Yu-Ning Teng1, Kun-I Lin2, Yu-Chao Lin3, Tran-Dinh Thang4, Yu-Hsuan Lan5, Chin-Chuan Hung6. 1. School of Medicine, College of Medicine, I-Shou University, 8 Yida Road, Kaohsiung 82445, Taiwan, R.O.C.. Electronic address: eunicegh520@gmail.com. 2. Department of Obstetrics and Gynecology, Chang Bing Show Chwan Memorial Hospital, Changhua, Taiwan, R.O.C.; Department of Cosmetic Science, Providence University, Taichung, Taiwan, R.O.C.. Electronic address: kunidg107@gmail.com. 3. 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. 4. Institute of Biotechnology and Food Technology, Industrial University of Ho Chi Minh City, Ho Chi Minh City, Vietnam. Electronic address: thangdhv@gmail.com. 5. Department of Pharmacy, College of Pharmacy, China Medical University, 100, Sec. 1, Jingmao Rd., Beitun Dist., Taichung City 406040, Taiwan, R.O.C.. Electronic address: lanyh@mail.cmu.edu.tw. 6. Department of Pharmacy, College of Pharmacy, China Medical University, 100, Sec. 1, Jingmao Rd., Beitun Dist., Taichung City 406040, Taiwan, R.O.C.; Department of Pharmacy, China Medical University Hospital, 2 Yude Road, Taichung 40447, Taiwan, R.O.C.; Department of Healthcare Administration, Asia University, 500, Lioufeng Rd., Wufeng, Taichung, 41354, Taiwan. Electronic address: cc0206hung@gmail.com.
Abstract
BACKGROUND: P-glycoprotein (P-gp) over-expression plays a vital role in not only systemic drug bioavailability but also cancer multi-drug resistance (MDR). Develop functional inhibitors of P-gp can conquer both problems. PURPOSE AND STUDY DESIGN: The aim of the present study was to research the P-gp modulating effects and MDR reversing ability of a novel flavonoid from Fissistigma cupreonitens, the underlying inhibitory mechanisms were further elucidated as well. METHODS: Calcein-AM, rhodamine 123, and doxorubicin were fluorescent substrates for the evaluation of P-gp inhibitory function and detailed drug binding modes. Docking simulation was performed to reveal the in silico molecular bonding. ATPase assay and MDR1 shift assay were adopted to reveal the ATP consumption and conformational change of P-gp. The MDR reversing effects were demonstrated through cytotoxicity, cell cycle, and apoptosis analyses. RESULTS: 5‑hydroxy‑7,8‑dimethoxyflavanone inhibited the efflux of rhodamine 123 and doxorubicin in a competitive manner, and increased the intracellular fluorescence of calcein at a concentration as low as 2.5 μg/ml. 5‑hydroxy‑7,8‑dimethoxyflavanone slightly changed P-gp's conformation and only stimulated ATPase at very high concentration (100 μg/ml). The docking results showed that 5‑hydroxy‑7,8‑dimethoxyflavanone and verapamil exhibited similar binding affinity to P-gp. The MDR reversing effects were prominent in the vincristine group, the reversal folds were 23.01 and 13.03 when combined with 10 μg/ml 5‑hydroxy‑7,8‑dimethoxyflavanone in the P-gp over-expressing cell line (ABCB1/Flp-In™-293) and MDR cancer cell line (KB/VIN), respectively. CONCLUSION: The present study demonstrated that 5‑hydroxy‑7,8‑dimethoxyflavanone was a novel effective flavonoid in the P-gp efflux inhibition and in vitro cancer MDR reversion.
BACKGROUND: P-glycoprotein (P-gp) over-expression plays a vital role in not only systemic drug bioavailability but also cancer multi-drug resistance (MDR). Develop functional inhibitors of P-gp can conquer both problems. PURPOSE AND STUDY DESIGN: The aim of the present study was to research the P-gp modulating effects and MDR reversing ability of a novel flavonoid from Fissistigma cupreonitens, the underlying inhibitory mechanisms were further elucidated as well. METHODS: Calcein-AM, rhodamine 123, and doxorubicin were fluorescent substrates for the evaluation of P-gp inhibitory function and detailed drug binding modes. Docking simulation was performed to reveal the in silico molecular bonding. ATPase assay and MDR1 shift assay were adopted to reveal the ATP consumption and conformational change of P-gp. The MDR reversing effects were demonstrated through cytotoxicity, cell cycle, and apoptosis analyses. RESULTS: 5‑hydroxy‑7,8‑dimethoxyflavanone inhibited the efflux of rhodamine 123 and doxorubicin in a competitive manner, and increased the intracellular fluorescence of calcein at a concentration as low as 2.5 μg/ml. 5‑hydroxy‑7,8‑dimethoxyflavanone slightly changed P-gp's conformation and only stimulated ATPase at very high concentration (100 μg/ml). The docking results showed that 5‑hydroxy‑7,8‑dimethoxyflavanone and verapamil exhibited similar binding affinity to P-gp. The MDR reversing effects were prominent in the vincristine group, the reversal folds were 23.01 and 13.03 when combined with 10 μg/ml 5‑hydroxy‑7,8‑dimethoxyflavanone in the P-gp over-expressing cell line (ABCB1/Flp-In™-293) and MDR cancer cell line (KB/VIN), respectively. CONCLUSION: The present study demonstrated that 5‑hydroxy‑7,8‑dimethoxyflavanone was a novel effective flavonoid in the P-gp efflux inhibition and in vitro cancer MDR reversion.