Treatment with β-elemene combined with paclitaxel inhibits growth, migration, and invasion and induces apoptosis of ovarian cancer cells by activation of STAT-NF-κB pathway

Fu Xiaomeng Lv Lei An Jinghong Jiang Juan Yue Qi Yuan Dandan About the authors

Abstract

In this study, we aimed to analyze the anti-cancer effects of β-elemene combined with paclitaxel for ovarian cancer. RT-qPCR, MTT assay, western blot, flow cytometry, and immunohistochemistry were used to analyze in vitro and in vivo anti-cancer effects of combined treatment of β-elemene and paclitaxel. The in vitro results showed that β-elemene+paclitaxel treatment markedly inhibited ovarian cancer cell growth, migration, and invasion compared to either paclitaxel or β-elemene treatment alone. Results demonstrated that β-elemene+paclitaxel induced apoptosis of SKOV3 cells, down-regulated anti-apoptotic Bcl-2 and Bcl-xl gene expression and up-regulated pro-apoptotic P53 and Apaf1 gene expression in SKOV3 cells. Administration of β-elemene+paclitaxel arrested SKOV3 cell cycle at S phase and down-regulated CDK1, cyclin-B1, and P27 gene expression and apoptotic-related resistant gene expression of MDR1, LRP, and TS in SKOV3 cells. In vivo experiments showed that treatment with β-elemene+paclitaxel significantly inhibited ovarian tumor growth and prolonged the overall survival of SKOV3-bearing mice. In addition, the treatment inhibited phosphorylated STAT3 and NF-κB expression in vitro and in vivo. Furthermore, it inhibited migration and invasion through down-regulation of the STAT-NF-κB signaling pathway in SKOV3 cells. In conclusion, the data suggested that β-elemene+paclitaxel can inhibit ovarian cancer growth via down-regulation of the STAT3-NF-κB signaling pathway, which may be a potential therapeutic strategy for ovarian cancer therapy.

Ovarian cancer; β-elemene; Paclitaxel; Apoptosis; STAT3; NF-κB


Introduction

Ovarian cancer is one of the most common gynecological tumors, which can be highly mutilating, invade the adjacent tissue, and its recurrence rate is relatively high (11. Kebapci E, Gulseren V, Tugmen C, Gokçu M, Solmaz U, Sert I, et al. Outcomes of patients with advanced stage ovarian cancer with intestinal metastasis. Ginekologia Pol 2017; 88: 537–542, doi: 10.5603/GP.a2017.0098.
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). The incidence of ovarian cancer in China has drastically increased since 2010, while the mortality rate has not been improved over the past 8 years (44. Wang B, Liu SZ, Zheng RS, Zhang F, Chen WQ, Sun XB. Time trends of ovarian cancer incidence in China. Asian Pac J Cancer Prev 2014; 15: 191–193, doi: 10.7314/APJCP.2014.15.1.191.
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). Due to a paucity of effective screening modalities, most patients are diagnosed at advanced stages (55. Merritt MA, Rice MS, Barnard ME, Hankinson SE, Matulonis UA, Poole EM, et al. Pre-diagnosis and post-diagnosis use of common analgesics and ovarian cancer prognosis (NHS/NHSII): a cohort study. Lancet Oncol 2018; 19: 1107–1116, doi: 10.1016/S1470-2045(18)30373-5.
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). Clinically, the most widely adopted standard treatment regimen for advanced ovarian cancer includes surgery, target therapy, and chemotherapy (88. Parikh R, Kurosky SK, Udall M, Chang J, Cappelleri JC, Doherty JP, et al. Treatment patterns and health outcomes in platinum-refractory or platinum-resistant ovarian cancer: a retrospective medical record review. Int J Gynecol Cancer 2018; 28: 738–748, doi: 10.1097/IGC.0000000000001222.
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1010. Moore KN, Martin LP, O'Malley DM, Matulonis UA, Konner JA, Vergote I, et al. A review of mirvetuximab soravtansine in the treatment of platinum-resistant ovarian cancer. Future Oncol 2018; 14: 123–136, doi: 10.2217/fon-2017-0379.
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). Tumor metastasis and resistance to apoptosis contributes to a poor survival rate in clinical ovarian cancer patients. However, the anti-apoptosis mechanisms of ovarian cancer remain incompletely understood.

Paclitaxel is isolated from Taxus brevifolia and it is regarded as an anti-tumor agent (1111. Knox SN, Robinson JB, Im DD, Logan L, Rosenshein NB. The use of paclitaxel and cisplatin in a patient with epithelial ovarian cancer and human immunodeficiency virus. Gynecol Oncol 2000; 76: 118–122, doi: 10.1006/gyno.1999.5590.
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). Currently, paclitaxel is effective for various human diseases, such as neurodegenerative diseases, cancer, and chronic kidney disease (1212. Niwa H, Nakahara Y, Yokoba M, Mitsufuji H, Sasaki J, Masuda N. Safety and efficacy of carboplatin plus nab-paclitaxel for treating advanced non-small-cell lung cancer with interstitial lung disease. Mol Clin Oncol 2017; 7: 604–608, doi: 10.3892/mco.2017.1359.
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13. Kitasato L, Shimohama T, Ikeda Y, Namba S, Hashikata T, Kameda R, et al. Clinical outcomes of chronic kidney disease patients treated with everolimus-eluting stents (EES) and paclitaxel-eluting stents (PES). Biomed Pharmacother 2015; 72: 6–10, doi: 10.1016/j.biopha.2015.03.002.
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1414. Meng Z, Lv Q, Lu J, Yao H, Lv X, Jiang F, et al. Prodrug Strategies for Paclitaxel. Int J Mol Sci 2016; 17. pii: E796, doi: 10.3390/ijms17050796.
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). Paclitaxel is one of the most effective drugs to treat cancer, and many reports have investigated the anti-cancer efficacy in ovarian cancer (1515. Ye H, Liu XJ, Hui Y, Liang YH, Li CH, Wan Q. USF1 gene polymorphisms may associate with the efficacy and safety of chemotherapy based on paclitaxel and prognosis in the treatment of ovarian cancer. Neoplasma 2018; 65: 153–160, doi: 10.4149/neo_2018_170322N205.
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16. Richardson DL, Sill MW, Coleman RL, Sood AK, Pearl ML, Kehoe SM, et al. Paclitaxel with and without pazopanib for persistent or recurrent ovarian cancer: a randomized clinical trial. JAMA Oncol 2018; 4: 196–202, doi: 10.1001/jamaoncol.2017.4218.
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1717. Pashaei-Asl F, Pashaei-Asl R, Khodadadi K, Akbarzadeh A, Ebrahimie E, Pashaiasl M. Enhancement of anticancer activity by silibinin and paclitaxel combination on the ovarian cancer. Artif Cells Nanomed Biotechnol 2018; 46: 1483–1487, doi: 10.1080/21691401.2017.1374281.
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). However, single paclitaxel has limited anti-cancer capacity and it needs a relatively higher dose to be effective compared to other anticancer drugs. In addition, pre-treatment microtubule stability correlates with paclitaxel response in ovarian cancer cell lines, and, while most ovarian cancer patients receive paclitaxel chemotherapy, less than half respond (1818. Boyd LR, Muggia FM. Carboplatin/paclitaxel induction in ovarian cancer: the finer points. Oncology (Williston Park) 2018; 32: 418–420, 422–424.). Furthermore, paclitaxel plus nedaplatin and paclitaxel plus carboplatin are more efficient in the treatment of women with epithelial ovarian cancer (1919. Li L, Zhuang Q, Cao Z, Yin R, Zhu Y, Zhu L, et al. Paclitaxel plus nedaplatin vs. paclitaxel plus carboplatin in women with epithelial ovarian cancer: A multi-center, randomized, open-label, phase III trial. Oncol Lett 2018; 15: 3646–3652, doi: 10.3892/ol.2018.7761.
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). Therefore, it is crucial to uncover the possible mechanism of ovarian cancer cell apoptosis induced by paclitaxel.

Elemene is extracted from a traditional herbal medicine and is widely used in the treatment of human cancer (2020. Edris AE. Anti-cancer properties of Nigella spp. essential oils and their major constituents, thymoquinone and beta-elemene. Curr Clin Pharmacol 2009; 4: 43–6, doi: 10.2174/157488409787236137.
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2222. Tao L, Zhou L, Zheng L, Yao M. Elemene displays anti-cancer ability on laryngeal cancer cells in vitro and in vivo. Cancer Chemother Pharmacol 2006; 58: 24–34, doi: 10.1007/s00280-005-0137-x.
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). A study indicates that β-elemene sensitizes chemoresistant ovarian carcinoma cells to cisplatin-induced apoptosis and that the augmented effect of β-elemene on cisplatin cytotoxicity and sensitivity in resistant ovarian tumor cells is mediated through a mitochondria- and caspase-dependent cell death pathway (2323. Li QQ, Lee RX, Liang H, Zhong Y, Reed E. Enhancement of cisplatin-induced apoptosis by beta-elemene in resistant human ovarian cancer cells. Med Oncol 2013; 30: 424, doi: 10.1007/s12032-012-0424-4.
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). In addition, β-elemene and taxanes synergistically induce cytotoxicity and inhibit proliferation in ovarian cancer and other tumor cells (2424. Zou B, Li QQ, Zhao J, Li JM, Cuff CF, Reed E. β-elemene and taxanes synergistically induce cytotoxicity and inhibit proliferation in ovarian cancer and other tumor cells. Anticancer Res 2013; 33: 929–940.). Furthermore, β-elemene effectively suppresses the growth and survival of platinum-sensitive and -resistant ovarian tumor cells (2525. Lee RX, Li QQ, Reed E. β-elemene effectively suppresses the growth and survival of both platinum-sensitive and -resistant ovarian tumor cells. Anticancer Res 2012; 32: 3103–3113.). Thus, understanding the mechanism of drug resistance of ovarian cancer to β-elemene plays a crucial role in the successful treatment of ovarian cancer; combined therapeutic strategies should also be explored.

Studies have found that STAT3 is a potential target for overcoming the cisplatin resistance in ovarian cancer (2626. Jin P, Liu Y, Wang R. STAT3 regulated miR-216a promotes ovarian cancer proliferation and cisplatin resistance. Biosci Rep 2018; 38. pii: BSR20180547, doi: 10.1042/BSR20180547.
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). NF-κB signaling pathway may be a promising therapeutic target for treating malignant ovarian cancers (2727. Lin Z, Li D, Cheng W, Wu J, Wang K, Hu Y. MicroRNA-181 functions as an antioncogene and mediates NF-kappaB pathway by targeting RTKN2 in ovarian cancers. Reprod Sci 2019; 26: 1071–1081, doi: 10.1177/1933719118805865.
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). Additionally, the STAT/NF-κB signal pathway is involved in human tumors and fulfills essential pathological features by allowing to translocate from the cytoplasm into the nucleus for the activation of oncoprotein promoter DNAs (2828. Sinkovics JG. The cnidarian origin of the proto-oncogenes NF-kappaB/STAT and WNT-like oncogenic pathway drives the ctenophores (Review). Int J Oncol 2015; 47: 1211–1129, doi: 10.3892/ijo.2015.3102.
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). In this study, we investigated the combined anti-cancer effect of β-elemene and paclitaxel on ovarian cancer and explored the association between β-elemene+paclitaxel and STAT3-NF-κB signal pathway in SKOV3 cells. This study also analyzed the in vitro and in vivo effect of β-elemene+paclitaxel on ovarian tumor growth.

Material and methods

Cell culture

SKOV3 cell line was purchased from ATCC (American Type Culture Collection, USA). SKOV3 cells were cultured in RPMI 1640 medium (Invitrogen, Life Technologies, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen, Life Technologies), 100 U/mL penicillin (Invitrogen, USA), 100 µg/mL streptomycin (Invitrogen), sodium pyruvate (Invitrogen), and L-glutamine (Invitrogen) at 37°C with 5% CO2. SKOV3 cells were exposed to platinum treatment and then incubated with β-elemene and/or paclitaxel.

MTT assay

SKOV3 cells (1×103/well) were incubated with or without β-elemene (50, 75, 100, 125 mg/mL, Holistic Integrative Pharmacy Institutes of Hangzhou Normal University, China), paclitaxel (10, 15, 20, 25 mg/mL, Nanjing Jingzhu Bio-technology Co., Ltd., China), or combined treatment (β-elemene, 100 mg/mL, paclitaxel, 20 mg/mL) in 96-well plates in triplicate at 37°C with 5% CO2. After culture for different times (24, 48, and 72 h), cell growth was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, Sigma-Aldrich, Germany) assay according to the manufacturer's instructions. Absorbance was measured at 490 nm (A490) with a spectrophotometer (BioRad, USA). Experiments were repeated at least three times.

Flow cytometry assay

Apoptosis of SKOV3 cells was assessed using a flow cytometer (FACSCalibur BD Biosciences, USA). In brief, the treated SKOV3 cells (1×105) were collected, washed with PBS, and stained with fluorochrome-conjugated annexin V (5 μL) and propidium iodide (5 μL) for 2 h at 4°C in darkness using an Annexin V Apoptosis Detection Kit (eBioscience, USA) according to the manufacturer's instructions. All the independent experiments by FACS were repeated at least 3 times. The apoptosis rate was calculated using the software Developer XD 1.2 (Definiens AG, Germany). Three replicates were performed for the flow cytometry assay.

Cell cycle assay

Cells were treated with β-elemene (100 mg/mL), paclitaxel (20 mg/mL), or combined treatment for 72 h at 37°C and fixed in 75% ethanol at 4°C overnight. Cells were washed with PBS three times and incubated with PI solution (Promega, Germany) according to the manufacturer's instructions. The cell cycle was analyzed using a FACSCalibur flow cytometer (BD Biosciences) and quantified using FlowJo 7.0 software. All experiments were performed in triplicate.

Real-time quantitative PCR (RT-qPCR) assay

Total RNA was extracted from SKOV3 cells using a TRIzol™ Plus RNA Purification Kit (Invitrogen, Life Technologies) according to the manufacturer's instructions. A total of two micrograms of total RNA were reverse-transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, USA). The cDNA (1 μg) was subjected to qRT-PCR using FastStart Universal SYBR Green Master (4913850001, Roche, USA) and a LightCycler 480 Real-Time PCR system (Roche, China). All primers were synthesized by Invitrogen (Table 1). PCR thermocycling conditions were 45 amplification cycles, denaturation at 95°C, primer annealing at 64°C with touchdown to 58°C, and applicant extension at 72°C. The relative mRNA expression was assessed using the 2−ΔΔCt method (2929. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25: 402–408, doi: 10.1006/meth.2001.1262.
https://doi.org/10.1006/meth.2001.1262...
). All experiments were performed in triplicate.

Table 1
Sequences of primers used in this study.

Cell invasion and migration assays

SKOV3 cells were grown at 37°C with 5% CO2 until 90% confluence. SKOV3 cells were then incubated with β-elemene (100 mg/mL), paclitaxel (20 mg/mL), or combined treatment for 24 h at 37°C. Matrigel-uncoated and -coated 8-µm pores (Corning Incorporated, USA) were used to evaluate cell migration and invasion, respectively. For invasion assay, SKOV3 cells were suspended at a density of 1×105 in 500 μL in serum-free RPMI1640. Cells were subjected to the upper chambers of BD BioCoat Matrigel Invasion Chambers (BD Biosciences) according to the manufacturer's instructions. For migration assay, cells were subjected to a control insert (BD Biosciences) instead of a Matrigel Invasion Chamber. The migration and invasion of the tumor cells were stained with crystal violet (Sigma-Aldrich) for 5 min at room temperature and counted using a microscope (Olympus, Japan) by randomly selecting at least three fields of view per membrane.

STAT3 transfection

Expression plasmid pcDNA-STAT3 was constructed by Invitrogen. The SKOV3 cells (5×105 per well) were cultured in a 12-well plate, washed with PBS, and then transfected with pcDNA-STAT3 or pcDNA-vector using Lipofectamine 2000 according to the manufacturer's protocol (Invitrogen). Cells were used for analysis of protein expression after 72-h transfection. The STAT3-overexpressed SKOV3 cells were incubated with β-elemene (100 mg/mL) and/or paclitaxel (20 mg/mL) for 12 h for further analysis.

Western blot analysis

The treated SKOV3 cells were collected and lysed using radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich). A BCA protein assay kit (Thermo Scientific, USA) was used to measure protein concentration. Cell extracts were boiled in loading buffer, and equal amounts of cell extracts (40 μg) were electrophoresed on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred into polyvinylidene fluoride membranes (Corning Incorporated), and incubated in blocking buffer (5% BSA) overnight at 4°C. The primary antibodies against MTA3 (ab176346, Abcam, USA), VEGF (ab11938, Abcam), STAT3 (ab68153, Abcam), pSTAT3 (ab76315, Abcam), NF-κB (ab220803, Abcam), pNF-κB (ab194908, Abcam), and β-actin (ab8226, Abcam) were diluted at a ratio of 1:2,000 according to manufacturer instructions and incubated overnight at 4°C. Subsequently, protein was incubated with horseradish peroxidase (HRP)-linked anti-rabbit IgG secondary antibody (1:2,000; ab6721, Abcam) for 2 h at 37°C followed by labeling with ECL Detection System (GE Healthcare, USA). The band density was determined by NIH-ImageJ software 1.2 USA).

Animal study

A total of 60 male BALB/c-nu/nu nude mice (6-8 weeks; body weight, 18–22 g) were purchased from Shanghai Laboratory Animal Co., Ltd. (SLAC, China). All animals were housed and fed in accordance with the guidelines established by the National Science Council of China. SKOV3 cells (1×106 cells) in 100 μL PBS were injected subcutaneously into the right axillary fossa and randomly divided into four groups: β-elemene (1.0 mg/kg), paclitaxel (1.0 mg/kg), combined treatment of β-elemene (1.0 mg/kg) and paclitaxel (1.0 mg/kg), and the same dose of PBS. Treatments were done intravenously once every day for a total of 24 days. The tumor diameters in each mouse were assessed every 3 days. The tumor volume was calculated using the formula: V (mm3) = L (mm) × W2 (mm) / 2, where L is the long diameter and W is the short diameter. All animals were sacrificed and the tumors were weighed 25 days after inoculation.

Immunohistochemical staining

Tumor tissues were isolated from experimental mice on day 25. Paraffin-embedded tumor tissues were cut into 4-μm sections. Tissue sections were deparaffinized in xylene, rehydrated in graded alcohol to TBS (Sigma-Aldrich), and incubated with 3% hydrogen peroxide for 10 min at 25°C. Tissues sections (4-μm thick) were deparaffinized in xylene, rehydrated through graded ethanol solutions, followed by blocking of endogenous peroxidase activity in 3% hydrogen peroxide for 10 min at room temperature. The sections were blocked with 5% BSA (Sigma-Aldrich) and then incubated with rabbit anti-mouse STAT3 (ab68153, Abcam), pSTAT3 (ab76315, Abcam), NF-κB (ab220803, Abcam), and pNF-κB (ab194908, Abcam) at a 1:2,000 dilution overnight at 4°C. All sections were washed with PBS and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:2,000, ab6785, Abcam) at a 1:10,000 dilution for 2 h at 37°C. Target protein expression was visualized using a Betazoid 3,3′-diaminobenzidine chromogen kit (Biocare Medical, USA) according to manufacturer's protocol.

TUNEL assay

Apoptosis in tumor cells was measured using the DeadEnd Colorimetric terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) System (Promega) according to the manufacturer's instructions. In brief, tumor sections (4-μm thick) were incubated using TUNEL (DeadEnd™ Colorimetric TUNEL System, Promega) for 2 h at 37°C. Finally, images were captured with a Zeiss LSM 510 confocal microscope (Germany) at 488 nm. The number and percentage of TUNEL-positive cells were determined by counting 1×103 cells from six random selected fields.

Statistical analysis

Data are reported as means±SD. Student's t-test was performed for two groups and one-way ANOVA followed by Tukey's multiple comparison post hoc test was performed for multiple groups using SPSS version 17.0 software (IBM Corporation, USA). A value of P<0.05 was considered statistically significant.

Results

Dose decision for β-elemene or paclitaxel on SKOV3 cell growth

To determine the anti-cancer effects of β-elemene or paclitaxel on SKOV3 cells growth, a dose-dependent assay was performed in vitro. As shown in Figure 1A, β-elemene at 100 mg/mL concentration demonstrated optimal inhibition effect on SKOV3 cells growth. Results showed that 20 mg/mL of paclitaxel presented the maximum inhibitory effect on SKOV3 cells growth (Figure 1B).

Figure 1
Dose-dependent assay of β-elemene (A) and paclitaxel (B) for SKOV3 cells growth. Data are reported as means±SD. *P<0.05, **P<0.01 (ANOVA followed by Tukey's multiple comparison post hoc test). ns: not significant.

β-elemene+paclitaxel suppressed growth and cell cycle of SKOV3 cells

As illustrated in Figure 2A, β-elemene+paclitaxel inhibited growth of SKOV3 cells compared to either β-elemene or paclitaxel. Data demonstrated that β-elemene+paclitaxel inhibited SKOV3 cell growth in a time-dependent manner (Figure 2B). β-elemene and paclitaxel arrested SKOV3 cells cycle at G1 and S phases, respectively (Figure 2C–E). As depicted in Figure 2F, administration of β-elemene+paclitaxel down-regulated CDK1, cyclin-B1, and P27 mRNA expression levels in SKOV3 cells compared to either β-elemene or paclitaxel.

Figure 2
Effect of β-elemene (100 mg/mL), paclitaxel (20 mg/mL), or their combination on cell growth of SKOV3 cells (A and B), and on cell cycle (C to E). F, Effect of β-elemene and/or paclitaxel on genes CDK1, cyclin-B1, and P27 expression in SKOV3 cells. Data are reported as means±SD. *P<0.05, **P<0.01 (ANOVA followed by Tukey's multiple comparison post hoc test). ns: not significant.

β-elemene+paclitaxel induced apoptosis of SKOV3 cells

The results in Figure 3A showed that β-elemene+paclitaxel resulted in more apoptotic SKOV3 cells than β-elemene or paclitaxel. Treatment with β-elemene+paclitaxel down-regulated anti-apoptotic mRNA expression of Bcl-2 and Bcl-xl compared to either β-elemene or paclitaxel (Figure 3B). However, β-elemene+paclitaxel up-regulated pro-apoptosis mRNA expression of P53 and Apaf1 in SKOV3 cells (Figure 3C). As illustrated in Figure 3D, β-elemene and β-elemene+paclitaxel decreased drug-resistant gene expression of MDR1, LRP, and TS in SKOV3 cells.

Figure 3
Effect of β-elemene (100 mg/mL), paclitaxel (20 mg/mL), or their combination on apoptosis of SKOV3 cells (A), and the relative mRNA expression of Bcl-2 and Bcl-xl (B), P53 and Apaf1 (C), and MDR1, LRP, and TS (D). Data are reported as means±SD. **P<0.01 (ANOVA followed by Tukey's multiple comparison post hoc test). ns: not significant.

β-elemene+paclitaxel inhibited migration and invasion of SKOV3 cells

As illustrated in Figure 4A and B, treatment with β-elemene+paclitaxel markedly suppressed the migration and invasion of SKOV3 cells compared to either β-elemene or paclitaxel. Treatment with β-elemene+paclitaxel down-regulated E-cadherin and MTA3 mRNA and protein expression in SKOV3 cells (Figure 4C and D).

Figure 4
Effect of β-elemene+paclitaxel on migration (A) and invasion (B) of SKOV3 cells (magnification bar, 100 μm). C and D, Relative E-cadherin and MTA3 mRNA (C) and protein (D) expression in SKOV3 cells after treatment with β-elemene and/or paclitaxel. Data are reported as means±SD. **P<0.01 (ANOVA followed by Tukey's multiple comparison post hoc test). ns: not significant.

β-elemene+paclitaxel down-regulated STAT3-NF-κB signal pathway in SKOV3 cells

Treatment with β-elemene+paclitaxel down-regulated STAT3 and NF-κB gene and protein expression levels in SKOV3 cells compared to β-elemene or paclitaxel (Figure 5A and B). STAT3 overexpression (STAT3OP) increased STAT3 and NF-κB expression and phosphorylation in SKOV3 cells (Figure 5C). However, results showed that STAT3 overexpression (STAT3OP) blocked β-elemene-inhibited growth (Figure 5D), migration (Figure 6A), and invasion (Figure 6B) of SKOV3 cells. As shown in Figure 7, STAT3 overexpression also blocked β-elemene+paclitaxel- and β-elemene-induced apoptosis of SKOV3 cells.

Figure 5
Effect of β-elemene+paclitaxel on relative STAT3 and NF-κB mRNA (A) and protein (B) expression levels in SKOV3 cells. C, Effects of STAT3 overexpression (STAT3OP) and NF-κB expression and phosphorylation in SKOV3 cells. D, Effects of STAT3 overexpression (STAT3OP) on β-elemene+paclitaxel-regulated growth. Data are reported as means±SD. *P<0.05, **P<0.01 (ANOVA followed by Tukey's multiple comparison post hoc test). ns: not significant.
Figure 6
Effects of STAT3 overexpression (STAT3OP) on β-elemene+paclitaxel-regulated migration (A) and invasion (B) of SKOV3 cells. Data are reported as means±SD. **P<0.01 (ANOVA followed by Tukey's multiple comparison post hoc test). ns: not significant.
Figure 7
Effects of STAT3 overexpression (STAT3OP) on β-elemene+paclitaxel-regulated apoptosis of SKOV3 cells. Data are reported as means±SD. **P<0.01 (ANOVA followed by Tukey's multiple comparison post hoc test). ns: not significant.

In vivo anti-cancer effect of β-elemene+paclitaxel in SKOV3-bearing mice

β-elemene+paclitaxel treatment presented smaller tumors compared to either β-elemene or paclitaxel (Figure 8A). Treatment with β-elemene+paclitaxel markedly increased the number of apoptotic cells in tumor tissue (Figure 8B). Treatment with β-elemene+paclitaxel up-regulated the expression levels of STAT3 and NF-κB in tumor tissue compared to β-elemene, paclitaxel, and the control group (Figure 8C). Compared with β-elemene and paclitaxel, β-elemene+paclitaxel prolonged survival of SKOV3-bearing mice in 120-day long-term observation (Figure 8D).

Figure 8
Anti-cancer effect of β-elemene+paclitaxel in SKOV3-bearing mice. A, Tumor growth in SKOV3-bearing mice after receiving β-elemene and/or paclitaxel. B, Apoptotic tumor cells in tumor tissue after treatment with β-elemene and/or paclitaxel determined by TUNEL assay (magnification bar: 50x). C, Protein expression of STAT3 and NF-κB in tumor tissue after treatment with β-elemene and/or paclitaxel (magnification bar: 50x). D, Survival time of tumor-bearing mice after treatment with β-elemene and/or paclitaxel. Data are reported as means±SD. **P<0.01 (ANOVA followed by Tukey's multiple comparison post hoc test).

Discussion

Previous reports have found that STAT3 and NF-κB signal pathways involved in growth and invasion of ovarian cancer cells (3030. Ning Y, Cui Y, Li X, Cao X, Chen A, Xu C, et al. Co-culture of ovarian cancer stem-like cells with macrophages induced SKOV3 cells stemness via IL-8/STAT3 signaling. Biomed Pharmacother 2018; 103: 262–271, doi: 10.1016/j.biopha.2018.04.022.
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31. Dorayappan KDP, Wanner R, Wallbillich JJ, Saini U, Zingarelli R, Suarez AA, et al. Hypoxia-induced exosomes contribute to a more aggressive and chemoresistant ovarian cancer phenotype: a novel mechanism linking STAT3/Rab proteins. Oncogene 2018; 37: 3806–3821, doi: 10.1038/s41388-018-0189-0.
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3333. Li X, Bao C, Ma Z, Xu B, Ying X, Liu X, et al. Perfluorooctanoic acid stimulates ovarian cancer cell migration, invasion via ERK/NF-kappaB/MMP-2/-9 pathway. Toxicol Lett 2018; 294: 44–50, doi: 10.1016/j.toxlet.2018.05.009.
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). Evidence has shown that paclitaxel can be regarded as an efficient therapy for human ovarian cancer and the therapy of paclitaxel combined with other drugs provides a potential approach to overcome the resistance of ovarian cancer (3434. Coleman RL, Brady MF, Herzog TJ, Sabbatini P, Armstrong DK, Walker JL, et al. Bevacizumab and paclitaxel-carboplatin chemotherapy and secondary cytoreduction in recurrent, platinum-sensitive ovarian cancer (NRG Oncology/Gynecologic Oncology Group study GOG-0213): a multicentre, open-label, randomised, phase 3 trial. Lancet Oncol 2017; 18: 779–791, doi: 10.1016/S1470-2045(17)30279-6.
https://doi.org/10.1016/S1470-2045(17)30...
35. Akiyama M, Sowa Y, Taniguchi T, Watanabe M, Yogosawa S, Kitawaki J, et al. Three combined treatments, a novel HDAC inhibitor OBP-801/YM753, 5-fluorouracil, and paclitaxel, induce G(2) phase arrest through the p38 pathway in human ovarian cancer cells. Oncol Res 2017; 25: 1245–1252, doi: 10.3727/096504017X14850164661097.
https://doi.org/10.3727/096504017X148501...
3636. Shen Y, Zhang XY, Chen X, Fan LL, Ren ML, Wu YP, et al. Synthetic paclitaxel-octreotide conjugate reverses the resistance of paclitaxel in A2780/Taxol ovarian cancer cell line. Oncol Rep 2017; 37: 219–226, doi: 10.3892/or.2016.5260.
https://doi.org/10.3892/or.2016.5260...
). Pashaei-Asl et al. (1717. Pashaei-Asl F, Pashaei-Asl R, Khodadadi K, Akbarzadeh A, Ebrahimie E, Pashaiasl M. Enhancement of anticancer activity by silibinin and paclitaxel combination on the ovarian cancer. Artif Cells Nanomed Biotechnol 2018; 46: 1483–1487, doi: 10.1080/21691401.2017.1374281.
https://doi.org/10.1080/21691401.2017.13...
) reported that combination of the chemotherapy drugs silibinin and paclitaxel can be more efficient in treatment of ovarian cancer cells. Combination therapy of bevacizumab and paclitaxel has been regarded as first-line adjuvant therapy for advanced stage ovarian cancer (3737. Fleming ND, Coleman RL, Tung C, Westin SN, Hu W, Sun Y, et al. Phase II trial of bevacizumab with dose-dense paclitaxel as first-line treatment in patients with advanced ovarian cancer. Gynecol Oncol 2017; 147: 41–46, doi: 10.3892/ijo.2015.3102.
https://doi.org/10.3892/ijo.2015.3102...
). Here, we reported the synergetic anti-cancer effects of β-elemene and paclitaxel on ovarian cancer cells and ovarian cancer cell-bearing mice. The results of the present study suggest that β-elemene+paclitaxel suppressed growth, migration, and invasion of ovarian cancer cells, induced apoptosis, and regulated apoptosis-related mRNA expression in SKOV3 cells. Importantly, we observed that β-elemene enhanced the anti-ovarian cancer effects of paclitaxel through regulation of the STAT3-NF-κB signaling pathway. In vivo experiment showed that β-elemene+paclitaxel treatment suppressed tumor growth and promoted apoptosis in tumor tissue.

Previously, β-elemene in combination with cisplatin increased apoptosis of prostate cancer through regulation of apoptosis-related gene in tumor cells (3838. Li QQ, Wang G, Reed E, Huang L, Cuff CF. Evaluation of cisplatin in combination with beta-elemene as a regimen for prostate cancer chemotherapy. Basic Clin Pharmacol Toxicol 2010; 107: 868–876, doi: 10.1111/j.1742-7843.2010.00580.x.
https://doi.org/10.1111/j.1742-7843.2010...
). A clinical trial indicated that elemene for the treatment of lung cancer is efficient and acceptable (2121. Rui D, Xiaoyan C, Taixiang W, Guanjian L. Elemene for the treatment of lung cancer. Cochrane Database Syst Rev 2007: CD006054.). Zhou et al. (3939. Zhou J, He LL, Ding XF, Yuan QQ, Zhang JX, Liu SC, et al. Combinatorial antitumor effect of rapamycin and beta-elemene in follicular thyroid cancer cells. Biomed Res Int 2016; 2016: 6723807, doi: 10.1155/2016/6723807.
https://doi.org/10.1155/2016/6723807...
) demonstrated that the novel combination of mTOR inhibitor with β-elemene synergistically attenuates tumor cell growth in follicular thyroid cancer. Elemene also inhibits the migration and invasion of breast cancer cells and it may be a promising agent targeting heparanase in the treatment of breast cancer (4040. Zhang Y, Sun X, Nan N, Cao KX, Ma C, Yang GW, et al. Elemene inhibits the migration and invasion of 4T1 murine breast cancer cells via heparanase. Mol Med Rep 2017; 16: 794–800, doi: 10.3892/mmr.2017.6638.
https://doi.org/10.3892/mmr.2017.6638...
). Results of the current study found that combined β-elemene and paclitaxel inhibited growth, migration, and invasion, which had inhibitory effects by down-regulation of E-cadherin and MTA3 expression in ovarian cancer cells. Notably, β-elemene+paclitaxel presented pro-apoptotic efficacy in the treatment of ovarian cancer both in vitro and in vivo.

The combined treatment for human cancer is more efficient in inhibiting growth and invasion of cancer patients than single treatment. A study indicated that combination therapy with β-elemene and taxanes has synergistic antitumor activity against ovarian and prostate carcinomas, which may be a promising new therapeutic combination that warrants further pre-clinical exploration for the treatment of chemoresistant ovarian cancer and other types of tumors (2424. Zou B, Li QQ, Zhao J, Li JM, Cuff CF, Reed E. β-elemene and taxanes synergistically induce cytotoxicity and inhibit proliferation in ovarian cancer and other tumor cells. Anticancer Res 2013; 33: 929–940.). In addition, combination of chemotherapy drugs of silibinin and paclitaxel were more efficient than a single drug in treatment of ovarian cancer cells (1717. Pashaei-Asl F, Pashaei-Asl R, Khodadadi K, Akbarzadeh A, Ebrahimie E, Pashaiasl M. Enhancement of anticancer activity by silibinin and paclitaxel combination on the ovarian cancer. Artif Cells Nanomed Biotechnol 2018; 46: 1483–1487, doi: 10.1080/21691401.2017.1374281.
https://doi.org/10.1080/21691401.2017.13...
). In addition, inhibiting NF-κB signaling pathway enhances cordycepin-induced apoptosis of ovarian tumor (3232. Cui ZY, Park SJ, Jo E, Hwang IH, Lee KB, Kim SW, et al. Cordycepin induces apoptosis of human ovarian cancer cells by inhibiting CCL5-mediated Akt/NF-kappaB signaling pathway. Cell Death Discov 2018; 4: 62, doi: 10.1038/s41420-018-0063-4.
https://doi.org/10.1038/s41420-018-0063-...
). Our data demonstrated that STAT3 overexpression inhibited paclitaxel-induced apoptosis promoted by β-elemene in SKOV3 cells. Drug combination of β-elemene and paclitaxel could be promising for future pre-clinical exploration of its potential usefulness as a treatment for ovarian cancer and possibly other tumor types. However, P-values alone from an effect-based strategy provided very limited evidence of synergism, and calculating the combination index (CI) would be useful for analyzing the inhibitory effect of β-elemene+paclitaxel on SKOV3 cells.

In conclusion, this study suggested that therapeutically effective combination of β-elemene+paclitaxel was a potential strategy for clinical ovarian cancer treatment and needs further preclinical study preceding human trials.

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Publication Dates

  • Publication in this collection
    08 May 2020
  • Date of issue
    2020

History

  • Received
    30 Sept 2019
  • Accepted
    9 Jan 2020
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