Comparative Study of ZnO-and-TiO2-Nanoparticles-Functionalized Polyvinyl Alcohol/Chitosan Bionanocomposites for Multifunctional Biomedical Applications.

This study aimed to synthesize chitosan/polyvinyl alcohol (CS/PVA)-based zinc oxide (ZnO) and titanium dioxide (TiO2) hybrid bionanocomposites (BNCs) and observe their comparative accomplishment against the skin cancer cell line, A431, and antioxidant potential. CS was blended with PVA to form polymeric films reinforced with the immobilization of ZnO and TiO2 nanoparticles (NPs), separately. The optimization of the BNCs was done via physicochemical studies, viz. moisture content, swelling ratio, and contact angle measurements. The free radical scavenging activity was observed for 1,1-diphenyl-2-picryl-hydrazyl, and the antibacterial assay against the Escherichia coli strain showed a higher zone of inhibition. Furthermore, the anticancer activity of the synthesized BNCs was revealed against the skin cancer cell line A431 under varying concentrations of 50, 100, 150, 200, and 300 µg/mL. The anticancer study revealed a high percent of cancerous cell inhibition (70%) in ZnO BNCs as compared to (61%) TiO2 BNCs in a dose-dependent manner.

Association of PARK-2 Non-synonyms Polymorphisms and Their In Silico Validation Among North Indian Colorectal Cancer Patients.

PURPOSE: PARK2 is a potential tumour suppressor gene and its genetic alterations (regionic loss) are common across many human cancers. The association of PARK2 germline variations (SNPs) with Parkinson's has been shown, but their association in development and progression of cancer remains elusive. The aim of this study was to identify association of PARK2 polymorphisms (rs1801474, rs1801334) with colorectal cancer in a case control study design. METHODS: This case control study included a total of 650 genetically unrelated subjects comprising 300 colorectal cancer cases and 350 healthy controls belonging to North Indian. Both SNPs were analyzed using the PCR-RFLP assay. Statistical analysis for describing risk and association was performed using SPSS-17 software. Structural deviations due to non- synonymous substitutions (S167N and D394N) were analyzed using MD simulations. RESULTS: The genotype distributions of both the SNPs were in Hardy-Weinberg equilibrium. For both the polymorphisms, the allelic model showed statistically significant risk with OR ~ 1.3. Many of the associations remained significant even after Bonferroni correction (P < 0.00125). The result suggested that both S167N and D394N were deviated from wild type and structures and were stable after 5 ns. The average value of RMSD for backbone atoms was calculated from 5 to 10 ns molecular dynamics simulation data. CONCLUSION: In conclusion, our study revealed a significant association of PARK2 SNPs with colorectal cancer as well as their relations with other clinical parameters highlighting their contribution towards colorectal cancer susceptibility in North Indian population.

Implication of alterations in Parkin gene among North Indian patients with colorectal cancer.

BACKGROUND/AIMS: Alterations in Parkin (PRKN) have been described in many cancers; however, the molecular mechanism that contributes to loss of Parkin expression in colorectal cancer (CRC) remains unclear. The aim of this study was to investigate the involvement of PRKN mutation and loss of heterozygosity (LOH) in loss of Parkin expression. To understand the role of PRKN in cancer progression, we also evaluated the association of Parkin expression with clinicopathological parameters in North Indian population. MATERIALS AND METHODS: We studied 219 CRC samples and their adjacent normal tissues (control) obtained from North Indian patients with CRC. The expression of Parkin was analyzed by immunohistochemistry (IHC). PRKN mutations were analyzed by single-stranded conformational polymorphism (SSCP) and sequencing. For loss of heterozygosity (LOH), we employed two intragenic, D6S305 and D6S1599, and one telomeric marker, D6S1008. RESULTS: In our study, we found four novel somatic mutations, namely, C166G, K413N, R420P (exon 4), and V425E (exon 11). Both mutation in Parkin (p = 0.0014) and LOH (p = 0.0140) were significantly associated with loss of Parkin expression. Additionally, Parkin mutations were not associated with the clinicopathological parameters of the patients. Furthermore, both, LOH in Parkin and Parkin expression were significantly correlated with different clinicopathological variables (p<0.05). CONCLUSION: Our results indicate that Parkin expression is not regulated by a single mechanism, but both mutation and LOH contribute to loss of Parkin expression. We also provide evidence of involvement of Parkin in metastasis and cancer progression. We, therefore, suggest Parkin as a potential prognostic marker and warrant further analysis in this direction.

DRP1 Promotes BRAFV600E-Driven Tumor Progression and Metabolic Reprogramming in Colorectal Cancer.

BACKGROUND: Mitochondria are highly dynamic organelles which remain in a continuous state of fission/ fusion dynamics to meet the metabolic needs of a cell. However, this fission/fusion dynamism has been reported to be dysregulated in most cancers. Such enhanced mitochondrial fission is demonstrated to be positively regulated by some activating oncogenic mutations; such as those of KRAS (Kristen rat sarcoma viral oncogene homologue) or BRAF (B- rapidly accelerated fibrosarcoma), thereby increasing tumor progression/ chemotherapeutic resistance and metabolic deregulation. However, the underlying mechanism(s) are still not clear, thus highlighting the need to further explore possible mechanism(s) of intervention. We sought to investigate how BRAFV600E driven CRC (colorectal cancer) progression is linked to mitochondrial fission/fusion dynamics and whether this window could be exploited to target CRC progression. METHODS: Western blotting was employed to study the differences in expression levels of key proteins regulating mitochondrial dynamics, which was further confirmed by confocal microscopy imaging of mitochondria in endogenously expressing BRAFWT and BRAFV600E CRC cells. Proliferation assays, soft agar clonogenic assays, glucose uptake/lactate production, ATP/ NADPH measurement assays were employed to study the extent of carcinogenesis and metabolic reprograming in BRAFV600E CRC cells. Genetic knockdown (shRNA/ siRNA) and/or pharmacologic inhibition of Dynamin related protein1/Pyruvate dehydrogenase kinase1 (DRP1/PDK1) and/or BRAFV600E were employed to study the involvement and possible mechanism of these proteins in BRAFV600E driven CRC. Statistical analyses were carried out using Graph Pad Prism v 5.0, data was analyzed by unpaired t-test and two-way ANOVA with appropriate post hoc tests. RESULTS: Our results demonstrate that BRAFV600E CRC cells have higher protein levels of mitochondrial fission factor- DRP1/pDRP1S616 leading to a more fragmented mitochondrial state compared to those harboring BRAFWT . This fragmented mitochondrial state was found to confer glycolytic phenotype, clonogenic potential and metastatic advantage to cells harboring BRAFV600E . Interestingly, such fragmented mitochondrial state seemed positively regulated by mitochondrial PDK1 as observed through pharmacologic as well as genetic inhibition of PDK1. CONCLUSION: In conclusion, our data suggest that BRAFV600E driven colorectal cancers have fragmented mitochondria which confers glycolytic phenotype and growth advantage to these tumors, and such phenotype is dependent at least in part on PDK1- thus highlighting a potential therapeutic target.

Parkin gene mutations are not common, but its epigenetic inactivation is a frequent event and predicts poor survival in advanced breast cancer patients.

BACKGROUND: Progression of breast cancer involves both genetic and epigenetic factors. Parkin gene has been identified as a tumor suppressor gene in the pathogenesis of various cancers. Nevertheless, the putative role of Parkin in breast cancer remains largely unknown. Therefore, we evaluated the regulation of Parkin through both genetic and epigenetic mechanisms in breast carcinoma. METHOD: A total of 156 breast carcinoma and their normal adjacent tissue samples were included for mutational analysis through SSCP, and sequencing. MS-PCR was employed for methylation study whereas Parkin protein expression was evaluated using immunohistochemistry and western blotting. For the survival analysis, Kaplan-Meier curve and Cox's proportional hazard model were used. RESULTS: In expression analysis, Parkin protein expression was found to be absent in 68% cases of breast cancer. We found that aberrant promoter methylation of Parkin gene is a frequent incident in breast cancer tumors and cell lines. Our MS-PCR result showed that Parkin promoter methylation has a significant role (p = 0.0001) in reducing the expression of Parkin protein. Consistently, expression of Parkin was rectified by treatment with 5-aza-2-deoxycytidine. We also found significant associations of both Parkin negative expression and Parkin promoter methylation with the clinical variables. Furthermore, we found a very low frequency (5.7%) of Parkin mutation with no clinical significance. In survival analysis, patients having Parkin methylation and Parkin loss had a worse outcome compared to those harboring none of these events. CONCLUSION: Overall, these results suggested that promoter methylation-mediated loss of Parkin expression could be used as a prognostic marker for the survival of breast cancer.

Assessment of epigenetic alterations and in silico analysis of mutation affecting PTEN expression among Indian cervical cancer patients.

Genetic and epigenetic anomalies accountable for genetic dysregulation are the most common aberrations that determine the underlying heterogeneity of the tumor cells. Currently, phosphatase and tensin homolog (PTEN) incongruity has emerged as potent and persuasive malfunctioning in varied human malignancies. In this study, we have analysed the promoter hypermethylation and expression status of PTEN. We identified different mutations in the exonic region of PTEN. Functional consequences of these mutations were explored using in silico techniques. Promoter hypermethylation of PTEN was detected using methylation-specific polymerase chain reaction (MS-PCR), expression analysis was performed with immunohistochemistry (IHC) and mutation by direct sequencing in a total of 168 uterine cervix tumor cases. The findings were statistically correlated with the clinical parameters. In addition, the effect of nonsynonymous mutations was studied with molecular dynamics simulations. PTEN promoter hypermethylation (45.8%) was found to be significantly associated with the of PTEN loss (57.14%, P < 0.0001). Tumor stages, tumor size, lymph node (LN) were found to be significantly correlated with both PTEN promoter hypermethylation and PTEN loss. Histological grade, however, showed a significant association with only PTEN loss. In total, 11.76% of tumors exhibited mutations in exon 5 and 7, out of which E150K of exon 5 showed the highest deviations in the crystal structure of PTEN by in silico analysis. This study provides valuable insights into oncology and paves the path in the development of efficient biomarker and/or imperative therapeutic tool for cervical cancer treatment.

Association of PARK2 promoter polymorphisms and methylation with colorectal cancer in North Indian population.

Different diseases have been associated with PARK2/PACRG overlapping promoter polymorphisms (rs2276201 and rs9347683) in the recent past. However association of these polymorphisms with cancer remains elusive till date. Thus in this study we evaluated association between these polymorphisms and colorectal cancer (CRC) incidences among North Indians. Genomic DNA was isolated using venous blood of 400 unrelated subjects (200 CRC cases and 200 healthy controls) of North Indian origin. Both SNPs were genotyped using PCR-RFLP method. Promoter methylation status in tumor DNA was checked using MS-PCR. Statistical analysis was performed using SPSS-17 software. In-silico predictions for transcription factor binding were performed using "PROMO" a freely available online tool. SNP rs2276201 showed statistically significant difference (P = 0.047) among cases and controls while rs9347683 did not (P = 0.113). The TC genotype (OR: 1.855, 95% CI: 1.021-3.369, P = 0.043), CC genotype (OR: 1.617, 95% CI: 1.042-2.510, P = 0.032), TT vs CT+CC genetic model (OR: 1.60, P = 0.0158) and allelic model (OR: 1.3931, 95% CI: 1.0498-1.8485, P = 0.0214) of rs2276201 showed significant risk for CRC. For rs9347683 AC genotype (OR: 1.604, 95% CI: 1.019-2.523, P = 0.041) and AA vs AC+CC genetic model (OR: 1.57, P = 0.039) showed significant risk. Haplotype CC provided significant risk (OR: 1.618, 95% CI: 1.112-2.352, P = 0.011) whereas haplotype TA provided significant protection (OR: 0.732, 95% CI: 0.543-0.987, P = 0.040) against CRC. Promoter methylation was significantly higher in tumor grade III + IV (OR: 2.37, P = 0.019), while PARK2 expression was lower in cancer tissues compared to normal tissue. Here we provide the first report where PARK2 promoter SNP's rs2276201 and rs9347683 are shown to be significantly associated with the risk of CRC development.

Association of mitochondrial copy number variation and T16189C polymorphism with colorectal cancer in North Indian population.

Globally, colorectal cancer is the third most common type of cancer. Genetic instability leading to cancer development is one of the major causes for development of cancer. Alterations in mitochondrial genome, that is, mutations, single-nucleotide polymorphisms, and copy number variations are known to contribute in cancer development. The aim of our study was to investigate association of mitochondrial T16189C polymorphism and copy number variation with colorectal cancer in North Indian population. DNA isolated from peripheral blood of 126 colorectal cancer patients and 114 healthy North Indian subjects was analyzed for T16189C polymorphism and half of them for mitochondrial copy number variation. Genotyping was done using polymerase chain reaction-restriction fragment length polymorphism, and copy number variation was estimated using real-time polymerase chain reaction, numbers of mitochondrial copies and found to be significantly higher in colorectal cancer patients than healthy controls (88 (58-154), p = 0.001). In the regression analysis, increased mitochondrial copy number variation was associated with risk of colorectal cancer (odds ratio = 2.885, 95% confidence interval = 1.3-6.358). However, T16189C polymorphism was found to be significantly associated with the risk of rectal cancer (odds ratio = 5.213, p = 0.001) and non-significantly with colon cancer (odds ratio = 0.867, p = 0.791). Also, false-positive report probability analysis was done to validate the significant findings. Our results here indicate that mitochondrial copy number variation may be playing an important role in the development of colorectal cancer, and detection of mitochondrial copy number variation can be used as a biomarker for predicting the risk of colorectal cancer in North Indian subjects.

Genetic and epigenetic alterations affecting PARK-2 expression in cervical neoplasm among North Indian patients.

The recent investigation on PARK-2, a putative tumor suppressor gene, has found that it has been altered in multiple human malignancies. However, the clinical impact of PARK-2 alteration in uterine cervix carcinoma has not yet been studied. Therefore, we aimed to examine mutations, promoter hypermethylation, and protein expression of PARK-2 among the North Indian patients and their association with clinical parameters to evaluate the implication of PARK-2 in the genesis of cervical cancer. A total of 168 patient samples were processed for mutational analysis by single-strand conformation polymorphism, sequencing, and further in silico analysis of the identified mutations. Promoter hypermethylation by methylation-specific polymerase chain reaction and expression of PARK-2 were performed using immunohistochemistry. Statistical correlation between molecular findings and the clinicopathological parameters was taken to figure out the meaningful outcome. As per our findings, 3.5% (6/168) tumors showed novel missense mutations in exon 11 of PARK-2. In silico analysis showed high structural deviations manifested by mutations, A398D and Y391N, in both mutant proteins as compared to wild type. Promoter hypermethylation was observed in total of 29% of (48/168) tumor samples. Furthermore, 46.43% tumors (78/168) exhibited loss of PARK-2 expression in cervical carcinoma. The loss of expression of PARK-2 when correlated with clinical parameters resulted in significant association with tumor stage (p = 0.002) and with histological grade (p = 0.025). However, only clinical stage remained significant after Bonferroni correction (p < 0.007). A trend was observed between PARK-2 promoter hypermethylation and its protein expression. Our study provided sufficient information and insight for investigation of PARK-2 and highlighted its role as a tumor suppressor gene in cervical cancer in North Indian population.