Kumar Umesh, Sharma Ujjawal, Rathi Garima
1 Molecular Oncology Division, Dr. B.R. Ambedkar Center for Biomedical Research (ACBR), University of Delhi (North Campus), Delhi, India.
2 Department of Biochemistry, Postgraduate Institute of Medical Education & Research (PGIMER), Chandigarh, India.
Tumour Biol. 2017 Feb;39(2):1010428317692258. doi: 10.1177/1010428317692258.
One of the mechanisms for epigenetic silencing of tumor suppressor genes is hypermethylation of cytosine residue at CpG islands at their promoter region that contributes to malignant progression of tumor. Therefore, activation of tumor suppressor genes that have been silenced by promoter methylation is considered to be very attractive molecular target for cancer therapy. Epigenetic silencing of glutathione S-transferase pi 1, a tumor suppressor gene, is involved in various types of cancers including breast cancer. Epigenetic silencing of tumor suppressor genes can be reversed by several molecules including natural compounds such as polyphenols that can act as a hypomethylating agent. Curcumin has been found to specifically target various tumor suppressor genes and alter their expression. To check the effect of curcumin on the methylation pattern of glutathione S-transferase pi 1 gene in MCF-7 breast cancer cell line in dose-dependent manner. To check the reversal of methylation pattern of hypermethylated glutathione S-transferase pi 1, MCF-7 breast cancer cell line was treated with different concentrations of curcumin for different time periods. DNA and proteins of treated and untreated cell lines were isolated, and methylation status of the promoter region of glutathione S-transferase pi 1 was analyzed using methylation-specific polymerase chain reaction assay, and expression of this gene was analyzed by immunoblotting using specific antibodies against glutathione S-transferase pi 1. A very low and a nontoxic concentration (10 µM) of curcumin treatment was able to reverse the hypermethylation and led to reactivation of glutathione S-transferase pi 1 protein expression in MCF-7 cells after 72 h of treatment, although the IC value of curcumin was found to be at 20 µM. However, curcumin less than 3 µM of curcumin could not alter the promoter methylation pattern of glutathione S-transferase pi 1. Treatment of breast cancer MCF-7 cells with curcumin causes complete reversal of glutathione S-transferase pi 1 promoter hypermethylation and leads to re-expression of glutathione S-transferase pi 1, suggesting it to be an excellent nontoxic hypomethylating agent.
肿瘤抑制基因表观遗传沉默的机制之一是其启动子区域CpG岛处胞嘧啶残基的高甲基化，这有助于肿瘤的恶性进展。因此，激活因启动子甲基化而沉默的肿瘤抑制基因被认为是癌症治疗中极具吸引力的分子靶点。谷胱甘肽S-转移酶pi 1是一种肿瘤抑制基因，其表观遗传沉默与包括乳腺癌在内的多种癌症有关。肿瘤抑制基因的表观遗传沉默可被多种分子逆转，包括多酚等天然化合物，这些化合物可作为去甲基化剂。姜黄素已被发现可特异性靶向多种肿瘤抑制基因并改变其表达。以剂量依赖方式检测姜黄素对MCF-7乳腺癌细胞系中谷胱甘肽S-转移酶pi 1基因甲基化模式的影响。为检测高甲基化的谷胱甘肽S-转移酶pi 1甲基化模式的逆转情况，用不同浓度的姜黄素处理MCF-7乳腺癌细胞系不同时间段。分离处理和未处理细胞系的DNA和蛋白质，使用甲基化特异性聚合酶链反应分析法分析谷胱甘肽S-转移酶pi 1启动子区域的甲基化状态，并使用抗谷胱甘肽S-转移酶pi 1的特异性抗体通过免疫印迹法分析该基因的表达。在处理72小时后，极低且无毒浓度（10µM）的姜黄素处理能够逆转高甲基化并导致MCF-7细胞中谷胱甘肽S-转移酶pi 1蛋白表达重新激活，尽管姜黄素的IC值为20µM。然而，低于3µM的姜黄素不能改变谷胱甘肽S-转移酶pi 1的启动子甲基化模式。用姜黄素处理乳腺癌MCF-7细胞可导致谷胱甘肽S-转移酶pi 1启动子高甲基化完全逆转并导致谷胱甘肽S-转移酶pi 1重新表达，表明它是一种极好的无毒去甲基化剂。
