3586 ONCOLOGY LETTERS 12: 3586-3590, 2016 Triptolide exerts pro‑apoptotic and cell cycle arrest activity on drug‑resistant human lung cancer A549/Taxol cells via modulation of MAPK and PI3K/Akt signaling pathways CHEN QIONG XIE1, PING ZHOU1, JIAN ZUO1, XIANG LI1,2, YONG CHEN1 and JIAN WEI CHEN1,3 1College of Pharmacy; 2Jiangsu Key Laboratory for Chinese Material Medical Processing; 3Jiangsu Key Laboratory for TCM Formulae Research, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210046, P.R. China Received June 29, 2015; Accepted August 9, 2016 DOI: 10.3892/ol.2016.5099 Abstract. Multidrug resistance (MDR) is a major obstacle in Introduction the effective chemotherapeutic treatment of cancers. Triptolide (TPL) is a diterpenoid isolated from Tripterygium wilfordii Lung cancer is the most commonly diagnosed cancer Hook. f., a traditional Chinese medicine. It was demonstrated in worldwide, contributing to 12.7% of the total incidence of our previous study that TPL exerts anti-MDR cancers on various cancers, and also the leading cause of mortality among all MDR cell lines (including A549/Taxol, MCF-7/ADR and tumors (18.2% of the total) (1). Multidrug resistance (MDR) Bel7402/5-Fu). The present study was designed to investigate its is a phenomenon whereby cancer cells exhibit simultaneous anti-proliferative activity on A549/Taxol cells, and explore the resistance to anti-cancer drugs, with various structures and underlying mechanism of action. The anti-proliferative activity mechanisms of action (2,3). MDR results in poor therapeutic of TPL on A549/Taxol cells was assessed by 3-(4,5-dimethyl- efficacy in the later stage of cancer treatments, and it is also the thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Its main obstacle in obtaining a satisfactory therapeutic outcome pro-apoptosis and cell cycle arrest activities were analyzed by in lung cancer (4). flow cytometry. Western blot assay was employed to investigate Extensive studies have revealed that MDR cancer cells the levels of mitogen-activated protein kinases (MAPKs) and are immune from anti-cancer drug-induced cell apoptosis apoptosis‑related proteins in cells. TPL efficiently suppressed through the upregulation of survival signaling pathways, the proliferation of A549/Taxol cells. Co-treatment with MAPK including phosphatidyl-3-phosphate kinase (PI3K) and inhibitors in the MTT assay indicated that the extracellular extracellular-regulated kinase-1 (ERK1) (5), or the suppres- signal-regulated kinase (ERK) and c-Jun N-terminal kinase sion of anti-proliferative signaling pathways, including p38 (JNK) pathways were involved in the process. Upregulation of mitogen-activated protein kinases (MAPKs) (6). Bcl-2 is an p-p38, p-ERK, p-GSK-3β, Bax and cleaved caspases-3 and -9, oncogene which contributes to tumor occurrence mainly due and downregulation of p-JNK, p-Akt and Bcl-2 were observed to the inhibition of apoptosis, and the overexpression of Bcl-2 upon treatment with TPL in the A549/Taxol cells. The results usually results in the resistance of cancer cells to anti-cancer from flow cytometry assay revealed that TPL induced apop- agents associated with abnormal changes of the pathways tosis and S-phase arrest in A549/Taxol cells. This occurred as discussed above (7,8). a result of the upregulation of p-ERK and p-GSK-3β, and the Preliminary observations by the present authors demon- downregulation of p-JNK and p-Akt, and was responsible for strated that the pro-apoptotic and cell cycle arrest activities the subsequent anti-proliferative activity. of triptolide (TPL) largely contribute to its antitumor effect, and were direct outcomes of the modulation of various important upstream pathways, including PI3K/Akt, MAPK, JAK/STAT and nuclear factor-κB, which subsequently modulated the expression of apoptosis-related proteins such as those of the Bcl-2 family (9-11). In our preliminary obser- vations, we demonstrated that TPL inhibited the proliferation Correspondence to: Professor Xiang Li or Dr Yong Chen, College of MDR A549/Taxol cells in vitro mainly through selective of Pharmacy, Nanjing University of Chinese Medicine, 138 Xian Lin Road, Nanjing, Jiangsu 210046, P.R. China modulation of MAPK signaling (9-11). However, until now, E-mail: [email protected] our understanding of the association between the inhibitory E-mail: [email protected] activity of TPL in MDR cells and the MAPK pathway has been limited. Hence, in the present study, we investigated Key words: multidrug resistance, triptolide, apoptosis, S-phase the modulatory effect of TPL on the MAPK pathway in arrest, mitogen-activated protein kinases, PI3K/Akt A549/Taxol cells along with its correlation with the inhibi- tory effect on the proliferation. XIE et al: MECHANISM OF ANTI-PROLIFERATIVE ACTIVITY OF TRIPTOLIDE IN A549/TAXOL CELLS 3587 Materials and methods (FACSCalibur, BD Biosciences, San Jose, CA, USA). For analysis of the cell cycle distribution, the supernatant was Drugs and reagents. TPL (≥98%, L‑004‑130304) was discarded, and attached cells were harvested and fixed in cold purchased from Chengdu Herb Purity Co., Ltd. (Chengdu, 70% ethanol overnight at -20˚C. Cell cycle distribution was China). It was dissolved in dimethyl sulfoxide (DMSO) at a analyzed using a flow cytometer according to the manufac- concentration of 10 mmol/l to obtain the stock solution and turer's instructions for the PI staining kit. kept below ‑20˚C, then diluted to various concentrations with phosphate‑buffered saline (PBS) prior to the assays. The final Protein extraction and western blot. A549/Taxol cells concentration of DMSO was less than 0.1%. 3-(4,5-dimeth- (3x105 cells/well) were treated with TPL at concentrations of ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 0.025 and 0.05 µM, or co-treated with MAPK inhibitors, for RPMI-1640, a bicinchoninic acid (BCA) protein assay kit, 24 h. Following the treatments, cells were collected, washed inhibitor of ERK U0126, propidium iodide (PI) staining kit twice with pre-chilled PBS, lysed, and then centrifuged at and Annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis 12,000 rpm for 10 min at 4˚C. The supernatant of the lysate detection kit were purchased from Nanjing KeyGen Biotech- was boiled, and total protein was measured using the BCA nology Co. Ltd. (Nanjing, China). Anti-p-p38, anti-p-JNK, protein assay kit. Proteins were separated by SDS-PAGE, anti-p-ERK antibodies, inhibitor of p38 SB202190 and inhib- and then transferred onto polyvinylidene fluoride (PVDF) itor of JNK SP600125 were purchased from Cell Signaling membranes (0.45 µm; Millipore). Non-specific protein binding Technology (Beverly, MA, USA). Anti-p-Akt (Ser473), sites were blocked with 5% bovine serum albumin diluted in anti-p-GSK-3β (Ser9), cleaved caspase (c-caspase)-3 (1:200), Tris-buffered saline buffer containing 0.1% Tween-20 (pH 7.4) c-caspase-9 (1:1,000), Bcl-2 (1:500) and COX IV (1:1,000) for 2 h at room temperature, followed by incubation with the were supplied by Bioworld Technology (Nanjing, China). An appropriate primary and secondary antibodies. The expression enhanced chemiluminescence kit was purchased from Thermo of proteins was detected using a Chemi Doc XRS+ system Fisher (Shanghai, China). Bovine calf serum was purchased (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The grey from Wisent corporation (Nanjing, China). All other chemi- level ratio of the target protein to COX IV was used to repre- cals and reagents used were of analytical grade. sent the relative protein expression level. Cell culture. The human lung cancer cell line A549 and the Statistical analysis. Results are expressed as the means ± stan- corresponding MDR cell line A549/Taxol were purchased dard deviation. Statistical differences among groups were from Nanjing KeyGen Biotechnology Co. Ltd., and cultured evaluated by the t-test using GraphPad Prism 6. P<0.05 and in RPMI-1640 medium supplemented with 10% bovine calf P<0.01 were considered to represent different levels of statis- serum, 100 U/ml penicillin and 100 µg/ml streptomycin at tical significance. 37˚C in a humidified atmosphere of 5% CO2. Paclitaxel (Taxol, 200 ng/ml) was added to the medium in order to maintain Results MDR in the A549/Taxol cells, and removed two weeks before the experiments. Cells were passaged every 2-3 days. Resistance fold of A549/Taxol cells. A549 and A549/Taxol cells were treated with Taxol for 48 h and the cytotoxicity was Cell viability. For the cell viability assay, A549 and assessed using MTT assay. The IC50 of Taxol against A549 A549/Taxol cells were seeded onto a 96-well plate at a density and A549/Taxol cells was 3.52±0.47 and 71.31±7.95 µM, of 8x103 cells per well. Following overnight incubation, the respectively. A549/Taxol cells exhibited more than a 20-fold culture medium was aspirated, and the cells were incubated resistance to Taxol in comparison with the drug-sensitive with various concentrations of TPL (the final concentrations A549 cell line. were 0.01, 0.02, 0.04, 0.06 and 0.08 µmol/l) or co-treated with MAPK inhibitors in complete culture medium for 48 h. The TPL inhibits proliferation of A549/Taxol cells. Various doses same volume of complete culture medium served as the nega- (0.01, 0.02, 0.04, 0.06, 0.08 µM) of TPL were used to treat tive control. Then 20 µl MTT solution (5 mg/ml) was added A549/Taxol cells for 48 h. The anti-proliferative effect of TPL to each well, and the plates were further incubated for 4 h. on A549/Taxol was assessed by MTT assay, and the results The medium was removed and 150 µl DMSO was added to revealed that TPL inhibited the proliferation of A549/Taxol solubilize the MTT formazan salt.