Butyrate Inhibits the Mitochondrial Complex Ι to Mediate Mitochondria Dependent Apoptosis of Cervical Cancer Cells

Abstract

Abstract Background Cervical cancer (CC) is a common gynecological malignant tumor with high morbidity worldwide. Butyrate, a short-chain fatty acid produced by the intestinal flora, was reported to inhibit cervical carcinogenesis. This study aimed to further investigate the pro-apoptotic effects of butyrate on CC and the underlying mechanisms. Methods Human Hela and Caski cells were used in this study. Cell proliferation was assessed using CCK-8 and EdU staining. Cell migration and invasion were detected by Transwell and wound healing assay. The cell cycle, mitochondrial membrane potential and apoptosis were evaluated by flow cytometry. Reactive oxygen species (ROS) assay was performed for the mitochondrial function. Western blot and RT-qPCR were carried out to examine the related genes and proteins of mitochondrial complex Ι and apoptosis. The metabolite changes were analyzed by energy metabolomics and assay kits. The association between G protein-coupled receptor 41, 43, 109a and CC prognosis was analyzed using data from The Cancer Genome Atlas (TCGA). Results CCK-8 results showed that the butyrate treatment significantly inhibited the proliferation of CC cells, which was confirmed by EdU staining and cell cycle. The data of Transwell and wound healing assay unraveled that the migration of cervical cells was dramatically reduced after butyrate treatment. Additionally, the invasion was also observed to be decreased by butyrate. The western blot analysis showed that cleaved-Caspase 3 and cleaved-PARP, the executors of apoptosis, were increased by butyrate treatment. The results of Annexin V/PI staining and TUNEL also found the increase of apoptotic cells induced by butyrate. The expression of Cytochrome C, Caspase 9, BAX, but not Caspase 12 or 8, were up-regulated under butyrate exposure. The accumulated ROS was observed in butyrate-treated cells. Furthermore, energy metabolism showed that levels of mitochondrial NADH and NAD + decreased after butyrate treatment, which was consistent with the results of the NAD+/NADH Assay Kit. Western blot results also demonstrated that mitochondrial complex Ι reduced by butyrate. Conclusions Collectively, our results revealed that butyrate can inhibit the proliferation, migration and invasion of CC cells, and induce mitochondrial dependent apoptosis by inhibiting mitochondrial complex Ι.