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.

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.


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 (1)(2)(3). 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 (4). Due to a paucity of effective screening modalities, most patients are diagnosed at advanced stages (5)(6)(7). Clinically, the most widely adopted standard treatment regimen for advanced ovarian cancer includes surgery, target therapy, and chemotherapy (8)(9)(10). Tumor metastasis and resistance to apoptosis contributes to a poor survival rate in clinical ovarian cancer patients. However, the antiapoptosis mechanisms of ovarian cancer remain incompletely understood.
Paclitaxel is isolated from Taxus brevifolia and it is regarded as an anti-tumor agent (11). Currently, paclitaxel is effective for various human diseases, such as neurodegenerative diseases, cancer, and chronic kidney disease (12)(13)(14). Paclitaxel is one of the most effective drugs to treat cancer, and many reports have investigated the anti-cancer efficacy in ovarian cancer (15)(16)(17). 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 (18). Furthermore, paclitaxel plus nedaplatin and paclitaxel plus carboplatin are more efficient in the treatment of women with epithelial ovarian cancer (19). 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 (20)(21)(22). A study indicates that b-elemene sensitizes chemoresistant ovarian carcinoma cells to cisplatin-induced apoptosis and that the augmented effect of b-elemene on cisplatin cytotoxicity and sensitivity in resistant ovarian tumor cells is mediated through a mitochondria-and caspase-dependent cell death pathway (23). In addition, b-elemene and taxanes synergistically induce cytotoxicity and inhibit proliferation in ovarian cancer and other tumor cells (24). Furthermore, b-elemene effectively suppresses the growth and survival of platinum-sensitive and -resistant ovarian tumor cells (25). Thus, understanding the mechanism of drug resistance of ovarian cancer to b-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 (26). NF-kB signaling pathway may be a promising therapeutic target for treating malignant ovarian cancers (27). Additionally, the STAT/NF-kB 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 (28). In this study, we investigated the combined anticancer effect of b-elemene and paclitaxel on ovarian cancer and explored the association between b-elemene +paclitaxel and STAT3-NF-kB signal pathway in SKOV3 cells. This study also analyzed the in vitro and in vivo effect of b-elemene+paclitaxel on ovarian tumor growth.

Flow cytometry assay
Apoptosis of SKOV3 cells was assessed using a flow cytometer (FACSCalibur BD Biosciences, USA). In brief, the treated SKOV3 cells (1 Â10 5 ) were collected, washed with PBS, and stained with fluorochrome-conjugated annexin V (5 mL) and propidium iodide (5 mL) 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 b-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 TRIzolt Plus RNA Purification Kit (Invitrogen, Life Technologies) according to the manufacturer's instructions. A total of two micrograms of total RNA were reversetranscribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystemst, USA). The cDNA (1 mg) 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 À DDCt method (29). All experiments were performed in triplicate.

Cell invasion and migration assays
SKOV3 cells were grown at 37°C with 5% CO 2 until 90% confluence. SKOV3 cells were then incubated with b-elemene (100 mg/mL), paclitaxel (20 mg/mL), or combined treatment for 24 h at 37°C. Matrigel-uncoated and Braz J Med Biol Res | doi: 10.1590/1414-431X20208885 Anti-cancer effect of b-elemene+paclitaxel for ovarian cancer 2/10 -coated 8-mm 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 Â10 5 in 500 mL 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 Â 10 5 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 b-elemene (100 mg/mL) and/or paclitaxel (20 mg/mL) for 12 h for further analysis.
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 Â10 6 cells) in 100 mL PBS were injected subcutaneously into the right axillary fossa and randomly divided into four groups: b-elemene (1.0 mg/kg), paclitaxel (1.0 mg/kg), combined treatment of b-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 (mm 3 ) = L (mm) Â W 2 (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.

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-mm thick) were incubated using TUNEL (DeadEndt 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 Â10 3 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 Po0.05 was considered statistically significant.

Dose decision for b-elemene or paclitaxel on SKOV3 cell growth
To determine the anti-cancer effects of b-elemene or paclitaxel on SKOV3 cells growth, a dose-dependent assay was performed in vitro. As shown in Figure 1A, b-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).

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

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

b-elemene+paclitaxel inhibited migration and invasion of SKOV3 cells
As illustrated in Figure 4A and B, treatment with b-elemene+paclitaxel markedly suppressed the migration and invasion of SKOV3 cells compared to either b-elemene or paclitaxel. Treatment with b-elemene+ paclitaxel down-regulated E-cadherin and MTA3 mRNA and protein expression in SKOV3 cells ( Figure 4C and D).
In vivo anti-cancer effect of b-elemene+paclitaxel in SKOV3-bearing mice b-elemene+paclitaxel treatment presented smaller tumors compared to either b-elemene or paclitaxel ( Figure 8A). Treatment with b-elemene+paclitaxel markedly increased the number of apoptotic cells in tumor tissue ( Figure 8B). Treatment with b-elemene+paclitaxel up-regulated the expression levels of STAT3 and NF-kB in tumor tissue compared to b-elemene, paclitaxel, and the control group ( Figure 8C). Compared with b-elemene and paclitaxel, b-elemene+paclitaxel prolonged survival of SKOV3-bearing mice in 120-day long-term observation ( Figure 8D).

Discussion
Previous reports have found that STAT3 and NF-kB signal pathways involved in growth and invasion of ovarian cancer cells (30)(31)(32)(33). 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 (34)(35)(36). Pashaei-Asl et al. (17) 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 (37). Here, we reported the synergetic anti-cancer effects of b-elemene and paclitaxel on ovarian cancer cells and ovarian cancer cell-bearing mice. The results of the present study suggest that b-elemene+paclitaxel suppressed growth, migration, and invasion of ovarian cancer cells, induced apoptosis, and regulated apoptosisrelated mRNA expression in SKOV3 cells. Importantly, we observed that b-elemene enhanced the anti-ovarian cancer effects of paclitaxel through regulation of the STAT3-NF-kB signaling pathway. In vivo experiment showed that b-elemene+paclitaxel treatment suppressed tumor growth and promoted apoptosis in tumor tissue.
Previously, b-elemene in combination with cisplatin increased apoptosis of prostate cancer through regulation of apoptosis-related gene in tumor cells (38). A clinical trial  indicated that elemene for the treatment of lung cancer is efficient and acceptable (21). Zhou et al. (39) demonstrated that the novel combination of mTOR inhibitor with b-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 (40). Results of the current study found that combined b-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, b-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 b-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 (24). In addition, combination of chemotherapy drugs of silibinin and paclitaxel were more efficient than a single drug in treatment of ovarian cancer cells (17). In addition, inhibiting NF-kB signaling pathway enhances cordycepin-induced apoptosis of ovarian tumor (32). Our data demonstrated that STAT3 overexpression inhibited paclitaxel-induced apoptosis promoted by b-elemene in SKOV3 cells. Drug combination of b-elemene and paclitaxel could be promising for future preclinical 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 b-elemene+paclitaxel on SKOV3 cells.
In conclusion, this study suggested that therapeutically effective combination of b-elemene+paclitaxel was a potential strategy for clinical ovarian cancer treatment and needs further preclinical study preceding human trials.