AA Homozygous Genotype of GSTP1 I105V Polymorphism in Oral Cancer: In Silico Screening, Molecular Dynamics Simulation and Activity Studies of Wild and Mutant GSTP1

Abstract

Background: Highest incidence of oral squamous cell carcinoma (OSCC) has been reported in north-eastern India and causal association between cancer and raw areca-nut (RAN) consumption are well evident. Earlier studies revealed that monitoring the occurrence of precocious anaphase and expression of securin in blood lymphocytes can be a good biomarkers for cancer risk in RAN+lime chewers. Glutathione S-transferases (GSTs) are considered an important cellular detoxification system that provide protection against the effects of toxins and determine individual's cancer susceptibility. Aim: Although GST gene family has been extensively studied, its role as susceptibility factor in oral cancer risk in the RAN chewing population in Meghalaya state, India has not been explored. The association of polymorphisms in GSTP1 (Ile105Val) gene with OSCC risk in Meghalaya, India, was evaluated. Earlier both AA reference and the GG/AG mutant genotype of GSTP1 gene showed an association with different types of cancers. Methods: Genetic polymorphism was evaluated by genotyping 129 cases and 156 controls using PCR-RFLP method and validated by Sanger sequencing in a hospital-based case-control study. GSTP1's interaction status with c-Jun Kinase (JNK) was evaluated through protein-protein docking analysis and this was also validated experimentally by immunohistochemistry and qRT-PCR. Results: Individuals with AA allele of GSTP1 showed significant association with the risk of OSCC compared with individuals with AG/GG mutant genotypes in habit-matched “RAN-only” and “RAN+Tobacco” group. The binding geometry between JNK and GSTP1 was disrupted in mutant combinations. It was demonstrated that AA homozygous genotype of oral epithelial cells showed reduced c-Jun-phosphorylation and proapoptotic genes expression than AG/GG genotypes. In silico docking revealed that homodimeric GSTP1 with AA genotype showed weak catalytic activity in detoxification of RAN and tobacco toxins compared with the AG/GG mutant proteins. Interestingly, the quantitation of 8-Oxo-2´-deoxyguanosine in DNA digests by ELISA-kit showed no differences in these genotypes, however the frequency of sister-chromatid exchanges was significantly higher in individuals with GSTP1 AA genotype than GG genotype. Conclusion: Thus in this population, specific traditional habit along with GSTP1 AA genotype play a significant role in predisposition to oral cancer risk by showing higher DNA-lesions, might be caused by some specific RAN/tobacco metabolites, and lower c-Jun phosphorylation which may inhibit apoptosis.