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

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.

SUBMITTER: Wang Z

PROVIDER: S-EPMC6417206 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Project description:Paclitaxel plays a major role in the treatment of ovarian cancer; however, resistance to paclitaxel is frequently observed. Thus, new therapy that can overcome paclitaxel resistance will be of significant clinical importance. We evaluated antiproliferative effects of an antimitotic and antivascular agent BPR0L075 in paclitaxel-resistant ovarian cancer cells. BPR0L075 displays potent and broad-spectrum cytotoxicity at low nanomolar concentrations (IC50 = 2-7 nM) against both parental ovarian cancer cells (OVCAR-3, SKOV-3, and A2780-1A9) and paclitaxel-resistant sublines (OVCAR-3-TR, SKOV-3-TR, 1A9-PTX10), regardless of the expression levels of the multidrug resistance transporter P-gp and class III β-tubulin or mutation of β-tubulin. BPR0L075 blocks cell cycle at the G2/M phase in paclitaxel-resistant cells while equal concentration of paclitaxel treatment was ineffective. BPR0L075 induces cell death by a dual mechanism in parental and paclitaxel-resistant ovarian cancer cells. In the parental cells (OVCAR-3 and SKOV-3), BPR0L075 induced apoptosis, evidenced by poly(ADP-ribose) polymerase (PARP) cleavage and DNA ladder formation. BPR0L075 induced cell death in paclitaxel-resistant ovarian cancer cells (OVCAR-3-TR and SKOV-3-TR) is primarily due to mitotic catastrophe, evidenced by formation of giant, multinucleated cells and absence of PARP cleavage. Immunoblotting analysis shows that BPR0L075 treatment induced up-regulation of cyclin B1, BubR1, MPM-2, and survivin protein levels and Bcl-XL phosphorylation in parental cells; however, in resistant cells, the endogenous expressions of BubR1 and survivin were depleted, BPR0L075 treatment failed to induce MPM-2 expression and phosphorylation of Bcl-XL. BPR0L075 induced cell death in both parental and paclitaxel-resistant ovarian cancer cells proceed through caspase-3 independent mechanisms. In conclusion, BPR0L075 displays potent cytotoxic effects in ovarian cancer cells with a potential to overcome paclitaxel resistance by bypassing efflux transporters and inducing mitotic catastrophe. BPR0L075 represents a novel microtubule therapeutic to overcome multidrug resistance and trigger alternative cell death by mitotic catastrophe in ovarian cancer cells that are apoptosis-resistant.

Project description:Ten human ovarian cancer cell lines were kindly provided as follows: an ovarian serous adenocarcinoma cell line, SKOV3 (Dr. N. Nagai, Hiroshima University, Hiroshima, Japan); KF28 and its TXL/CDDP-resistant variant (Dr. Y. Kikuchi, National Defense Medical College, Saitama, Japan); KF, SHIN3 and 5 ovarian clear cell adenocarcinoma cell lines, KK, TAYA, RMG-1, OVISE and OVTOKO (Dr. M. Suzuki, Jichi Medical School, Tochigi, Japan). All cell lines were cultured in RPMI 1640 medium (Life Technologies, Inc., Grand Island, NY) containing 10% heat-inactivated fetal bovine serum (FBS; BioWhittaker, Verviers, Belgium) in a humidified atmosphere of 5% CO2 and maintained in continuous exponential growth by passage every 3 days. Exponentially growing cultured cells were collected after two-washings with PBS. The cell pellets were immediately frozen in liquid nitrogen, and stored until use. Total RNA was extracted using QIAGEN RNeasy mini kit (QIAGEN, Inc., Valencia, CA). The quality of the RNA was checked using Agilent Technologies 2100 Bioanalyzer (Agilent, Palo Alto, CA). CodeLink Expression Bioarray System (Amersham Bioscience, Tokyo, Japan) was used according to the manufacturer’s protocol. Briefly, first-strand cDNA was generated from 1 microgram of total RNA of each cell line using reverse transcriptase and a T7 primer, and then second-strand cDNA was produced using DNA polymerase mix and RNase H. Complementary RNA (cRNA) was generated via an in vitro transcription reaction using T7 RNA polymerase and biotin-11-UTP (Perkin Elmer, Boston, MA), which was quantified by spectrometry and checked using Agilent Technologies 2100 Bioanalyzer (Agilent). Ten-microgram of cRNA was then fragmented and hybridized to a Uniset Human 20K I Bioarray containing 19,881 probes with positive and negative bacterial control probes. After hybridization, the arrays were rinsed and labeled with Streptavidin-Cy5, scanned using Agilent DNA Microarray Scanner (Agilent), then analyzed with CodeLink Expression Analysis Software ver.2.3. Expression levels were normalized to the median expression value of the whole array spots. Keywords: parallel sample

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

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