Wei-Chung Hsu1, Samiraj Ramesh2, Marthandam Asokan Shibu3, Ming-Cheng Chen4, Tso-Fu Wang5, Cecilia Hsuan Day6, Ray-Jade Chen7, V Vijaya Padma8, Chi-Cheng Li9, Yu-Chen Tseng10, Chih-Yang Huang11. 1. Department of Radiation Oncology, Chung-Kang Branch, Cheng-Ching General Hospital, Taichung 40764, Taiwan; Department of Occupational Therapy, Asia University, Taichung 41354, Taiwan. 2. Cardiovascular and Mitochondrial Related Disease Research Center, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien 970, Taiwan; Department of Microbiology, PRIST Deemed to be University, Thanjavur 614 904, Tamil Nadu, India. 3. Cardiovascular and Mitochondrial Related Disease Research Center, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien 970, Taiwan. 4. Department of Surgery, Division of Colorectal Surgery, Taichung Veterans General Hospital, Taichung, Taiwan; Faculty of Medicine, National Yang-Ming University, Taipei, Taiwan. 5. Department of Hematology and Oncology, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien 970, Taiwan; School of Medicine, Tzu Chi University, 701, Section 3, Chung-Yang Road, Hualien 97004, Taiwan. 6. Department of Nursing, MeiHo University, Pingtung, Taiwan. 7. Department of Surgery, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan. 8. Department of Biotechnology, Bharathiar University, Coimbatore 641046, Tamil Nadu, India. 9. School of Medicine, Tzu Chi University, 701, Section 3, Chung-Yang Road, Hualien 97004, Taiwan; Center of Stem Cell & Precision Medicine, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien 970, Taiwan. 10. Division of Gastroenterology and Hepatology, Department of Internal Medicine, Taichung Armed Forces General Hospital, Taichung, Taiwan; Division of Gastroenterology and Hepatology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan. 11. Cardiovascular and Mitochondrial Related Disease Research Center, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien 970, Taiwan; Center of Stem Cell & Precision Medicine, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien 970, Taiwan; Graduate Institute of Biomedical Sciences, China Medical University, Taichung 404, Taiwan; Department of Biological Science and Technology, Asia University, Taichung, Taiwan; Center of General Education, Buddhist Tzu Chi Medical Foundation, Tzu Chi University of Science and Technology, Hualien 970, Taiwan; Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 404, Taiwan. Electronic address: cyhuang@mail.cmu.edu.tw.
Abstract
BACKGROUND: Chemoresistance remains the main obstacle in hepatocellular carcinoma (HCC) therapy. Despite significant advances in HCC therapy, HCC still has a poor prognosis. Thus, there is an urgent need to identify a treatment target to reverse HCC chemotherapy resistance. Platycodon grandiflorus (PG) is a perennial herb that has been used as food and traditional Chinese medicine for thousands of years in Northeast Asia. Platycodin D (PD), a main active triterpenoid saponin found in the root of PG, has been reported to possess anticancer properties in several cancer cell lines, including HCC; however, the reversal effect of this molecule on HCC chemoresistance remains largely unknown. PURPOSE: This study aimed to investigate the role and the mechanism of PD-mediated reversal of the histone deacetylase inhibitor (HDACi) resistance in HCC cells. METHODS: Human HCC cells (HA22T) and HDACi-resistant (HDACi-R) cells were used. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Combination index was used to calculate the synergism potential. Expression of ERK1/2 (total/phospho), cofilin-1 (total/phospho) and apoptosis-related protein was determined using western blotting. Mitochondrial membrane potential was assessed using the JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide) probe. Apoptosis was detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Mitochondrial reactive oxygen species generation was measured using the MitoSOX Red fluorescent probe. RESULTS: We found that PD treatment inhibited cell viability both in HA22T HCC and HDACi-R cells. Inhibition of ERK1/2 by PD98059 could reverse drug resistance in HDACi-R cells treated with PD98059 and PD. Nevertheless, pre-treatment with U46619, an ERK1/2 activator, rescued PD-induced apoptosis by decreasing levels of apoptosis-related proteins in HCC cells. The combined treatment of PD with apicidin a powerful HDACi, dramatically enhanced the apoptotic effect in HDACi-R cells. CONCLUSION: For the first time, we showed that PD reversed HDACi resistance in HCC by repressing ERK1/2-mediated cofilin-1 phosphorylation. Thus, PD can potentially be a treatment target to reverse HCC chemotherapy resistance in future therapeutic trials.
BACKGROUND: Chemoresistance remains the main obstacle in hepatocellular carcinoma (HCC) therapy. Despite significant advances in HCC therapy, HCC still has a poor prognosis. Thus, there is an urgent need to identify a treatment target to reverse HCC chemotherapy resistance. Platycodon grandiflorus (PG) is a perennial herb that has been used as food and traditional Chinese medicine for thousands of years in Northeast Asia. Platycodin D (PD), a main active triterpenoidsaponin found in the root of PG, has been reported to possess anticancer properties in several cancer cell lines, including HCC; however, the reversal effect of this molecule on HCC chemoresistance remains largely unknown. PURPOSE: This study aimed to investigate the role and the mechanism of PD-mediated reversal of the histone deacetylase inhibitor (HDACi) resistance in HCC cells. METHODS:Human HCC cells (HA22T) and HDACi-resistant (HDACi-R) cells were used. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Combination index was used to calculate the synergism potential. Expression of ERK1/2 (total/phospho), cofilin-1 (total/phospho) and apoptosis-related protein was determined using western blotting. Mitochondrial membrane potential was assessed using the JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide) probe. Apoptosis was detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Mitochondrial reactive oxygen species generation was measured using the MitoSOX Red fluorescent probe. RESULTS: We found that PD treatment inhibited cell viability both in HA22T HCC and HDACi-R cells. Inhibition of ERK1/2 by PD98059 could reverse drug resistance in HDACi-R cells treated with PD98059 and PD. Nevertheless, pre-treatment with U46619, an ERK1/2 activator, rescued PD-induced apoptosis by decreasing levels of apoptosis-related proteins in HCC cells. The combined treatment of PD with apicidin a powerful HDACi, dramatically enhanced the apoptotic effect in HDACi-R cells. CONCLUSION: For the first time, we showed that PD reversed HDACi resistance in HCC by repressing ERK1/2-mediated cofilin-1 phosphorylation. Thus, PD can potentially be a treatment target to reverse HCC chemotherapy resistance in future therapeutic trials.