Добірка наукової літератури з теми "3211 Oncology and carcinogenesis"

The work was undertaken to examine the effects of nutritional deficiencies on cancer induction. Two of the most common and widely distributed nutrients, iron and folic acid, were examined to evaluate the effects of their deficiency on animals. The Syrian golden hamster was the animal model for all experimental work. In the first part of the study an attempt was made to induce iron deficiency in young adult male hamsters by feeding iron deficient diet coupled with repeated venesection of 1.5ml every two weeks. Following twelve weeks on this regime a superficial biopsy was taken, on week 13, from the medial wall of one pouch in each hamster in order to evaluate the effect of iron depletion on the epithelial compartment thicknesses. After allowing the biopsy sites to heal for two weeks, a solution of 0.25% DMBA in acetone was painted, three times per week, for eight weeks, on a defined one square centimetre area in each pouch of each hamster of the experimental and control groups. The hamsters were then maintained on the same dietary regimes for twelve weeks before being killed at the beginning of week 37 for analysis. Iron deficiency anaemia could not be induced in the experimental animals of this study. The effect of the iron deficient diet on epithelial compartment thicknesses at the stage of the biopsy was not clear. However, restriction of iron intake did cause animals to develop significantly fewer grossly seen tumours and histologically identified carcinomas than control animals. In the second part of this thesis an attempt was made to investigate alternative hamster dietary components that have less iron contamination than the diet given in the first part of this thesis. Casein and calcium lactate were the main contributers to iron in the hamster diet. Casein could not be substituted by another source of protein for hamsters. However, other sources for calcium with less iron contamination were available and therefore investigated in this part of the study. Three groups of young adult male and female hamsters were given the fully nourishing powdered diet used in previous studies. However, calcium lactate was substituted for by either calcium acetate, calcium chloride or calcium sulphate in each group. None of the three diets was accepted by the animals and many of them died of starvation. When calcium salts were replaced by calcium lactate the surviving animals accepted the diet and recovered quickly afterwards. This study proved that calcium lactate could not be substituted by any other calcium salt with less iron content and therefore iron contamination in the hamster diet could not be further reduced by this method. In the third part of this thesis the effect of nutritional folate deficiency on cancer induction was studied. A group of young adult female hamsters was given folate deficient diet for four weeks. On week 5, DMBA in acetone at a concentration of 0.25% was painted on a defined one square centimetre area of the medial wall of each pouch in each hamster in folate deficient and control groups. The carcinogen was applied three times per week for eight weeks following which animals were maintained on the same dietary regimes for a further 13 weeks before being killed at the beginning of week 27 for the final analysis of the study. It was found that nutritional folate deficiency had significantly reduced the number of animals developing grossly counted tumours and histologically identified carcinomas. The folate deficient animals also developed significantly less tumours and carcinomas compared to control groups. In the last part of this thesis, the effect of combined iron and folate deficiency was examined for its role in carcinogenesis of the hamster cheek pouch. Two groups of young adult male hamsters were fed powdered diet lacking iron and folic acid and a third group was fed diet lacking iron only. One of the combined deficiency groups and the iron deficiency group were bled 1.0-1.3ml every week. On week 6 of the study DMBA in acetone at a concentration of 0.25% was painted three times per week for eight weeks on the same area of the pouch used in the previous studies. The animals were then maintained on the same experimental regimes for a further eleven weeks before being sacrificed, on week 25, for the final analysis of the study. In this study, iron deficiency anaemia was induced in animals of the bleeding groups. Animals in the group with combined iron and folate deficiency without bleeding showed low normal folate levels and normal haemoglobin levels. The two groups that were bled repeatedly showed iron deficiency anaemia. In all groups, the numbers of tumours counted grossly and the numbers of carcinomas identified histologically were significantly reduced compared to control animals in the previous studies. The folate deficient diet did not appear to influence the induction of iron deficiency. The studies reported in this thesis proved that nutritional folate deficiency not only reduces the incidence, but it also reduces the numbers of tumours and carcinomas in the hamster cheek pouch. Iron deficiency anaemia was also found to significantly reduce the numbers of tumours and carcinomas of the hamster cheek pouch. It was not possible to produce combined iron and folate deficiency under the conditions of these studies. However, animals fed on a diet lacking iron and folic acid had significantly reduced numbers of grossly seen tumours and histologically identified carcinomas in the cheek pouch in response to DMBA applications. In each of the reported studies, the nutritional deficiency of iron and folic acid, whether individually or combined was found to significantly reduce the growth rate of affected animals.

Introduction Oesophageal cancer has a poor prognosis. Early diagnosis and the use of chemotherapy and surgery for local disease are key to improving survival. This study was designed to see if plasma and tissue metabolic profiles could be used to identify oesophago-gastric malignancy, indicate the presence of unstable pre-malignant (Barrett’s) epithelium or predict response to chemotherapy. Methods Patients were recruited from University Hospitals Birmingham between May 2009 and March 2010. Nuclear Magnetic Resonance (NMR) metabolomics was performed on filtered plasma and extracted tissue samples. Results Some 258 participants were recruited. NMR metabolomics discriminated between normal, Barrett’s and neoplastic epithelium. Tissue levels of hypoxanthine were highest in oesophageal adenocarcinoma compared to adjacent normal mucosa. Levels in Barrett’s mucosa in the presence of cancer fell between normal and neoplastic mucosa. 3-hydroxybutyrate levels were elevated both in cancer tissues and plasma compared to controls. Plasma levels of 3-hydroxybutyrate were higher in patients with node positive and full thickness tumours compared to those who were node negative with early local disease. Conclusion NMR metabolomics identified metabolic profiles that characterized different histologic tissue types. Metabolites involved in oesophageal carcinogenesis might influence diagnostic and management strategies in these patients.

BACKGROUND: The incidence of skin cancer in Australia has increased rapidly in the last few decades. Ultraviolet radiation (UV) is a major risk factor for skin carcinogenesis. UV, particularly the UVB spectrum, causes formation of cyclobutane pyrimidine dimers (CPD) in cellular DNA. Persistent and incorrectly repaired CPDs lead to DNA mutations and consequently, formation of cutaneous lesions. Interestingly, recent epidemiological studies have shown a significant increase in skin cancer incidence in geographical locations with high environmental temperatures. Thus, heat stress may potentiate the effects of UV exposure and act as an additional risk factor for skin cancer. Previous studies in mice have shown that repeated and concurrent exposure to UVB and heat stress, increases the rate and incidence of cutaneous tumour formation relative to UVB alone. However, the effects of UVB plus heat on human epidermal cells have yet to be determined. Furthermore, the exact mechanisms responsible for the observed effects of heat stress need to be characterised in skin keratinocytes to increase knowledge of its risk in skin cancer. Heat stress induces upregulation of heat shock proteins (HSPs), particularly HSP72 and HSP90 which are known to affect the activity of the p53 protein. Furthermore, heat stress has been linked with increased Sirtuin1 (SIRT1) protein activity. SIRT1 is an important histone deacetylase that helps maintain chromosomal integrity but can also induce post-translational modifications of the p53 protein. By mediating deacetylation of the p53 protein, SIRT1 can diminish the ability of p53 to bind to its downstream gene targets. The p53 protein is an integral mediator of the cellular stress response in skin cells, particularly keratinocytes. Thus, impairment of p53 transcription factor functions could compromise the ability of epidermal cells to mount an appropriate response to DNA damage. Moreover, loss of p53 function may induce survival of cells harbouring DNA lesions. We hypothesise, therefore, that exposure to UVB plus heat induces survival of DNA damaged keratinocytes and that these cells escape apoptosis surveillance as a result of heat-mediated alteration to the p53 signalling pathway. Thus, exposure to heat stress could exacerbate the carcinogenic effects of UV and increase the risk of skin tumour formation in humans AIMS: In this study, we aimed to determine whether repeated exposure to UVB followed immediately by heat stress (39°C) has a more damaging effect on human keratinocytes than UVB alone. In particular, we assessed the effects on DNA damage, apoptosis, cell cycle and DNA repair. Furthermore, we aimed to unravel the mechanism through which heat mediates the survival of UVB DNA-damaged keratinocytes, focusing on the effects on the p53 signalling pathway. MATERIALS AND METHODOLOGY: Primary adult human epidermal keratinocytes (NHEK) and ex vivo punch biopsies of normal human skin called NativeSkin® (Genoskin, France), were used as experimental models for this study. A UV cabinet fitted with a TLUVB Narrowband lamp (Philips, GERMANY), with a spectral output of 290 -315 nm, was used to administer UVB irradiation at a dose of 1 KJ/m². Heat stress involved culture in a normal CO2 incubator, with temperature maintained at 39°C for three hours. The temperature used in the experiments was based on previous measurements of skin surface temperature of open cut miners, who are prone to intense heat stress, in the Pilbara region of Western Australia. For UVB plus heat exposures, cells and skin models were sequentially exposed to 1 KJ/m2 of UVB, (at room temperature), followed immediately by 3 hours incubation at 39°C once per day, for four consecutive days. Unexposed skin models and NHEK, maintained at 37ºC, were used as experimental controls. Cell proliferation, apoptosis and whole genome expression profiles were analysed at four hours post day 4 exposure, to understand earlier events, and at 2 days post-exposure, to assess persistent outcomes of these exposures. Treated primary NHEK cells were counted in a Vi-CellTM Viability Analyser and the level of apoptosis for exposed primary cells was determined using Annexin V/Propidium Iodide apoptosis assay at 4 hours and 2 days post exposure. To determine the presence of DNA damage, total and active p53 protein, as well as total and active SIRT protein, in the skin models and primary NHEKs, immunohistochemistry and/or immunocytochemistry was performed. Skin FFPE and primary NHEKs were incubated with antibodies to thymine dimers (CPD, DNA damage) and p53 (total), acetylated p53-382 (active), SIRT1 (total) or SIRT1-p (active) antibodies. To measure apoptosis in skin, an anti-pan-cytokeratin marker was used to label keratinocytes and active-caspase-3 antibodies were used to identify apoptotic cells. To determine the expression of p53-downstream target genes at 4 hours, quantitative RT-PCR was performed using TaqMan probes for BAX, Survivin (BIRC5), ERCC1 and XPC genes, with Human 18S gene as the endogenous reference gene. Relative quantification of the expression levels of each transcript in each sample were calculated using the Delta-Delta CT method relative to untreated controls. A whole genome expression analysis was performed at 2 days postexposure using the Human HT-12 Expression v4 BeadChip (Illumina, USA). The Ingenuity Pathway Analysis (IPA) (Qiagen, USA) software was used to annotate the effects of altered gene expression on cell function and upstream signalling pathways. Two-way ANOVA was used to analyse differences across treatment groups, while parametric unpaired t-tests were used to detect differences between specific treatment groups in all experimental categories, i.e. proliferation, apoptosis and gene expression, with p-values RESULTS: Outcome 1 –Using ex vivo skin models and NHEKs, we show for the first time that UVB plus heat treated keratinocytes exhibit DNA damage, as observed after UVB treatment alone. However, apoptosis was significantly reduced, possibly as a result of inactivation of the p53-mediated stress response, in DNA damaged cells of UVB plus heat treated samples. Furthermore, whole genome expression and IPA upstream analysis showed that heat induces SIRT1 activation, which was confirmed via immunohistochemistry assays. Heat-induced SIRT1 expression was linked to a decrease in acetylated p53 and consequently, downregulation of p53-regulated pro-apoptotic and DNA damage repair genes. These results suggest that p53-mediated cell cycle arrest and apoptosis, known to be induced by UVB, are ablated with the addition of heat, leading to survival of DNA damaged cells after UVB plus heat treatment. Outcome 2 – We further confirmed that SIRT1 activation did not inhibit the transcription of the p53 protein but mediated deacetylation of p53, resulting in significant deregulation of expression of p53 downstream gene targets and decreased keratinocyte apoptosis in UVB plus heat treated samples. Importantly, chemical inhibition of SIRT1 by Ex-527, a known chemical inhibitor of SIRT1, in UVB plus heat exposed keratinocytes, resulted in reactivation of the p53 signalling pathway and increased apoptosis of DNA damaged keratinocytes. This clearly demonstrated the role of heat-mediated SIRT1 activation in the survival of DNA damaged keratinocytes after exposure to UVB plus heat. CONCLUSION: In this study, we showed that the efficiency of cellular stress response to UVB-induced DNA damage is diminished in the presence of heat and, for the first time, provide a molecular mechanism that explains these effects. With the novel use of an ex vivo human skin model, this study showed that heat stress prevents human keratinocytes, damaged by UV irradiation, from undergoing apoptosis and/or necrosis. We found UV plus heat exposure mediates SIRT1 activation which has been found to induce deacetylation of p53 and, consequently, the inactivation of the p53 signalling pathway. SIRT1 inhibition precluded the downregulation of p53 signalling by UV plus heat exposure, restoring apoptosis levels to those observed in UVB-only exposures. Thus, we demonstrated that SIRT1 activation is the main molecular mechanism driving UVB plus heat-induced survival of DNA damaged keratinocytes. Overall, the results of this study suggest that by allowing the survival of DNA damaged keratinocytes, via induction of SIRT1 activation, heat stress can exacerbate the carcinogenic effects of UVB radiation. Exposure to heat stress, in addition to UV, could therefore increase the accumulation of mutations in keratinocytes, possibly leading to the transformation of normal cells into pre-cancerous cells. Further research is warranted to determine the role of UVB plus heat in skin cancer pathogenesis. Such knowledge could be utilised in public health campaigns to decrease risk, particularly for people exposed to combinations of these environmental hazards in workplaces such as in the mining, construction and petroleum industries.

Prostate cancer (PC) is characterised by dependence upon androgen receptor (AR) as its driving oncogene. When organ-confined, radical treatment can be curative, however there is no cure for advanced, castration-resistant prostate cancer (CRPC). There is therefore a need to better understand the biology of PC, and how influencing AR can modify disease progression. Estrogen is essential for prostate carcinogenesis with evidence from epidemiological, in vitro, human tissue and animal studies. Most suggests that estrogen receptor beta (ERβ) is tumour-suppressive, but trials of ERβ-selective agents have not improved clinical outcomes. ERβ has also been implicated as an oncogene, therefore its role remains unclear. Additional evidence suggests interplay between ERβ and AR, the mechanisms of which are uncertain. The study hypothesis ‘ERβ is an important modulator of prostate carcinogenesis’ was developed to establish whether targeting ERβ could affect PC progression. Much of the confusion around ERβ stems from use of inadequately validated antibodies and cell line models. The first phase of this work was to test ERβ antibodies using an ERβ-inducible cell system. Eight ERβ antibodies were assessed by multiple techniques, showing that commonly used antibodies are either non-specific or only specific in one modality. Two reliable antibodies were identified. Next, cell lines previously used to study ERβ were assessed using validated antibodies and independent approaches. No ERβ expression was detected; an important finding that casts doubt on previously published ERβ biology. Subsequently, a PC cell line with inducible ERβ expression (LNCaP-ERβ) was developed and validated to enable controlled experiments on the effects of ERβ on proliferation, gene expression and ERβ/AR genomic cross-talk. Phase three of this work focused on ERβ biology in PC and its relationship to AR. Interrogation of clinical datasets showed that greater ERβ expression associated with favourable prognosis. Gene expression data from men treated with androgen deprivation therapy revealed that AR represses ERβ. This was confirmed in vitro. The LNCaP-ERβ cell line was treated with androgen and/or ERβ-selective estrogen. Activated ERβ in the presence of androgen-stimulated AR inhibited cell proliferation and down-regulated androgen-dependent genes. Genome-wide mapping of ERβ binding sites reveals that ERβ antagonises AR through competition for shared DNA binding sites. In conclusion, ERβ expression is down-regulated by AR during malignant transformation of prostate epithelium. We reveal an antagonistic relationship between ERβ and AR whereby sustaining or replacing ERβ may inhibit tumour growth through down-regulation of AR-target genes. In future, an ERβ-selective compound may be used to slow or abrogate PC progression.

Ionizing radiation is a physical agent that is tumorigenic in all exposed tissues. These radiation-induced secondary neoplasms tend to be more aggressive and carry a poor prognosis. Our knowledge of the molecular mechanisms of ionizing radiation carcinogenesis is not as advanced as compared to chemical carcinogenesis. We have used repeated exposure to low LET radiation in the mouse skin model to study the molecular mechanisms of ionizing radiation as a complete carcinogen and as a tumor progression agent. Shaved backs of CD-1 mice were treated with fractionated doses of beta-irradiation in a complete carcinogenesis experiment. A total of 27 carcinomas and sarcomas were seen. Cell lines were established from four sarcomas and one squamous cell carcinoma. Biochemical studies revealed that three sarcoma cell lines were derived from rhabdomyosarcornas. All four sarcoma cell lines had a p53 null phenotype. We screened cDNA expression libraries from three cell lines for dominant transforming activities. GAPDH was isolated as a candidate transforming gene in the squamous cell carcinoma cell line. Using a papilloma producing mouse keratinocyte cell line, we have shown that repeated doses of ionizing radiation are equally effective as a tumor progression agent when compared to N-methyl N'-nitro-N-nitrosoguanidine (MNNG). In this model, elevated reactive oxygen species levels were seen in both radiation and MNNG progressed cells. Elevated transcription factor transactivation as well as constitutive activation of Erk-1/2 and p38 MAP kinase activities were found to be potential mediators of the reactive oxygen species mediated mitogenic signaling in the progressed phenotype. Analyses of the anti-oxidant defense mechanisms showed that attenuation of catalase activity was a potentially important mechanism for the establishment of the pro-oxidant state. Forced re-expression of catalase in the malignant variants resulted in a reduction in transcription factor transactivation. Taken together, the results from experiments presented in this dissertation suggest that inactivation of gene products that maintain genomic stability, such as p53, may be an important step during neoplastic transformation with fractionated doses of ionizing radiation. Altered expression patterns of genes related to cell metabolism and oxidative stress can be functionally involved during the later stages of ionizing radiation-induced malignant transformation.

Epidemiological data suggest that non-steroidal anti-inflammatory drugs (NSAIDs) have anti-tumorigenic activities against colorectal cancer. NSAIDs work by inhibiting cyclooxygenases (COX) enzyme. Sulindac, a NSAID prodrug, is metabolized into pharmacologically active sulfide and sulfone derivatives. Microarray analysis was used to detect COX independent effects of sulindac on gene expression in human colorectal cells. Spermidine/spermine N 1-acetyl transferase (SSAT) gene, which encodes a polyamine catabolic enzyme, was one of the genes induced by clinically relevant sulindac sulfone concentrations. Promoter analysis and mutational studies were done to map the sulindac sulfone dependent response sequences in SSAT 5' flanking sequences, which led to the identification of two Peroxisome Proliferator Activated Receptors (PPARs) response elements (PPREs) in the SSAT gene. PPRE-2 is required for the induction of SSAT by sulindac sulfone and is specifically bound by PPARgamma in the Caco-2 cells, while PPRE-1 is not required for the induction of SSAT by sulindac sulfone, but can be bound by both PPARdelta and PPARgamma. Clinically relevant concentrations of sulfone reduced intracellular polyamine levels, inhibited cell growth and induced apoptosis in colon cancer cells. Further, only sulindac sulfone induced apoptosis could be partially rescued by exogenous polyamines. Upon evaluating other NSAIDs for their action on SSAT gene expression, it was found that they induce SSAT mRNA in either a COX dependent or independent mechanism in colon cancer cells. Studies with physiologically relevant concentrations of aspirin show that these concentrations can induce SSAT expression thereby leading to a decrease in polyamine levels. Activating mutations in K-ras, which is a late process in colon carcinogenesis, led to the suppression of SSAT expression in the Caco-2 cells due to the inhibition of PPARgamma by ERK. K-ras didn't have any effect on the induction of SSAT by sulindac sulfone but partially abolished the apoptosis caused by sulindac sulfone, indicating a possible role of mutant K-ras in sulindac resistant colon polyps. Sulindac sulfone, or Exisulin(TM) have been recently used in clinical trials for the prevention of colon, lung and prostate cancer. The data shown here, suggest that one of the mechanisms, by which sulindac sulfone could act as a chemopreventive agent is to induce the expression of SSAT thereby leading to a decrease in the intracellular polyamines. This reduction in polyamines plays an important part in the apoptosis induced by sulindac sulfone in the colon cancer cells. Further, induction of SSAT seems to a general mechanism for different NSAIDs like aspirin, indomethacin, ibuprofen, sulindac and celecoxib in colon cancer. Aspirin is able to induce SSAT and decrease intracellular polyamines at physiological concentrations, which can lead to a significant reduction in adenoma recurrence. Also, activated K- ras suppressed SSAT, but was not able to abolish the induction of SSAT by sulindac sulfone indicating the potential of using sulindac sulfone in colon cancer chemoprevention.

Introduction: Breast cancer (BC) is one of the most prevalent cancers and considered one of the main causes of death among women worldwide. The genetic contribution to the incidence of BC is of great relevance, however, modifiable risk factors seem to be related to the development of this neoplasm. Several studies have provided evidence on the role of blood pressure (BP) levels in the carcinogenesis process. Goals assess the association between BP levels and the occurrence of BC. Methods: This is a case-control study nested in a cohort conducted between December 2013 and August 2014 in a municipality in the south of Brazil. Patients referred to oncology referral centers before starting adjuvant or neoadjuvant therapy for BC participated in the study. The control group was composed of women who visited the gynecology clinic during the same period. For each newly diagnosed case of BC, a control matched for age (±5 years) and menopausal status was included. Data were obtained through measurements with standardized techniques of BP levels and waist circumference (WC) measurement. Individuals with systolic BP ≥ 130 mmHg and/or diastolic BP ≥ 85 mmHg were considered to have altered blood pressure. Results: Eighty two patients with BC and 82 controls (n=164) were evaluated. Slightly different characteristics were found between the two groups: skin color (p=0.097), months of breastfeeding (p=0.185) and physical activity (p=0.160). These variables were included in the adjusted analysis. Regarding the BP measurement, patients with SBP ≥130 mmHg and with DBP ≥85 mmHg were 7.30 and 4.56 times more likely to have MF (Malignant), respectively (95%CI 2.43–21.91, p <0.001 and 95%CI 1.82–11.44, p=0.003). This relationship remained significant after adjustment for WC ≥80 cm (OR = 6.75, 95%CI 2.22–20.54 and OR=6.75, 95%CI 1.67–10.81). Conclusions: The results of this study corroborate the current findings in the literature, showing evidence of an association between BP and BC levels. BP assessment, already incorporated into clinical practice and considered a predictor of several comorbidities, may gain additional importance in the female population as a modifiable risk factor for the prevention of BC. Thus, the importance of interventions in the management of BP levels in this population is elucidated.