ONCOLOGY LETTERS 6: 1084-1092, 2013
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Triptolide avoids cisplatin resistance and induces apoptosis via the reactive oxygen species/nuclear factor‑κB pathway in SKOV3PT platinum‑resistant human ovarian cancer cells YAN‑YING ZHONG1,2, HE‑PING CHEN1*, BU‑ZHEN TAN2*, HAI‑HONG YU1 and XIAO‑SHAN HUANG1 1
The Key Laboratory of Basic Pharmacology, School of Pharmaceutical Science, Nanchang University; 2 Department of Obstetrics and Gynecology, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China Received January 29, 2013; Accepted July 10, 2013 DOI: 10.3892/ol.2013.1524
Abstract. An acquired resistance to platinum‑based drugs has emerged as a significant impediment to effective ovarian cancer therapy. The present study explored the anticancer mechanisms of triptolide (TPL) in SKOV3PT platinum‑resistant human ovarian cancer cells and observed that TPL activated caspase 3 and induced the dose‑dependent apoptosis of the SKOV3PT cells. Furthermore, TPL inhibited complex I of the mitochondrial respiratory chain (MRC) followed by an increase of reactive oxygen species (ROS), which further inhibited nuclear factor (NF)‑κ B activation and resulted in the downregulation of anti‑apoptotic proteins, Bcl‑2 and X‑linked inhibitor of apoptosis protein (XIAP). Notably, the pre‑treatment with N‑acetyl‑L‑cysteine (NAC) abolished the TPL‑induced ROS generation, NF‑κ B inhibition and cell apoptosis, but did not affect the inhibitory effect of TPL on complex I activity. These results suggested that TPL negatively regulated the NF‑κ B pathway through mitochondria‑derived ROS accumulation, promoting the apoptosis of the SKOV3PT cells. Furthermore, TPL synergistically enhanced the cytotoxicity of cisplatin against platinum‑resistant ovarian cancer cells. Collectively, these findings suggest that TPL is able to overcome chemoresistance and that it may be an effective treatment for platinum‑resistant ovarian cancer, either alone or as an adjuvant therapy.
Correspondence to: Dr He‑Ping Chen, The Key Laboratory of
Basic Pharmacology, School of Pharmaceutical Science, Nanchang University, 461 Bayi Road, Nanchang, Jiangxi 330006, P.R. China E‑mail:
[email protected] *
Contributed equally
Key words: triptolide, reactive oxygen species, nuclear factor‑κ B, platinum resistance, ovarian cancer
Introduction Ovarian cancer is currently the leading cause of mortality among gynecological malignant tumors, with epithelial ovarian cancer (EOC) being the most common, accounting for >85% of all cases (1). The majority of ovarian cancers are diagnosed at an advanced stage, mostly due to a lack of effective screening strategies and difficulties in obtaining a diagnosis (2). Despite the progress that has been made in prolonging remission by the combination of surgical resection and platinum‑based chemotherapy, the overall survival of patients with advanced disease is rarely >30%. The poor prognosis in the treatment of ovarian cancer is mainly attributed to chemoresistance (3). Tumor cells may dampen the cytotoxic effects of anticancer drugs via several mechanisms, including increased drug efflux, drug inactivation, alteration in the drug target and increased DNA repair (4,5). As a result, efforts have been directed towards the development of novel agents in an attempt to ameliorate the lethality of this malignancy. Recent studies on the chemoresistance of ovarian cancer have indicated that a decreased susceptibility of the cancer to apoptosis is strongly associated with drug resistance. Constitutively activated nuclear factor (NF)‑κ B may be critical in the development of drug resistance in ovarian cancer cells (6). NF‑κ B is known to suppress apoptosis through the induction of anti‑apoptotic proteins, including Bcl‑2 and X‑linked inhibitor of apoptosis protein (XIAP), leading to a resistance to cancer therapy and a poor prognosis (7‑9). Intriguingly, numerous anticancer drugs, including the DNA‑damaging agent cisplatin, are able to simultaneously stimulate NF‑κ B activation, as they trigger the cell death process in neoplasm cells (7,8,10). Therefore, the inhibition of NF‑κ B may be useful in increasing the sensitivity of cells to chemotherapy‑dependent apoptosis and reversing drug resistance in ovarian cancer. Triptolide (TPL), a purified component extracted from Tripterygium wilfordii Hook f (TwHf; Lei Gong Teng), has been identified as the main active element that is responsible for immunosuppressive and anti‑inflammatory properties (11). A number of in vitro and in vivo studies have revealed that TPL exhibits a wide spectrum of anticancer effects toward various cancer models (12‑17). However, the underlying
ZHONG et al: TRIPTOLIDE AFFECTS SKOV3PT CELL GROWTH AND AVOIDS CISPLATIN RESISTANCE
molecular mechanisms are complicated and remain vague. In human anaplastic thyroid carcinoma cells, TPL has been shown to induce apoptosis through the inhibition of NF‑κ B in a p53‑independent pathway (13). TPL has also previously been shown to enhance tumor necrosis factor (TNF)‑related apoptosis‑inducing ligand (TRAIL)‑mediated apoptosis of lung cancer cells by the inhibition of NF‑κ B (18). Furthermore, TPL induces the production of reactive oxygen species (ROS), leading to apoptosis in human adrenal cancer NCI‑H295 cells (16). While certain NF‑κ B‑regulated genes, including Bcl‑2, play a major role in regulating the amount of ROS in the cell, ROS have various inhibitory and stimulatory roles in NF‑κ B signaling (19,20). The present study aimed to investigate whether TPL sensitized platinum‑resistant SKOV3PT ovarian cancer cells to apoptosis, along with the molecular signaling pathway triggered by TPL in platinum‑resistant cells. The study further hypothesized that TPL inactivated the NF‑κB pathway through ROS accumulation, promoting the apoptosis of the SKOV3PT cells. Materials and methods Materials. TPL (Sigma Aldrich Chemical Co., St. Louis, MO, USA) was dissolved as a stock solution in dimethyl sulfoxide (DMSO) and freshly diluted in 10 mM culture medium prior to use. Cisplatin, N‑acetyl‑L‑cysteine (NAC), 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide (MTT), Annexin V‑fluorescein isothiocyanate (FITC) and propidium iodide (PI) were obtained from Sigma. 2',7'‑Dichlorodihydrofluorescein diacetate (H 2 DCF‑DA) was purchased from Calbiochem (San Diego, CA, USA). The Mitochondrial Isolation kit was bought from Thermo Scientific (Pierce, Rockford, IL, USA) and the Mitochondrial Respiratory Chain (MRC) Complexes Activity Assay kits were purchased from Genmed Scientifics (Shanghai, China). Rabbit polyclonal anti-Bcl‑2 (1:600), rabbit polyclonal antiNF‑κ B (p65; 1:400) and goat polyclonal anti‑β‑actin (1:1000) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and rabbit monolonal anti‑caspase 3 (1:300) and rabbit monoclonal anti‑XIAP (1:500) antibodies were perchased from Cell Signaling Technology (San Diego, CA, USA). Cell culture. The human ovarian carcinoma‑derived platinum resistant SKOV3PT cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). To maintain the acquired resistance to cisplatin, the cells were cultured in RPMI‑1640 medium supplemented with fetal bovine serum (10%), penicillin/streptomycin (100 U/ml) and cisplatin (0.3 µg/ml) in a 5% humidified CO2 atmosphere at 37˚C. Cell viability assay. Cell viability was evaluated using the MTT assay. Briefly, 1x104 cells/well were seeded in 96‑well microtiter plates. Following the drug treatment, the cells were incubated with 20 µl MTT (5 mg/ml) for an additional 4 h. The MTT solution in the medium was discarded and the formazan crystals, which were formed in the viable cells, were dissolved in 150 µl DMSO. The optical density of each well was measured at 490 nm using a Microplate Reader (Molecular Devices, CA, USA).
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Apoptosis analysis. Early‑stage apoptosis cells that expressed phosphatidylserine on the outer layer of the cell were detected using the binding properties of fluoresceinated Annexin V (Annexin V‑FITC). Briefly, the treated cells were harvested and washed twice with cold phosphate‑buffered saline (PBS). The cells were suspended with a binding buffer and stained with Annexin V‑FITC and PI. The cell mixture was incubated for 15 min at room temperature in the dark followed by fluorescence‑activated cell sorting (FACS) cater‑plus flow cytometry (Becton‑Dickinson Co., Heidelberg, Germany). ROS detection. The changes in the intracellular ROS levels were determined using the fluorescent H 2DCF‑DA probe. Non‑fluorescent H 2DCF‑DA is cell‑permeable, cleaved by non‑specific esterases and oxidized in the presence of ROS to form fluorescent 2'7'‑dichlorofluorescein (DCF). ROS production is proportional to the fluorescence ratio of the treatment to the control. The cells were incubated with 10 µM H2DCF‑DA for 20 min at 37˚C prior to being harvested and analyzed for fluorescence intensity using flow cytometry. Western blotting. Following the treatment of the cells, the nuclear and cytoplasmic proteins were prepared according to the method described by Liu et al (21) and the protein concentrations were measured using a Bicinchoninic Acid (BCA) Protein Assay kit (Pierce). Equal amounts of proteins were electrophoresed through denaturing polyacrylamide gels, transferred onto polyvinylidene difluoride (PVDF) membranes and probed with primary antibodies against NF‑κ B (p65), Bcl‑2, XIAP and caspase 3. Subsequent to being washed with TBST, the membranes were incubated with peroxidase‑conjugated secondary antibodies for 1 h. The blots were detected with an Enhanced Chemiluminescence Detection kit (Pierce), following the manufacturer's instructions. Isolation of mitochondria. The mitochondria were isolated from the cultured SKOV3PT cells using a Mitochondrial Isolation kit. The cells were suspended in ice‑cold Mito‑Cyto isolation buffer and immediately homogenized. The homogenates were centrifuged at 600 x g at 4˚C for 10 min. The supernatant was transferred to a new tube and centrifuged at 11,000 x g at 4˚C for 10 min. The pellet was lysed with Laemmli Buffer (Bio‑Rad Laboratories, Hercules, CA, USA) to extract the mitochondrial protein. The mitochondrial protein concentration was determined by the BCA Protein Assay kit (Pierce). Measurement of mitochondrial complexes I, II and III activi‑ ties. The activities of the MRC complexes were determined using MRC Complexes Activity Assay kits. Mitochondrial complex I (NADH‑ubiquinone oxidoreductase) activity was measured by monitoring the decrease in NADH absorbance at 340 nm. The activity of complex I was calculated using the rotenone‑sensitive rate and expressed as µmol/min/mg protein. Complex II (succinate‑ubiquinone oxidoreductase) activity was determined in extracted mitochondria proteins through the reduction of 2,6‑dichloropheno‑lindophenol (DCIP) at 600 nm. The activity of complex II was calculated using the 2‑thenoytrifluoroacetone‑sensitive rate and the results were presented as µmol/min/mg protein. Mitochondrial complex III
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Figure 1. Effects of TPL on cell viability and apoptosis in SKOV3PT cells. (A) SKOV3PT cells were exposed to varying concentrations of TPL (0‑100 nM) for 24‑48 h. Cell viability was assessed by MTT assay. (B) SKOV3PT cells were cultured for 24 h with varying doses of TPL (0‑100 nM). Apoptosis was identified by Annexin V/PI staining and analyzed by flow cytometry. (C) Western blot analysis of caspase 3 in SKOV3PT cells that were treated for 24 h with the indicated concentrations of TPL (12.5‑100 nM) or at various time points with 50 nM TPL treatment. β‑Actin served as a loading control. Results are presented as the mean ± SD for three independent experiments; *P
