Dulbecco's modified Eagle's medium (DMEM), trypsin and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). PBS was obtained from Boster Biological Technology (Pleasanton, CA, USA). TPL was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Cisplatin and MTT were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit was obtained from BD Biosciences (Franklin Lakes, NJ, USA). The primary antibodies used for western blot analysis were purchased from multiple companies: Anti-Bcl-2 and anti-Bax were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA) and anti-GAPDH was purchased from Sigma-Aldrich, Merck KGaA. Dichlorofluorescin-diacetate (DCFH-DA) was obtained from Sigma-Aldrich, Merck KGaA.

OVCAR3 and SKOV3 human ovarian cancer cell lines were purchased from Southern Medical University Cancer Institute (Guangzhou, China). Cells were cultured in DMEM supplemented with 10% FBS at 37°C in a humidified incubator at 5% CO₂. Cells were routinely subcultured using 0.05% trypsin-EDTA solution.

Briefly, cells were seeded into 96-well plates at the density of 5×10³ cells/well. Following incubation at 37°C in humidified incubator for 24 h, the DMEM was removed and replaced with a fresh medium containing increasing concentrations of TPL (2, 4, 6, 8 or 10 mM) and cultured at 37°C for 48 h. In order to estimate the effect of TPL on cell proliferation, growth curve analyses of OVCAR3 and SKOV3 cells were performed. Cells were seeded into 96-well plates at the density of 1×10³ cells/well and cultured at 37°C with TPL (1.5 Mm in OVCAR3 cells and 1 mM in SKOV3 cells) for 5 days. To investigate the effect of combination treatment of TPL and DDP on OVCAR3 cells proliferation, cells were cultured with 6 concentrations of DDP (1, 2, 4, 8, 16 or 32 µM) at 37°C for 48 h, and then 3 µM DDP combined with 1.5 and 2 mM TPL, respectively at 37°C for 48 h. Following treatment, the medium was discarded, cells were washed with PBS once and 100 µl MTT solution (0.5 mg/ml), which was diluted with 10% FBS and phenol red-free DMEM (Thermo Fisher Scientific, Inc.), was added to each well and incubated for 4 h at 37°C. Subsequently, the solution was discarded, 50 µl dimethyl sulfoxide was added to each well and the absorbance was evaluated at 490 nm. All experiments were performed in triplicate.

OVCAR3 cells were seeded in 6-well plates (2×10⁶ cells/well) and treated with 1.5 mM TPL, 3 µM DDP or a combination of TPL and DDP (1.5 mM TPL and 3 µM DDP) at 37°C for 48 h. Following a 48 h exposure to drug or control treatments, the cells were observed at ×10 magnification in 22 fields of view (18 mm) using an inverted phase contrast microscope. Images were captured at magnification, ×200.

Induction of apoptosis was determined using an Annexin V-FITC Apoptosis Detection kit, according to the manufacturer's protocol. OVCAR3 cells were seeded in culture dishes at the density of 2×10⁵ cells/well and incubated at 37°C for 48 h with media containing 1.5 Mm TPL or 3 µM DDP or a combination of TPL and DDP (1.5 mM TPL and 3 µM DDP). Annexin V-FITC-positive staining is a hallmark of early apoptosis, whereas late apoptosis and necrosis are indicated by additional positive nuclear staining with propidium iodide (PI). The cells were immediately detected with the probes by FCM analysis using FACS Aria II (BD Biosciences) and analyzed by FlowJo Software (FlowJo LLC, Ashland, OR, USA). Each sample was analyzed three times in three independent experiments.

OVCAR3 cells were treated with TPL (1 mM), DDP (3 µM) or combination of 1 mM TPL and 3 µM DDP at 37°C for 48 h. Following incubation, cells were digested with 0.05% trypsin-EDTA solution at 37°C for 3 min, collected into a centrifuge tube and washed twice with cold PBS. Protein lysis buffer (50 µl/dish) made up of 1X protease inhibitors (Hangzhou Fude Biological Technology Co., Ltd., Hangzhou, China) and 1 mM phenylmethanesulfonyl fluoride (Fude Biological Technology Co., Ltd.) was added to centrifuge tubes and cells were lyzed on ice for 10 min. The concentration of the prepared protein was detected according to the manufacturer's protocol of the Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Inc.). The whole cellular proteins (50 µg) were then mixed with 5X SDS-PAGE sample loading buffer (Fude Biological Technology Co., Ltd.) and boiled at 95°C for 10 min. The treated samples were subjected to 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Nonspecific binding was blocked using 5% non-fat powdered milk (dissolved in PBS) at room temperature for 1 h and the blots were incubated overnight at 4°C with the following antibodies: Bcl-2 (cat. no. 3498; 1:1,000), Bax (cat. no. 14796; 1:3,000) and GAPDH (cat. no. G9545; 1:5,000). The following day, all the membranes were washed three times with PBS supplemented with 0.1% Tween-20 prior to incubation with the horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (cat. no. FD0128; 1:5,000; Fude Biological Technology Co., Ltd.) for 1 h at room temperature. Finally, the membranes were washed with PBS supplemented with 0.1% Tween-20 three times for 5 min each. The bands were visualized by chemiluminescence reagents according to the manufacturer's protocols (EMD Millipore) and visualized on the ChemiDoc XRS+ Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Image Lab Software (version 3.0; Bio-Rad Laboratories, Inc.) was used to quantify band densitometry. A total of three independent experiments were performed.

For the evaluation of the expression levels of ROS, OVCAR3 cells that had been treated with 1.5 mM TPL at 37°C for 12 h were harvested and washed twice with PBS. The cells were suspended in 0.5 ml PBS and incubated with 20 µM DCFH-DA, a cell membrane permeable fluorescence probe, for 30 min at 37°C. Subsequently, the cells were washed and suspended in 0.5 ml PBS for analysis. Fluorescence levels were detected by FCM analysis using FACS Aria II (BD Biosciences) and analyzed by FlowJo Software. Each sample was analyzed three times in three independent experiments.

Results represent data from triplicate experiments for each treatment group and are presented as the mean ± standard deviation. Student's t-test was performed for comparisons between 2 groups and one-way analysis of variance with the Student-Newman-Keuls post-hoc test for comparisons between >2 groups. P<0.05 was considered to indicate a statistically significant difference.

Fig. 1A depicted the chemical structure of TPL. In order to investigate the effect of TPL on cell proliferation, the present study performed an MTT assay to evaluate cell viability following TPL treatment with concentrations >1 mM. The cell viabilities of OVCAR3 and SKOV3 cells were suppressed by TPL in a dose-dependent manner. The half-maximal inhibitory concentration (IC₅₀) values of TPL in OVCAR3 and SKOV3 cells were 3.72 and 2.32 mM, respectively, after 48 h incubation (Fig. 1B). Based on the IC₅₀, a low concentration of drug without cytotoxicity was used. Therefore, the present study selected the concentration of 1.5 mM TPL in the OVCAR3 group and 1 mM TPL in the SKOV3 group to analyze the effect of TPL on cell proliferation. Cell growth curves indicated that the proliferation rate of OVACR3 cells was significantly inhibited by TPL, compared with the control group, from the third day (third day, P=0.0097; fourth day, P=0.0113; fifth day, P=0.0155); the proliferation rate of SKOV3 cells was also inhibited by TPL, compared with the control group, from the third day (third day, P=0.0286; fourth day, P=0.0239; fifth day, P=0.0174) (Fig. 1C). These results suggested that TPL at concentrations of ≥1 mM were toxic and suppressed cell proliferation in OVCAR3 and SKOV3 cells.

It is universally recognized that DDP is the chemotherapeutic drug for ovarian cancer in clinical practice (28). MTT assays were used in the present study determined the IC₅₀ of DDP in the experimental system. Fig. 2A indicated that the IC₅₀ of DDP was 4.71 µM following incubation for 48 h. To investigate whether TPL increased the effect of DDP on cell proliferation inhibition, OVCAR3 cells were incubated with a combination of 3 µM DDP and 1.5 or 2.0 mM TPL. Each concentration of TPL significantly increased DDP-induced cell proliferation inhibition, compared with the signal DDP treatment group (Fig. 2B), suggesting that TPL improved the effect of DDP on inhibiting cell proliferation. When incubated with TPL alone, the higher concentration of 2.0 mM revealed a slightly higher toxicity compared with low concentration of 1.5 mM TPL, but no significant statistical difference was observed. Following treatment of TPL combined with DDP, the high TPL concentration group exhibited significant proliferation inhibition in OVCAR3 cells compared with the low TPL concentration group (P<0.05), indicating that the proliferation inhibition effect of TPL was more marked at higher concentrations.

Morphological observations of OVCAR3 cells following treatment with TPL or DDP or the combination of two drugs are demonstrated in Fig. 3. OVCAR3 cells, cultured in the control group, were fibroblasts-like which had a long and flat shape with irregular protrusions. Following TPL treatment for 48 h, the cell shape was not altered significantly, while the cell number was decreased compared with the control group. In the DDP treatment group, the cell shape changed, including some cells shriveling and others appearing round in shape. In the combination treatment group, the cell number was markedly decreased compared with the DDP-only treatment, and the majority of the cells were round in shape and had not attached to the culture dish. The combination treatment decreased cell proliferation of OVCAR3 cells more effectively compared with treatment with TPL or DDP alone.

The effect of DDP in cancer therapy is dependent on the induction of cell apoptosis. To evaluate the apoptotic stages of the cells following stimulation with the combination treatment, FCM analysis was used to distinguish the early apoptotic cells stained with Annexin V-FITC and the late apoptotic cells stained with PI. Representative cytograms for each treatment group are presented in Fig. 4A. Stimulation of OVCAR3 cells with the combination of 1.5 mM TPL and 3 µM DDP for 48 h produced a significant increase in the percentage of early apoptotic cells (Annexin+/PI-; 74.1±2.9%) compared with the DDP alone treatment (59.52±1.58%; Fig. 4B; P=0.0121). The percentage of late apoptotic cells in the combination group (Annexin+/PI+; 5.43±0.58%) was similar to the DDP group (7.29±2.3%; Fig. 4B). This experiment indicated that TPL accelerated DDP-induced apoptosis, primarily reflected in the increase in proportion of early apoptotic cells.

The expression ratio of Bcl-2:Bax serves an important role in cellular apoptosis. To verify whether the combination treatment was able to increase the apoptosis rate of OVCAR3 cells, western blot analysis was used to detect the expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax, and the expression ratio of Bcl-2:Bax. Fig. 5A depicted the protein expression after TPL treatment, DDP treatment and combination treatment. The data in Fig. 5B indicates that the combination treatment decreased the protein level of Bcl-2 and the expression ratio of Bcl-2:Bax induced by DDP (P=0.028). These results suggest that the TPL treatment significantly promoted DDP-induced apoptosis in OVCAR3 cells.

To investigate the potential mechanism of TPL-increased DDP-induced apoptosis in OVCAR3 cells, the cellular ROS generation induced by TPL and DDP in OVCAR3 cells was examined with DCFH-DA staining. As depicted in Fig. 6A, following treatment with TPL or DDP for 12 h, TPL and DDP increased the fluorescence intensity of ROS in OVCAR3 cells compared with the control group. In addition, the fluorescence intensity of ROS in the combined treatment group was significantly increased compared with the DDP alone group (P=0.0035; Fig. 6B). Therefore, it was concluded that TPL improved the cellular ROS production induced by DDP, suggesting that TPL-increased DDP-induced apoptosis may be associated with an increase in DDP-induced ROS generation.