proliferation der Tumorzellen dosisabhängig hemmen. Von diesen wurde dann der mTOR Inhibitor RAD001 für die in vivo Anwendung ausgewählt. RAD001 konnte dabei die Entwicklung der Tumore signifikant hemmen und verlängerte das Überleben der Tiere dramatisch. Der Effekt der mTOR Inhibition bestand dabei vor allem in der Hemmung des mTOR-p70S6K und mTOR-4EBP1 Pathways, was im Westernblot und der Immunhistochemie gezeigt werden konnte. Trotzdem muss festgehalten werden, dass einige Tumorzellen der Therapie entkommen konnten. Um nun Informationen über den Transformations- und den Escape-Mechanismus zu bekommen, wurde versucht die Tumorursprungszelle zu beschreiben. Mögliche Kandidaten dafür sind sowohl die jüngst beschriebenen intestinalen Epithelstammzellen als auch mesenchymale Stammzellen. Dazu wurden Marker-Gene an den etablierten Zelllinien und den Tumoren mit Hilfe von RT-PCR, Immunhistochemie, Immunfluoreszenz, Westernblot und Microarray untersucht. Dabei fand sich in den Tumorzellen eine hohe Expression des möglichen epithelialen Stammzellmarkers Bmi1. Dies konnte auch im Westernblot und in der Immunfärbung bestätigt werden. Folgerichtig fand sich dieser Marker auch in den wachsenden Tumoren. Experimente, in denen mit siRNA die Expression des SV40-T- Antigen blockiert wurde, ergaben eine Reduktion der Bmi1-Expression und weisen damit auf einen engen Zusammenhang hin. Gleichzeitig fand sich allerdings auch eine hohe Expression des mesenchymalen Stammzell-Markergenes Etv1 im Tumor und in den etablierten Zelllinien. Etv1 stieg dabei im Verlauf der Tumorentwicklung im Gewebe deutlich an. Deshalb könnte es sich bei der ursprünglich transformierten Zelle auch um eine mesenchymale Stammzelle handeln. Da der CEA Promotor, der die Expression des SV40-T-Antigens in den transgenen Mäusen regulieren soll, einige Bindungsstellen für den Transkriptionsfaktor Etv1 hat, liegt die Möglichkeit der Induktion des T-Antigens über Etv1 nahe. Dazu stehen aber noch weitere Experimente aus. Zusammenfassend zeigen die hier präsentierten Daten, dass es sich bei dem CEA424-SV40 Tag-Mausmodell um ein klinisch relevantes Modell für einen neuroendokrinen Tumor handelt. Zusammen mit den etablierten Tumorzelllinien können daran neue Therapieansätze getestet werden. Damit bietet es die Möglichkeit sowohl den Zusammenhang zwischen dem T-Antigen und der Entwicklung des neuroendokrinen Phänotyps als auch neue Therapieformen zu untersuchen.

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39

ADANTI, SARA. "Molecular mechanisms of carcinogenesis: identification of new targets for diagnosis and therapy." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2014. http://hdl.handle.net/2108/203330.

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La proteina Translationally Controlled Tumor Protein (TCTP) svolge un importante ruolo come fattore di sopravvivenza nelle cellule tumorali ed è overespressa nelle cellule di carcinoma mammario scarsamente differenziato. TCTP è un target specifico della Diidroartemisinina (DHA). La DHA è il principale metabolita dell’Artemisinina, il principio attivo estratto dalla Artemisia annua L. La DHA è attualmente utilizzata come farmaco antimalarico. Recentemente, è stata inoltre dimostrata l’efficacia della DHA come antitumorale. In questo studio abbiamo valutato l’effetto antitumorale della DHA su lineee cellulari di carcinoma della mammella presentanti fenotipo altamente aggressivo, come le linee cellulari MDA-MB-231 ed SKBR3. I nostri risultati mostrano che la DHA inibisce la crescita delle cellule tumorali della mammella ed induce apoptosi, attraverso la riduzione dei livelli di espressione della forma fosforilata della TCTP. Inoltre, abbiamo dimostrato che la DHA aumenta l’efficacia dei farmaci comunemente utilizzati nella terapia antitumorale, come la Doxorubicina e il Trastuzumab. I dati ottenuti da questo studio suggeriscono che la fosfo-TCTP può rappresentare un nuovo bersaglio terapeutico per il trattamento del cancro della mammella. Inoltre, il trattamento combinatoriale con la DHA e i convenzionali chemioterapici potrebbe rappresentare una nuova potenziale strategia terapeutica per il cancro della mammella.
Translationally Controlled Tumor Protein (TCTP) is a survival factor in tumor cells overexpressed in poorly differentiated breast cancer cells. TCTP is a specific target of Dihydroartemisinin (DHA). DHA is a metabolite of Artemisinin, the active principle of Artemisia annua L. DHA is an anti-malaria drug with antitumor properties. We studied the effect of DHA on human breast cancer cell lines (such as MDA-MB-231 and SKBR3 cells) with more aggressive phenotype. Our results show that DHA inhibits breast cancer cells growth and induces apoptosis by reducing the levels of the phosphorylated form of TCTP. We also show that DHA improves the antitumor effect of the conventional chemotherapy drugs, such as Doxorubicin and Trastuzumab. Altogether, these results suggest that phospho-TCTP is a novel therapeutic target for breast cancer cells. DHA in combination with conventional chemotherapeutics is a novel strategy to treat breast cancer

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Lopes, da Rosa-Spiegler Jessica. "Targeting the Histone Acetyl-Transferase, RTT109, for Novel Anti-Fungal Drug Development: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/624.

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Discovery of new antifungal chemo-therapeutics for humans is limited by the large degree of conservation among eukaryotic organisms. In recent years, the histone acetyl-transferase Rtt109 was identified as the sole enzyme responsible for an abundant and important histone modification, histone H3 lysine 56 (H3K56) acetylation. In the absence of Rtt109, the lack of acetylated H3K56 renders yeast cells extremely sensitive to genotoxic agents. Consequently, the ability to sustain genotoxic stress from the host immune system is crucial for pathogens to perpetuate an infection. Because Rtt109 is conserved only within the fungal kingdom, I reasoned that Rtt109 could be a novel drug target. My dissertation first establishes that genome stability provided by Rtt109 and H3K56 acetylation is required for Candida albicans pathogenesis. I demonstrate that mice infected with rtt109 -/- cells experience a significant reduction in organ pathology and mortality rate. I hypothesized that the avirulent phenotype of rtt109 -/- cells is due to their intrinsic hypersensitivity to the genotoxic effects of reactive oxygen species (ROS), which are utilized by phagocytic cells of the immune system to kill pathogens. Indeed, C. albicans rtt109 -/- cells are more efficiently killed by macrophages in vitro than are wild-type cells. However, inhibition of ROS generation in macrophages renders rtt109 -/- and wild-type yeast cells equally resilient to killing. These findings support the concept that ability to resist genotoxic stress conferred by Rtt109 and H3K56 acetylation is a virulence factor for fungal pathogens and establish Rtt109 as an opportune drug- target for novel antifungal therapeutics. Second, I report the discovery of a specific chemical inhibitor of Rtt109 catalysis as the initial step in the development of a novel antifungal agent. We established a collaboration with the Broad Institute (Cambridge, MA) to perform a high-throughput screen of 300,000 compounds. From these, I identified a single chemical, termed KB7, which specifically inhibits Rtt109 catalysis, with no effect on other HAT enzymes tested. KB7 has an IC50 value of approximately 60 nM and displays noncompetitive inhibition regarding both acetyl-coenzyme A and histone substrates. With the genotoxic agent camptothecin, KB7 causes a synergistic decrease in C. albicans growth rate. However, this effect is only observed in an efflux-pump mutant, suggesting that this compound would be more effective if it were better retained intracellularly. Further studies through structure-activity relationship (SAR) modifications will be conducted on KB7 to improve its effective cellular concentration.

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Kerouedan, Christelle. "Adhesion molecule expression in corneal transplantation : a target for therapy?" Thesis, Imperial College London, 2003. http://hdl.handle.net/10044/1/11792.

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Teunissen, Jacobus Johannes Maria. "Endocrine tumours molecular radiation on target peptide receptor radionuclide therapy with lutetium-octreotate." [S.l.] : Rotterdam : [The Author] ; Erasmus University [Host], 2008. http://hdl.handle.net/1765/14119.

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43

Phadke, Manali. "GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE: A NEW MOLECULAR TARGET IN CHEMOTHERAPY." Diss., Temple University Libraries, 2012. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/214810.

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Pharmaceutical Sciences
Ph.D.
Cancer therapy traditionally seeks to achieve complete tumor eradication via induction of cancer cell death by chemotherapeutic agents or radiation. An alternative strategy is to induce cytostasis, i.e. to arrest proliferation of cancer cells, perhaps in parallel with conventional chemotherapy. Such an alternative strategy could provide prolonged survival with less severe consequences of cytotoxic agents. To be truly effective, a chemotherapeutic drug should exert its action on biochemical targets specific for neoplastic cells while leaving the normal cells unaffected. Therefore, the knowledge of tumor cell-specific biochemical and signaling pathways is a pre-requisite for development of new, prospective anticancer drugs. In this study, we concentrated on the energy metabolism which is remarkably different in tumor and healthy cells. Cancer cells generate ATP mainly through the glycolytic pathway, and depend far less on oxidative phosphorylation (the Warburg effect). The way cancer cells generate energy reflects their need for energy as well as building blocks required for fast biosynthesis. Glycolysis, in contrast to oxidative phosphorylation, enhances biosynthetic pathways thus accelerating progression of tumor cells through the cell cycle. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) occupies a central position in the glycolytic pathway thus playing a critical role in the energy metabolism of cancer cells. Along with its enzymatic activity, GAPDH is a multifunctional protein which acts as a signaling and regulatory molecule in several cellular mechanisms. Based on the fact that glycolysis plays a pivotal role in survival of cancer cells, we hypothesized that down-regulation of GAPDH protein would alter the cancer cell proliferation, and cellular sensitivity of cancer cells to chemotherapy. The goal of this study was to evaluate GAPDH as a potential molecular target for treatment of cancer. In this project, our aims were: 1) To determine the effect of GAPDH level on cell proliferation and cell cycle progression of human carcinoma cells; 2) To elucidate the molecular mechanism(s) causing proliferation arrest in GAPDH-depleted cells; 3) To identify the chemotherapeutic agents exhibiting cytotoxic effect against non-dividing, senescent cells; 4) To analyze molecular dynamics of nuclear GAPDH and its mutant variants in the context of chemotherapy-induced stress. Towards these aims, we developed an experimental model where the level of GAPDH in human carcinoma cells was modulated by RNA interference (RNAi) technology. In vitro experiments were performed in this model system to evaluate the energy status, and signaling pathways in cancer cells after GAPDH depletion. Human carcinoma isogenic cell lines with different levels of GAPDH protein were analyzed for the sensitivity to various chemotherapeutic agents. Using site-mutagenesis, we prepared mutated variants of GAPDH and estimated their enzymatic activity. We also prepared constructs where GAPDH cDNA was fused with green fluorescent protein (EGFP) cDNA, and transiently expressed them in human cancer cells, to assess GAPDH localization and biological effects. We analyzed intranuclear localization and dynamic characteristics of GAPDH and its variants in the live cells using image confocal technologies (e.g. FRAP). In our study, we demonstrated that GAPDH is a molecular target with clinical potential for senescence-based tumor suppression. Our experiments revealed that depletion of GAPDH induces energy crisis and proliferation arrest in human carcinoma cells. We elucidated the molecular mechanisms initiated by GAPDH depletion, and demonstrated that GAPDH-depleted cells acquire the accelerated senescence phenotype. Moreover, we found chemotherapeutic agents cytotoxic to the senescent cells, a finding that opens a way to combination chemotherapy with therapy-induced senescence agents. Our results on dynamic characteristics of intranuclear GAPDH and its mutant forms indicate that in the nucleus, GAPDH interacts with biomolecules yet to be identified. The results of this study suggest a novel, prospective molecular target for pharmacotherapeutic intervention in cancer management.
Temple University--Theses

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Garcia, Jessica. "Évaluation du patrimoine tumoral circulant dans la prise en charge thérapeutique des patients atteints de cancer broncho-pulmonaire." Thesis, Lyon, 2019. http://www.theses.fr/2019LYSE1275.

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Le cancer broncho-pulmonaire (CBP) le 4ème cancer le plus répandu au niveau mondial après les cancers de la prostate, du sein et du côlon. Diagnostiqués à des stades tardifs, il est la première cause de cancer par décès. Cependant, une meilleure compréhension des mécanismes moléculaires sous-jacents au cancer a permis de développer des thérapies personnalisées pour chaque patient. L’émergence des thérapies ciblées et de l’immunothérapie a révolutionné la prise en charge thérapeutique, permettant d’améliorer la survie globale, la survie sans progression et les effets secondaires des patients en comparaison avec les traitements de chimiothérapies conventionnelles. La prescription des thérapies personnalisées est basée sur les caractéristiques moléculaires de la tumeur et, par conséquent, nécessite des analyses moléculaires innovantes. Néanmoins, entre 10 et 30% des analyses moléculaires des patients atteints de CBNPC sont non contributives et l’accès aux thérapies ciblées est compromis. De plus, même si l’analyse anatomo-pathologique reste utile pour l’évaluation du stade ou encore de l’histologie, elle reste peu adaptée en cas d’actes répétitifs, tout au long de la maladie. La « biopsie liquide », est un concept émergeant, correspondant à l’analyse des acides nucléiques circulants mais aussi des cellules tumorales circulantes (CTC), issus de la tumeur. Cette méthode faiblement invasive, basée sur un prélèvement sanguin, permet d’analyser le patrimoine tumoral circulant et donne accès aux informations moléculaires de la tumeur primaire. Le développement de nouvelles activités de diagnostic est donc primordial pour répondre à ses nouvelles demandes cliniques. Depuis 2015, les Hospices Civils de Lyon (HCL) ont déployé un programme de recherche translationnelle, appelé CIRCAN « CIRculating Cancer » dans lequel s’inscrit ce travail de thèse. De nombreuses méthodes de détection des biomarqueurs pertinents en oncologie thoracique dans l’ADNcir ont été développées et été validées au laboratoire pour plus de 1500 patients actuellement, leur permettant de bénéficier de thérapies ciblées. L’optimisation et la validation des technologies de biologie moléculaire telles que le séquençage à haut débit et la PCR digitale ultra-sensible ont été réalisées durant ce travail de thèse et valorisées dans des journaux internationaux. Au-delà des thérapies ciblées, les immunothérapies représentent les nouveaux traitements prometteurs pour ces patients dont le taux d’expression PD-L1 sur biopsie tissulaire est le biomarqueur de choix. Etant donné les contraintes qu’occasionne la biopsie tissulaire, nous avons développé un protocole de caractérisation phénotypique de PD-L1 dans les CTC. De plus, de nombreuses études montrent la pertinence clinique de l’utilisation de la charge mutationnelle (TMB) comme marqueur prédictif de réponse à l’immunothérapie. En parallèle, nous avons développé des outils moléculaires en cours de validation pour le calcul du TMB dans l’ADNcir et dans les CTC en comparaison avec celui calculé dans le tissu, et le taux de PD-L1 évalué par immunohistochimie. Toutefois, pour environ 50% des patients atteints de CBP, aucun biomarqueur n’est retrouvé, bloquant l’accès à des thérapies personnalisées et réduisant les chances de survie du patient. A la recherche de nouveaux biomarqueurs, nous avons développé un protocole permettant d’accéder à la signature transcriptomique des CTC à un niveau « single-cell » afin de caractériser l’hétérogénéité tumorale de ces cellules et de mieux comprendre les mécanismes de résistance mis en place. Les échantillons cliniques de patients sont en cours d’analyse, avec ce protocole validé avec une lignée cellulaire modèle. En effet, les résultats de la validation de méthode mettent en exergue la possibilité d’évaluer l’hétérogénéité tumorale et les voies de signalisation impliquées dans la dissémination métastasique, telles que la transition épithélio-mésenchymateuse
Broncho-pulmonary cancer (PBC) is the 4th most common cancer worldwide after prostate, breast and colon cancer. Diagnosed at late stages, it is the leading cause of cancer death. However, a better understanding of the molecular mechanisms underlying cancer has led to the development of personalized therapies for each patient. The emergence of targeted therapies and immunotherapy has revolutionized the therapeutic management, improving the overall survival, progression-free survival and side effects of patients compared to conventional chemotherapy treatments. The prescription of personalized therapies is based on the molecular characteristics of the tumor and, therefore, requires innovative molecular analyzes. Nevertheless, between 10 and 30% of the molecular analyzes of NSCLC patients are non-contributory and access to targeted therapies is compromised. Moreover, even if the pathological analysis remains useful for stadium evaluation or histology, it remains unsuitable for repetitive actions throughout the illness. The "liquid biopsy", is an emerging concept, corresponding to the analysis of circulating nucleic acids but also circulating tumor cells (CTC), derived from the primary tumor. This low-invasive method, based on a blood sample, makes it possible to analyze the circulating tumor inheritance and gives access to the molecular information of the primary tumor. The development of new diagnostic activities is therefore essential to meet its new clinical demands. Since 2015, Hospices Civils de Lyon (HCL) has deployed a translational research program, called CIRCAN "CIRculating Cancer" in which this thesis. Many methods for detecting relevant biomarkers in thoracic oncology in circulating free DNA (cfDNA) have been developed and validated in the laboratory for more than 1500 patients currently, allowing them to benefit from targeted therapies. The optimization and validation of molecular biology technologies such as high-throughput sequencing and ultra-sensitive digital PCR were performed during this thesis work and published in international journals. Beyond targeted therapies, immunotherapies represent promising new treatments for these patients whose PD-L1 expression level on tissue biopsy is the biomarker of choice. Given the constraints of tissue biopsy, we have developed a phenotypic characterization protocol for PD-L1 in CTCs. In addition, numerous studies show the clinical relevance of the use of mutational load (TMB) as a predictive marker of response to immunotherapy. In parallel, we have developed molecular tools undergoing validation for the calculation of TMB in cfDNA and in CTC compared with the value calculated in tissue, and the PD-L1 level evaluated by immunohistochemistry. However, for about 50% of patients with CBP, no biomarker is found, blocking access to personalized therapies and reducing the patient's chances of survival. In search of new biomarkers, we have developed a protocol allowing access to the transcriptomic signature of CTCs at a "single-cell" level in order to characterize the tumor heterogeneity of these cells and to better understand the resistance mechanisms implemented. The clinical samples of patients are being analyzed, with this protocol validated with a model cell line. Indeed, the results of the method validation highlight the possibility of evaluating tumor heterogeneity and the signaling pathways involved in metastatic spread, such as the epithelial-mesenchymal transition

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Wiench, Benjamin [Verfasser]. "Molecular biology, pharmacogenomics and pharmacoproteomics of shikonin for target oriented cancer therapy / Benjamin Wiench." Mainz : Universitätsbibliothek Mainz, 2014. http://d-nb.info/1046482254/34.

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Kauntz, Henriette. "Cellular and molecular targets of silibinin, a natural flavonoid, in colorectal cancer prevention and therapy." Thesis, Strasbourg, 2012. http://www.theses.fr/2012STRAJ052/document.

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Le cancer colorectal est la deuxième cause de mortalité due au cancer en Europe et aux États-Unis. Etant donné l’efficacité limitée et la toxicité élevée des agents de chimiothérapie, de nouvelles approches sont nécessaires. Le flavanolignane silybine représente le principal constituant actif du chardon-marie (Silybum marianum). Les mécanismes moléculaires des propriétés anticancéreuses de la silybine ont été étudiés dans un modèle cellulaire de progression du cancer colorectal humain : les cellules SW480 issues d’un adénocarcinome, et leurs dérivées métastatiques les cellules SW620. Les effets chimiopréventifs de la silybine ont été étudiés dans un modèle de cancérogenèse colique induite par l’azoxyméthane chez le rat. La silybine induit une mort apoptotique avec activation de la caspase-3 dans les deux lignées. L’expression des récepteurs de mort TRAIL est augmentée, et la caspase-8 activée. Le potentiel mitochondrial est perturbé provoquant une libération du cytochrome c et une activation de la caspase-9. En plus de l’activation des voies apoptotiques extrinsèque et intrinsèque la silybine induit une réponse autophagique. La combinaison de la silybine et de TRAIL, un agent anti-cancéreux prometteur, provoque une mort cellulaire synergique dans les deux lignées. Un effet synergique est aussi observé avec la combinaison de la silybine et des inhibiteurs des histones déacétylases (HDAC) : TSA et SAHA. Dans le modèle chez le rat, la silybine réduit de 50% le nombre des lésions prénéoplasiques. En conclusion, la silybine est un agent naturel intéressant pour la prévention du cancer colorectal et dans le cadre d’une combinaison avec TRAIL/des inhibiteurs d’HDACs
Colorectal cancer (CRC) is the second most common cause for cancer-related deaths in Europe and in the USA. Because of the limited efficacy and considerable toxicity of chemotherapeutic agents, new approaches are needed. The hepatoprotective flavonolignan silibinin is the major biologically active compound of the milk thistle (Silybum marianum).The molecular mechanisms of the anticancer properties of silibinin in CRC were studied in an in vitro model of cancer progression consisting of the adenocarcinoma cell line SW480 and its derived metastatic cell line SW620. Its chemopreventive effects were assessed in an in vivo model of azoxymethane-induced colon carcinogenesis in the rat. Silibinin induced apoptotic cell death with activation of caspase-3 in both cell lines. The expression of death receptors was upregulated, and caspase-8 was activated. The potential of the mitochondrial membrane was perturbed permitting the release of cytochrome c and the activation of caspase-9. Besides the activation of the extrinsic and the intrinsic apoptotic pathway, silibinin induced an autophagic response. Combination of silibinin and TRAIL, a promising anticancer agent selectively inducing apoptosis in cancer cells, induced synergistic cell death in both cell lines. Synergy in cell death induction was also observed by the combination of silibinin and the histone deacetylase (HDAC) inhibitors TSA and SAHA. In the preclinical model in the rat, silibinin administration was able to reduce by half the number of preneoplastic lesions present in the colon. In conclusion, silibinin is a promising natural agent for colon cancer chemoprevention and for combination therapy with TRAIL/HDAC inhibitors

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LAIRES, Raquel de Sá da Silva. "Trypanosoma brucei peptidase inhibitors.Immunolocalization, secretion and potential use as targets for therapy." Doctoral thesis, Instituto de Higiene e Medicina Tropical, 2013. http://hdl.handle.net/10362/19282.

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As peptidases encontram-se envolvidas em diversas funções biológicas possuindo um papel importante na patogenicidade de várias infecções parasitárias. Em mamíferos, a actividade peptidica é controlada por inibidores endógenos, como as cistatinas e as serpinas. Os genes que codificam para inibidores homólogos às cistatinas e serpinas de mamíferos, encontram-se ausentes do genoma de tripanossomatídeos. A ecotina é uma proteína de Escherichia coli, capaz de inibir uma grande variedade de peptidases serínicas da família S1A, tais como a tripsina. Existem duas proteínas do tipo da ecotina em T. brucei, ISP1 e ISP2. A ausência de peptidases sensíveis à acção dos ISPs no genoma de Trypanosoma brucei, sugere que estes tenham como alvo as peptidases serínicas do hospedeiro. Linhas celulares de T. brucei deficientes em ISP1 (Δisp1), ISP2 (Δisp2) e em ambos os ISPs (Δisp1/2) foram produzidas com sucesso e a ausência dos inibidores comprovada com o uso de anticorpos monoclonais específicos contra o ISP1 e o ISP2, que reconhecem a proteina alvo em extractos de proteína total de parasitas selvagens mas não em extractos de parasitas mutantes. O efeito da ausência dos ISPs nas células foi avaliado in vitro e in vivo, verificando-se que a delecção individual de cada ISP não produz qualquer efeito nos parasitas que revelam um crescimento normal em cultura e padrões de infectividade em murganhos idênticos aos de parasitas selvagens. Em contraste, os parasitas Δisp1/2, embora possuam um crescimento normal in vitro com ausência de alterações morfológicas grosseiras, são caracterizados por um alteração na mobilidade e pela acumulação de micro-vesiculas na região da bolsa flagelar, consistente com um defeito no processo de endocitose/exocitose. Adicionalmente, por imunofluorescência foi possível localizar os ISPs no citoplasma e na perto da bolsa flagelar. Em conjunto, estes dados sugerem uma função intracelular, independente da actividade inibitória dos ISPs e provavelmente associada ao flagelo ou à bolsa flagelar. O papel biológico dos ISPs na interação parasita-hospedeiro foi também avaliada através da infecção de murganhos com os parasitas Δisp1/2.A monitorização da infecção demonstrou que a deleção de ambos os inibidores resulta em sobrevivência prolongada dos murganhos com cargas parasitárias reduzidas, sugerindo que os ISPs possuem um papel importante na sobrevivência do parasita. Em contraste com os resultados obtidos em Leishmania que atribuem diferentes funções a ISP1 e ISP2, este estudo sugere que existe uma redundância funcional dos mesmos em T. brucei, sendo necessária a presença de ambos para que o parasita estabeleça eficientemente a infecção no hospedeiro mamifero. Foram também realizados estudos de imuno-protecção em murganhos de modo a determinar o potencial dos ISPs como alvos terapêuticos. A imunização com ISP recombinante e o tratamento com anticorpos específicos contra ISP1 e ISP2 não produzem qualquer efeito protector ou neutralizante da infecção de murganhos, sugerindo que estes inibidores não constituem bons candidatos para o desenvolvimento de uma vacina ou terapia baseada em anticorpos.
Peptidases are involved in several biological functions playing an important role in the pathogenicity of many parasitic infections. In mammals, one way in which the activity of these peptidases is controlledisby interaction with natural inhibitors, such as cystatins and serpins. In trypanosomatids, the genes encoding forendogenousinhibitors homologous to mammalian cystatins and serpins, are absent. Ecotin is an Escherichia coliprotein capable of inhibiting a wide range of serine peptidases from S1A family such as trypsin. Ecotin orthologues can be found in a restricted range of bacterial pathogens and also in trypanosomatid parasitic protozoa.In Trypanosomabruceitwo proteins of 19.7 kDa and 17.8 kDa were identified as ecotin-like inhibitors and given the generic name of Inhibitors of Serine Peptidases (ISP). The absence of peptidases predicted to be sensitive to the action of ISPs in T. bruceisuggests that these inhibitors are targeting the host’s serine peptidases, although their exact biological role was unknown. T. bruceimutant cell linesdeficientin ISP1 (Δisp1),ISP2 (Δisp2) and both ISPs (Δisp1/2) weresuccessfully generatedby targeted gene disruption. This was confirmed using monoclonal antibodies generated specifically against ISP1 and ISP2 that recognize the target protein in wild type and re-expressor cell lysates but not in the respective mutant cell line. Theeffect of ISP deletion in the parasiteswasdeterminedboth in culture andin micein vivo. The deletion of ISPgenes individually was shown to have no effect on the parasites in vitroor in vivo. However, different results were obtained for the simultaneous deletion of ISP1and ISP2. Although Δisp1/2parasites have normal growth and morphology in culture, they exhibit a motility defect and are characterized by the accumulation of small vesicles outside the flagellar pocket consistent with an endocytosis/exocytosis defect. Furthermore, immunofluorescence shows that both ISPs are localized in the cytosol and in small punctuated structures near the flagellar pocket region. These data suggests that ISPs have an intracellular function independent of their inhibitory activityand probably associated with the flagellar pocket.The biological role of ISP1 and ISP2 in the host-parasite interaction was also evaluated by infecting mice with Δisp1/2parasites. Parasitemia monitoring shows that the deletion of both ISPsresults in prolonged host survival with reduced or undetectable parasite loads, suggesting an important role for these inhibitors in the parasite’s survival. In contrast with recent findings of ISPs functionin Leishmania, that assigns different biological roles to ISP1 and ISP2, the present study reveals some functional redundancy of ISPs in T. brucei,with both inhibitors being required for the parasite to infect the mammalian host efficiently. Finally, immuno-protection studies were made, to assess the potential of ISPs as targets for therapy. Immunization with recombinant ISPs and treatment with antibodies specific against ISP1 and ISP2 had no protective or neutralizing effecton T. bruceiinfected mice, revealing that these inhibitors are poor candidates for an anti-disease vaccine or for antibody-based therapy.

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Premnauth, Gurdat. "Design, Synthesis and Biological Evaluation of New Molecules to Selectively Target Specific Cancers." University of Cincinnati / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1613744938434214.

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DI, SILVESTRE ALESSIA. "Identification of new molecular targets for personalized therapy in pediatric patients with inflammatory bowel disease (IBD)." Doctoral thesis, Università degli Studi di Trieste, 2018. http://hdl.handle.net/11368/2922616.

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Le malattie infiammatorie corniche intestinali (MICI) sono un gruppo di malattie infiammatorie immunomediate che comprendono il morbo di Crohn e la rettocolite ulcerosa. Nella popolazione pediatrica le MICI sono di particolare interesse a causa della aumentata incidenza della malattia e, sebbene siano stati sviluppati diversi approcci terapeutici, è molto difficile individuare il trattamento ottimale. I glucocorticoidi (GC) sono farmaci prescritti per indurre la remissione ma alcuni pazienti risultano resistenti al trattamento o richiedono terapie prolungate e tali pazienti sono soggetti a numerosi reazioni avverse. Il long non-coding RNA (lncRNA) growth-arrest specific 5 (GAS5) interagisce con il complesso GC-recettore dei glucocorticoidi (GR) inibendo l’attività trascrizionale dei geni responsivi ai GC. La prima parte del mio progetto di tesi si occupa di studiare il ruolo di GAS5 come marker molecolare della resistenza farmacologica ai GC. L’associazione tra il lncRNA e l’efficacia degli steroidi, espressa in termini di inibizione della proliferazione, è stata valutata su due linee cellulari tumorali di colon e ovaio che sono state identificate rispettivamente come modello di resistenza e sensibilità farmacologica ai GC. Inoltre, il ruolo di GAS5 è stato osservato nelle cellule mononucleate del sangue periferico di pazienti pediatrici affetti da MICI sia alla diagnosi che dopo 4 settimane di trattamento con GC; una maggiore espressione di GAS5 è stata osservata nei pazienti con una risposta sfavorevole agli steroidi. Questi risultati preliminari indicano che GAS5 potrebbe essere considerato un nuovo biomarker di resistenza farmacologica ai GC. I livelli di espressione di GAS5 sono stati valutati anche nelle biopsie di colon di pazienti pediatrici affetti da MICI anche rispetto ai livelli di espressione proteica e genica di due metalloproteasi (MMP) coinvolte nel danno tissutale. La downregolazione di GAS5 osservata nei tessuti infiammati rispetto ai tessuti non infiammati è inversamente correlata all’espressione delle MMP suggerendo che il lncRNA potrebbe controllare l’attività di queste proteine. Nella seconda parte del mio progetto di tesi abbiamo valutato i livelli di espressione proteica della tristetraprolin (TTP) nelle MICI. La TTP e una proteina zinc finger capace di interagire e inibire le citochine pro-infiammatorie attraverso il legame con gli elementi ricchi di AU sul 3’ UTR degli mRNA target. Abbiamo considerato anche il ruolo della sua fosforilazione, poiché questa modificazione post-traduzionale interferisce con l’attività della TTP che in questo stato è responsabile della stabilizzazione delle citochine d’interesse. L’espressione proteica della TTP è stata valutata nei tessuti di colon e nei macrofagi dei pazienti pediatrici affetti da MICI. L’espressione della TTP risulta upregolata sia nei tessuti di colon che nei macrofagi. I risultati inoltre confermano il coinvolgimento della fosforilazione nell’attività della TTP. Questi risultati preliminari, se confermati con ulteriori esperimenti, potrebbero aprire nuove prospettive nello studio delle IBD e nella formulazione di una nuova terapia farmacologica mirata in grado di modulare la fosforilazione della TTP attraverso l’uso di fosfatasi e favorire così la degradazione delle citochine pro-infiammatorie.
Inflammatory bowel disease (IBD) is a chronic immune-mediated condition of the gastrointestinal tract that includes Crohn disease and ulcerative colitis. Pediatric IBDs are of particular interest since their incidence is rising and, even if different pharmacological strategies are used, the optimal treatment is far from being achieved. Glucocorticoid (GCs) are prescribed for inducing remission but there is a high risk of adverse effects especially in subjects that poorly respond to these agents and require long treatments. The long non-coding RNA (lncRNA) growth arrest-specific 5 (GAS5) interacts with the activated glucocorticoid receptor (GR), inhibiting the transcription of GCs responsive genes. The first part of my thesis project is focused on the study of GAS5 as a molecular marker of GCs resistance. We evaluated the association between the lncRNA and the efficacy of steroids, in terms of inhibition of proliferation, in two immortalized cell lines from colon and ovarian cancers, a GC-resistant and GC-sensitive model, respectively. After GCs treatment in the GC-resistant cells GAS5 upregulation was observed and, in response to the drug, the lncRNA accumulated more in the cytoplasm compared to the nucleus. Furthermore, we evaluated GAS5 levels in the peripheral blood mononuclear cells of pediatric IBD patients at diagnosis and after 4 weeks of GCs administration. Gene expression analysis have shown an upregulation of the lncRNA in patients with unfavourable steroid response. These preliminary results suggest that GAS5 could be considered a novel pharmacogenomic marker useful for the personalization of GC therapy. GAS5 expression was also measured in IBD patients’ colon biopsies and its levels have been evaluated with respect to the gene and protein expression of two metalloproteinases (MMP-2, MMP-9) involved in tissue damage in IBDs. The GAS5 downregulation observed in inflamed tissues compared with the non-inflamed one is inversely related to MMPs expression suggesting a role of this lncRNA in controlling the activity of these molecules. In the second part of my thesis project we evaluated the role of the tristetraprolin (TTP) protein in IBDs. TTP is a zinc finger protein able to interact and inhibit pro-inflammatory cytokines through the binding with AU-rich elements on the 3’ untranslated region on mRNA. The role of phosphorylation on TTP activity was also evaluated, since this post-translational modification could impair protein activity and consequently the stabilization of cytokines levels. TTP protein expression was studied in pediatric IBDs patients’ colon tissues and in macrophages differentiated from peripheral blood mononuclear cells. An upregulation of TTP expression in both inflamed colon tissues and in macrophages of IBD patients was observed, and was closely related to the phosphorylation of the protein. These preliminary results, if confirmed with further experiments, could open new perspectives in the study of IBDs and in the investigation of new target therapy based on the modulation of TTP phosphorylation by phosphatases to favour pro-inflammatory cytokines degradation.

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Pan, Jie [Verfasser], and Georg [Akademischer Betreuer] Enders. "Molecularly targeted therapy on a new preclinical mouse model for gastric neuroendocrine tumors / Jie Pan. Betreuer: Georg Enders." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2013. http://d-nb.info/1038152062/34.

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Dissertations / Theses on the topic 'Molecular Targeted Therapy'

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