Chia-Liang Lin1, Chien-Hsing Lee2,3, Chien-Min Chen4,5, Chun-Wen Cheng1, Pei-Ni Chen1, Tsung-Ho Ying6,7, Yi-Hsien Hsieh1,8,9. 1. Institute of Biochemistry, Microbiology and Immunology, Chung Shan Medical University, Taichung, Taiwan. 2. School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan. 3. Division of Pediatric Surgery, Department of Surgery, China Medical University Children's Hospital, Taichung, Taiwan. 4. Division of Neurosurgery, Department of Surgery, Changhua Christian Hospital, Changhua, Taiwan. 5. School of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan. 6. Department of Obstetrics and Gynecology, Chung Shan Medical University Hospital, Taichung, Taiwan. 7. Department of Obstetrics and Gynecology, School of Medicine, College of Medicine, Chung Shan Medical University, Taichung, Taiwan. 8. Department of Biochemistry, School of Medicine, Chung Shan Medical University, Taichung, Taiwan. 9. Clinical laboratory, Chung Shan Medical University Hospital, Taichung, Taiwan.
Abstract
BACKGROUND/AIMS: Protodioscin (PD) is a steroidal saponin with anti-cancer effects on a number of cancer cells, but the anti-tumor effects and mechanism of action of PD on human cervical cancer cells is unclear. METHODS: We determined cell viability using the MTT assay. Cell death, mitochondrial membrane potential (MMP), intracellular reactive oxygen species (ROS) generation, and endoplasmic reticulum (ER) stress were measured on a flow cytometry. Caspase activation, ER stress, and MMP-dependent apoptosis proteins in cervical cancer cells in response to PD were determined by Western blot analysis. The ability of ATF4 binding to ChIP promoter was measured using the ChIP assay. RESULTS: We demonstrated that PD inhibits cell viability, causes a loss of mitochondrial function, and induces apoptosis, as evidenced by up-regulation of caspase-8, -3, -9, -PARP, and Bax activation, and down-regulation of Bcl-2 expression. PD was shown to induce ROS and the ER stress pathway, including GRP78, p-eIF-2α, ATF4, and CHOP. Pre-treatment with NAC, a ROS production inhibitor, significantly reduced ER stress and apoptosis-related proteins induced by PD. Transfection of GRP78/CHOP-siRNA effectively inhibited PD-induced ER stress-dependent apoptosis. Moreover, treatment with PD significantly increased p38 and JNK activation. Co-administration of a JNK inhibitor (SP600125) or p38 inhibitor (SB203580) abolished cell death and ER stress effects during PD treatment. In addition, PD induced the expression of nuclear ATF4 and CHOP, as well as the binding ability of ATF4 to the CHOP promoter. CONCLUSION: Our results suggest that PD is a promising therapeutic agent for the treatment of human cervical cancer.
BACKGROUND/AIMS: Protodioscin (PD) is a steroidal saponin with anti-cancer effects on a number of cancer cells, but the anti-tumor effects and mechanism of action of PD on human cervical cancer cells is unclear. METHODS: We determined cell viability using the MTT assay. Cell death, mitochondrial membrane potential (MMP), intracellular reactive oxygen species (ROS) generation, and endoplasmic reticulum (ER) stress were measured on a flow cytometry. Caspase activation, ER stress, and MMP-dependent apoptosis proteins in cervical cancer cells in response to PD were determined by Western blot analysis. The ability of ATF4 binding to ChIP promoter was measured using the ChIP assay. RESULTS: We demonstrated that PD inhibits cell viability, causes a loss of mitochondrial function, and induces apoptosis, as evidenced by up-regulation of caspase-8, -3, -9, -PARP, and Bax activation, and down-regulation of Bcl-2 expression. PD was shown to induce ROS and the ER stress pathway, including GRP78, p-eIF-2α, ATF4, and CHOP. Pre-treatment with NAC, a ROS production inhibitor, significantly reduced ER stress and apoptosis-related proteins induced by PD. Transfection of GRP78/CHOP-siRNA effectively inhibited PD-induced ER stress-dependent apoptosis. Moreover, treatment with PD significantly increased p38 and JNK activation. Co-administration of a JNK inhibitor (SP600125) or p38 inhibitor (SB203580) abolished cell death and ER stress effects during PD treatment. In addition, PD induced the expression of nuclear ATF4 and CHOP, as well as the binding ability of ATF4 to the CHOP promoter. CONCLUSION: Our results suggest that PD is a promising therapeutic agent for the treatment of human cervical cancer.
Authors: Jong-Il Shin; Yong-Joon Jeon; Sol Lee; Yoon Gyeong Lee; Ji Beom Kim; Hak Cheol Kwon; Sung Hun Kim; Inki Kim; Kyungho Lee; Ye Sun Han Journal: Biomed Res Int Date: 2018-07-09 Impact factor: 3.411