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Introduction

Epithelial ovarian cancer (EOC) is regarded as the most common type of ovarian cancers, which is usually diagnosed at an advanced stage due to the ineffective screening strategies, and causes immense morbidity and mortality worldwide (1). Although the surgical resection combined with cisplatin-based chemotherapy has greatly benefited cancer patients, the total survival of patients with advanced disease is <30% due to chemoresistance (2,3). Therefore, it is meaningful to find the sensitizer of EOC to cisplatin and clarify its mechanisms.

Numerous studies indicate that the mechanism of tumor occurrence, development, invasion, metastasis and resistance is complex (4–9). As one of the most well-known natural products, triptolide (TPL) had been used as an anti-inflammatory agent for rheumatoid arthritis for a long time in China, and was also recognized as a potential medicine for various types of cancers (10–16) although some researchers showed its potential toxicity on animal liver, kidney, testes, ovary and heart (17). Recent studies indicated that TPL induced cell apoptosis by inhibiting nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) in a p53-independent pathway, producing reactive oxygen species (ROS) and inactivating the PI3K/Akt signal pathway (9,16,18–21). In previous studies, our group proved that the TPL effectively inhibited cell growth, proliferation, metabolism, survival and cancer genesis by regulating the PI3K/Akt pathway (22). However, scarce study is carried out to evaluate the antitumor effect of TPL on cellular immunity and angiogenesis.

In the present study, we investigated the TPL anticancer effects using SKOV3/DDP cell line and a mouse model, and studied the TPL sensitization effects via inhibiting protein expression of angiogenesis and immunology.

Materials and methods

Cell experiments

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.

Cellular migration and invasion assays

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.

Apoptosis analysis

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).

Western blotting

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.

Mouse model of ovarian cancer

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.

ELISA

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).

Immunohistochemical staining

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.

Statistical analysis

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

Results

Effects of TPL and DDP + TPL on migration and invasion of SKOV3/DDP cells

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).

Figure 1. Effects of DDP, TPL and DDP + TPL on (A) cancer cell invasion and (B) cancer cell migration. The DDP, TPL and DDP + TPL reduce the cellular invasion and migration of SKOV3/DDP cells, and the DDP + TPL possessed the best inhibition effect. Compared with blank group, *P<0.05; compared with TPL group, ∆P<0.05; compared with DDP group, ▲P<0.05.

TPL + DDP increases the apoptosis rate of SKOV3/DDP cells

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).

Figure 2. Effect of DDP, TPL and DDP + TPL on apoptosis of SKOV3/DDP cells (x±s, n=3). Both the DDP group and TPL group can promote the apoptosis rate of SKOV3/DDP cells, and TPL + DDP had the best apoptosis rate of 24.733% among the groups. Compared with blank group, *P<0.05; compared with TPL group, ∆P<0.05; compared with DDP group, ▲P<0.05.

Effects of TPL and DDP + TPL on protein expression of SKOV3/DDP cells

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).

Figure 3. (A) Effects of DDP, TPL and DDP + TPL on protein expression profiles of MMP-2, MMP-9, ITGβ1 and β-actin; (B) Effects of DDP, TPL and DDP + TPL on protein expression profiles of Smac, survivin, cleaved caspase-3 and β-actin. The DDP + TPL significantly reduced the production of ITGβ1, MMP-2 and MMP-9, and significantly enhanced the yield of cleaved caspase-3 and Smac. Compared with blank group, *P<0.05; compared with TPL group, ∆P<0.05; compared with DDP group, ▲P<0.05.

Effects of TPL and DDP + TPL on IL-2 and TNF-α expression

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.

Figure 4. (A) Effects of DDP, TPL and DDP + TPL on the expression of IL-2. (B) Effects of DDP, TPL and DDP + TPL on the expression of TNF-α. Data indicate that DDP + TPL significantly enhanced the production of IL-2 and TNF-α. Compared with blank group, *P<0.05; compared with TPL group, ∆P<0.05; compared with DDP group, ▲P<0.05.

Effects of TPL and DDP + TPL on protein expression of cellular immunity and angiogenesis

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).

Figure 5. Effects of DDP, TPL and DDP + TPL on the expression of CD16, CD56, CD31 and CD105 using immunohistochemical method (at 4*C). The combination of TPL and DDP can greatly increase the NK cell related proteins (CD16 and CD56) and can obviously reduce the vascular endothelial growth factor (VEGF) related proteins (CD31 and CD105) to inhibit tumor growth.

Discussion

The platinum-based chemotherapy combined with curative resection was widely used in various cancer treatments, while drug resistance to DDP has emerged as the major hinderence to this method (26), thus naturally-occurring, plant-derived compounds have become a research hotspot in cancer therapies, which have been proven to influence multiple signaling pathways and can enhance the activity of conventional chemotherapy and radiation therapy (27).

As one of the well-known phytochemicals, TPL has been investigated for its pleiotropic anticancer activities by inhibiting cancer cell proliferation and inducing apoptosis of various cancers (11,12,16,28–35), while few studies were carried out on the effect of TPL on the cellular immunity and angiogenesis of human epithelial ovarian cancer (EOC).

In the present study, we first evaluated the combination of DDP and TPL on cell invasion, migration and apoptosis of cisplatin-resistance cell SKOV3/DDP in vitro, and determined the combined effects of DDP and TPL. The expected value of combination effect between DDP and TPL was calculated as: [(observed DDP value)/(control value)] × [(observed TPL value)/(control value)] × (control value); and the combination index is calculated as the ratio of (expected value)/(observed DDP + TPL value), and the ratio of >1 indicated a synergistic effect (36). As the ratio of the combination of DDP + TPL was >1, thus, they presented a synergistic effect on the inhibition of human EOC. Our results indicated that the DDP + TPL group significantly inhibited the invasion and migration of SKOV3/DDP cells compared with DDP and TPL group, and the apoptosis rate in DDP + TPL group was as high as (24.733±2.009)% (Figs. 1 and 2). Moreover, the western blot results confirmed that addition of TPL to DDP group greatly enhanced the yields of apoptosis-promoting proteins of cleaved caspase-3 and Smac, and obviously reduced the production of ITGβ1, survivin, MMP-2 and MMP-9 (P<0.05). As is known, caspase-3 belongs to the cysteine protease family, playing a key role in apoptotic pathways via cleaving a series of key cellular proteins, while survivin is a member of the inhibitor of apoptosis (IAP) family to inhibit caspase activation (37), and Smac is a member of promoter for caspase activation via binding to inhibitors of apoptosis-related proteins (38), therefore the negative regulation of survivin and positive regulation of Smac to caspase-3 lead to apoptosis (programmed cell death) of SKOV3/DDP cells.

In addition, the significant reduction of the membrane protein ITGβ1 (39) (a key protein in tumor invasion and metastasis, via mediating the adhesion of cells to the matrix and regulating the adhesion growth, migration, invasion and angiogenesis and chemotherapy resistance of many tumor cells) and MMP-2/MMP-9 (40) (whose altered expression and activity levels had been strongly implicated in the progression and metastasis of many forms of cancers) contributed to tumor apoptosis and tumor suppression. Fig. 4 shows the DDP + TPL greatly enhanced expression of IL-2 (which promotes the differentiation of T cells into effector T cells and into memory T cells to enhance the immunity of host) and TNF-α (whose primary role is to regulate immune cells, and is able to inhibit tumorigenesis), creating a inflammatory environment to promote the death of cancer cells (41,42).

In conclusion, our results indicated that both the TPL and DDP + TPL greatly enhanced cell apoptosis and tumor suppression via adjusting cellular immunity and angiogenesis of human EOC. Therefore, we proposed that TPL can lower the resistance of EOC to cisplatin, and can serve as a promising reagent for the treatment of human ovarian cancer.

Acknowledgements

The present study was supported by grants from the National Natural Science Foundation of China (nos. 81760729, 81503364 and 31560264), and the Jiangxi Government (20161BBG70218, 2016A078, 20171BCB23028 and 20175526).

References