Ying-Tzu Chang1, Yu-Chao Lin2, Lijuan Sun3, Wei-Chieh Liao4, Charles C N Wang5, Che-Yi Chou6, Susan L Morris-Natschke7, Kuo-Hsiung Lee8, Chin-Chuan Hung9. 1. Department of Pharmacy, College of Pharmacy, China Medical University, Taichung, Taiwan 40402, R.O.C.. Electronic address: tel22336978@hotmail.com. 2. Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan, R.O.C.; Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, China Medical University Hospital, Taichung, Taiwan, R.O.C.. Electronic address: cmchlyc@yahoo.com.tw. 3. National & Local Joint Engineering Research Center for High-throughput Drug Screening Technology, Hubei Province Key Laboratory of Biotechnology of Chinese Traditional Medicine, Hubei University, China. Electronic address: lijuansun1212@163.com. 4. Department of Pharmacy, College of Pharmacy, China Medical University, Taichung, Taiwan 40402, R.O.C. 5. Department of Bioinformatics and Medical Engineering, Asia University, Taichung 41354, Taiwan, R.O.C.. Electronic address: chaoneng.wang@gmail.com. 6. Division of Nephrology, Asia University Hospital, Taichung, Taiwan, R.O.C.; Department of Post-baccalaureate Veterinary Medicine, Asia University, Taichung, Taiwan, R.O.C.. Electronic address: cychou.chou@gmail.com. 7. Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, United States. Electronic address: susan_natschke@unc.edu. 8. Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, United States; Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan 40447, R.O.C.. Electronic address: khlee@unc.edu. 9. Department of Pharmacy, College of Pharmacy, China Medical University, Taichung, Taiwan 40402, R.O.C.; Department of Pharmacy, China Medical University Hospital, Taichung, Taiwan 40447, R.O.C.. Electronic address: cc0206hung@gmail.com.
Abstract
BACKGROUND AND PURPOSE: Multidrug resistance (MDR) remains the main obstacle in cancer treatment and overexpression of P-glycoprotein (P-gp) is one of the most common causes of chemoresistance. The development of novel P-gp inhibitors from natural products is a prospective strategy to combat MDR cancers. Among the natural sesquiterpene compounds, sesquiterpene pyridine alkaloids exhibit various biological properties. Therefore, in the present study, we evaluated the modulatory effects of wilforine on P-gp expression and function. The molecular mechanisms and kinetic models of wilforine-mediated P-gp inhibition were further investigated. METHODS: The human P-gp stable expression cells (ABCB1/Flp-InTM-293) and human cervical cancer cells (sensitive: HeLaS3; MDR: KBvin) were used. The cell viability was assessed by SRB assay. The inhibitory effect of wilforine on P-gp efflux and the underlying mechanism were evaluated by assays for calcein-AM uptake, rhodamine123 and doxorubicin efflux, ATPase activity, real-time quantitative RT-PCR, apoptosis, and cell cycle analysis. Molecular docking was performed by the docking software CDOCKER with BIOVIA Discovery Studio 4.5 (D.S. 4.5). RESULTS: We found that wilforine significantly inhibited the efflux activity of P-gp in a concentration-dependent manner. Further kinetic analysis demonstrated that wilforine significantly inhibited P-gp efflux function by competitive inhibition and stimulated the basal P-gp ATPase activity. In addition, wilforine re-sensitized MDR cancer cells to chemotherapeutic drugs. The docking model indicated that wilforine was bound to residues of P-gp such as LEU884, LYS887, THR176 and ASN172. CONCLUSION: These results suggest a novel future therapeutic strategy for MDR cancer using wilforine as an adjuvant treatment with chemotherapy.
BACKGROUND AND PURPOSE: Multidrug resistance (MDR) remains the main obstacle in cancer treatment and overexpression of P-glycoprotein (P-gp) is one of the most common causes of chemoresistance. The development of novel P-gp inhibitors from natural products is a prospective strategy to combat MDR cancers. Among the natural sesquiterpene compounds, sesquiterpene pyridine alkaloids exhibit various biological properties. Therefore, in the present study, we evaluated the modulatory effects of wilforine on P-gp expression and function. The molecular mechanisms and kinetic models of wilforine-mediated P-gp inhibition were further investigated. METHODS: The humanP-gp stable expression cells (ABCB1/Flp-InTM-293) and human cervical cancer cells (sensitive: HeLaS3; MDR: KBvin) were used. The cell viability was assessed by SRB assay. The inhibitory effect of wilforine on P-gp efflux and the underlying mechanism were evaluated by assays for calcein-AM uptake, rhodamine123 and doxorubicin efflux, ATPase activity, real-time quantitative RT-PCR, apoptosis, and cell cycle analysis. Molecular docking was performed by the docking software CDOCKER with BIOVIA Discovery Studio 4.5 (D.S. 4.5). RESULTS: We found that wilforine significantly inhibited the efflux activity of P-gp in a concentration-dependent manner. Further kinetic analysis demonstrated that wilforine significantly inhibited P-gp efflux function by competitive inhibition and stimulated the basal P-gp ATPase activity. In addition, wilforine re-sensitized MDR cancer cells to chemotherapeutic drugs. The docking model indicated that wilforine was bound to residues of P-gp such as LEU884, LYS887, THR176 and ASN172. CONCLUSION: These results suggest a novel future therapeutic strategy for MDR cancer using wilforine as an adjuvant treatment with chemotherapy.