Journal articles: 'PrestoBlue'

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Gaucher, Sonia, and Mohamed Jarraya. "Technical note: comparison of the PrestoBlue and LDH release assays with the MTT assay for skin viability assessment." Cell and Tissue Banking 16, no. 3 (2014): 325–29. http://dx.doi.org/10.1007/s10561-014-9478-1.

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Shaikh, Zahab N., Abdullah Alqahtani, Thafar Almela, Kirsty Franklin, Lobat Tayebi, and Keyvan Moharamzadeh. "Effects of electronic cigarette liquid on monolayer and 3D tissue-engineered models of human gingival mucosa." Journal of Advanced Periodontology & Implant Dentistry 11, no. 2 (2019): 54–62. http://dx.doi.org/10.15171/japid.2019.010.

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Background. There is limited data available on potential biological effects of E-cigarettes on human oral tissues. The aim of this study was to evaluate the effects of E-cigarette liquid on the proliferation of normal and cancerous monolayer and 3D models of human oral mucosa and oral wound healing after short-term and medium-term exposure. Methods. Normal human oral fibroblasts (NOF), immortalized OKF6-TERET-2 human oral keratinocytes, and cancerous TR146 keratinocyte monolayer cultures and 3D tissue engineered oral mucosal models were exposed to different concentrations (0.1%, 1%, 5% and 10%) of E-cigarette liquid (12 mg/ml nicotine) for 1 hour daily for three days and for 7 days. Tissue viability was monitored using the PrestoBlue assay. Wounds were also produced in the middle surface of the monolayer systems vertically using a disposable cell scraper. The alterations in the cell morphology and wound healing were visualized using light microscopy and histological examination. Results. Statistical analysis showed medium-term exposure of TR146 keratinocytes to 5% and 10% E-liquid concentrations significantly increased the viability of the cancer cells compared to the negative control. Short-term exposure of NOFs to 10% E-liquid significantly reduced the cell viability, whereas medium-term exposure to all E-liquid concentrations significantly reduced the NOF cells’ viability. OKF6 cells exhibited significantly lower viability following short-term and mediumterm exposure to all E-cigarette concentrations compared to the negative control. 3D oral mucosal model containing normal oral fibroblasts and keratinocytes showed significant reduction in tissue viability after exposure to 10% E-liquid, whereas medium-term exposure resulted in significantly lower viability in 5% and 10% concentration groups compared to the negative control. There was a statistically significant difference in wound healing times of both NOF and OKF6 cells after exposure to 1%, 5% and 10% E-cigarette liquid. Conclusion. Medium-term exposure to high concentrations of the E-cigarette liquid had cytotoxic effects on normal human oral fibroblasts and OKF6 keratinocytes, but a stimulatory cumulative effect on the growth of cancerous TR146 keratinocyte cells as assessed by the PrestoBlue assay and histological evaluation of 3D oral mucosal models. In addition, E-liquid exposure prolonged the wound healing of NOF and OKF6 oral mucosa cells.

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Cheah, Joshua U.-Jin, Heng Boon Low, Yongliang Zhang, and James Chen Yong Kah. "Light-independent M1 macrophage polarization by photosensitizer-loaded protein corona on gold nanorods." Nanomedicine 15, no. 24 (2020): 2329–44. http://dx.doi.org/10.2217/nnm-2020-0249.

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Aim: To establish a light-independent functionality of gold nanorods (AuNRs) with a human serum (HS) protein corona loaded with photosensitizer Chlorin e6 (AuNR-HS-Ce6) in M1 polarization of macrophages. Methods: RT-qPCR and ELISA were used to determine gene and protein expression, respectively. Uptake of AuNR-HS-Ce6 was determined via flow cytometry, inductively coupled plasma mass spectrometry and fluorescence microscopy. Cell viability was determined using PrestoBlue® cell viability assay. Results: An increase in M1 gene and protein expression was observed in AuNR-HS-Ce6-treated macrophages. Delivery of high Ce6 payload via AuNR-HS-Ce6 was the primary contributor toward M1 polarization. Finally, DLD-1 cells treated with conditioned media from AuNR-HS-Ce6-treated macrophages showed significantly reduced proliferation. Conclusion: Our study suggests an immunomodulatory potential of Ce6 in inducing light-independent M1 polarization outside of its role as a photosensitizer.

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Sianipar, N. F., Y. E. Hadisaputri, K. Assidqi, P. Simanjuntak, and R. Purnamaningsih. "A STUDY OF ANTICANCER ACTIVITY FROM THE FRACTIONS OF RODENT TUBER SUPERIOR MUTANT EXTRACT (Typhonium flagelliforme) BY PRESTOBLUE ASSAY METHOD." Rasayan Journal of chemistry 13, no. 03 (2020): 1992–98. http://dx.doi.org/10.31788/rjc.2020.1335703.

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Xu, Manlong, David J. McCanna, and Jacob G. Sivak. "Use of the viability reagent PrestoBlue in comparison with alamarBlue and MTT to assess the viability of human corneal epithelial cells." Journal of Pharmacological and Toxicological Methods 71 (January 2015): 1–7. http://dx.doi.org/10.1016/j.vascn.2014.11.003.

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Boncler, Magdalena, Marek Różalski, Urszula Krajewska, Anna Podsędek, and Cezary Watala. "Comparison of PrestoBlue and MTT assays of cellular viability in the assessment of anti-proliferative effects of plant extracts on human endothelial cells." Journal of Pharmacological and Toxicological Methods 69, no. 1 (2014): 9–16. http://dx.doi.org/10.1016/j.vascn.2013.09.003.

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Oeschger, Taylor Mae, and David Carl Erickson. "Visible colorimetric growth indicators of Neisseria gonorrhoeae for low-cost diagnostic applications." PLOS ONE 16, no. 6 (2021): e0252961. http://dx.doi.org/10.1371/journal.pone.0252961.

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N. gonorrhoeae is one of the most pressing antibiotic resistant threats of our time and low-cost diagnostics that can easily identify antibiotic resistance are desperately needed. However, N. gonorrhoeae responds so uniquely to growth conditions that it cannot be assumed gonorrhea will respond to common microbiological methods used for other pathogenic organisms. In this paper, we explore visual colorimetric indicators of N. gonorrhoeae growth that can be seen without a microscope or spectrophotometer. We evaluate growth media, pH indicators, resazurin-based dyes, and tetrazolium-based dyes for their use in simple colorimetric system. Overall, we identified Graver Wade media as the best at supporting robust gonococcal growth while also providing the least background when analyzing results of colorimetric tests. XTT, a tetrazolium-based dye, proved to show to brightest color change over time and not negatively impact the natural growth of N. gonorrhoeae. However, other dyes including PrestoBlue, MTT, and NBT are less expensive than XTT and work well when added after bacterial growth has already occurred. By identifying the specific use cases of these dyes, this research lays the groundwork for future development of a color-based antibiotic susceptibility low-cost test for N. gonorrhoeae.

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Lauritano, Dorina, Giulia Moreo, Fedora Della Vella, Annalisa Palmieri, Francesco Carinci, and Massimo Petruzzi. "Biology of Drug-Induced Gingival Hyperplasia: In Vitro Study of the Effect of Nifedipine on Human Fibroblasts." Applied Sciences 11, no. 7 (2021): 3287. http://dx.doi.org/10.3390/app11073287.

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Background: It has been proven that the antihypertensive agent nifedipine can cause gingival overgrowth as a side effect. The aim of this study was to analyze the effects of pharmacological treatment with nifedipine on human gingival fibroblasts activity, investigating the possible pathogenetic mechanisms that lead to the onset of gingival enlargement. Methods: The expression profile of 57 genes belonging to the “Extracellular Matrix and Adhesion Molecules” pathway, fibroblasts’ viability at different drug concentrations, and E-cadherin levels in treated fibroblasts were assessed using real-time Polymerase Chain Reaction, PrestoBlue™ cell viability test, and an enzyme-linked immunoassay (ELISA), respectively. Results: Metalloproteinase 24 and 8 (MMP24, MMP8) showed significant upregulation in treated cells with respect to the control group, and cell adhesion gene CDH1 (E-cadherin) levels were recorded as increased in treated fibroblasts using both real-time PCR and ELISA. Downregulation was observed for transmembrane receptors ITGA6 and ITGB4, the basement membrane constituent LAMA1 and LAMB1, and the extracellular matrix protease MMP11, MMP16, and MMP26. Conclusions: The obtained data suggested that the pathogenesis of nifedipine-induced gingival overgrowth is characterized by an excessive accumulation of collagen due to the inhibition of collagen intracellular and extracellular degradation pathways.

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Hernandez-Patlan, D., B. Solis-Cruz, A. Méndez-Albores, et al. "Comparison of PrestoBlue® and plating method to evaluate antimicrobial activity of ascorbic acid, boric acid and curcumin in an in vitro gastrointestinal model." Journal of Applied Microbiology 124, no. 2 (2018): 423–30. http://dx.doi.org/10.1111/jam.13659.

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Strzempek, Weronika, Aleksandra Korzeniowska, Andrzej Kowalczyk, Wieslaw J. Roth, and Barbara Gil. "Detemplated and Pillared 2-Dimensional Zeolite ZSM-55 with Ferrierite Layer Topology as a Carrier for Drugs." Molecules 25, no. 15 (2020): 3501. http://dx.doi.org/10.3390/molecules25153501.

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The present studies were conducted to show the potential of 2D zeolites as effective and non-toxic carriers of drugs. Layered zeolites exhibit adjustable interlayer porosity which can be exploited for controlled drug delivery allowing detailed investigation of the drug release because the structure of the carrier is known exactly. This study was conducted with model drugs ciprofloxacin and piracetam, and ZSM-55 with ca 1 nm thick layers, in detemplated and pillared forms. The release profiles differed from the commercial, crystalline forms of drugs—the release rate increased for ciprofloxacin and decreased for piracetam. To understand the dissolution mechanisms the release data were fitted to Korsmeyer-Peppas equation, showing Fickian (for pillared) and anomalous (for detemplated sample) transport. FT-IR studies showed that strong interaction carrier-drug may be responsible for the modified, slowed down release of piracetam while better solubility and faster release of ciprofloxacin was attributed to formation of the protonated form resulting in weaker interaction with the zeolite than in the pure crystalline form. Two independent tests on L929 mice fibroblasts (ToxiLight and PrestoBlue) showed that ZSM-55, in moderate concentrations may be safely used as a carrier of drug molecules, not having negative effect on the cells viability or proliferation rate.

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Schäfer, Benedikt, Caroline Emonts, Nikola Glimpel, et al. "Warp-Knitted Spacer Fabrics: A Versatile Platform to Generate Fiber-Reinforced Hydrogels for 3D Tissue Engineering." Materials 13, no. 16 (2020): 3518. http://dx.doi.org/10.3390/ma13163518.

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Mesenchymal stem cells (MSCs) possess huge potential for regenerative medicine. For tissue engineering approaches, scaffolds and hydrogels are routinely used as extracellular matrix (ECM) carriers. The present study investigated the feasibility of using textile-reinforced hydrogels with adjustable porosity and elasticity as a versatile platform for soft tissue engineering. A warp-knitted poly (ethylene terephthalate) (PET) scaffold was developed and characterized with respect to morphology, porosity, and mechanics. The textile carrier was infiltrated with hydrogels and cells resulting in a fiber-reinforced matrix with adjustable biological as well as mechanical cues. Finally, the potential of this platform technology for regenerative medicine was tested on the example of fat tissue engineering. MSCs were seeded on the construct and exposed to adipogenic differentiation medium. Cell invasion was detected by two-photon microscopy, proliferation was measured by the PrestoBlue assay. Successful adipogenesis was demonstrated using Oil Red O staining as well as measurement of secreted adipokines. In conclusion, the given microenvironment featured optimal mechanical as well as biological properties for proliferation and differentiation of MSCs. Besides fat tissue, the textile-reinforced hydrogel system with adjustable mechanics could be a promising platform for future fabrication of versatile soft tissues, such as cartilage, tendon, or muscle.

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Paterson, Thomas E., Rui Shi, Jingjing Tian, et al. "Electrospun Scaffolds Containing Silver-Doped Hydroxyapatite with Antimicrobial Properties for Applications in Orthopedic and Dental Bone Surgery." Journal of Functional Biomaterials 11, no. 3 (2020): 58. http://dx.doi.org/10.3390/jfb11030058.

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Preventing the development of osteomyelitis while enhancing bone regeneration is challenging, with relatively little progress to date in translating promising technologies to the clinic. Nanoscale hydroxyapatite (nHA) has been employed as a bone graft substitute, and recent work has shown that it may be modified with silver to introduce antimicrobial activity against known pathogens. The aim of this study was to incorporate silver-doped nHA into electrospun scaffolds for applications in bone repair. Silver-doped nHA was produced using a modified, rapid mixing, wet precipitation method at 2, 5, 10 mol.% silver. The silver-doped nHA was added at 20 wt.% to a polycaprolactone solution for electrospinning. Bacteria studies demonstrated reduced bacterial presence, with Escherichia coli and Staphylococcus aureus undetectable after 96 h of exposure. Mesenchymal stem cells (MSCs) were used to study both toxicity and osteogenicity of the scaffolds using PrestoBlue® and alkaline phosphatase (ALP) assays. Innovative silver nHA scaffolds significantly reduced E. coli and S. aureus bacterial populations while maintaining cytocompatibility with mammalian cells and enhancing the differentiation of MSCs into osteoblasts. It was concluded that silver-doped nHA containing scaffolds have the potential to act as an antimicrobial device while supporting bone tissue healing for applications in orthopedic and dental bone surgery.

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Shyu, Peter T., Glenn G. Oyong та Esperanza C. Cabrera. "Cytotoxicity of Probiotics from Philippine Commercial Dairy Products on Cancer Cells and the Effect on Expression ofcfosandcjunEarly Apoptotic-Promoting Genes andInterleukin-1βandTumor Necrosis Factor-αProinflammatory Cytokine Genes". BioMed Research International 2014 (2014): 1–9. http://dx.doi.org/10.1155/2014/491740.

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This study determined cytotoxicity of probioticLactobacillusspp. from Philippine dairy products on cancer cells and normal fibroblasts and their effects on expression of early apoptotic-promotingcfos, cjunand proinflammatory cytokineIL-1β,TNF-αgenes. Cultures were from Yakult, Bear Brand Probiotic Drink, Nido3+ Powdered Milk. Filter-sterilized supernatants from cultures ofLactobacillusspp. were evaluated for cytotoxicity to colon cancer cells (HT-29 and HCT116), leukemia cells (THP-1), and normal human dermal fibroblasts (HDFn) using PrestoBlue. Bleomycin was the positive control. Absolute quantification of transcript levels was conducted using qRT-PCR. Cytotoxicity index profiles on HDFn, THP-1 of all probiotic supernatants and negative controls suggest nontoxicity to the cells when compared to bleomycin, whereas all probiotic supernatants were found to be cytotoxic to HT-29 and HCT-116 colon cancer cell lines. Expression ofcfos, cjuntranscripts was significantly upregulated in HT-29 and HCT116 cells treated with probiotic supernatants compared to untreated baseline levels (P<0.05). Expression ofIL-1βandTNF-αby lipopolysaccharide-treated macrophages was significantly downregulated in cells with probiotic supernatants compared to those exposed to MRS medium (P<0.05). Results provide strong support for the role ofLactobacillusspp. studied in anticancer therapy and in prevention of inflammation that may act as precursor to carcinogenesis.

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Xiao, Li, Mai Mochizuki, Taka Nakahara, and Nobuhiko Miwa. "Hydrogen-Generating Silica Material Prevents UVA-Ray-Induced Cellular Oxidative Stress, Cell Death, Collagen Loss and Melanogenesis in Human Cells and 3D Skin Equivalents." Antioxidants 10, no. 1 (2021): 76. http://dx.doi.org/10.3390/antiox10010076.

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Ultraviolet-A (UVA) irradiation induces harmful effects on skin cells and accelerates skin aging through oxidative stress. In this study, the effects of a hydrogen-generating silica material named ULH-002 against UVA injuries in human cells and 3D skin equivalents were investigated. The oxygen radical absorption capacity (ORAC) assay showed that both freshly prepared ULH-002 solutions and 7-day-old solutions exhibited equal peroxyl radical (ROO·) scavenging activities concentration-dependently. CellROX® green/orange staining showed that ULH-002 could reduce UVA-induced oxidative stress in human keratinocytes HaCaT and human gingival fibroblasts (HGFs). ULH-002 significantly prevented UVA-induced apoptotic/necrotic cell death and cell-viability decline in HGFs and keratinocytes, as shown by Annexin V/PI apoptosis assay and PrestoBlue assay, respectively. Immunostaining showed that ULH-002 prevented the UVA-induced deterioration of expression of both type IV and I collagens in the 3D skin equivalents, and similarly in monolayer HGFs. UVA-enhanced melanogenesis was observed in human melanocytes HMV-II and HMV-II cell-containing 3D skin equivalents, but markedly prevented by ULH-002 as demonstrated by Fontana–Masson’s staining. In conclusion, our data suggested that ULH-002 could protect human keratinocytes and fibroblasts from UVA-induced injuries, prevent the loss of type IV and I collagens, as well as reduce melanogenesis. ULH-002 might be developed as a skin care reagent in the cosmetic industry.

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Nowak, Adriana, Małgorzata Zakłos-Szyda, Dorota Żyżelewicz, Agnieszka Koszucka, and Ilona Motyl. "Acrylamide Decreases Cell Viability, and Provides Oxidative Stress, DNA Damage, and Apoptosis in Human Colon Adenocarcinoma Cell Line Caco-2." Molecules 25, no. 2 (2020): 368. http://dx.doi.org/10.3390/molecules25020368.

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Acrylamide (AA) toxicity remains an interesting subject in toxicological research. The aim of the research performed in this paper was to determine mechanisms of cyto- and genotoxic effects of AA on the human colon adenocarcinoma cell line Caco-2, to estimate the inhibitory concentration (IC)50 values in cell viability assays, to measure the basal and oxidative DNA damage as well as the oxidative stress leading to apoptosis, and to assess the morphological changes in cells using microscopic methods. It has been proven that AA induces cytotoxic and genotoxic effects on Caco-2 cells. Higher cytotoxic activity was gained in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay compared with the PrestoBlue assay, with IC50 values of 5.9 and 8.9 mM after 24 h exposure, respectively. In the single-cell gel electrophoresis assay, the greatest DNA damage was caused by the highest concentration of acrylamide equal to 12.5 mM (89.1% ± 0.9%). AA also induced oxidative DNA damage and generated reactive oxygen species (ROS), which was concentration dependent and correlated with the depletion of mitochondrial membrane potential and apoptosis induction. In the microscopic staining of cells, AA in the dosage close to the IC50 induced morphological changes typical for apoptosis. Taken together, these results demonstrate that AA has a pro-oxidative effect on Caco-2 cells, leading to apoptotic cell death.

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Xiao, Li, Mai Mochizuki, Taka Nakahara, and Nobuhiko Miwa. "Hydrogen-Generating Silica Material Prevents UVA-Ray-Induced Cellular Oxidative Stress, Cell Death, Collagen Loss and Melanogenesis in Human Cells and 3D Skin Equivalents." Antioxidants 10, no. 1 (2021): 76. http://dx.doi.org/10.3390/antiox10010076.

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Ultraviolet-A (UVA) irradiation induces harmful effects on skin cells and accelerates skin aging through oxidative stress. In this study, the effects of a hydrogen-generating silica material named ULH-002 against UVA injuries in human cells and 3D skin equivalents were investigated. The oxygen radical absorption capacity (ORAC) assay showed that both freshly prepared ULH-002 solutions and 7-day-old solutions exhibited equal peroxyl radical (ROO·) scavenging activities concentration-dependently. CellROX® green/orange staining showed that ULH-002 could reduce UVA-induced oxidative stress in human keratinocytes HaCaT and human gingival fibroblasts (HGFs). ULH-002 significantly prevented UVA-induced apoptotic/necrotic cell death and cell-viability decline in HGFs and keratinocytes, as shown by Annexin V/PI apoptosis assay and PrestoBlue assay, respectively. Immunostaining showed that ULH-002 prevented the UVA-induced deterioration of expression of both type IV and I collagens in the 3D skin equivalents, and similarly in monolayer HGFs. UVA-enhanced melanogenesis was observed in human melanocytes HMV-II and HMV-II cell-containing 3D skin equivalents, but markedly prevented by ULH-002 as demonstrated by Fontana–Masson’s staining. In conclusion, our data suggested that ULH-002 could protect human keratinocytes and fibroblasts from UVA-induced injuries, prevent the loss of type IV and I collagens, as well as reduce melanogenesis. ULH-002 might be developed as a skin care reagent in the cosmetic industry.

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Kanpittaya, Kasama, Aroon Teerakapong, Noppawan Phumala Morales, et al. "Inhibitory Effects of Erythrosine/Curcumin Derivatives/Nano-Titanium Dioxide-Mediated Photodynamic Therapy on Candida albicans." Molecules 26, no. 9 (2021): 2405. http://dx.doi.org/10.3390/molecules26092405.

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This study focuses on the role of photosensitizers in photodynamic therapy. The photosensitizers were prepared in combinations of 110/220 µM erythrosine and/or 10/20 µM demethoxy/bisdemethoxy curcumin with/without 10% (w/w) nano-titanium dioxide. Irradiation was performed with a dental blue light in the 395–480 nm wavelength range, with a power density of 3200 mW/cm2 and yield of 72 J/cm2. The production of ROS and hydroxyl radical was investigated using an electron paramagnetic resonance spectrometer for each individual photosensitizer or in photosensitizer combinations. Subsequently, a PrestoBlue® toxicity test of the gingival fibroblast cells was performed at 6 and 24 h on the eight highest ROS-generating photosensitizers containing curcumin derivatives and erythrosine 220 µM. Finally, the antifungal ability of 22 test photosensitizers, Candida albicans (ATCC 10231), were cultured in biofilm form at 37 °C for 48 h, then the colonies were counted in colony-forming units (CFU/mL) via the drop plate technique, and then the log reduction was calculated. The results showed that at 48 h the test photosensitizers could simultaneously produce both ROS types. All test photosensitizers demonstrated no toxicity on the fibroblast cells. In total, 18 test photosensitizers were able to inhibit Candida albicans similarly to nystatin. Conclusively, 20 µM bisdemethoxy curcumin + 220 µM erythrosine + 10% (w/w) nano-titanium dioxide exerted the highest inhibitory effect on Candida albicans.

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Lauritano, Dorina, Giulia Moreo, Luisa Limongelli, Annalisa Palmieri, and Francesco Carinci. "Drug-Induced Gingival Overgrowth: The Effect of Cyclosporin A and Mycophenolate Mophetil on Human Gingival Fibroblasts." Biomedicines 8, no. 7 (2020): 221. http://dx.doi.org/10.3390/biomedicines8070221.

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Drug-induced gingival overgrowth may occur after a chronic administration of three classes of systemic drugs: Anticonvulsants, immunosuppressants, and calcium channel blockers. This study aimed to investigate how cyclosporin A and mycophenolate mophetil (immunosuppressive drugs) could interfere with human gingival fibroblasts functions, leading to gingival enlargement. Human gingival fibroblasts derived from the tissue of a 60-year-old female were cultured in a DMEME medium. A stock solution with 1 mg/mL of mycophenolate and 1 mg/mL of cyclosporine were prepared and dissolved in a DMEM medium to prepare a serial dilution at the concentrations of 5000, 2000, 1000, 500, and 100 ng/mL, for both treatments. Cell viability was measured using the PrestoBlue™ Reagent Protocol. Quantitative real-time RT-PCR was performed in order to analyze the expression of 57 genes coding for gingival fibroblasts “Extracellular Matrix and Adhesion Molecules”. Mycophenolate and cyclosporine had no effect on fibroblast cell viability at 1000 ng/mL. Both the treatments showed similar effects on the expression profiling of treated cells: Downregulation of most extracellular matrix metalloproteases genes (MMP8, MMP11, MMP15, MMP16, MMP24) was assessed, while CDH1, ITGA2, ITGA7, LAMB3, MMP12, and MMP13 were recorded to be upregulated in fibroblasts treated with immunosuppressive drugs. It has been demonstrated that gingival overgrowth can be caused by the chronic administration of cyclosporin A and mycophenolate mophetil. However, given the contrasting data of literature, further investigations are needed, making clear the possible effects of immunosuppressive drugs on fibroblasts.

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Ragasa, Consolacion Y., Glenn G. Oyong, Maria Carmen S. Tan, Mariquit M. De Los Reyes, and Maria Ellenita G. De Castro. "Cytotoxic Sterols from Philippine Mushrooms." Asian Journal of Chemistry 32, no. 5 (2020): 1197–202. http://dx.doi.org/10.14233/ajchem.2020.22591.

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Ergosterol peroxide (1) and ergosterol (2) were commonly isolated as the major compounds of Philippine mushrooms. Sterols 1 and 2 from the dichloromethane extract of Geastrum triplex and Termitomyces clypeatus, respectively, were evaluated for their cytotoxic activities against four human cancer cell lines, viz., breast cancer (MCF-7), colon cancer (HT-29), leukemia (THP-1), and small lung cell carcinoma (H69PR), and a human normal cell line, human dermal fibroblast-neonatal (HDFn), using the PrestoBlue® cell viability assay. Compounds 1 and 2 exhibited the strongest activities against HT-29 with IC50 values of 1.79 and 2.98 μg/mL, respectively, while Zeocin gave an IC50 of 4.89 μg/mL. These compounds also exhibited strong antiproliferative effects against MCF-7 with IC50 values of 4.13 for 1 and 4.20 μg/mL for compound 2, comparable to Zeocin with IC50 = 3.68 μg/mL. Only moderate cytotoxicity resulted when compounds 1 and 2 were tested against H69PR with IC50 values of 7.78 and 6.83 μg/mL, respectively, while Zeocin exhibited an IC50 of 9.81 μg/mL. Furthermore, compounds 1 and 2 showed no effects against THP-1 (IC50 > 100 μg/mL), while Zeocin showed an IC50 of 4.73 μg/mL. Although compounds 1 and 2 have been reported to exhibit different bioactivities in previous studies, the cancer cell lines tested and/or the polarities of the solvents for extraction varied. Therefore, comparisons of the cytotoxic activities of compounds 1 and 2 with earlier studies could not be made extensively.

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Manogaran, Manimegalai, Vuanghao Lim, and Rafeezul Mohamed. "Phytoconstituents of the Gynura procumbens ethanol leaf extract and its fractions and their effects on viability of macrophages." Journal of Herbmed Pharmacology 8, no. 3 (2019): 224–30. http://dx.doi.org/10.15171/jhp.2019.33.

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Introduction: Gynura procumbens (GP) is a medicinal plant with numerous beneficial pharmacological activities. The aim of this study was to identify the bioactive phytoconstituents in GP ethanol extract and hexane, chloroform, ethyl acetate, and aqueous fractions of GP, and also to evaluate the cell viability of GP ethanol extract and its fractions-treated RAW264.7 cells.Methods: The ethanol GP leaf extract was prepared and further subjected to fractionation. The cell viability of GP ethanol extract and its fractions-treated RAW264.7 cells were measured by PrestoBlue. The phytoconstituents of GP ethanol extract and its fraction were determined by using liquid chromatography-mass spectrometry (LC-MS).Results: RAW264.7 cells exposed to the GP ethanol extract and its fractions showed significantly high proliferation and weak cytotoxic effect on the macrophages, with an average inhibitory concentration of 90% at 24, 48, and 72 hours of incubation. However, at a concentration of 10 μg/mL, the aqueous GP fraction clearly displayed anti-proliferative properties because the cell viability of aqueous GP fraction-treated RAW264.7 cells reduced to 64%, 29% and 4% after 24, 48 and 72 hours of incubation, respectively. The GP extracts and its fractions contained mainly fatty acids, flavonoids, sesquiterpenoids, and products of chlorophyll breakdown.Conclusion: GP ethanol extract and its fractions at certain concentrations may act as immunomodulators, as they induced promising proliferation activity of macrophages. Further studies are needed to determine either the identified chemical compounds influenced on the proliferation of macrophages solely or cooperatively.

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Chauvin, Julie, Florian Judee, Nofel Merbahi, and Patricia Vicendo. "Effects of Plasma Activated Medium on Head and Neck FaDu Cancerous Cells: Comparison of 3D and 2D Response." Anti-Cancer Agents in Medicinal Chemistry 18, no. 6 (2018): 776–83. http://dx.doi.org/10.2174/1871520617666170801111055.

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Objective: The aim of this work is to investigate the inhibitory effect of Plasma Activated Medium (PAM) on Head and Neck cancerous cells (FaDu). The response of FaDu cells in monolayer cultures and Multi Cellular Tumor Spheroids (MCTS) after treatment with different PAMs will be compared. Background: Head and Neck squamous cell carcinoma is a widespread cancer that responds poorly to anticancer treatments such as chemotherapy and radiotherapy. Nowadays there is a growing interest in cold plasmas and their applications in cancer therapy. Methods: A homemade helium plasma jet is used to produce PAM. The effects of PAM and hydrogen peroxide H2O2 on FaDu 2D cells cultures and MCTS were characterized by evaluating the cell viability with PrestoBlue test and by measuring the size of MCTS. Results: One treatment with PAM induce cell detachment from MCTS since the first day in a PAM exposure dependent manner. This is due to the presence of H2O2 in PAM. However, a rapid spheroids regrowth is observed attributed to a resistance of FaDu cells to H2O2. After multiple treatments of MCTS with PAM we obtained an inhibition of cell growth. MCTS are brought out when comparing PAM effect on 2D versus MCTS. Inversely, PAM induces cell death in the case of 2D cell culture. Conclusion: PAM may be considered as a potentially efficient agent in the therapy of head and neck cancer. We also point out that MCTS is a more valuable model than 2D cell culture for the evaluation of the anti-cancer activity of PAM.

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Jarquin-Yáñez, Katia, Efrain Rubio-Rosas, Gabriela Piñón-Zárate, Andrés Castell-Rodríguez, and Martha Poisot. "Cellulose-Chitosan-Nanohydroxyapatite Hybrid Composites by One-Pot Synthesis for Biomedical Applications." Polymers 13, no. 10 (2021): 1655. http://dx.doi.org/10.3390/polym13101655.

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The development of organic–inorganic hybrid materials deserves special interest for bone tissue engineering applications, where materials must have properties that induce the survival and activation of cells derived from the mesenchyme. In this work, four bio-nanocomposites based on cellulose and variable content of chitosan, from 15 to 50 w% based on cellulose, with nanohydroxyapatite and β-Glycerophosphate as cross-linking agent were synthesized by simplified and low-energy-demanding solvent exchange method to determine the best ratio of chitosan to cellulose matrix. This study analyzes the metabolic activity and survival of human dermal fibroblast cells cultivated in four bio-nanocomposites based on cellulose and the variable content of chitosan. The biocompatibility was tested by the in vitro cytotoxicity assays Live/Dead and PrestoBlue. In addition, the composites were characterized by FTIR, XRD and SEM. The results have shown that the vibration bands of β-Glycerophosphate have prevailed over the other components bands, while new diffraction planes have emerged from the interaction between the cross-linking agent and the biopolymers. The bio-nanocomposite micrographs have shown no surface porosity as purposely designed. On the other hand, cell death and detachment were observed when the composites of 1 and 0.1 w/v% were used. However, the composite containing 10 w% chitosan, against the sum of cellulose and β-Glycerophosphate, has shown less cell death and detachment when used at 0.01 w/v%, making it suitable for more in vitro studies in bone tissue engineering, as a promising economical biomaterial.

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Koppelmann, Tal, Yulia Pollak, Yoav Ben-Shahar, Gregory Gorelik, and Igor Sukhotnik. "The Mechanisms of the Anti-Inflammatory and Anti-Apoptotic Effects of Omega-3 Polyunsaturated Fatty Acids during Methotrexate-Induced Intestinal Damage in Cell Line and in a Rat Model." Nutrients 13, no. 3 (2021): 888. http://dx.doi.org/10.3390/nu13030888.

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Background: The aim of this study was to examine the anti-inflammatory and anti-apoptotic patterns of omega-3 polyunsaturated fatty acids (n-3 PUFAs) during methotrexate (MTX) induced intestinal damage in cell culture and in a rat model. Methods: Non-treated and treated with MTX HT 29 and HCT116cells were exposed to increasing doses of n-3 PUFAs and cell viability was evaluated using PrestoBlue® assay. Male Sprague-Dawley rats were divided into 4 experimental groups: Control rats, CONTR+n-3 PUFA rats that were treated with oral n-3 PUFA, MTX rats were treated with MTX given IP, and MTX+n-3 PUFA rats were treated with oral n-3 PUFA before and following injection of MTX. Intestinal mucosal parameters and mucosal inflammation, enterocyte proliferation and apoptosis, TNF-α in mucosal tissue and plasma (ELISA), NF-κB, COX-2, TNF-α, Fas, FasL, Fadd, Bid, Bax and Bcl-2gene and protein levels were determined 72 h following MTX injection. Results: Exposure of HT 29 and HCT116cells to n-3 PUFA attenuated inhibiting effects of MTX on cell viability. MTX-n-3 PUFA rats demonstrated a lower intestinal injury score and enhanced intestinal repair. A significant decrease in enterocyte apoptosis in MTX+n-3 PUFA rats was accompanied by decreased TNF-α, FAS, FasL, FADD and BID mRNA levels. Decreased NF-κB, COX-2 and TNF-α levels in mucosa was accompanied by a decreased number of IELs and macrophages. Conclusions: n-3 PUFAs inhibit NF-κB/COX-2 induced production of pro-inflammatory cytokines and inhibit cell apoptosis mainly by extrinsic pathway in rats with MTX-induced intestinal damage.

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Chen, Cheng-Yu, Ming-You Shie, Alvin Kai-Xing Lee, Yun-Ting Chou, Chun Chiang, and Chun-Pin Lin. "3D-Printed Ginsenoside Rb1-Loaded Mesoporous Calcium Silicate/Calcium Sulfate Scaffolds for Inflammation Inhibition and Bone Regeneration." Biomedicines 9, no. 8 (2021): 907. http://dx.doi.org/10.3390/biomedicines9080907.

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Bone defects are commonly found in the elderly and athletic population due to systemic diseases such as osteoporosis and trauma. Bone scaffolds have since been developed to enhance bone regeneration by acting as a biological extracellular scaffold for cells. The main advantage of a bone scaffold lies in its ability to provide various degrees of structural support and growth factors for cellular activities. Therefore, we designed a 3D porous scaffold that can not only provide sufficient mechanical properties but also carry drugs and promote cell viability. Ginsenoside Rb1 (GR) is an extract from panax ginseng, which has been used for bone regeneration and repair since ancient Chinese history. In this study, we fabricated scaffolds using various concentrations of GR with mesoporous calcium silicate/calcium sulfate (MSCS) and investigated the scaffold’s physical and chemical characteristic properties. PrestoBlue, F-actin staining, and ELISA were used to demonstrate the effect of the GR-contained MSCS scaffold on cell proliferation, morphology, and expression of the specific osteogenic-related protein of human dental pulp stem cells (hDPSCs). According to our data, hDPSCs cultivated in GR-contained MSCS scaffold had preferable abilities of proliferation and higher expression of the osteogenic-related protein and could effectively inhibit inflammation. Finally, in vivo performance was assessed using histological results that revealed the GR-contained MSCS scaffolds were able to further achieve more effective hard tissue regeneration than has been the case in the past. Taken together, this study demonstrated that a GR-containing MSCS 3D scaffold could be used as a potential alternative for future bone tissue engineering studies and has good potential for clinical use.

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Cheong, Ye-Whang, Young Choi Kim та Tae-Hoon Kim. "Relationship of anti-proliferative activity of TAS-108, a steroid anti-estrogen, and high expression of ERα in MCF7 cells." Journal of Clinical Oncology 31, № 15_suppl (2013): e11566-e11566. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.e11566.

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e11566 Background: TAS-108 is an anti-estrogen that binds to both ERα and ERβ and has potent anti-proliferative activity in vitro and in vivo assay. Recently, the phase II clinical study of TAS-108 showed that it has competitive clinical benefic with lower level of adverse effects. However, the effect of TAS-108 on molecular mechanism, including ER dimerization, remains largely uninvestigated. Methods: The plasmids containing ERα or ERβ fused with N- and C-terminal fragments of firefly luciferase were constructed for split luciferase complementation assays. The resulting plasmids were transfected in HEK293 cells for 24 hr and TAS-108 was added to the media for 6 hr. For RT-qPCR of AREG and TFF1 and proliferation assay, ERβ was transfected in MCF7 cells for 24hr. The proliferation level was determined by measuring the fluorescent level induced by Prestoblue reagent. Results: Using split luciferase complementation assay, we determined that TAS-108 induced ERα/β heterodimerization about 100-fold more potently than ERα/α homodimerization. Increasing the ERβ level by transfection in MCF7 cells (MCF7-ERβ), potency of TAS-108 was increased significantly; 30nM TAS-108 was suitable to block the E2 mediated increase of AREG and TFF1 expression and significantly decrease proliferation of MCF7-ERβ. Conclusions: Taken together, these data suggest that TAS-108 has a higher affinity for ERα/β heterodimerization than ERα/α homodimerization and TAS-108 shows more effective anti-proliferative activity by blocking the expression of E2-induced proliferative genes when coupled with increased levels of ERβ. These findings also underscore the molecular role of ERβ in the biology and suggest the possibility of the compound inducing ERα/β heterodimerization as anti-breast cancer drugs.

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Basok, Y. B., A. M. Grigoryev, L. A. Kirsanova, et al. "Comparative study of chondrogenesis of human adipose-derived mesenchymal stem cells when cultured in collagen-containing media under in vitro conditions." Russian Journal of Transplantology and Artificial Organs 23, no. 3 (2021): 90–100. http://dx.doi.org/10.15825/1995-1191-2021-3-90-100.

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In terms of method of production, collagen carriers are subdivided into materials obtained on the basis of extracellular matrix (ECM) components, particularly collagen-containing hydrogels and decellularized tissue.Objective: to compare in vitro the ability of biopolymer microheterogeneous collagen-containing hydrogel (BMCH) and tissue-specific matrix from decellularized porcine articular cartilage (DPAC) to support adhesion, proliferation and chondrogenic differentiation of human adipose-derived mesenchymal stem cells (hAMSCs).Materials and methods. For cartilage decellularization, we carried out treatment with surfactants (sodium dodecyl sulfate, Triton X-100) followed by exposure in DNAase. The metabolic activity of hAMSCs was assessed by PrestoBlue™ (Invitrogen, USA) staining. The morphological study of cell-engineered constructs (CECs) formed by culturing hAMSCs in the presence of matrices was performed using histological staining and scanning electron microscopy (SEM) with lanthanide contrasting.Results. The number of cells on the surface of both BMCH and DPAC increased within 14 days. Mitochondrial activity of the cells was 1.7, 1.7, and 1.3 times higher on days 3, 10, and 14 when cultured on DPAC compared to BMCH, respectively. On day 14 of cultivation in the chondrogenic culture medium, hAMSCs formed cell layers on the DPAC surface and on the BMCH surface. Cytoplasm of the cells included numerous granules, which, when stained, resembled the matrix itself. On the DPAC matrix surface, cells were more evenly distributed, whereas in the case of BMCH, cell adhesion and proliferation were observed only in certain areas. The ECM produced by the cells contained collagen and glycosaminoglycans (GAGs).Conclusion. The ability of DPAC obtained according to the developed protocol to form CECs with hAMSCs with uniform distribution of cells and their production of specific collagen- and GAG-containing ECM suggests that DPAC is effective in regeneration of damaged cartilage. Chondrogenic differentiation of hAMSCs was observed both when cultured with BMCH and with DPAC. When creating a tissue equivalent of cartilage in vitro, the advantage of using tissue-specific matrix over BMCH should be considered.

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Grigoriev, A. M., Yu B. Basok, A. D. Kirillova, et al. "Experimental approaches to creating a tissue-specific matrix for a bioartificial liver." Russian Journal of Transplantology and Artificial Organs 22, no. 3 (2020): 123–33. http://dx.doi.org/10.15825/1995-1191-2020-3-123-133.

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Shortage of donor organs for liver transplantation in the treatment of end-stage liver disease dictates the need to develop alternative methods that include technologies on tissue engineering and regenerative medicine. Objective: to study the ability of a tissue-specific matrix from decellularized human liver fragments (DHLF) to maintain adhesion and proliferation of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) and HepG2 under static conditions and in a flow-through bioreactor. Materials and methods. Treatment with surfactants (SAS) – sodium dodecyl sulfate, Triton X-100 – followed by exposure to DNase was used for decellularization of human liver fragments (no more than 8 mm3). Biochemical screening included the determination of DNA quantity in the test samples. Efficiency of surfactant washing was assessed by the cytotoxicity of the matrix in the NIH 3T3 fibroblast culture. Viability and metabolic activity of cells were assessed via vital staining with a complex of fluorescent dyes LIVE/DEAD ® and PrestoBlue™ (Invitrogen, USA). Morphological examination of the liver cell-engineered constructs was carried out through histological staining and scanning electron microscopy with lanthanide contrast. Results. It was shown that the liver decellularization method used allows to obtain a biocompatible matrix with a residual DNA quantity <1%, which is capable of maintaining adhesion and proliferation of hAT-MSCs and HepG2. On day 7 of cultivation in the bioreactor, there was formation of a single conglomerate of the DHLF matrix with numerous groups of viable cells with a high nuclear-cytoplasmic ratio. The urea content in the culture medium is 1.5 ± 0.1 mmol/L, exceeding that of samples obtained under static conditions. This indicates the metabolic activity of HepG2 in the composition of the obtained culture systems. It was shown that constant flow of the culture medium in the perfusion bioreactor increased the proliferative activity of HepG2 and allowed to provide a more uniform colonization by matrix cells in comparison with static cultivation conditions. Conclusion. The conditions for uniform colonization of DHLFs in a flow-through bioreactor with cell cultures were established. The ability of the matrix to maintain adhesion and proliferation of hADSCs and HepG2 for 11 days indicates that it could be used in liver tissue engineering.

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Kislitsyna, N. M., S. V. Novikov, N. V. Perova, S. V. Kolesnik, A. I. Kolesnik, and M. P. Veselkova. "Experimental in vitro Comparative Study of Chromovitectomy Agents Cyto- and Phototoxicity." Ophthalmology in Russia 17, no. 3 (2020): 473–80. http://dx.doi.org/10.18008/1816-5095-2020-3-473-480.

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Intravitreal use of vital dyes in combination with the action of endoillumination can induce a cyto- and phototoxic effect on posterior eye segment structures. The search for a staining agent with a maximum safety profile to retinal structures, intensively and selectively coloring vitreous body and vitreoretinal interface structure, remains relevant.Objective: to determine comparative viability of NIH / 3T3 mouse fibroblast cell culture with traditional agents for chromovitrectomy and “Vitreocontrast” suspension with and without endovitreal illumination.Materials and methods. NIH / 3T3 mouse fibroblast cultures contacted with agents for chromovitrectomy (MembraneBlue® Dual, Triamcinolone acetonide, “Vitreocontrast” suspension) and the corresponding controls in a volume of 50 μl / well. The test plate was irradiated with a Photon II illuminator (Synergetics, USA), working distance of 5 mm. The control tablet with the introduced preparations was not exposed to light. Next, the cells were washed and incubated, after which the morphology and lysis of the cells, as well as the number of proliferating relatively negative control of fibroblasts, were evaluated using the vital dye PrestoBlue Cell Viability Reagent. Negative control was the complete growth medium for the cultivation of mouse fibroblasts of the NIH / 3T3 line. The results of the cytotoxic reaction of a culture of mouse fibroblasts of the NIH / 3T3 line were interpreted using the table “The degree of cell response”.Results. Studies have shown that exposure to a source of endovitual illumination does not affect the cytotoxic effect of TA suspension and MembraneBlue® Dual dye. The TA suspension, both after light source and without it, has a moderate cytotoxic effect, and MembraneBlue® Dual has no cytotoxic effect on the culture of mouse fibroblasts of the NIH / 3T3 strain. Without light, “Vitreocontrast” suspension does not have cytotoxic effect on mouse fibroblasts culture NIH / 3T3 line. Light irradiation for 1 h increases the cytotoxicity of “Vitreocontrast” suspension to the level of unsharp cytotoxicity allowed by ISO Standard 10993-5-2011.Conclusion. The safety profile of MembraneBlue® Dual and “Vitreocontrast” suspension allows them to be recommended for use in endovitreal surgery. The cyto- and phototoxicity demonstrated in the experiment with TA suspension can reduce the functional outcomes of retinal surgery.

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Thompson, Laurie, Thais Oliveira, Evan Hermann, Mckale Montgomery, Winyoo Chowanadisai, and Stephen Clarke. "TP53 Mutation Status Influences Iron Regulatory Protein RNA Binding Activity and Sensitivity to Ferroptotic Cell Death." Current Developments in Nutrition 4, Supplement_2 (2020): 1277. http://dx.doi.org/10.1093/cdn/nzaa058_035.

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Abstract Objectives The tumor suppressor gene TP53 is the most commonly mutated gene in human cancer, but mutations in TP53 do not just result in loss of tumor suppressor function, they can also promote cancer progression by altering cellular iron acquisition and metabolism. A newly identified role for TP53 in the mediation of iron homeostasis and cancer cell survival lies in the ability for TP53 to protect against ferroptosis, a form of iron mediated cell death. The purpose of this study was to determine the extent to which TP53 mutation status effects iron-mediated cell death in response to ferroptosis induction. We also measured TP53 dependent differences in iron regulatory protein (IRP) RNA binding activity to begin to clarify the mechanisms by which TP53 mutation status may influence sensitivity to ferroptosis. Methods Using H1299 cells, which are null for TP53, we generated cell lines expressing either a tetracycline inducible wild-type TP53 gene, or a representative mutated TP53 gene from exemplary “hotspot” mutations in the DNA binding domain (R248, R273, R282, G245, R249 and R175). These six mutation types were selected because they represent 25% of all TP53 mutations in human cancer. To determine the influence of TP53 mutation status on sensitivity to ferroptotic cell death, we treated cells with erastin, a potent inducer of ferroptosis and measured differences in cell viability between these cell lines using PrestoBlue cell viability reagent. To assess mutant TP53-depenent differences in IRP RNA binding activity during ferroptosis we measured differences in IRP RNA binding activity via Electrophoretic Mobility-Shift Assay. Results We found that TP53 mutants (R273, R248, R175, G245, and R249) were significantly less viable (P &lt; 0.05) after initiation of ferroptosis compared to cells expressing WT TP53. Following ferroptosis induction, we observed a significant (P &lt; 0.05) increase in IRP RNA binding in G245, R248, and R175 mutants. Conclusions Our preliminary analyses indicate that TP53 mutants may be more sensitive to ferroptosis, but IRPs do not seem to be solely responsible for the increase in iron during ferroptotic cell death. Furthermore, ferroptosis may be a potential therapeutic target for cancers with these TP53 mutations but further investigation is warranted. Funding Sources Internal funding at Oklahoma State University.

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Adebayo, Ismail Abiola, Adamu Ibrahim Usman, Fatimah Bukola Shittu, et al. "Boswellia dalzielii-Mediated Silver Nanoparticles Inhibited Acute Myeloid Leukemia (AML) Kasumi-1 Cells by Inducing Cell Cycle Arrest." Bioinorganic Chemistry and Applications 2020 (September 22, 2020): 1–11. http://dx.doi.org/10.1155/2020/8898360.

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Background. Acute myeloid leukemia (AML) persists to be a major health problem especially among children as effective chemotherapy to combat the disease is yet to be available. Boswellia dalzielii is a well-known herb that is traditionally used for treatment and management of many diseases including degenerative diseases. In this study, silver nanoparticles were synthesized from the phytochemicals of B. dalzielii stem bark aqueous extract. The silver nanoparticles were characterized by carrying out Fourier Transform Infrared (FTIR) spectroscopy, Energy Filtered Scanning Electron Microscopy (FESEM), X-ray diffraction, and Dynamic Light Scattering (DLS) analyses. Antioxidant capacity of the nanoparticles was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and the antiproliferative effect of the nanoparticles on Kasumi-1 leukemia cells was investigated using PrestoBlue assay. Flow cytometry analysis was performed to observe the effect of the nanoparticles on the leukemia cell cycle progression. Results. Our findings revealed that the synthesized silver nanoparticles were formed from electrons of the plant phytochemicals which include aromatic compounds, ethers, and alkynes. FESEM analysis revealed that the sizes of the nanoparticles range from 12 nm to 101 nm; however, DLS analysis estimated a larger average size of the nanoparticles (108.3 nm) because it measured the hydrodynamic radii of the nanoparticles. The zeta potential of the nanoparticles was −16 nm, and the XRD pattern of the nanoparticles has distinct peaks at 38.02°, 42.94°, 64.45°, 77.20°, and 81.47°, which is typical of face-centered cubic (fcc) structure of silver. The Trolox Equivalence Antioxidant Capacity (TEAC) of the nanoparticles was estimated to be 300.91 μM Trolox/mg silver nanoparticles. The nanoparticles inhibited Kasumi-1 cell proliferation. The half minimal inhibitory concentrations (IC50s) that inhibited Kasumi-1 cell proliferation are 49.5 μg/ml and 13.25 μg/ml at 48 and 72 hours, respectively. The nanoparticles induced cell cycle arrest in the Kasumi-1 cells at S (5% increase) and G2/M (3% increase) phases. Conclusion. The nanoparticles synthesized from the stem bark extract of B. dalzielii inhibit the growth of Kasumi-1 leukemia cells by activating cell cycle arrest; thus, they are potential antileukemic agents.

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Tan, Maria Carmens, Glenn G. Oyong, Chien Chang Shen, and Consolacion Y. Ragasa. "CYTOTOXIC LABDANE DITERPENOIDS FROM ANDROGRAPHIS PANICULATA (BURM.F.) NEES." Asian Journal of Pharmaceutical and Clinical Research 10, no. 12 (2017): 99. http://dx.doi.org/10.22159/ajpcr.2017.v10i12.19194.

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Objective: The primary objective of this study was to probe the cytotoxic capacity of the labdane diterpenoids andrographolide (1), 14-deoxyandrographolide (2), 14-deoxy-12-hydroxyandrographolide (3), and neoandrographolide (4) on mutant and wild-type immortalized cell lines.Methods: Breast adenocarcinoma (MCF-7), colon carcinomas (HCT-116 and HT-29), small cell lung carcinoma (H69PR), human acute monocytic leukemia (THP-1), and wild-type primary normal human dermal fibroblasts - neonatal cells (HDFn) were incubated with 1-4, and the degree of cytotoxicity was analyzed by employing the in vitro PrestoBlue® cell viability assay. Working solutions of 1-4 were prepared in complete cell culture medium to a final non-toxic dimethyl sulfoxide concentration of 0.2%. The plates were incubated at 37°C with 5% CO2 in a 98% humidified incubator throughout the assay. Nonlinear regression and statistical analyses were done to extrapolate the half maximal inhibitory concentration 50% (IC50). One-way ANOVA (p<0.05) and multiple comparison, Tukey’s post hoc test (p<0.05), were used to compare different pairs of data sets. Results were considered statistically significant at p<0.05.Results: The highest cytotoxicity index was exhibited by the H69PR and 1 trials which displayed the lowest IC50 value of 3.66 μg/mL, followed by HT-29 treated with 2, HCT-116 and 1 trials, and H69PR treated with 4 (IC50=3.81, 3.82, and 4.19 μg/mL, respectively). Only 1 and 4 were detrimental toward MCF-7, while 1, 3, and 4 were degenerative against H69PR. Tukey’s post hoc multiple comparison indicated no significant difference in the cytotoxicity of 1-4 on HCT-116 cells which afforded IC50 values ranging from 3.82 to 5.12 μg/mL. Evaluation of the two colon carcinoma cell lines showed that HCT-116 was categorically more susceptible to cellular damage caused by treatments with 1-4 than was HT-29. Cytotoxicity was not detected in THP-1 and HDFn cells (IC50>100 μg/mL).Conclusion: Diterpenoids 1-4 isolated from the dichloromethane extract of the leaves of A. paniculata exhibited different cytotoxic activities against MCF-7, HCT-116, HT-29, and H69PR. All constituents had comparable action on HCT-116 cells but were not found to be cytotoxic to normal HDFn cells and mutant THP-1 cells.

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Morris, Benjamin B., Lisa G. Gray, Ryan D. Gentzler, David Randolph Jones, and Marty W. Mayo. "Omics guided small molecule inhibitor screen for the identification of therapeutic vulnerabilities in metastatic lung adenocarcinoma." Journal of Clinical Oncology 39, no. 15_suppl (2021): e21015-e21015. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.e21015.

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e21015 Background: Recent studies from our lab and others have demonstrated that double-strand DNA break accumulation, error-prone repair, and genomic instability are strongly linked to increased metastasis to distant organs. Our lab has shown that lung adenocarcinomas overexpressing the transcription factor MYBL2 display chronic replication stress, elevated error-prone repair, and widespread genomic instability. Importantly, this MYBL2-driven phenotype accounts for ̃21% of all lung adenocarcinomas and identifies aggressive disease enriched for metastases to brain, liver, and kidney. This study was performed to identify clinically actionable therapeutic vulnerabilities in MYBL2-driven metastatic lung adenocarcinoma. Methods: RNA-sequencing and proteomic data from the TCGA and ORIEN consortiums were mined to identify highly expressed druggable targets in MYBL2-driven lung adenocarcinomas. Identified targets were used to assemble a custom inhibitor library of 50 small molecules targeting DNA repair effectors, epigenetic factors, protein translation, kinase-signaling, autophagy, and post-translational modifications. Omics data from the Cancer Cell Line Encyclopedia was used to identify human cell line models (H1650 (pleura), H1568 (lymph node), H1299 (lymph node)) of MYBL2-driven metastatic disease. Inhibitors were tested at two doses, 5 uM and 500 nM, in triplicate, across replicate experiments. The PrestoBlue HS viability reagent was used to quantify cell viability in a 96 well plate format. The primary endpoint of this study was statistically significant cancer cell death, compared to vehicle controls. Results: Screen results demonstrated that, at nanomolar doses, inhibitors of protein translation have strong anti-tumor effects in MYBL2-driven disease. Interestingly, small molecules targeting EIF4G1 (SBI-0640756), ribosome biogenesis/RNA export (YM155), and rRNA synthesis (CX5461) were effective in inducing cell death while inhibitors blocking mTOR signaling, EIF2A phosphorylation, and EIF4F complex assembly were not. Bioinformatic analysis revealed that ̃60% of MYBL2-driven transcripts are dependent on EIF4G1 for translation. Importantly, effected transcripts include effectors controlling DNA repair, cell cycle coordination, and cell survival. Conclusions: Our data demonstrates that MYBL2-driven metastatic disease is uniquely sensitive to inhibitors of the protein translation machinery. Importantly, these inhibitors significantly outperform current standard-of-care agents cisplatin and pemetrexed. To our knowledge, our study is the first to demonstrate that blocking EIF4G1 effectively induces widespread cell death in metastatic lung adenocarcinoma models. Collectively, our work supports initiation of clinical trials testing the efficacy of SBI-0640756 in MYBL2-driven metastatic lung adenocarcinoma.

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Chen, Lih-Ren, Yu-Hsin Chen, and Hsiu-Lien Lin. "Application of PrestoBlueTM to evaluate boar semen quality." Theriogenology 137 (October 2019): 135. http://dx.doi.org/10.1016/j.theriogenology.2019.05.074.

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Gu, Zhimin, Yi Zhou, He Wang, et al. "Elevated Expression Of CKS1B Inhibits Senescence Thorough Enhanced Degradation Of p21 In Multiple Myeloma." Blood 122, no. 21 (2013): 1882. http://dx.doi.org/10.1182/blood.v122.21.1882.1882.

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Abstract Background High expression of CKS1B gene as a result of amplification of region 1q21 identifies a subset of patients with poor prognosis in multiple myeloma (MM). We have previously shown that over-expression of CKS1B enhances degradation of p27 and activates both MEK/ERK and JAK/STAT3 signaling pathways, promotes MM cell drug-resistance. However, other mechanisms of drug resistance induced by high expression of CKS1B remain to be identified. Materials and methods CKS1B was over-expressed or knocked-down by shRNA in OCI-MY5 and KMS28PE myeloma cell lines. Cell viability was detected by PrestoBlue assay; Annexin-V assay was employed to detect cell apoptosis. Colony formation was assessed in soft agar. The direct dye efﬂux to assess multidrug resistance was performed using eFluxx-ID Multidrug resistance assay. Senescence was detected by SA-β-galactosidase (β-Gal) staining. Expression of SCF-Skp2 substrates was evaluated by western blots. Results We demonstrated that overexpression of CKS1B significant decreased sensitivity to bortezomib and increased the clonogenic capacity compared to cells transfected with empty vector in the presence of bortezomib (p < 0.05). A prominent mechanism of drug resistance involved upregulation of multidrug resistance associated proteins (MRPs). We investigated whether the level of MDR1, BCRP, and MRP1, mitotic checkpoint protein MAD2 and ABC transporter family members was increased in cells with elevated CKS1B. However, none of drug pump proteins was up-regulated or stabilized by over-expression of CKS1B in OCI-MY5 and KMS28PE. Accordingly, over-expression of CKS1B did not promote efflux of the hydrophilic eFluxx-ID™ gold fluorescent dye from MM cells any greater than control cells, suggesting alternative mechanisms. We therefore tested whether CKS1B can inhibit drug-induced senescence in MM cells after treatment with bortezomib by quantifying the percentage of cells expressing senescence-associated β-Gal. Interestingly, MM cells with elevated CKS1B were significantly less β-Gal positive following treatment with bortezomib compared to control cells (p < 0.05). To explore molecular mechanism underlying CKS1B induced anti-senescence, expression of SCF-Skp2 substrates involved in senescence pathway was examined as CKS1B is an essential cofactor for SCF-Skp2 complex. Over-expression of CKS1B significantly reduced expression of p21, p27, and p57 while knock-down of CKS1B stabilized these proteins. Notably, cells with high expression of CKS1B failed to accumulate p21 protein in response to bortezomib. Conclusion Our findings suggest that anti-senescence is a novel mechanism in CKS1B-induced drug resistance in MM and the turnover of p21 regulated by CKS1B could have important implications not only for bortezomib therapy but also to develop therapies aimed at enhancing p21 function independent of p53. Disclosures: No relevant conflicts of interest to declare.

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Gu, Juan, Lianjuan Yang, Dennis C. Gaughan, et al. "GSK458 Is a Novel Dual PI3K/mTOR Inhibitor with Preclinical Antitumor Activity in T Cell Lymphomas As a Single Agent and in Combination Therapy." Blood 132, Supplement 1 (2018): 5378. http://dx.doi.org/10.1182/blood-2018-99-113997.

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Abstract Introduction: T-cell lymphoma (TCL) accounts approximately 15% of all Non-Hodgkin's lymphoma cases. TCL are often divided into either systemic or cutaneous TCL (CTCL). The management of TCL systemic or cutaneous is challenging because the emergence of chemotherapy resistance that lead to early death (systemic T-cell lymphomas) or chronic debilitating clinical course (CTCL). To improve the clinical outcomes and reduce treatment-related toxicity, research need to be done focusing on understanding and targeting the molecular mechanism driving TCL subtypes. Recently, studies showed that the PI3K AKT/mTOR pathway was activated in TCL. GSK458 is a potent oral dual inhibitor of pan PI3K (α, β, γ and δ) and mTOR (mTOR1 and mTOR2). Preclinical studies in B-cell lymphomas showed GSK458 had broad antitumor activity in vitro and in vivo. In 2016, Phase I clinical trial of GSK458 was competed at the maximal dosage of 2.5mg twice daily. However, the effects of GSK458 on T-cell malignancies remain utterly unknown. Here, we evaluated the activity of GSK458 in preclinical T cell lymphoma models. Methods: We used a panel of T-cell lymphoma cell lines representing PTCL (J45), T-cell lymphoblastic lymphoma (SupT-1), and Mycosis Fungoides (MF)(MJ, HH and H9). TCL cell lines were exposed to escalating doses of GSK458 (1nM-100µM) without or with chemotherapeutic agents (doxorubicin, cisplatin, carboplatin, and dexamethasone); Bcl-2 inhibitor (Venetoclax); proteasome inhibitors ( bortezomib, carfilzomib, Ixazomib); or HDAC inhibitors (SAHA) for 48 and 72 hrs. Differences in cell viability, ATP levels, low mitochondria potential, glucose update, apoptosis and cell cycle distribution were evaluated utilizing PrestoBlue, Cell-Titer Glo assays, DiOC6, 2-NDG, Annexin V and propidium iodide staining followed by flow cytometric analysis, respectively. IC50 was calculated by GraphPad. PI3K and mTOR downstream pathway phosphorylation status, such as p-AKT Ser473, p-AKT Thr308, p-mTOR and p-GSK3β were detected by internally staining of FITC conjugated-antibodies followed by flow cytometry. Apoptosis proteins (MCL-1, PARP, p53, XIAP etc.) were detected by western blot. The additive/synergistic activity of GSK458 was detected by presto blue assay and Coefficient of synergy was calculated using CalcuSyn. Results:In vitro exposure of TCL cell lines to GSK458 demonstrated a dose- and time-dependent cell death. The IC50 of the cells were ranged from 3nM to 1.05uM at 72 hours. At 72h, GSK458 10nM lowered cellular mitochondrial potential, ATP levels and glucose uptake. GSK458 induced apoptosis and arrested the cell cycle at G1. At molecular level, GSK458 reduced phosphorylation status of AKT ser473 and Thr308, mTOR and GSK3β. Interestingly, GSK458 inhibited Mcl-1 expression level. GSK458 exhibited synergistic activity when combined with doxorubicin and dexamethasone. To a lesser degree, GSK458 enhanced the anti-tumor activity of Venetoclax, proteasome and HDAc inhibitors. Conclusion: GSK458 is active as a single agent or in combination with chemotherapy agents or small molecule inhibitors in a variety of T-cell pre-clinical models representing forms of systemic or cutaneous T-cell lymphoma. GSK458 was able to inhibit phosphorylation of AKT, mTOR and GSK3β, which may be the mechanism to reduce ATP production and glucose uptake in the cancer cells. Moreover, GSK458 arrested cell cycle at G1 arrest. Our data supports the clinical evaluation of GSK458 in relapsed/refractory T-cell lymphoma patients. (Supported by Roswell Park Cancer Institute Alliance Foundation Grant) Disclosures No relevant conflicts of interest to declare.

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Man, Cheuk-Him, Chae-Yin Cher, Stephen S. Y. Lam, Eric S. K. Ho, Nelson K. L. Ng, and Anskar Y. H. Leung. "Novel Therapeutic Strategy Targeting NHE1 and Its Upstream Activators in Acute Myeloid Leukemia." Blood 124, no. 21 (2014): 913. http://dx.doi.org/10.1182/blood.v124.21.913.913.

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Abstract Increase in Tescalcin (TESC) gene expression and intracellular pH (pHi) have been associated with drug resistance in acute myeloid leukemia (AML). Tescalcin was shown to stabilize the membrane sodium/hydrogen exchanger (NHE1) that maintains a high pHi by H+ efflux in exchange for Na+. NHE1 has also been shown to be activated by PDGFR, PKC, calmodulin, p90-RSK and ROCK-RhoA, but their relevance to leukemogenesis and drug resistance in AML was unknown. We hypothesized that targeting NHE1 and its upstream activators might offer a novel and effective therapeutic strategy in AML. AML cell lines and mononuclear cell fraction from peripheral blood (PB) or bone marrow (BM) of AML patients (comprising primarily myeloblasts as shown by microscopic review of cytospin preparations) were treated with inhibitors for 3 days (concentrations: 0.1nM to 10mM) that target potential activators of NHE1. The anti-leukemia effects of these inhibitors were evaluated by PrestoBlue® Cell Viability Reagent as a measure of viable cell number. Their effects on pHi and apoptosis were evaluated by SNARF-1 and Annexin V/7-AAD staining respectively by flow cytometry. AML cell lines ML2, Kasumi-1, MOLM-13 and MV4-11 (IC50 in mM: 12.2, 13.1, 11.6 and 9.2 respectively) were more sensitive than KG1, NB4, THP-1 and OCI-AML3 (IC50 in mM: 30.7, 24.8, 119.2 and 49.4 respectively) to the growth inhibitory effects of NHE1 inhibitor, 5-(N,N-hexamethylene) amiloride (HMA), accompanied with a larger extent of cellular acidification and apoptosis induction in those 4 HMA-sensitive lines. To look for the upstream activators of NHE1 relevant to AML, the cell lines were treated with specific inhibitors targeting potential NHE1 activators. Both HMA-sensitive and insensitive cell lines were susceptible to the intracellular acidification and growth inhibition by PDGFR and p90-RSK inhibitors. Furthermore, FLT3 inhibitors, sorafenib and quizartinib, also reduced pHi of FLT3-ITD+ (Fms-Like Tyrosine Kinase 3 - Internal Tandem Duplication) AML cell lines, MOLM-13 and MV4-11, suggesting that FLT3-ITD might also activate NHE1, resulting in high pHi of FLT3-ITD+ AML. Different primary AML samples were treated with inhibitors to NHE1 (n=50), PDGFR (n=50) and p90-RSK (n=36) (Concentration: 100nM to 10mM) in vitro. Their response to the growth inhibitory effect of HMA, accompanied by effective pHi reduction (n=10), correlated with that of PDGFR and p90-RSK inhibitors (Pearson r=0.74, p&lt;0.001 and r=0.73, p&lt;0.001 respectively), supporting the proposition that these signaling pathways might be the critical and common activators of NHE1. Synergism of anti-leukemia effects could also be demonstrated between HMA and PDGFR inhibitors, calculated by Excess over Bliss Additivism (EOBA). To evaluate the clinical relevance of the study, serum was obtained from medical patients treated with high dose amiloride (20 mg daily), an NHE1 inhibitor, for underlying congestive heart failure. Compared with the serum of healthy volunteers, the amiloride-containing serum significantly reduced the pHi (n=10, p=0.001), induced apoptosis (n=4, p=0.04) and potentiated the inhibitory effects of PDGFR inhibitors (n=4, p=0.04) in primary AML samples. NHE1 might be a potential target in drug-resistant AML and activated by PDGFR, PKC, p90-RSK or both in a patient-specific fashion. Therefore, employing specific inhibitors to target NHE1 and its upstream activators should be explored as novel therapeutic strategy in this group of patients. Disclosures No relevant conflicts of interest to declare.

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Gu, Juan J., Samuel J. Thompson, Cory Mavis, Matthew J. Barth, Pallawi Torka, and Francisco J. Hernandez-Ilizaliturri. "Targeting MDM2 and XIAP By Idasanutlin in Diffuse Large B-Cell Lymphoma." Blood 134, Supplement_1 (2019): 5301. http://dx.doi.org/10.1182/blood-2019-129009.

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Purpose: The combination of rituximab and chemotherapy has improved overall survival of diffuse large B-cell lymphoma (DLBCL) patients in recent decades. Relapsed/refractory DLBCL patients previously treated with rituximab-containing regimen had significantly poorer response to salvage therapy. To study the mechanisms of this resistance, our laboratory developed several rituximab resistant cell lines. Previous study from our group found demonstrated an aberrant imbalance in the levels of pro- and anti-apoptotic proteins in these cell lines, including Bak/Bak, Mcl-1/BCLxL/Survivin and inhibitor of apoptosis (IAP) family proteins resulting in acquired chemotherapy resistance. MDM2 is an E3 ligase which regulates the degradation of multiple cellular protein targets such as pRB, HIF-1, p73, NF-kB, and E2F1 as well as FOXO3a. It is also the major negative regulator of p53. Recently, we found that MX69 (a MDM2 inhibitor), which blocks the MDM2 protein-XIAP RNA interaction, led to both MDM2 and XIAP degradation, and induced apoptosis-dependent cell killing activity. Moreover, MX69 re-sensitized resistant DLBCL cell lines to chemotherapy agents or small inhibitors (i.e. Venetoclax) in vitro. However, MX69 cannot be used clinically stressing the need to use a clinically available MDM2 inhibitor. To this end, we evaluated Idasanutlin, an investigational Nutlin family MDM2 antagonist, in B-cell lymphoma pre-clinical models. Methods: We used a panel of different DLBCL subtypes cell lines including activated B-cell lymphoma cell lines (TMD8, U2932); germinal center B-cell lymphoma (DOHH2, VAL, OCILY2, DH4, DH6, ROS50); rituximab-sensitive cell lines (Raji and RL); and rituximab/-resistant cell lines (Raji 4RH and RL 4RH). Cells were exposed to escalating doses of Idasanutlin (1nM-100µM) without or with other chemotherapeutic agents for 24, 48 and 72 hrs. Differences in cell viability were evaluated utilizing PrestoBlue. IC50 was calculated by GraphPad and Coefficient of synergy was calculated using CalcuSyn. Low mitochondrial potential was detected by staining cell with DiOC6 10ng/ml for 30 minutes and followed by flow cytometric analysis. Western blot was used to detect the changes of MDM2, XIAP and PUMA expression before and after exposure to Idasanutlin 1uM for 24h. Results:In vitro exposure Idasanutlin to DLBCL cell lines demonstrated a dose- and time-dependent cell death. IC50 dosage of the cells was ranged from 0.7uM to 63.07uM at 48h. Low dose of Idasanutlin (1uM) was able to disrupt mitochondria and induced low mitochondrial membrane potential in both ABC and GCB cell lines. At molecular level, Idasanutlin reduced expression level of MDM2 in TMD8, U2932, VAL, OCILy2 DHL4 and ROS50 cell lines. XIAP was reduced in Val and DHL4. Meanwhile, PUMA, the downstream of p53 activation, was increased after Idasanutlin exposure. Idasanutlin enhanced the anti-tumor activity of proteasome inhibitors (carfilzomib, ixazomib), Bcl-2 inhibitor venetoclax and Bryton kinase inhibitor ibrutinib. Conclusion: Idasanutlin showed potent anti-tumor activity as a single agent in DLBCL pre-clinical setting. Idasanutlin was able to decrease cellular mitochondrial membrane potential. At molecular level, Idasanutlin decreased MDM2 and XIAP protein level and induced PUMA expression. Moreover, Idasanutlin enhanced anti-tumor activities of other small inhibitors. Taking together, Idasanutlin could be used as a novel and promising drug in the clinical setting of DLBCL with relapsed/refractory disease. Disclosures No relevant conflicts of interest to declare.

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Thavhana, M. P., T. L. Nedambale, L. J. Shai, and M. L. Mphaphathi. "44 Exploring the use of silver and diamond nanoparticles on sperm cell invitro and chicken embryo in ovo." Reproduction, Fertility and Development 33, no. 2 (2021): 129. http://dx.doi.org/10.1071/rdv33n2ab44.

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In poultry industry, chick viability is a crucial factor determining profitability from fertilized egg to placement at the farm. However, decreases in fertility and hatchability have been observed. Recently, there has been renewed interest in the use of silver nanoparticles (Ag-NPs) due to their antimicrobial properties and growth-promoting ability, and diamond nanoparticles (D-NPs) due to their biocompatibility properties. The aim of the study was to evaluate the effect of silver and diamond nanoparticles on chicken embryo oxidative status, biochemical indices, and expression of immune-related genes and on sperm cell viability. The experiment was conducted in Ross 308 chicken embryos and Ross 308 cockerels. One hundred and fifty fertilized eggs were divided randomly into 5 groups (5×30). Fertilized eggs were injected with 50 mg/L Ag-NPs at volumes of 100μL (group 1), 200μL (group 2) or 50 mg/L D-NPs at volumes of 100μL (group 3) or 200μL (group 4), or received no nanoparticles (control; group 5) and incubated at 37°C and 55% humidity for 20 days. Then, chicken blood was collected and centrifuged to evaluate alkaline phosphatase (ALP), alanine transaminase (ALT), lactate dehydrogenase (LDH), glucose, urea, and free haemoglobin. Chicken embryo liver was used to evaluate antioxidant capacity (TAC) and chicken embryo spleen was used to evaluate expression of the immune-related genes interleukin-1β (IL-1β), toll-like receptor (TLR)4, TLR2, and TLR15. Semen was randomly divided into 1 control and 8 treatment groups and treated with 50 mg/L Ag-NPs: group A (0.1ppm), group B (1ppm), group C (5ppm), group D (10ppm) or 50 mg/L D-NPs: group E (1ppm), group F (5ppm), group G (10ppm), and group H (20ppm). Sperm viability was analysed using prestoblue metabolic assay. Data were analysed using PROC in GLM procedure of SAS 2014 (SAS Institute Inc.). Decrease in sperm cell viability was recorded in a dose-dependent manner. Sperm cell viability decreased (P&lt;0.005) as the concentration of Ag-NP or D-NP increased. Addition of 100μL of Ag-NPs increased the growth rate of chicken embryo but not 200μL of Ag-NPs or addition of D-NPs. Increases in ALP, ALT, LDH, glucose and urea enzyme were observed in a dose-dependent manner in both Ag-NPs and D-NPs. Addition of 50 mg/L Ag-NPs or 50 mg/L D-NPs increased (P&lt;0.001) TAC of chicken embryo as the volume increased. Additions of 200μL of Ag-NPs, 100μL of D-NPs, and 200μL of D-NPs were haemolytic (P&lt;0.001) but addition of 100μL of Ag-NPs was not. Additions of 100 or 200μL of Ag-NPs or 100μL of D-NPs downregulated IL-1β and 200μL of D-NPs upregulated IL-1β compared with the untreated control group. Additions of 100 or 200μL of Ag-NPs or 200μL of D-NPs induced expression of TLR4 and TLR15. Furthermore, addition of Ag-NPs did not result in expression of TLR2. We concluded that administration of 50 mg/L Ag-NPs and 50 mg/L D-NPs in ovo improve immune status and administration of 100μL of Ag-NPs improved the growth rate of chicken embryo. However, toxicity associated with 50 mg/L Ag-NPs and 50 mg/L D-NPs remains a concern and need to be addressed before use.

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Thavhana, M. P., T. L. Nedambale, L. J. Shai, and M. L. Mphaphathi. "44 Exploring the use of silver and diamond nanoparticles on sperm cell invitro and chicken embryo in ovo." Reproduction, Fertility and Development 33, no. 2 (2021): 129. http://dx.doi.org/10.1071/rdv33n2ab44.

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In poultry industry, chick viability is a crucial factor determining profitability from fertilized egg to placement at the farm. However, decreases in fertility and hatchability have been observed. Recently, there has been renewed interest in the use of silver nanoparticles (Ag-NPs) due to their antimicrobial properties and growth-promoting ability, and diamond nanoparticles (D-NPs) due to their biocompatibility properties. The aim of the study was to evaluate the effect of silver and diamond nanoparticles on chicken embryo oxidative status, biochemical indices, and expression of immune-related genes and on sperm cell viability. The experiment was conducted in Ross 308 chicken embryos and Ross 308 cockerels. One hundred and fifty fertilized eggs were divided randomly into 5 groups (5×30). Fertilized eggs were injected with 50 mg/L Ag-NPs at volumes of 100μL (group 1), 200μL (group 2) or 50 mg/L D-NPs at volumes of 100μL (group 3) or 200μL (group 4), or received no nanoparticles (control; group 5) and incubated at 37°C and 55% humidity for 20 days. Then, chicken blood was collected and centrifuged to evaluate alkaline phosphatase (ALP), alanine transaminase (ALT), lactate dehydrogenase (LDH), glucose, urea, and free haemoglobin. Chicken embryo liver was used to evaluate antioxidant capacity (TAC) and chicken embryo spleen was used to evaluate expression of the immune-related genes interleukin-1β (IL-1β), toll-like receptor (TLR)4, TLR2, and TLR15. Semen was randomly divided into 1 control and 8 treatment groups and treated with 50 mg/L Ag-NPs: group A (0.1ppm), group B (1ppm), group C (5ppm), group D (10ppm) or 50 mg/L D-NPs: group E (1ppm), group F (5ppm), group G (10ppm), and group H (20ppm). Sperm viability was analysed using prestoblue metabolic assay. Data were analysed using PROC in GLM procedure of SAS 2014 (SAS Institute Inc.). Decrease in sperm cell viability was recorded in a dose-dependent manner. Sperm cell viability decreased (P&lt;0.005) as the concentration of Ag-NP or D-NP increased. Addition of 100μL of Ag-NPs increased the growth rate of chicken embryo but not 200μL of Ag-NPs or addition of D-NPs. Increases in ALP, ALT, LDH, glucose and urea enzyme were observed in a dose-dependent manner in both Ag-NPs and D-NPs. Addition of 50 mg/L Ag-NPs or 50 mg/L D-NPs increased (P&lt;0.001) TAC of chicken embryo as the volume increased. Additions of 200μL of Ag-NPs, 100μL of D-NPs, and 200μL of D-NPs were haemolytic (P&lt;0.001) but addition of 100μL of Ag-NPs was not. Additions of 100 or 200μL of Ag-NPs or 100μL of D-NPs downregulated IL-1β and 200μL of D-NPs upregulated IL-1β compared with the untreated control group. Additions of 100 or 200μL of Ag-NPs or 200μL of D-NPs induced expression of TLR4 and TLR15. Furthermore, addition of Ag-NPs did not result in expression of TLR2. We concluded that administration of 50 mg/L Ag-NPs and 50 mg/L D-NPs in ovo improve immune status and administration of 100μL of Ag-NPs improved the growth rate of chicken embryo. However, toxicity associated with 50 mg/L Ag-NPs and 50 mg/L D-NPs remains a concern and need to be addressed before use.

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Thomas, Gregory S., Junwei Huang, Yi Zhou, et al. "Targeting Neddylation to Overcome Drug Resistance in Multiple Myeloma." Blood 126, no. 23 (2015): 1804. http://dx.doi.org/10.1182/blood.v126.23.1804.1804.

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Abstract Background: The overexpression of CKS1B resulting from chromosomal amplification of the 1q21 region identifies a subset of multiple myeloma (MM) patients with poor clinical outcomes. Bortezomib (Btz) is an FDA-approved first-in-class proteasome inhibitor that has greatly improved clinical outcomes in MM. However, toxicities affiliated with pan-proteasomal inhibition and resistant populations remain a problem, highlighting the need for improved therapies. We have previously demonstrated that elevated expression of CKS1B results in the destabilization of p21 and contributes to insensitivity to bortezomib. MLN4924 is an inhibitor of the NEDD8-Activating Enzyme E1 (NAE1) and prevents the neddylation and subsequent activation of Cullin-1, a crucial component in the activation of SCF-driven ubiquitin-mediated degradation. We therefore explored if MLN4924 might serve as an alternative inhibitor to proteasomal degradation in MM. Materials and Methods: CKS1B was overexpressed or knocked down in myeloma cell lines using lentiviral vectors and shRNA. Cell proliferation and viability were assessed by cell counts and using PrestoBlue reagent. Clonogenicity was assessed by colony formation in soft agar. SA-b-galactosidase staining was used to assess senescence. Gene expression profiling was performed using the publicly available databases Total Therapy 2 (TT2) and APEX trials. Results: To examine the efficacy of Btz and MLN4924 in elevated CKS1B expression environments, we compared treatment with the MLN4924 to Btz in CKS1B OE cells in vitro. Cells with basal levels of CKS1B were sensitive to treatment with either Btz or MLN4924, with treatment leading to decreased proliferation and cellular viability. In the CKS1B OE background, we found cells were resistant to Btz repression of proliferation but sensitive to MLN4924. We also found that MLN4924 could more potently reduce colony formation in soft agar and more potently induce senescence in CKS1B OE cells compared to treatment with Btz. Immunoblot analyses confirmed a correlation between CKS1B expression and Cullin-1 neddylation. Further immunoblotting of known SCF-mediated ubiquitin targets in cells with and without CKS1B OE demonstrated a stabilization of p21 in all cells upon MLN4924 treatment that was not exhibited upon Btz treatment. To investigate the role of p21 in CKS1BOE cell sensitivity to MLN4924, we stably knocked down expression of p21. We found the knockdown of p21 partially abrogated the sensitivity of CKS1B OE cells to MLN4924, increasing cell viability, increasing colony formation in soft agar, and decreasing senescence induction. The importance of neddylation in the clinic was confirmed using GEP. We found that expression of the neddylation-related genes (i.e. NAE1, UBA3, and UBC12) is significantly upregulated (p<0.05) in MM patients relative to patients in MGUS or to healthy donors. Expression of NEDD8 was also significantly elevated in patients unresponsive to either Btz treatment or treatment with the combination of Btz and dexamethasone compared to responsive patients. The elevated expression of CKS1B highlights a population of patients with poor clinical outcomes. Considering the elevated expression of neddylation-related genes in MM progression, we performed Kaplan Meier survival analyses looking at patients segregated on two axes by CKS1B expression levels (high/low) and neddylation-related gene expression levels (either UBA3 or UBC12). We found patients with high/high expression of CKS1B and neddylation-related genes had significantly decreased survival relative to patients with mixed high/low expression from either axis. Together, these data suggest the importance of neddylation contributions to MM progression and clinical outcomes. Conclusions: These results shed light on the subtle differences in the blockade of proteasomal inhibition, illustrating the distinct effects of pan inhibition from inhibition of SCF-driven ubiquitin-mediated degradation. Mechanistically, we demonstrate at least a partial role for p21 in mediating cell sensitivity to MLN4924 in CKS1B overexpressing cells. Our findings highlight the important contributions of neddylation to MM disease progression and suggest the utility of targeting neddylation as a means of overcoming drug resistance in MM. Disclosures No relevant conflicts of interest to declare.

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Zimmerman, Jessica A. O., Mimi Fang, Arunkumar Modi, Adam Dupuy, Sarah K. Tasian та Miles A. Pufall. "PI3Kδ Inhibition Enhances Sensitivity of Primary High-Risk Childhood B-Cell Acute Lymphoblastic Leukemia Cells to Glucocorticoid Chemotherapy". Blood 134, Supplement_1 (2019): 2572. http://dx.doi.org/10.1182/blood-2019-129205.

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Introduction: Despite excellent 5-year survival for most patients with B-cell acute lymphoblastic leukemia (B-ALL), those with high-risk (HR) B-ALL continue to have inferior outcomes. The prognosis is even worse for patients who respond poorly to chemotherapy, and improvements to current therapy approaches are needed. Glucocorticoids (GCs) are a key component of chemotherapy regimens. Rapid response to GC monotherapy is a well-documented predictor of favorable outcomes, while slow response to GCs correlates with poor outcomes. We hypothesized that enhancing specific GC toxicity to B-ALL cells can improve clinical outcomes for high-risk patients without increasing systemic toxicity. PI3Kδ is a promising target for this purpose. PI3Kδ is a critical component of the B-cell receptor cell-survival pathway; it restrains GC signaling, while GCs repress its expression. PI3Kδ expression is restricted to lymphoid cells, making it an appealing precision target to enhance GC toxicity in lymphoid cells while minimizing risk of increased systemic GC-associated toxicity. We previously demonstrated synergy between dexamethasone (dex) and the FDA-approved isoform-selective PI3Kδ inhibitor idelalisib (idela) in B-ALL cell lines, patient specimens, and patient-derived xenograft models (Kruth et al Blood 2017). In clinical practice, both dex and prednisone (pred) are essential components of childhood ALL chemotherapy. In this study, we sought to elucidate the extent to which idela in combination with dex or pred could induce synergistic killing of HR B-ALL specimens with various genetic backgrounds. Methods: Peripheral blood or bone marrow from patients ages 1-31 years with newly-diagnosed NCI HR or standard-risk (SR) B-ALL at the University of Iowa Stead Family Children's Hospital was obtained after informed consent on an Institutional Review Board-approved research study. Mononuclear cells were isolated from the specimens via Ficoll gradients and cultured in vitro in 384-well plates. Immediately after isolation, cells were treated with serial dilutions of the GCs dex or pred and idela in RPMI1640+10% FBS medium. Cell viability was measured with PrestoBlue (Invitrogen) after 72 hours of treatment. Survival curves were plotted to determine the LD50 of each drug using GraphPad. Dose effects around the LD50 for each drug were analyzed using Compusyn to determine if the drug combination was synergistic (combination index, CI <1), additive (CI=1), or antagonistic (CI >1). Results: As of August 1, 2019, 5 HR B-ALL patient specimens of various genetic alterations have been tested. Two patients were high-risk at diagnosis due to steroid pre-treatment (MAP011) or by NCI risk criteria (age >10 years and/or WBC count >50K/mm3; MAP014). Three patients with initially SR B-ALL were upstaged due to minimal residual disease (MRD) >1% at day 8 in peripheral blood (MAP012) or marrow MRD ≥0.01% at end of induction (MAP009, MAP010). In our in vitro studies, only one sample (MAP014) showed reduced viability with GC alone, while 4 specimens (all except MAP012) responded to idela alone. Most specimens showed a synergistic response to dex or pred in combination with idela with greater reduction in cell viability compared to GC monotherapies. Surprisingly, one specimen (MAP010) had an antagonistic response with pred and idela combination. This near-haploid B-ALL specimen had reduced viability with idela monotherapy, but an antagonistic response with single-agent pred, raising the question of whether GC/idela synergy is influenced by genetic background. Conclusions: In pilot studies, idela sensitized HR B-ALL cells to GC chemotherapy and is a promising strategy for further evaluation in a larger cohort of specimens. Our results highlight the potential impact of genetic heterogeneity within childhood B-ALL upon differential therapeutic responses to standard steroid chemotherapy and demonstrate a potential precision medicine approach to augment GC sensitivity via combination with selective PI3Kδ inhibition. Disclosures Tasian: Gilead Sciences: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees; Incyte Corportation: Research Funding.

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Lam, Stephen Sze Yuen, Chae Yin Cher, Nelson Ka Lam Ng, et al. "Development of a Clinically Relevant in Vitro Drug Screening Platform for Chemo-Refractory AML Patients." Blood 124, no. 21 (2014): 3624. http://dx.doi.org/10.1182/blood.v124.21.3624.3624.

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Abstract Acute myeloid leukaemia (AML) is a heterogeneous group of diseases with distinct clinicopathologic, cytogenetic and genetic characteristics. The heterogeneity has made unified and regimental approach unsuccessful for most patients, and outcome with standard chemotherapy and allogeneic haematopoietic stem cell transplantation (HSCT) was unsatisfactory with an overall cure rate of 30-40%. We hypothesised that an optimised in vitro drug screening platform for primary AML samples might help to identify the best personalised therapeutic agents for AML patients from whom the samples were obtained at real time. Primary mononuclear cells isolated from peripheral blood (PB) or bone marrow (BM) of AML patients at different stages of disease were seeded onto 96-well plates and treated with a panel of 25 selected drugs at 1000-fold concentration range for 3 days. The drugs included 17 tyrosine kinase inhibitors: axitinib, crizotinib, pazopanib, erlotinib, gefitinib, lapatinib, vandetanib, vemurafenib, sorafenib, quizartinib, ponatinib, lestaurtinib, nilotinib, dasatinib, ruxolitinib, TG101209, tofacitinib; 2 differentiation agents: arsenic trioxide, all-trans retinoic acid; a protein translation inhibitor: homoharringtonine; a proteasome inhibitor: bortezomib; a chemotherapy: cytarabine; a histone deacetylase inhibitor: vorinostat; a DNA methyltransferase inhibitor: azacitidine; and an mTOR inhibitor: rapamycin. The blast percentage in each sample was above 50% as confirmed by film review of the cytospin preparation. The inhibitory effect of each drug was evaluated by a high throughput PrestoBlue® fluorometric assay that was a measure of viable cell number. The results were expressed with reference to the vehicle control (0.1% DMSO) for each sample. There was significant cell death when primary AML cells were cultured in IMDM medium supplemented with 10% FBS (IMDM10) for 3 days (Annexin V+ cells: 53.8% ± 4.5% , n=33). To identify the optimal culture condition, multiple culture conditions were tested for each sample (n=53 samples). The 3-day post-culture survival of primary AML cells improved significantly in a 1:1 mix of IMDM10 and medium conditioned by a mixture of mouse fibroblast lines engineered to produce human G-CSF, SCF, IL-3 and Flt3L (50%CM) (47.1% ± 6.5% improvement in Annexin V/7AAD -/- cell count normalised to day 0 input compared to IMDM10, p<0.0001). Assay readout was highly reproducible between replicates of the same samples (r=0.9681). There was significant correlation in drug response between PB and BM myeloblasts (r=0.9356, p<0.0001, 16 pairs), supporting the feasibility of drug screen based on PB samples. The readout might be predictive of clinical drug response in patients, as exemplified by the superior in vitro response to sorafenib in FLT3-ITD+ than FLT3 wild type AML samples (p<0.01 at 1 and 10 µM Sorafenib) that corroborated with the clinical observation for these patients treated with sorafenib. Furthermore, sorafenib-naïve FLT3-ITD+ AML samples were more sensitive to sorafenib in vitro than the resistant samples collected from the same patients during subsequent leukaemia progression. To identify personalised agents effective for individual patients, a total of 85 samples from 60 AML patients were screened in 50%CM. Axitinib and vemurafenib had minimal cytotoxic effect on all samples whereas bortezomib, ponatinib, lestaurtinib, homoharringtonine and vorinostat exhibited significant anti-leukaemia effect (>75%) to over 90% samples at concentrations around or below the peak plasma concentrations in respective pharmacokinetic studies. The remaining 18 drugs exhibited variable effects on different samples. Further experiments and analyses are underway to identify distinct combinations and correlate with in vivo efficacy in xenotransplantation mouse model. This optimised and clinically relevant in vitro drug screening platform might provide the laboratory basis for the selection of personalised treatment regime for AML patients with a short turnover time. Disclosures No relevant conflicts of interest to declare.

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Qin, Shuchao, Xinyu Zhang, Qimei Han, et al. "Transcriptome Sequencing Reveals Alternative Splicing Patterns and an Increased Sensitivity to Spliceosome Inhibition Associated MYD88 L265P Mutation in Chronic Lymphocytic Leukemia." Blood 132, Supplement 1 (2018): 5535. http://dx.doi.org/10.1182/blood-2018-99-118667.

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Abstract Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the Western countries but is relatively rare in East Asia. Mutations in myeloid differentiation primary response gene 88 (MYD88) occur in 2.0-4.4% of Caucasian patients with CLL. However, Asian subjects showed a higher MYD88 mutation frequency of 8% and MYD88 mutations were associated with unfavorable prognosis in Asian CLL patients with mutated IGHV gene. To further explore the molecular and pathological mechanisms of the MYD88 L265P mutation in CLL, we performed a pilot study to investigate the transcriptome-wide differential expression between CLL patients with and without MYD88 L265P mutation. Methods: mRNA sequencing was performed on a cohort of 15 Chinese CLL patients with MYD88 L265P mutation (n=5) or wild-type MYD88 gene (n=10). Differential expression and pathway enrichment analyses were performed based on FPKM values after processing the raw sequencing reads. The alternative splicing events were analyzed using JuncBASE and the differences in splicing patterns were compared between the two groups. The MYD88 L265P and wild-type genes were overexpressed in the MEC-1 cell line using lentiviral vectors, and stable cell lines were established after puromycin selection. Real time quantitative PCR was used to determine the mRNA expression of spliceosome pathway genes. The protein expression of TLR pathway was assessed with Western Blot. The PrestoBlue™ assay was used for determining IC-50 of spliceosome inhibitor-treated CLL cell lines, and flow cytometry was utilized for apoptosis and cell death analyses. Results: mRNA sequencing analysis revealed that 4231 genes showed significant differences in mRNA expression between MYD88 mutant and wild-type CLL patients. Of these genes, 3033 were up regulated and 1198 were down regulated in patients with MYD88 L265P mutation. Pathway analysis demonstrated that tumor necrosis factor (TNF) signaling, cell cycle, spliceosome, ribosome, p53 signaling, and NF-κB signaling pathways were significantly enriched among differentially expressed genes. Interestingly, about two thirds (19 out of 30) of differentially expressed genes in the spliceosome pathway were down regulated in CLL samples with MYD88 mutation. Quantitative RT-PCR confirmed the down regulation of spliceosome genes including SF3A1, SF3A2, SF3B4, SF3B5 and PUF60 in MYD88 mutated samples in an independent CLL patient cohort. Analysis of alternative splicing events revealed a total of 180 genes with inter-group splicing differences, among which differential splicing events were most common in the genes involved in immune system regulation, i. e. CD22, CD79B, CD200 and IKZF1 etc. The MEC1 cells stably overexpressing MYD88 L265P demonstrated decreases in RelA and TRAF6 expression as compared to wild type MYD88-overexpressing cells, which was in agreement with RNA-seq analysis results in CLL patient samples. In addition, we found that overexpression of MYD88 L265P mutant resulted in decreased mRNA expression of several spliceosome genes such as SF3A1, SF3A2, SF3B4, SF3B5 and PUF60 in the stable cell line. Furthermore, MYD88 L265P increased the sensitivity of MEC1 cells to spliceosome inhibitor sudemycin D6 (SD6) by 3 fold, while reducing their sensitivity to Ibrutinib by two fold. Conclusion: We discovered the differential mRNA expression landscapes related to MYD88 mutation status in CLL and their contributions to the progression of CLL through multiple pathways. Of particular interest was the down regulation of spliceosome pathway genes. In a CLL cell line model, we found that, compared with the MYD88 wild-type CLL cell line, CLL cells overexpressing the mutant MYD88 were more sensitive to spliceosome inhibitor SD6, suggesting that inhibiting spliceosome function may be a potential therapeutic target for CLL patients with mutant MYD88 gene. In summary, our findings provide novel insights for further understanding of the molecular and pathological mechanisms of MYD88 mutation in CLL. Disclosures Lagisetti: St. Jude Children's Research Hospital: Patents & Royalties: Chandraiah Lagisetti is listed as inventors on patents assigned to St. Jude Children's Research Hospital covering sudemycin D6 and was employed by SRI Biosciences at the time these experiments were carried out. Webb:St. Jude Children's Research Hospital: Patents & Royalties: Thomas R. Webb is listed as inventors on patents assigned to St. Jude Children's Research Hospital covering sudemycin D6 and was employed by SRI Biosciences at the time these experiments were carried out..

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Daly, Tara, Thomas Ippolito, Juan J. Gu, et al. "MCL-1 Inhibition By the Selective MCL-1 Inhibitor AMG-176 Induces in Vitro Activity Against Burkitt Lymphoma Cell Lines and Synergistically Enhances the Cytotoxic Effect of Chemotherapy and BH3 Mimetics." Blood 134, Supplement_1 (2019): 5303. http://dx.doi.org/10.1182/blood-2019-129052.

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Background: Although >90% of Burkitt Lymphoma (BL) patients will be cured by intensive multi-agent immunochemotherapy, relapsed and refractory BL patients have poor prognoses demonstrating the need to identify novel therapies. Myeloid cell leukemia-1 (MCL-1) is a pro-survival protein within the BCL-2 family that has been investigated as a therapeutic target in several leukemia and lymphoma pre-clinical models and clinical studies. Analysis of pediatric BL tumors has implicated MCL-1 in BL pathogenesis (Giulino-Roth et al., Blood 2012) and targeting MCL-1 may be effective in MYC-driven lymphomas (Kelly et al., Genes Dev 2014). High MCL-1 expression has also been associated with therapy resistance and can confer resistance to BH3 mimetic agents (Carrington et al., Cell Death Differ 2017). AMG176, a selective MCL-1 inhibitor, is an effective promoter of apoptosis and is currently under investigation in clinical trials for relapsed and refractory multiple myeloma, lymphoma, and acute myeloid leukemia. Hypothesis: We hypothesized that MCL-1 inhibition with AMG176 will promote apoptosis and increase sensitivity of BL therapy-sensitive (TSCL) and therapy-resistant (TRCL) cell lines to other chemotherapy drugs. Methods: A panel of TSCLs (Raji, Ramos and Daudi) and one TRCL (Raji 4RH) BL cell lines were exposed to increasing concentrations of AMG176 as a single agent and in combination with Doxorubicin or Venetoclax at different time points. Cell viability was analyzed using PrestoBlue. Synergy in drug combination exposures was determined by Calcusyn software and reported as a combination index (CI) value with values increasingly <1 indicating increasing synergistic activity. Apoptosis induction was analyzed by flow cytometry for Annexin V (AV) and Sytox-7AAD and by western blotting for PARP. Effects of MCL-1 inhibition on the expression of other pro- and anti-apoptotic BCL-2 family member proteins was assessed by western blotting. AMG176 mechanism(s) of action was evaluated by co-immunoprecipitation followed by western blotting for pro-apoptotic BCL-2 family members known to be bound by MCL-1. Results: Single agent AMG176 exposure of BL cell lines exhibited a dose and time-dependent decrease in viable cells. 48hr IC50 values of Ramos (0.3097uM) and Raji (1.652uM) showed an increased sensitivity to AMG176 as compared to Daudi (9.616uM) and Raji 4RH (12.45uM). Exposure of cells to AMG176 also resulted in altered expression levels of pro-survival and pro-apoptotic proteins of the BCL-2 family proteins. Co-immunoprecipitation performed after 48hrs of AMG176 exposure exhibited a decrease in MCL-1:BIM and MCL-1:BAK complexes in Raji and Ramos cells promoting a pro-apoptotic environment. After 48hr exposure to AMG176, induction of apoptosis was observed in a dose-dependent manner in all cell lines as evidenced by increased PARP cleavage on western blot and AV-Sytox positivity on flow cytometry. To investigate the ability of MCL-1 inhibition to enhance the activity of cytotoxic chemotherapy, BL cells were exposed to AMG176 in combination with Doxorubicin. Daudi and Raji exhibited strong synergy with peak synergistic activity observed at 72hrs (Daudi CI values <0.1 at all AGM176 concentrations with 0.1-0.2uM Doxorubicin; Raji CI ranged from 0.233-0.295 at multiple AMG176 concentrations with <0.4uM Doxorubicin) while a minimal effect was observed in Ramos and Raji 4RH. As high MCL-1 is associated with resistance to Venetoclax by sequestering free BIM following Venetoclax exposure, the combination of AMG176 and Venetoclax was investigated and also showed significant synergy in BL cells. A minimal effect was observed in Daudi and Raji 4RH in Venetoclax combination but Ramos and Raji, the more AMG176 sensitive cell lines, showed peak synergy at 24hrs at high doses of Venetoclax (Ramos CI values ranged from 0.3-0.478; Raji CI ranged from 0.511 to 0.67). Conclusion: MCL-1 inhibition with AMG176 demonstrates dose- and time-dependent anti-lymphoma activity impairing in vitro proliferation and inducing apoptosis in BL cells. AMG176 exhibits synergistic activity in combination with cytotoxic chemotherapy in both therapy-sensitive and therapy-resistant BL cell lines and synergistically enhances response to the BH3 mimetic agent Venetoclax. These findings highlight the potential of MCL-1 inhibition as a therapeutic option in BL. This work was supported by T35 NIH training grant (T35AI089693). Disclosures No relevant conflicts of interest to declare.

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Journal articles on the topic 'PrestoBlue'

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