The Glutathione S-transferases (GSTs) protects cellular DNA against oxidative damage. The role of GSTP1 polymorphism (A313G; Ile105Val) as a susceptibility factor in oral cancer was evaluated in a hospital-based case-control study in NorthEast India, because the habit of chewing raw areca-nut (RAN) with/without tobacco is common in this region. Genetic polymorphism was investigated by genotyping 445 cases and 444 controls. Individuals with the GSTP1 AA-genotype showed association with the oral cancer (OR = 3.1, 95% CI = 2.4-4.2, p = 0.0002). Even after adjusting for age, sex and habit the AA-genotype is found to be significantly associated with oral cancer (OR = 2.4, 95% CI = 1.7-3.2, p = 0.0001). A protein-protein docking analysis demonstrated that in the GG-genotype the binding geometry between c-Jun Kinase and GSTP1 was disrupted. It was validated by immunohistochemistry in human samples, showing lower c-Jun-phosphorylation and down-regulation of pro-apoptotic genes in normal oral epithelial cells with the AA-genotype. In silico docking revealed that AA-genotype weakly detoxifies the RAN/tobacco metabolites. In addition, experiments revealed a higher level of 8-Oxo-2′deoxyguanosine induction in tumor samples with the AA-genotype. Thus, habit of using RAN/tobacco and GSTP1 AA-genotype together play a significant role in predisposition to oral cancer risk by showing higher DNA-lesions and lower c-Jun phosphorylation that may inhibit apoptosis. The oral squamous cell carcinoma (OSCC) is the most common cancer in India, with highest occurrence in the northeastern part of the country 1. The traditional habit of chewing raw rather than dry areca-nut with lime appears to be an important causative factor in addition of tobacco 2,3. In NorthEast India, particularly in Meghalaya, the betel-quid contains raw and unprocessed areca-nut (RAN), lime paste and small portion of betel-leaf but without tobacco. It has been noted that alkaloids, and polyphenols and tannic acid of RAN that are released in the saliva may contribute to carcinogenicity 4-6. It has been demonstrated that areca-nut alkaloids cause depression of antioxidants including glutathione and glutathione-S-transferases (GST) that are known to neutralize reactive oxygen species 7. Earlier studies indicate that RAN and lime together induce oral, esophagous and stomach cancers both in mouse and human and highlighted the occurrence of precocious anaphase (premature separation of sister-chromatids) and higher expression of p53 and Securin as a potential screening marker for identification of mitotic checkpoint defects during early days of RAN exposure 8,9. There are enough data to view lifestyle as well as genetic factors as important contributors for an individual's susceptibility to cancer 10. Glutathione redox and GST are supposed to play important roles in cellular
People of north-eastern states of India consume raw areca-nut (RAN) and lime which could lead to oral, esophageal and gastric cancers. However, the incidence of these cancers are significantly lesser in those who consume pieces of Potentilla fulgens root along with RAN. Since evaluation of anticancer role, if any, of P. fulgens on RAN-mediated genetic alterations in human is difficult because of other compounding factors, this study was undertaken in mice to focus on gastric carcinogenesis since ad libitum administration of RAN extract with lime in drinking water induced stomach cancer due to greater exposure of its lining. A total of 160 mice were used at different time points and either methanol extract of P. fulgens roots (PRE) or mixture of four compounds of ethyl-acetate fraction (EA-mixture) was mixed with mice feed. Histological studies revealed that RAN + lime induced cancer in all the mice and interestingly only 20% developed cancer when PRE/EA-mixture was provided along with RAN + lime. Higher frequency of precocious anaphase and over expression of p53 and Securin genes were significantly reduced by PRE/EA-mixture. Thus PRE/EA-mixture mitigates the RAN-induced tumor-initiating process in stomach by maintaining expression of tumor suppressor and check-point genes under control.
Background Raw areca-nut (RAN) consumption induces oral, esophageal and gastric cancers which are significantly associated with the overexpression of pituitary tumor transforming gene1/Securin and chromosomal instability (CIN). Since the molecular mechanism underlying Securin upregulation remains unclear, this study is intended to investigate the association of Securin upregulation with Rb-E2F1 circuit and epigenetic histone (H3) modifications pattern both globally and in the promoter region of Securin gene. Methods Six groups of mice were used and in the treated group each mouse consumed 1 mg of RAN-extract with lime per day ad libitum in the drinking water for 60 days after which the dose was increased by 1 mg after every 60 days. Histopathological evaluation of stomach tissues was done and Securin expression was analysed by immunoblotting as well as by immunohistochemistry. ChIP-qPCR assay was performed for evaluating the recruitment of different histone modifications in the two regions of Securin promoter. Results All mice developed gastric cancer with Securin overexpression after 300 days of feeding. Immunohistochemistry data revealed hyperphosphorylation of Rb and upregulation of E2F1 in the RAN-treated samples. Increased trimethylation of H3 Lysine4 and acetylation of H3 Lysine9 and 18 both globally and in the promoter region of Securin gene was observed by increasing the level of lysine-N-methyltransferase2A, lysine-acetyltransferase, EP-300 and PCAF after RAN-treatment. ChIP-qPCR data show an increased recruitment of H3K4me3, H3K9ac and H3K18ac in the promoter of Securin gene after RAN-exposure Conclusions In light of the present results, it seems that RAN-mediated pRb-inactivation induced Securin upregulation, a putative E2F1 target, by inducing misregulation in chromatin remodeling in its promoter region which led to transcriptional activation and subsequently develop chromosomal instability. Therefore, the present results have led to the hypothesis that RAN-induced changes in the epigenetic landscape, Securin overexpression and subsequent elevation of chromosomal instability are probably a by-product of the inactivation of the pRb pathway.
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