Found programs: National Natural Science Foundation of China (No.82273957);Guizhou Province Science and Technology Plan Project (Nos.Qiankehe Platform Talent [2021]5632,Qiankehe Foundation-ZK [2023] Keynote034);Research Project on Traditional Chinese Medicine and Ethnic Medicine Science and Technology in Guizhou Province (No.QZYY-2022-025);Science and Technology Fund Project of Guizhou Provincial Health Commission(No.gzwkj2021-519);Guizhou Medical University Student Innovation and Entrepreneurship Training Program(No.S202210660139)
Authors：Liu Xing; Fan Shuangqin; Yan Xiaomin; Zhao Shijie ;Wang Rong; Shen Xiangchun; Zhou Xue; Zhang Yue; Chen Yan
Keywords：triple-negative breast cancer;migration;invasion;POLG inhibitor;mitochondrial biogenesis;ATP synthesis
DOI：DOI：10.19405/j.cnki.issn1000-1492.2024.10.005
〔Abstract〕 Objective To investigate the effects of zalcitabine(ddC), a mitochondrial DNA polymerase γ(POLG) inhibitor, on the migration, invasion, and to preliminarily explore mitochondrial biogenesis of human triple-negative breast cancer MDA-MB-231 cells. Methods The effect of ddC on cell viability was detected using the MTT assay. The migration and invasion abilities of the cells were evaluated using the cell scratch and Transwell invasion assays. Cell apoptosis was determined using flow cytometry and a V-FITC/PI cell apoptosis detection kit. The protein expression of POLG, NADH dehydrogenase subunit Ⅰ(NADH1), NADH dehydrogenase subunit Ⅱ(NADH2), ATP synthase subunit 6(ATPase6), cytochrome c oxidase subunit Ⅰ(COX-1) and cytochrome c oxidase subunit Ⅲ(COX-3) were determined using Western blot. The POLG mRNA level and mtDNA copy number were determined using qPCR. The mitochondrial content and ATP levels were determined using MitoTracker Green fluorescent probe staining and an ATP determination kit. MDA-MB-231 cells were transfected with pcDNA3.1-EGFP-POLG plasmids to overexpress POLG. The inhibitory effects of ddC on cell migration and invasion were detected in POLG-overexpressed MDA-MB-231 cells. Results POLG expression was higher in MDA-MB-231 cells than in normal mammary epithelial cells(MCF-10A)(P<0.01). ddC inhibited cell viability in a dose-dependent manner. ddC inhibited the migration(P<0.01) and invasion(P<0.01) of MDA-MB-231 cells; however, it displayed no significant inhibitory effects on cell viability in normal mammary epithelial cells(MCF-10A) at the same concentration. ddC downregulated the protein(P<0.01) and mRNA(P<0.01) levels of POLG, reduced mtDNA copy number(P<0.01) and downregulated mtDNA-coded NADH1, NADH2, ATPase6, COX-1 and COX-3 protein expression(P<0.01) in MDA-MB-231 cells. Furthermore ddC inhibited mitochondrial content(P<0.01) and ATP(P<0.01) levels in MDA-MB-231 cells. POLG overexpression increased the migration(P<0.05) and invasion(P<0.05) abilities of MDA-MB-231 cells, while ddC did not significantly inhibit the migration and invasion abilities of MDA-MB-231 cells overexpressing POLG. Conclusion ddC downregulates POLG expression in MDA-MB-231 cells and inhibits mitochondrial biogenesis and ATP levels, thereby inhibiting the migration and invasion of MDA-MB-231 cells.