Abstract：
Cancer cells modulate their metabolism, creating an acidic microenvironment that, in turn, can favor tumor progression and chemotherapy resistance. Tumor cells adopt strategies to survive a drop in extracellular pH (pHe). In the present manuscript, we investigated the contribution of mitochondrial sirtuin 3 (SIRT3) to the adaptation and survival of cancer cells to a low pHe. SIRT3-overexpressing and silenced breast cancer cells MDA-MB-231 and human embryonic kidney HEK293 cells were grown in buffered and unbuffered media at pH 7.4 and 6.8 for different times. mRNA expression of SIRT3 and CAVB, was measured by RT-PCR. Protein expression of SIRT3, CAVB and autophagy proteins was estimated by western blot. SIRT3-CAVB interaction was determined by immunoprecipitation and proximity ligation assays (PLA). Induction of autophagy was studied by western blot and TEM. SIRT3 overexpression increases the survival of both cell lines. Moreover, we demonstrated that SIRT3 controls intracellular pH (pHi) through the regulation of mitochondrial carbonic anhydrase VB (CAVB). Interestingly, we obtained similar results by using MC2791, a new SIRT3 activator. Our results point to the possibility of modulating SIRT3 to decrease the response and resistance of tumor cells to the acidic microenvironment and ameliorate the effectiveness of anticancer therapy.
摘要：
癌细胞调节它们的新陈代谢，创造一个酸性微环境，反过来,有利于肿瘤进展和化疗耐药。肿瘤细胞采取策略以在细胞外pH(pHe)下降中存活。在目前的手稿中，我们研究了线粒体沉默蛋白3（SIRT3）对癌细胞对低pHe的适应和存活的贡献。SIRT3过表达和沉默的乳腺癌细胞MDA-MB-231和人胚肾HEK293细胞在pH7.4和6.8的缓冲和未缓冲培养基中生长不同的时间。SIRT3和CAVB的mRNA表达,通过RT-PCR测量。通过蛋白质印迹评估SIRT3，CAVB和自噬蛋白的蛋白表达。通过免疫沉淀和邻近连接测定(PLA)确定SIRT3-CAVB相互作用。通过蛋白质印迹和TEM研究自噬的诱导。SIRT3过表达增加两种细胞系的存活。此外，我们证明SIRT3通过调节线粒体碳酸酐酶VB（CAVB）来控制细胞内pH（pHi）。有趣的是,我们通过使用新的SIRT3激活剂MC2791获得了类似的结果。我们的结果表明，调节SIRT3可能降低肿瘤细胞对酸性微环境的反应和抗性，并改善抗癌治疗的有效性。
