Oncometabolic role of mitochondrial sirtuins in glioma patients.

Mitochondrial sirtuins have diverse role specifically in aging, metabolism and cancer. In cancer, these sirtuins play dichotomous role as tumor suppressor and promoter. Previous studies have reported the involvement of sirtuins in different cancers. However, till now no study has been published with respect to mitochondrial sirtuins and glioma risks. Present study was purposed to figure out the expression level of mitochondrial sirtuins (SIRT3, SIRT4, SIRT5) and related genes (GDH, OGG1-2α, SOD1, SOD2, HIF1α and PARP1) in 153 glioma tissue samples and 200 brain tissue samples from epilepsy patients (taken as controls). To understand the role of selected situins in gliomagenesis, DNA damage was measured using the comet assay and oncometabolic role (oxidative stress level, ATP level and NAD level) was measured using the ELISA and quantitative PCR. Results analysis showed significant down-regulation of SIRT4 (p = 0.0337), SIRT5 (p<0.0001), GDH (p = 0.0305), OGG1-2α (p = 0.0001), SOD1 (p<0.0001) and SOD2 (p<0.0001) in glioma patients compared to controls. In case of SIRT3 (p = 0.0322), HIF1α (p = 0.0385) and PARP1 (p = 0.0203), significant up-regulation was observed. ROC curve analysis and cox regression analysis showed the good diagnostic and prognostic value of mitochondrial sirtuins in glioma patients. Oncometabolic rate assessment analysis showed significant increased ATP level (p<0.0001), NAD+ level [(NMNAT1 (p<0.0001), NMNAT3 (p<0.0001) and NAMPT (p<0.04)] and glutathione level (p<0.0001) in glioma patients compared to controls. Significant increased level of damage ((p<0.04) and decrease level of antioxidant enzymes include superoxide dismutase (SOD, p<0.0001), catalase (CAT, p<0.0001) and glutathione peroxidase (GPx, p<0.0001) was observed in patients compared to controls. Present study data suggest that variation in expression pattern of mitochondrial sirtuins and increased metabolic rate may have diagnostic and prognostic significance in glioma patients.

Expression variation of OGG1 and HPRT gene and DNA damage in arsenic exposed industrial workers.

Arsenic exposure alters redox balance, induces DNA damage, and deregulates many genes. OGG1 gene involved in base repair mechanism, for excision of 8-oxoguanine (8-oxoG) from DNA formed as a result of accumulation of ROS in cell. HPRT gene encode transferase enzymes involved in purine recycling mechanism. The main focus of the study was to evaluate the expression variation in HPRT, OGG1 gene expression, and DNA damage of industrial workers. Blood samples of 300 occupational workers were collected from welding, brick kiln, furniture, pesticide, and paint industry (n = 60/industry) to evaluate the expression variation in HPRT, OGG1 gene expression, and DNA damage in blood cells by comet assay along with age and gender matched 300 control individuals. Blood arsenic content was higher (P<0.001) in an industrial group compared to the control. OGG1 and HPRT expression were (P<0.05) downregulated in exposed workers compared to controls. Spearman correlation analysis showed a significant positive correlation between HPRT vs OGG1 (P< 0.0001) in exposed workers compared to controls. Altered expression of both genes was observed between workers with <25years and >25years of age as well as between workers with <10years and >10year exposure. Reduced expression (P<0.05) of both genes and a high extent of DNA damage was evident in exposed smokers compared to respective non-smokers. DNA fragmentation was higher (P<0.05) in the furniture, welding and brick kiln group compared to control, and other industries. The present study suggests that altered expression of OGG1 and HPRT gene induce oxidative stress, showed a negative impact on the recycling of purines leading to DNA damage which increase the vulnerability of workers to carcinogenicity.

Genetic and expression variations of cell cycle pathway genes in brain tumor patients.

The present study was designed to determine the association between the genetic polymorphisms/expression variations of RB1 and CCND1 genes and brain tumor risk. For this purpose, 250 blood samples of brain tumor patients along with 250 controls (cohort I) and 96 brain tumor tissues (cohort II) with adjacent control section were collected. Mutation analysis of RB1 (rs137853294, rs121913300) and CCND1 (rs614367, rs498136) genes was performed using ARMS-PCR followed by sequencing, and expression analysis was performed using real-time PCR and immunohistochemistry. The results showed homozygous mutant genotype of RB1 gene polymorphism, rs121913300 (P=0.003) and CCND1 gene polymorphism rs614367 (P=0.01) were associated significantly with brain tumor risk. Moreover, significant down-regulation of RB1 (P=0.005) and up-regulation of CCND1 (P=0.0001) gene was observed in brain tumor sections vs controls. Spearman correlation showed significant negative correlation between RB1 vs proliferation marker, Ki-67 (r = -0.291*, P<0.05) in brain tumors. Expression levels of selected genes were also assessed at protein level using immunohistochemical analysis (IHC) and signification down-regulation of RB1 (P=0.0001) and up-regulation of CCND1 (P=0.0001) was observed in brain tumor compared with control sections. In conclusion, it is suggested that polymorphisms/expression variations of RB1 and CCND1 genes may be associated with increased risk of brain tumor.

Expression deregulation of DNA repair pathway genes in gastric cancer.

This study was designed to check correlation of mRNA and protein expression of BER pathway genes(XRCC1, OGG1) and a proliferation marker (Ki-67) in 100 gastric tissue samples and controls (adjacent uninvolved area). The expression was estimated usingreal time PCR and immunohistochemistry. Genomic instability was also calculated in the same study cohort using 8-OHdG assay, DNA fragmentation assay and comet assay. A significant downregulation of XRCC1 (p < 0.0001) and OGG1 (p < 0.0001) expression was observed in gastric cancer tumors vs controls. When analyzed with spearman correlation, significant positive correlation was observed between OGG1 vs XRCC1 (r = 0.319*, p < 0.02) and significant negative correlation was observed between OGG1 vs Ki-67 (r = -0.462**, p < 0.001) and XRCC1 vs Ki-67 (r = -0.589**, p < 0.001) in gastric cancer tumors. Significantly higher level of 8-OHdG, when compared to controls, was observed in gastric cancer tumors (p < 0.0001). DNA fragmentation assay and comet assay showed the formation of increased ladder patterns and comets in gastric cancer tumors when compared with controls These findings suggest that dysregulation of XRCC1, OGG1 combined with overexpression of Ki-67 may contribute to progression of gastric cancer and may help to sub-classify patients within diverse risk groups for therapeutic advantages.

PARP1: A potential biomarker for gastric cancer.

Gastric cancer (GC) is the third leading cause of cancer mortality worldwide, with an overall 5-y survival rate of 25%. The majority of GCs are caused by infectious agents, including the bacterium Helicobacter pylori (H. pylori) and Epstein-Barr virus (EBV). Furthermore, inappropriate repair of DNA damage can also result in genomic instability, which has shown to be a key factor in carcinogenesis of different regions including gastric region. Present study was designed to explore the association between base excision repair pathway genes, PARP1 and APEX1 and gastric pathology and H. pylori infection. Two hundred gastric cancer tissue samples (114 H. pylori positive and 86 H. pylori negative) and adjacent uninvolved area taken as controls was used for expression analysis of BER pathway genes at mRNA level and protein levels using quantitative PCR (qPCR) and immunohistochemistry (IHC) respectively. Oxidative stress and DNA damage was also determined by measuring the level of antioxidant enzymes and comet assay respectively. Significant upregulation in PARP1 (p < 0.001) and APEX1 (p < 0.02) was observed in GC tissue samples compared to controls and this upregulation was more pronounced in H. pylori positive cases (HPGC) (PARP1, p < 0.02: APEX1, p < 0.04) than H. pylori negative cases (HNGC). Upregulation of BER pathway genes in HPGC was found correlated with smoking status (p < 0.0001), T stage (p < 0.01) and lymph node metastasis (p < 0.03). Moreover, immunohistochemical staining of BER pathway genes was found correlated with a number of clinicopathological characteristics such as tumor type (p < 0.03), tumor size (p < 0.01) and lymph node metastasis (p < 0.01). Expression levels of APEX1 and PARP1 gene also correlated with increased oxidative burden (p < 0.0001) and DNA damage (p < 0.001) in GC patients. Survival analysis showed that upregulation of PARP1 gene was associated with poor overall survival outcome of gastric cancer patients (HR = 2.04 (95% CI = 1.10-3.76; p < 0.02). Univariate and multivariate cox regression analysis showed the upregulated PARP1 gene (HR = 5.03; 95%CI (2.22-11.35); p = 0.0001), positive smoking status (HR = 3.58; 95%CI (1.67-7.65); p = 0.001), positive status for H pylori infection (HR = 4.38; 95%CI (1.82-10.56); p = 0.001) and advance N-stage (HR = 5.29; 95%CI (2.28-12.24); p = 0.0001) were independent prognostic factors for gastric cancer and may serve as a valuable biomarker for the diagnosis and progression of GC and can be helpful in developing individualized treatment strategies for treating GC.

Redox balance and DNA fragmentation in arsenic-exposed occupational workers from different industries of Pakistan.

AbstractOccupational exposure accounts for a contact between workers and different toxicants. Present study was designed to measure the arsenic-induced DNA fragmentation and oxidative stress in exposed workers. Blood, hair, and nail samples were collected from welding, brick kiln, furniture, pesticide, and paint industries (n = 50/industry) of Pakistan along with 200 controls. DNA damage was calculated using DNA fragmentation assay. Antioxidant enzymes (CAT, SOD, GPx) were measured using ELISA. Results revealed that arsenic exposure induced DNA fragmentation in brick kiln, furniture, and welding industries. Enzyme activity was reduced in five industries compared to control. In exposed group, significant depletion of enzymes was observed in furniture, welding, and brick kiln workers. Based on age and time of exposure, significant difference was observed in welding and brick kiln group. Smokers of exposed group showed significantly reduced levels of enzymes compared to controls. Arsenic deposition was observed higher in the hair, nail, and blood samples of exposed group (P < 0.001) compared to control. Likewise, lead and cadmium contents were higher in the blood samples of industrial workers compared to control. This study suggests increased trend of cellular damage and oxidative stress in occupational workers profoundly in welding, furniture, and brick kiln industries. Moreover, this study recognizes the contribution of age, exposure time, and smoking status toward arsenic-induced oxidative stress and DNA fragmentation.

CYP1A1 and GSTP1 gene variations in breast cancer: a systematic review and case-control study.

In first part of this study, a systematic review was designed to explore the involvement of CYP1A1 and GSTP1 genes in breast cancerogenesis. Based on systematic review, we designed a study to screen CYP1A1 and GSTP1 genes for mutation and their possible association with breast carcinogenesis. A total of 400 individuals were collected and analyzed by PCR-SSCP. After sequence analysis of coding region of CYP1A1 we identified eleven mutations in different exons of respective gene. Among these eleven mutations, ~3 folds increased breast cancer risk was found associated with Asp82Glu mutation (OR 2.99; 95% CI 1.26-7.09), with Ser83Thr mutation (OR 2.99; 95% CI 1.26-7.09) and with Glu86Ala mutation (OR 3.18; 95% CI 1.27-7.93) in cancer patients compared to controls. Furthermore, ~4 folds increase in breast cancer risk was found associated with Asp347Glu, Phe398Tyr and 5178delT mutations (OR 3.92; 95% CI 1.35-11.3) in patients compared to controls. The sequence analysis of GSTP1 resulted in identification of total five mutations. Among these five mutations, ~3 folds increase in breast cancer risk was observed associated with 1860G>A mutation, with 1861-1876delCAGCCCTCTGGAGTGG mutation (OR 2.70; 95% CI 1.10-6.62) and with 1861C>A mutation (OR 2.97; 95% CI 1.01-8.45) in cancer patients compared to controls. Furthermore, ~5 folds increase in breast cancer risk was associated with 1883G>T mutation (OR 4.75; 95% CI 1.46-15.3) and ~6 folds increase in breast cancer risk was found associated with Iso105Val mutation (OR 6.43; 95% CI 1.41-29.3) in cancer patients compared to controls. Our finding, based on systematic review and experimental data suggest that the polymorphic CYP1A1 and GSTP1 genes may contribute to risk of developing breast cancer.

Adverse effects of arsenic exposure on uterine function and structure in female rat.

The present investigation examined the adverse effects of arsenic exposure on uterine function and structure of female rat at 56 days of age, exposed to different doses (50, 100, and 200ppm) of sodium arsenite in drinking water at immature age (28 days) for 28 days. Dose-dependent decrease (P<0.001) was observed in mean uterine weight and length in all treated groups compared to control. Higher arsenic deposition was found in uterine tissue against increased doses of arsenite. Arsenite treatment altered the histomormphology of the uterus. Uterine epithelium in 50ppm group was lined by cuboidal cells instead of columnar cells observed in control epithelium. In 100 and 200ppm groups, no demarcation was observed between epithelial cells and endometrial stroma. No basement membrane was seen in these groups; even in 50ppm, basement membrane was disturbed. The endometrial stroma in 100 and 200ppm groups was very dense in appearance and contained irregular-shaped cells. In myometrium, loosening of cells was observed in 100 and 200ppm groups. Dose-dependent decrease (P<0.001) was observed in mean uterine diameter, epithelial height, thickness of endometrium, myometrium, and in plasma levels of estradiol, progesterone, FSH and LH in all the treatment groups compared to control. In summary, arsenic is a major threat to female reproductive health acting as a reproductive toxicant and as an endocrine disruptor, restricted the function and structure of uterus, by altering the gonadotrophins and steroid levels, not only at high dose concentration but also at low (50ppm) levels, when they become mature.