Abstract
BackgroundRaw areca nut (RAN) consumption induces oral, esophageal and gastric cancers, which are significantly associated with the overexpression of pituitary tumor transforming gene 1/securin and chromosomal instability (CIN). An association of Securin/PTTG1 upregulation and gastric cancer in human was also demonstrated earlier. Since the molecular mechanism underlying securin upregulation remains unclear, this study intended to investigate the association of securin upregulation with the Rb-E2F1 circuit and epigenetic histone (H3) modification patterns both globally and in the promoter region of the securin gene.MethodsSix 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 every 60 days. Histopathological evaluation of stomach tissues was performed and securin expression was analysed by immunoblotting as well as by immunohistochemistry. ChIP-qPCR assays were performed to evaluate the recruitment of different histone modifications in the core promoter region of securin gene as well as its upstream and downstream regions.ResultsAll 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 lysine 4 and acetylation of H3 lysine 9 and 18 both globally and in the promoter region of the securin gene were observed by increasing the levels of lysine-N-methyltransferase 2A, lysine-acetyltransferase, EP-300 and PCAF after RAN treatment. ChIP-qPCR data revealed that the quantity of DNA fragments retrieved from the immunoprecipitated samples was maximum in the -83 to -192 region than further upstream and the downstream of the promoter for H3K4Me3, H3K9ac, H3K18ac and H3K9me3.ConclusionsRAN-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 subsequent development of chromosomal instability. Therefore, present results have led to the hypothesis that RAN-induced changes in the epigenetic landscape, securin overexpression and subsequent elevation of chromosomal instability is probably byproducts of inactivation of the pRb pathway.
Highlights
Mammalian securin which was initially isolated from rat pituitary tumor cells as pituitary tumor transforming gene 1 (PTTG1) [1], is an oncogene and has been implicated in the development and progression of several malignancies [2]
It has been demonstrated that higher expression of securin, induction of precocious anaphase and chromosomal instability have been associated with an increased risk of raw areca nut (RAN)-induced oral, esophageal and gastric cancers in both humans and mice [6, 7]
Securin overexpression through immunohistochemical staining The expression of securin was evaluated by immunostaining in a panel of mouse stomach samples collected from untreated mice and treated with Raw areca nut (RAN) + lime for 60, 100, 180, 240 and 300 days (Fig. 1B)
Summary
Mammalian securin which was initially isolated from rat pituitary tumor cells as pituitary tumor transforming gene 1 (PTTG1) [1], is an oncogene and has been implicated in the development and progression of several malignancies [2]. It has been demonstrated that higher expression of securin, induction of precocious anaphase (premature separation of sister chromatids) and chromosomal instability have been associated with an increased risk of raw areca nut (RAN)-induced oral, esophageal and gastric cancers in both humans and mice [6, 7]. Higher DNA damage, p53 overexpression, greater delay in cell kinetics and lower GSH levels in peripheral blood lymphocytes have been demonstrated in heavy RAN chewers than in nonchewers [8] Such observations prompted us to propose that in addition to cytogenetic parameters, the levels of endogenous GSH and p53 protein could act as effective biomarkers for RAN chewers [8]. Since the molecular mechanism underlying securin upregulation remains unclear, this study intended to investigate the association of securin upregulation with the Rb-E2F1 circuit and epigenetic histone (H3) modification patterns both globally and in the promoter region of the securin gene
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