Combination of shikonin with paclitaxel overcomes multidrug resistance in human ovarian carcinoma cells in a P-gp-independent manner through enhanced ROS generation

Abstract

Abstract Background Shikonin (SKN), a naphthoquinone compound, is isolated from Chinese herbal medicine Lithospermum root and has been studied as an anticancer drug candidate in human tumor models. This study is designed to investigate whether SKN can sensitize the therapeutic effect of paclitaxel (PTX) in drug-resistant human ovarian carcinoma cells. Methods Human ovarian carcinoma A2780 cell along with the paired PTX-resistant A2780/PTX cells were used. The effects of SKN, PTX or their combination on cell viability were conducted using Sulforhodamine B assay. P-glycoprotein (P-gp) expression was analyzed by flow cytometry after staining with P-gp-FITC anti-body. P-gp activity was determined by a fluorometric MDR assay kit or a rhodamine 123-based efflux assay, respectively. Apoptosis was evaluated by flow cytometry after Annexin V-FITC/PI co-staining. The effect of SKN, PTX or their combination on reactive oxygen species (ROS) generation and expression of pyruvate kinase M2 (PKM2) were investigated using flow cytometry or western blotting, respectively. PKM2 activity was detected by a Pyruvate Kinase Assay Kit. Results SKN/PTX co-treatment led to synergistically enhanced cytotoxicity and apoptosis in PTX-resistant ovarian cancer cells, indicating the circumvention of multidrug resistance (MDR) of PTX by SKN. Further study indicated that the MDR reversal effect of SKN was independent of inhibiting activity of the efflux transporter P-gp. Notably, SKN/PTX significantly increased the generation of intracellular ROS in A2780/PTX cells, and scavenging intracellular ROS blocked the sensitizing effects of SKN in PTX-induced cytotoxicity and apoptosis in A2780/PTX cells, but not in A2780 cells. Furthermore, SKN/PTX-induced downregulation of PKM2 (a key enzyme in glycolysis) and the suppression of its activity were inhibited by a ROS scavenger N-acetyl cysteine (NAC), suggesting that the synergy of the SKN/PTX combination may be not rely on PKM2 suppression. Conclusions These results reveal a P-gp-independent mechanism through ROS generation for the SKN/PTX combination to overcome MDR in ovarian cancer.

Keywords

• Shikonin
• Paclitaxel
• Multidrug resistance
• Reactive oxygen species
• Pyruvate kinase M2
• P-glycoprotein