Platinum-resistant SKOV3/DDP cell line (purchased from China Center for Type Culture Collection, Wuhan, China), which was derived from human ovarian carcinoma, was cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin. Cells were cultured at 37°C in a 5% humidified CO₂ atmosphere, and 0.3 µg/ml cisplatin was added to the culture media to maintain the acquired resistance to DDP.

To evaluate the effect of TPL on cell migration, a scratch assay was applied. SKOV3/DDP cells were seeded onto 6-well plates to make a confluent monolayer, and then a p200 pipette tip was used to create a straight line to make a ‘scratch’. Suspended cells were washed using PBS, then 200 µl RPMI-1640 medium containing 2% FBS was added. SKOV3/DDP cells were co-cultured with 10 µg/ml DDP, 8 ng/ml TPL and 10 µg/ml DDP + 8 ng/ml TPL for 24 h. The wound area was calculated using Image-Pro Plus software (IPP; Media Cybernetics, Rockville, MD, USA).

For cell invasion assay, SKOV3/DDP cells (5×10⁴ cells/well) were seeded to the upper chamber of the Transwell plates (Corning Life Sciences, Lowell, MA, USA) and co-cultured with RPMI-1640 media containing 2% FBS, and 500 µl RPMI-1640 media supplemented with 10% FBS were added into the bottom wells. Then, 10 µg/ml DDP, 8 ng/ml TPL and 10 µg/ml DDP + 8 ng/ml TPL were added to the chambers, respectively. Incubated for 24 h, the SKOV3/DDP cells that invaded through the Matrigel matrix membrane were stained with crystal violet for 30–40 min, and their number was counted using an inverted microscope.

The treated SKOV3/DDP cells were digested using trypsin and washed twice using cold Hanks' solution. Then, SKOV3/DDP cells were suspended in a binding buffer containing Annexin V-FITC and PI. The cell mixture was incubated at room temperature (RT) (in dark) for 15 min, and were then sorted by cell flow cytometry (Becton-Dickinson, San Jose, CA, USA).

Cell lysis buffer supplemented with protease inhibitor cocktail and 1 mM phenylmethanesulfonyl fluoride (PMSF) were used to prepare whole-cell lysates, and protein concentrations were measured (10). Then, samples were resolved by polyacrylamide electrophoresis, the polyvinylidene difluoride membranes were blocked with 5% non-fat milk in TBST for 1 h at RT, and were incubated with primary antibodies for 3 h at RT, and then incubated with the appropriate HRP-conjugated secondary antibody for another 1 h.

To establish primary tumor xenografts, five million SKOV3/DDP cells were injected in the BALB/c-nu nude mice in a volume of 20 µl of PBS. When tumors reached 100 mm³, PBS (50 ml/kg/day, every day), DDP (4 mg/kg/day on the 1st and 8th days), TPL (0.15 mg/kg/day every day), DDP + TPL (4 mg/kg/day of DDP on the 1st and 8th days, 0.15 mg/kg/day of TPL every day) was injected i.p. into the mice. In the end, all mice were cervically sacrificed and their orbital blood were collected.

The present study was approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University, and all the research was carried out based on the approved guidelines.

The yields of IL-2 and TNF-α in mouse sera were evaluated using the IL-2 ELISA kit) and TNF-α ELISA kit (both from eBioscience, San Diego, CA, USA).

Resected tumors were fixed in the 10% buffered formalin, and then embedded in paraffin and mounted on slides. Tumor sections were paraffinized and suppressed in endogenous peroxidase activity incubation in 3% hydrogen peroxide, and were microwaved in 10-mM sodium citrate (pH 6.0) to achieve the antigen retrieval. Then, 2.5% horse serum were used to block sections, and corresponding antibodies were used and incubated for 16 h at 4°C. 3,3′-Diaminobenzidine and hematoxylin were used to stain slides, and detection was achieved using Avidin-Biotin Complex System (Vector Laboratories, Burlingame, CA, USA), which was analyzed using the IPP based on their density mean, area sum and integrated optical density.

The data are presented as mean ± SD, P<0.05 was considered statistically significant (23–25).

To the best of our knowledge, cellular invasion is an important part of cellular migration. Cells with high migration usually possess high invasion, while cells with high invasion may not possess high migration. So we simultaneously tested the migration and invasion of SKOV3/DDP cells. As shown in Fig. 1, the addition of DDP, TPL and DDP + TPL significantly reduced the cellular invasion and migration of SKOV3/DDP compared with the control group (NC) at 24 h (P<0.05), and the synergistic effect of TPL and DDP had significantly enhanced the inhibition effect on cellular invasion and migration compared with DDP and TPL group (P<0.05).

When treated with different concentrations of reagents, the apoptosis rates in the control, DDP, TPL and TPL + DDP group were (4.863±0.930), (8.333±0.965), (19.823±2.558) and (24.733±2.009)%, respectively. For the single drug group, DDP and TPL greatly enhanced the apoptosis rate of SKOV3/DDP cells (P<0.05), and the TPL + DDP had the best promoting effect on apoptosis of SKOV3/DDP cells compared with DDP and TPL group (P<0.05) (Fig. 2).

As tumor development is a complex process, so cancer-related proteins were tested. As shown in Fig. 3, the treatment of DDP, TPL and DDP + TPL significantly reduced the expression of adhesion-related protein integrin β1 (ITGβ1) and apoptosis-inhibiting proteins survivin, matrix metalloproteinase 2 (MMP-2) and MMP-9 (DDP + TPL group >TPL >DDP; P<0.05), and significantly increased the yields of apoptosis-promoting proteins cleaved caspase-3 and Smac (DDP + TPL group > TPL > DDP; P<0.05).

To further study the anticancer effects of TPL and DDP + TPL on tumors, the DDP, TPL and DDP + TPL were intraperitoneally injected or orally administered to mice, and their effects on related cytokines were evaluated. As shown in Fig. 4, the 4 mg/kg/day DDP, 0.15 mg/kg/day TPL, 4 mg/kg/day TPL and 0.15 mg/kg/day TPL greatly enhanced the inflammatory factors IL-2 and TNF-α in serum, and the DDP + TPL possessed the best enhancement effects compared to the other two groups.

To find the synergistic mechanisms of TPL and DDP, proteins related to tumor immunity and angiogenesis were studied in control, DDP, TPL, and TPL + DDP groups. As shown in Fig. 5, the NK cell-related protein levels of CD16 and CD56 obviously increased in treatment groups, and the TPL + DDP had the best effect. Moreover, the addition of DDP, TPL and DDP + TPL greatly inhibited the production of vascular endothelial growth factor (VEGF) related proteins CD31 and CD105 (DDP + TPL group >TPL group >DDP group).