Abstract
BackgroundShikonin (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.MethodsHuman 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.ResultsSKN/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.ConclusionsThese results reveal a P-gp-independent mechanism through ROS generation for the SKN/PTX combination to overcome MDR in ovarian cancer.
Highlights
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
Combinational treatment of SKN with PTX significantly enhances cytotoxicity to PTX‐resistant ovarian cancer cells We demonstrated that PTX at 1, 10, 100 and 1000 nM significantly inhibited viability of A2780 cells by 49.2, 54.0, 64.7 and 73.2% (Fig. 1a), respectively, while only about 7.8 and 34.5% of growth inhibition on A2780/PTX cells was observed after treatment of PTX at 100 and 1000 nM (Fig. 1b), respectively, indicating that A2780/ PTX cells were resistant to PTX
SKN at a concentration of 1 or 2 μM was used in Combination of SKN and PTX induces enhanced apoptosis Based on the above results, we treated A2780 or A2780/ PTX cells with 1 μM SKN and/or 1 μM PTX in most of the subsequent experiments
Summary
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. Cancer cells usually are sensitive to drug-induced apoptosis at early stages, and become resistant eventually through abnormal regulation of apoptotic machinery [7, 8] or overexpression of efflux transporters such as P-glycoprotein (P-gp) to actively pump out anticancer drugs from cancer cells [9, 10]. To circumvent MDR relevant to apoptotic defects is usually much more difficult than that relevant to transporters, because apoptotic machinery is regulated by hundreds of antiapoptotic and proapoptotic proteins [13]. To overcome these apoptotic defects to re-sensitize anticancer agents to MDR tumors has been always a primary goal to achieve successful cancer treatment
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