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Introduction

Ovarian cancer (OC) is a type of gynaecological malignant tumour and is the eighth leading cause of cancer-associated mortality in women worldwide (1). The high mortality rate associated with OC may be due to its late detection, as the vast majority of patients with OC are diagnosed at an advanced stage (2), and thus encounter recurrence and drug resistance. Although a significant proportion of patients with advanced disease can achieve a complete response with tumour resection and adjuvant chemotherapy, the majority of patients will relapse within 2 years (2,3). The 5-year survival rate of patients with advanced OC is ~40% in the United States (4,5). The combination of paclitaxel (PTX) and carboplatin is the first-line medication for postoperative treatment in patients with OC. PTX can bind to tubulin proteins and enhance their polymerization to induce mitotic G2/M phase arrest, which results in cell cycle arrest (6). However, PTX application is limited because of its serious side effects and multidrug resistance in tumour cells (7,8). Over the past decade, there has been little improvement in the cure rate for advanced OC; therefore, it is necessary to develop effective therapeutics to overcome chemoresistance.

In recent years, adjuvant chemotherapy with traditional Chinese medicine has been widely considered. Schisandra chinensis is a perennial woody vine of the Magnoliaceae family, which is commonly used as a traditional Chinese medicine. It is commonly called Wuweizi or ‘five-flavour fruit’ in China. Fructus Schizandra has a high medicinal value because of its protective effect on a number of organs, including the heart, liver, kidney, nervous system and gastrointestinal tract (9). The active ingredient of Schisandra chinensis, Schisandrin B, has been used to treat human lung carcinoma cells (10) and murine breast cancer 4T1 cells (11). In addition, it has been demonstrated that the major bioactive constituents of Fructus Schizandra can significantly enhance the doxorubicin-induced apoptosis of cancer cells, such as SMMC7721, a human hepatic carcinoma cell line, and MCF-7, a human breast cancer cell line (12). Crude extracts of S. chinensis have been shown to reverse multidrug resistance and improve the sensitivity of cancer cells to chemotherapeutic drugs by inhibiting the expression of P-glycoprotein and protein kinase C (13). Furthermore, previous studies have indicated that an effective component of Schisandra, Gomisin A (GA), serves a role in reversing drug resistance (14), inhibiting proliferation, and exerting antioxidant and anti-inflammatory activities (15). In addition, as one of the major bioactive constituents of Fructus Schizandra, GA has been shown to possess anticancer and antiangiogenic activity (16). Although GA has exhibited its inhibitory effects against various tumour cell lines, such as melanoma (17) and colorectal cancer (18) cell lines, its cytotoxicity in OC cells has not yet been evaluated. Furthermore, the benefit of the combination of GA and PTX on OC cells is unknown.

It has previously been reported that PTX can promote the production of reactive oxygen species (ROS) (19). ROS are a series of chemical compounds containing oxygen produced during cellular metabolism, including superoxide anion, hydroxyl radical and hydrogen peroxide; these chemicals can act on lipids, proteins and DNA (20). Oxidative stress induced by potential ROS causes irreversible cell damage and death; however, it has also been suggested that ROS can initiate carcinogenesis and help tumour cells survive (21). Radiation, tobacco and xenobiotics have long been some of the best-known sources of ROS and are associated with tumour-induced events (22). Therefore, the present study hypothesized that GA could enhance the sensitivity of OC cells to PTX by reducing ROS production.

In the present study, the human ovarian cancer cell lines SKOV3 and A2780 were used to investigate the effects of an effective component of Schisandra, GA, with PTX in vitro and in vivo. The present study assessed whether GA could increase the antitumor effects of PTX. Moreover, by observing the changes in ROS in cells, the possible mechanism underlying the effects of GA-induced ROS inhibition on PTX sensitivity was discussed.

Materials and methods

Chemicals and reagents

GA (purity ≥98%), PTX (purity ≥98%), and N-acetyl cysteine (NAC; purity ≥98%) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. An MTT reagent kit and dimethyl sulfoxide (DMSO) were purchased from MilliporeSigma. Wright-Giemsa stain solution, and haematoxylin and eosin (H&E) Staining Kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. Propidium iodide (PI), RNase-A, Triton X-100, ROS DCFH-DA assay kit (cat. no. S0033S), and radioimmunoprecipitation assay (RIPA) lysis buffer were purchased from Beyotime Institute of Biotechnology. An Annexin V-FITC/PI cell apoptosis detection kit was purchased from Beijing TransGen Biotech Co., Ltd. Dihydroethidium (DHE; cat. no. KGAF019) assay kit was purchased from Nanjing KeyGen Biotech Co., Ltd. Antibodies against epidermal growth factor receptor (EGFR; cat. no. 18986-1-AP), PI3K (cat. no. 20584-1-AP), Akt (cat. no. 10176-2-AP), phosphorylated (p)-Akt (cat. no. 66444-1-Ig), β-actin (cat. no. 20536-1-AP), cyclin-dependent kinase 4 (CDK4; cat. no. 11026-1-AP), cyclin B1 (cat. no. 28603-1-AP), GAPDH (cat. no. 10494-1-AP), matrix metallopeptidase 2 (MMP-2; cat. no. 10373-2-AP), Nrf2 (cat. no. 16396-1-AP), Keap-1 (cat. no. 10503-2-AP), NQO1 (cat. no. 11451-1-AP), and Ki-67 (cat. no. 27309-1-AP) were purchased from Wuhan Sanying Biotechnology.

Cell culture

SKOV3 and A2780 cell lines were obtained from the Prostate Disease Prevention and Control Centre, Basic Medical College of Jilin University; the SKOV3 (cat. no. CL-0215) and A2780 (cat. no. CL-0013) cell lines were originally purchased from Procell Life Science & Technology Co., Ltd. The SKOV3 cells were cultured in Iscove's modified Dulbecco's medium (HyClone; Cytiva) and the A2780 cells were cultured in Dulbecco's modified Eagle's medium (HyClone; Cytiva) both supplemented with 10% foetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2.

Animals

A total of 20 female BALB/c-Nu mice (age, 6 weeks; weight, 16–20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and were reared under specific pathogen-free conditions at a temperature of 22±1°C and 45–55% humidity. Mice were maintained under a 12-h light/dark cycle and were provided with ad libitum access to food and water. The feeding and handling of experimental animals followed the regulations of Jilin University and the in vivo experimental protocol was approved by the Experimental Animal Ethics Committee of Jilin University Second Hospital (approval no. 2018106; Changchun, China).

A2780 cells (2×106 cells) were subcutaneously injected into the right back of each mouse. When the volume reached 100–150 mm3, the mice were distributed into four groups (n=5 mice/group) as follows: i) Control group, in which mice were intraperitoneally injected with 200 µl PBS once every 2 days; ii) GA group, in which mice were intraperitoneally injected with 200 µl GA (10 mg/kg) once every 2 days; iii) PTX group, in which mice were intraperitoneally injected with 200 µl PTX (10 mg/kg) once every 2 days; and iv) PTX combined with GA group, in which mice received the same dose of each drug as the monotherapy group. PTX and GA were dissolved in saline. For each mouse, a volume of 200 µl solution containing the drug was injected each time.

Tumour volume was calculated daily as follows: Volume=L × W2/2, where L and W represent the length and width of the tumour, respectively. In addition, the body weight of each mouse was calculated daily. No animals were withdrawn from the study. After 14 days of treatment, the mice were euthanized by cervical dislocation under inhalation of excess ether (mice were anaesthetized until their muscles were completely relaxed and their eyelid reflexes disappeared, the ether was then removed and mice were sacrificed by cervical dislocation), and the tumours were excised and weighed. The heart, liver, spleen, lung and kidney were also removed for further analysis. The specific humane endpoints used to determine euthanasia were when the tumours were >2,000 mm3, tumour diameter was >20 mm, or body weight loss was >20%. Death was verified by observation of the cessation of breath and heartbeat, and areflexia.

MTT assay

The in vitro cytotoxicity of GA, NAC and PTX in SKOV3 and A2780 cells was measured using the MTT assay, as previously described (23). Briefly, the cells were cultured in 96-well plates at a density of 3,000 cells/well. SKOV3 and A2780 cells were treated with 0.04 µΜ GA, 16 µg/ml PTX or their combination (GA + PTX group) for 6 and 24 h at 37°C. In addition, for additional experiments, SKOV3 and A2780 cells were incubated with 0.5 mM NAC for 1 h, and then 16 µg/ml PTX was added to the NAC + PTX group for 6 and 24 h at 37°C. After being exposed to the drugs, MTT (5 mg/ml) was added to each well. After incubated at 37°C for 4 h, DMSO was added to dissolve the precipitate. The absorbance of each well was measured the optical density at 490 nm (OD490). All experiments were repeated three times. The inhibition rates were calculated as following: Inhibition rate (%)=(ODc-Odt)/ODc, where ODt and ODc represent the OD490 values of the treatment group and control group, respectively.

Colony formation

SKOV3 and A2780 cells were seeded into 6-well plates at 300 cells/well and were treated with GA, PTX, NAC, NAC + PTX or GA + PTX for 2 weeks at 37°C. The drug concentrations were the same as those used prior to the MTT assay, and these concentrations were used for the subsequent experiments in vitro. After treatment, the cells were fixed with 4% paraformaldehyde for 15 min at 37°C and washed three times with PBS. Finally, the cells were stained with Wright-Giemsa stain solution according to the manufacturer's protocols. The colonies containing >50 cells were counted. The cell clonality rates were calculated as following: Cell clonality (%)=colony number/seeded cells number.

Cell cycle and apoptosis assays

SKOV3 and A2780 cells were seeded into 6-well plates at 4×105 cells/well and were divided into different groups with the indicated treatments for 24 h at 37°C, after which cell cycle progression and apoptosis were analysed. For the cell cycle assay, after being harvested, the cells were fixed in 70% precooled ethanol overnight at −20°C and then suspended in PBS containing 50 µg/ml PI, 100 µg/ml RNase-A and 0.2% Triton X-100 for 30 min at 4°C in the dark. The stained cells were analysed by a FACScan flow cytometer (BD Biosciences), and the results were analysed by Modfit LT 5 software (Verity Software House, Inc.).

For apoptosis detection. all cells, both floating and adherent, were harvested. Subsequently, the cells were stained with 5 µl Annexin V-FITC and 5 µl PI for 15 min at room temperature in the dark according to the manufacturer's instructions. The stained cells were analysed by a FACScan flow cytometer and the results were analysed using BD Diva 3 software (BD Biosciences).

Wound scratch assay and Transwell invasion assay

For the wound scratch assay, SKOV3 and A2780 cells were seeded into 12-well plates at 3×105 cells/well. When the cells reached 80–90% confluence, a sterile 200-µl pipette tip was used to scratch the cell monolayer. The wounds were observed at 0 and 48 h after being treated with GA, NAC, PTX, NAC + PTX or GA + PTX in serum-free medium at 37°C. Cell images were captured by an optical microscope (Motic Incorporation, Ltd.). Semi-quantification of the wound scratch assay was conducted by measuring the gap distance. Migration rate (%) was calculated as follows: (0 h gap distance-48 h gap distance)/0 h gap distance.

For the invasion assay, 24-well Transwell chambers (pore size, 8.0 µm; cat. no. 3422; Corning, Inc.) were used to analyse the invasive ability of the cells in each treatment group. The chamber was coated with Matrigel (BD Biosciences) according to the manufacturer's instructions. A total of 3×104 cells/well were seeded into the upper chamber with 100 µl serum-free media-based drugs (0.04 µΜ GA, 16 µg/ml PTX and 0.5 mM NAC), whereas 600 µl 10% FBS-containing media-based drug was added into the lower chamber. After 24 h of treatment at 37°C, the chambers were removed and stained with Wright-Giemsa staining solution as aforementioned for colony formation. Images were captured by an optical microscope (Motic Incorporation, Ltd.).

Western blot analysis

Briefly, cells were collected after different treatments and maintained on ice. The cells were resuspended in RIPA lysis buffer, according to the manufacturer's instructions, for protein extraction, and protein concentrations were quantified using the bicinchoninic acid method. Western blot analysis was conducted as previously described (24). A total of 20 µg proteins were loaded per lane into a 4–15% Precast Tris-Glycine Gel (TransGen Biotech Co., Ltd.); the proteins were then separated electrophoretically and transferred to PVDF membranes. The PVDF membranes were blocked with 5% skim milk at room temperature for 30 min, followed by incubation with the primary antibodies overnight at 4°C. The membranes were then incubated with HRP-tagged goat anti-rabbit and anti-mouse antibodies secondary antibodies (1:10,000; cat. nos. SA00001-2 and SA00001-1; Proteintech Group, Inc.) for 1 h at room temperature. Finally, the bands were visualized using the ECL method (BeyoECL Moon kit; cat. no. P0018FS; Beyotime Institute of Biotechnology) on a ChemiScope 6000 Exp chemiluminescence imaging system (Clinx Science Instruments Co., Ltd.) and were semi-quantified with ImageJ software (Version 2.1.0/1.53c; National Institutes of Health). The following dilutions of primary antibodies were used: Anti-EGFR (1:3,000), anti-PI3K (1:500), anti-Akt (1:5,000), anti-p-Akt (1:5,000), anti-β-actin (1:3,000), anti-CDK4 (1:4,000), anti-cyclin B1 (1:4,000), anti-GAPDH (1:10,000), anti-MMP-2 (1:500), anti-Nrf-2 (1:3,000), anti-Keap-1 (1:5,000) and anti-NQO1 (1:4,000).

DHE staining

Cells were plated into 6-well cell culture plates at a density of 4×105 cells/well were treated according to the aforementioned methods. After being washed with PBS twice, the cells were incubated with 2 ml medium containing 10% FBS and 5 µM DHE for 30 min at 37°C in the dark and were then visualized under a fluorescence microscope (Motic Incorporation, Ltd.). Red staining indicating oxidative stress was semi-quantified (ROS-positive staining) using ImageJ software (Version 2.1.0/1.53c).

DCHF-DA staining

Cells were plated on 6-well cell culture plates at a density of 4×105 cells/well and were then treated with GA, NAC, PTX, NAC + PTX, and GA + PTX. All of the cells were harvested in medium supplemented with 10% FBS, centrifuged at 300 × g for 5 min at room temperature, and resuspended in PBS containing 10 µM DCHF-DA. Cells were maintained at 37°C in the dark for 30 min. Subsequently, the cells were pelleted by centrifugation at 300 × g for 5 min at room temperature and resuspended in preheated PBS. The cells were finally analysed using a FACScan flow cytometer and the results were analysed by BD Diva 3 software.

Immunohistochemical (IHC) and H&E staining

IHC staining and H&E staining were conducted as previously described (25). Briefly, tumour, heart, liver, spleen, lung and kidney tissues were fixed in 10% formalin at room temperature overnight, embedded in paraffin and then cut into 4-µm consecutive sections. For H&E staining, following deparaffinization and rehydration, the sections were stained with H&E, according to the manufacturer's protocol. Images were captured under an optical microscope (Motic Incorporation, Ltd.). For IHC staining, following deparaffinization and rehydration, the slides were heated in a microwave oven at 97°C for 20 min with EDTA retrieval buffer (Beijing Solarbio Science & Technology Co., Ltd.) for epitope retrieval. Nonspecific blinding was conducted with PBS containing 2% BSA (cat. no. AR1006; Boster Biological Technology) for 2 h at room temperature. Subsequently, the sections were incubated with anti-Ki-67 (1:6,000), anti-p-Akt (1:200), anti-MMP-2 (1:200) and anti-Nrf2 (1:200) primary antibodies at 4°C overnight. Slides were then incubated with HRP-conjugated goat anti-rabbit IgG (1:200) and goat anti-Mouse IgG (1:200) secondary antibodies, for 30 min at room temperature, and were then visualized with a DAB Detection Kit (Thermo Fisher Scientific, Inc.). Images were captured under an optical microscope (Motic Incorporation, Ltd.) and were semi-quantified with ImageJ software (Version 2.1.0/1.53c).

Routine blood examination

Whole blood samples (200 µl) were collected from retro-orbital venous sinus of each mouse after inhalation of ether at the final day of the experiment. The white blood cell (WBC) count, red blood cell (RBC) count, haemoglobin (HGB) count and blood platelet (PLT) count were analysed using a haematology analyser (Shinova).

Statistical analysis

All data are presented as the mean ± SD. One-way ANOVA followed by Bonferroni's post hoc test was used for comparisons between multiple groups. All experiments were repeated three times. ANOVA was performed using SPSS (version 26; IBM Corp.) software and graphs were constructed using GraphPad Prism 5 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Results

GA enhances the proliferation-inhibiting effect of PTX on OC cells

The structure of GA is shown in Fig. 1A. The inhibitory effects of GA and PTX on two OC cell lines, SKOV3 and A2780, were detected by MTT assay. The results showed that the inhibition rates of SKOV3 and A2780 cells treated with GA + PTX were significantly increased compared with the inhibition rates of the PTX group at 6 and 24 h (Fig. 1B). These data indicated that GA could reinforce the sensitivity of SKOV3 and A2780 cells to PTX. The antioxidant effect of GA has been widely confirmed (26). To identify whether a GA-induced reduction in ROS levels was associated with the inhibition of cell proliferation, GA was replaced with NAC. The MTT results showed that the inhibition rates of SKOV3 and A2780 cells treated with NAC + PTX were almost identical to the inhibition rates of the GA + PTX group. To verify the effect of GA and PTX on the proliferation of OC cells, a colony formation assay was performed (Fig. 1C). Treatment with GA or NAC combined with PTX resulted in a significantly reduced colony formation rate in both OC cell lines compared with that in the PTX group.

Figure 1. GA enhances the proliferation-inhibiting effect of PTX on ovarian cancer cells. (A) Structure of GA. (B) Inhibitory effects of different treatments on SKOV3 and A2780 cells. (C) Colony formation assay of cell clonality of SKOV3 and A2780 cells. (D) Flow cytometric and statistical analysis of apoptosis. (E) Flow cytometric and statistical analysis of cell cycle progression. Data are expressed as the mean ± SD from three independent experiments. **P<0.01 vs. Ctrl; #P<0.05, ##P<0.01 vs. PTX. Ctr, control; GA, Gomisin A; NAC, N-acetyl cysteine; PTX, paclitaxel.

To gain insight into the mechanism by which GA enhances the sensitivity of SKOV3 and A2780 cells to PTX, flow cytometry was used to detect the rate of apoptosis (Fig. 1D). The results revealed that apoptotic rate was very similar between the PTX group and both combined groups in both cell lines. The apoptotic rate of the GA + PTX group was significantly increased compared with that in control group in both SKOV3 and A2780 cells, whereas the apoptotic rate of the NAC + PTX group was only increased compared with the control group in SKOV3 cells. These results indicated that the apoptotic process may not be involved in the mechanism by which GA mediates the enhancement of sensitivity to PTX in OC cells.

In addition, to investigate whether the mechanism was related to the cell cycle, PI staining was employed to observe cell cycle progression. According to the results of flow cytometric analysis (Fig. 1E), the treatment of both cell lines with PTX significantly increased the proportion of cells in the G2/M phase of the cell cycle and decreased the proportion of cells in the G0/G1 phase of the cell cycle. In addition, the two combination treatments, GA + PTX and NAC + PTX, enhanced the accumulation of cells in the G2/M phase compared with PTX treatment alone.

To explore the mechanism underlying the effects of NAC, GA and PTX on G2/M phase arrest, western blot analysis was performed to measure the expression levels of the cell cycle-regulating proteins cyclin B1 and CDK4. CDK4 has been reported to play an important role in regulating cell G1, as CDK4 mediates the progression of cells from G1 to S phase (27), and cyclin B1 regulates the G2/M phase (28). In the present study, the expression levels of CDK4 in the control, NAC, PTX, NAC + PTX, and GA + PTX groups in both cells and GA group in A2780 cell were consistent with the proportion of cells in G1 phase, in that the lower the proportion of cells in G1 phase, the lower the expression of CDK4 detected. However, in the GA group of SKOV3 cells, the proportion of cells in G1 phase showed no difference with that of the control group, whereas the expression of CDK4 was significantly decreased compared with that in the control group. It was hypothesized that these results were caused by the higher sensitivity of SKOV3 cells to GA than A2780 cells, and that the change in molecular level (CDK4 protein) needed more time to cause a change in the reduction of the proportion of cells in G1 phase. The expression of cyclin B1 was significantly upregulated following treatment with PTX alone in A2780 cells, but not in SKOV3 cells, and it was hypothesized that these results were associated with the low-dose ROS stimulation produced by PTX. Compared with in the PTX group, the expression levels of cyclin B1 and CDK4 were significantly attenuated in the groups treated with NAC + PTX and GA + PTX (Fig. 2A and C).

Figure 2. Effects of GA and PTX treatment on protein expression in ovarian cancer cells. Expression levels of (A) CDK4 and cyclin B1, and (B) EGFR, PI3K, Akt and p-Akt were detected by western blotting. (C and D) Semi-quantification of the western blot analysis. Data are expressed as the mean ± SD from three independent experiments. *P<0.05, **P<0.01 vs. Ctrl; #P<0.05, ##P<0.01 vs. PTX. Ctr, control; EGFR, epidermal growth factor receptor; GA, Gomisin A; NAC, N-acetyl cysteine; p, phosphorylated; PTX, paclitaxel.

In addition, the PI3K/Akt signalling pathway was analysed by western blotting. The situation of cyclin B1 also occurred in the PI3K/AKT pathway. Although PTX downregulated the expression levels of PI3K, the low-dose ROS stimulation produced by PTX upregulated the expression levels of p-AKT, which is an important downstream protein of the PI3K pathway. Similarly, the expression levels of PI3K, AKT and p-AKT were downregulated in the NAC + PTX and GA + PTX groups. Subsequently, the ratio of p-Akt/Akt was assessed in each group. The ratio of p-Akt/Akt in the PTX group was significant increased compared with that in the control group. Although there was no difference in the p-Akt/Akt ratio between the PTX group and the GA + PTX group, total Akt was markedly lower in the GA + PTX group, which resulted in a higher ratio of p-Akt/Akt in the GA + PTX group (Fig. 2B and D).

These data suggested that GA and PTX could block the G2/M phase and induce cell cycle arrest in OC cells, indicating that the inhibitory effect of PTX enhanced by GA was mediated by blocking the cell cycle, but not apoptosis.

GA enhances the inhibitory effects of PTX on OC cell invasion and migration

To clarify whether GA or NAC could increase the inhibitory effects of PTX on SKOV3 and A22780 cell invasion and migration, wound scratch and Transwell invasion assays were used to detect the migration and invasion of both cell lines, respectively. The migratory ability of both SKOV3 and A2780 cells was decreased in the GA, NAC and PTX groups compared with that in the control group. Following treatment with GA + PTX and NAC + PTX, the migratory ability of SKOV3 and A2780 cells was further decreased compared with that in cells treated with PTX or GA alone, and the difference was statistically significant (Fig. 3A and C). Chambers coated with Matrigel were used to mimic the extracellular matrix and simulate the invasion process of tumour cells in vivo. The invasion rate, relative to the control group, of SKOV3 and A2780 cells in the GA + PTX treatment groups was significantly lower than the relative invasion rate of both SKOV3 and A2780 cells in the PTX group (Fig. 3B and D).

Figure 3. GA enhances the inhibitory effect of PTX on ovarian cancer cell migration and invasion. (A and C) Migratory ability of SKOV3 and A2780 cells following different treatments, as determined by the wound scratch assay. (B and D) Invasive ability of SKOV3 and A2780 cells following different treatments, as determined by Transwell assay. (E) Expression levels of MMP-2, as detected by western blot analysis. Data are expressed as the mean ± SD from three independent experiments. *P<0.05, **P<0.01 vs. Ctrl; #P<0.05, ##P<0.01 vs. PTX. Ctr, control; GA, Gomisin A; NAC, N-acetyl cysteine; PTX, paclitaxel; MMP-2, matrix metallopeptidase 2.

Western blotting results showed that following treatment with NAC + PTX or GA + PTX, the expression levels of MMP-2 were downregulated compared with those in the PTX group (Fig. 3E).

GA enhances the inhibitory effects of PTX on OC cells by downregulating the levels of ROS

As shown in the aforementioned results, the NAC + PTX group exhibited a similar inhibitory effect to the GA + PTX group. To explore whether this was mediated by a GA-induced reduction in ROS levels, ROS were detected by DHE staining. The PTX group exhibited a marked increase in ROS production; ROS levels were presented as fluorescence intensity relative to the control group. However, the relative fluorescence intensity was decreased in the GA + PTX and NAC + PTX groups compared with that in the PTX group (Fig. 4A). In addition, flow cytometry was employed to detect the ROS content in each group. The results were in accordance with the DHE staining results (Fig. 4B and C). Furthermore, the Keap-1/Nrf-2 signalling pathway was detected by western blotting (Fig. 4D). Compared with in the control group, the expression levels of Keap-1 were downregulated in the PTX group, whereas they were upregulated in the GA + PTX and NAC + PTX groups. The expression levels of Nrf-2 and NQO1 were upregulated in the PTX group but were markedly downregulated in the GA + PTX and NAC + PTX groups. These results indicated that GA strengthened the sensitivity of SKOV3 and A2780 cells to PTX, which was mediated by ROS inhibition regulated by GA.

Figure 4. GA enhances the inhibitory effect of PTX by downregulating the levels of ROS. (A) DHE staining and statistical analysis of ROS in SKOV3 and A2780 cells following different treatments. (B and C) Flow cytometric and statistical analysis of ROS levels in SKOV3 and A2780 cells following different treatments, as determined by DCHF-DA staining. (D) Expression levels of Keap-1, Nrf-2 and NQO1, as detected by western blot analysis. Data are expressed as the mean ± SD from three independent experiments. *P<0.05, **P<0.01 vs. Ctrl; #P<0.05, ##P<0.01 vs. PTX. Ctr, control; DHE, dihydroethidium; GA, Gomisin A; NAC, N-acetyl cysteine; PTX, paclitaxel; ROS, reactive oxygen species.

GA enhances the inhibitory effects of PTX on mouse ovarian subcutaneous xenograft tumours

When tumour volume reached 100–150 mm3, the mice were distributed into each group and the in vivo antitumor effect study was initiated. Notably, there was no significant difference in the mean graft volume among the four groups at the start of the study. A direct visual representation of the tumours is shown in Fig. 5A. The suppressive effect of GA on tumour volume and weight was only slightly smaller than that in the control group and no statistically significant difference was observed in terms of antitumor effect, which may be due to the low dose of GA used. By contrast, the PTX group and the combined group exhibited an obvious reduction in tumour growth compared with that in the control group. Moreover, the combination group further enhanced the tumour inhibitory effects of the PTX group.

Figure 5. GA enhances the inhibitory effect of PTX on transplanted tumours. (A) Volume (left and middle panels) and weight (right panel of transplanted tumours. (B) H&E staining of transplanted tumours. H&E staining showed more tumour necrosis and the tumour tissue was impaired with nuclear pyknosis and fragmentation, with some nuclei disappearing (arrows), in the GA, PTX and combined groups compared with in the control group. (C) Immunohistochemical staining of Ki-67, p-Akt, MMP-2 and Nrf-2. (D) Semi-quantification of immunohistochemical staining analysis. **P<0.01 vs. Ctrl; #P<0.05, ##P<0.01 vs. PTX, Ctr, control; Com, combined; GA, Gomisin A; p, phosphorylated; PTX, paclitaxel; MMP-2, matrix metallopeptidase 2; H&E, haematoxylin and eosin.

To further validate the antitumor activity of GA and PTX, pathological analysis of tumour tissues was performed by H&E staining (Fig. 5B). Tumour necrosis was not obvious and most tissue structures were intact in the control group. In the GA, PTX and combined groups, the tumour tissue was impaired to varying degrees, and the pathological differences were noticeable, with nuclei in the necrotic area showing pyknosis and fragmentation, and with some nuclei disappearing.

Immunohistochemical staining was further used to detect the expression of the cell proliferation markers Ki-67 and p-Akt, the cell migration marker MMP-2 and the ROS marker Nrf2 (Fig. 5C and D). The highest Ki-67, MMP-2 and p-Akt expression levels were both detected in the control group of A2780 tumour-bearing mice, whereas the lowest were detected in the combined group. Nrf2 expression was markedly upregulated in the PTX group compared with that in the control group, whereas it was obviously downregulated in the GA and GA + PTX groups; the expression of Nrf2 was much lower in the GA + PTX than that in the GA group. The immunohistochemistry results for Ki-67, p-Akt, MMP-2 and Nrf2 expression were consistent with those of western blot analysis of in vitro results.

Additionally, biosafety assessments were performed by H&E staining to detect histological changes in the heart, liver, spleen, lung and kidney (Fig. 6A), and routine blood examinations of WBC, RBC, HGB and PLT (Fig. 6B). The results of H&E staining showed that no obvious pathological damage was detected, and no significant differences were identified in the routine blood examinations of each group.

Figure 6. Biosafety assessments of GA and PTX treatments. (A) Haematoxylin and eosin staining of the histological changes in the heart, liver, spleen, lung and kidney. (B) Routine blood examinations of WBC, RBC, HGB and PLT counts. Ctr, control; GA, Gomisin A; Com, combined; HGB, haemoglobin; PLT, platelet; PTX, paclitaxel; RBC, red blood cell; WBC, white blood cell.

Discussion

OC is a malignant tumour of the female reproductive system, which is associated with a high mortality rate (29). Platinum-based chemotherapy combined with PTX is the most important adjuvant therapy for OC. Although PTX has strong antiangiogenic activity, the drug resistance of PTX, which may be related to the production of ROS induced by it, limits its clinical application to a certain extent (19). Notably, the antioxidant activity of GA has been widely proven (13) and the present study confirmed this. In recent years, GA has been widely used in several research domains due to its minimal side effects (30,31); however, the exact effect of GA on human OC is unclear. In the present study, OC cells treated with GA or PTX were observed, and the results provided a theoretical and experimental basis for the practical application of PTX-based combined chemotherapy in the clinic.

The sensitivity of OC cells to PTX is based on the growth inhibition rate. Previous studies have shown that EGFR overexpression in a number of solid tumours is associated with tumour cell proliferation, angiogenesis, tumour invasion, metastasis and inhibition of apoptosis (32,33). The present study demonstrated that the expression of EGFR was significantly decreased after GA combined with PTX treatment, indicating that GA + PTX could significantly enhance the inhibition of proliferation of the OC cell lines A2780 and SKOV3 compared with PTX alone. As GA has an antioxidant effect, it was hypothesized that GA could decrease the level of ROS produced by PTX. In addition, a specific inhibitor of ROS, NAC, was used in the present study. As an antioxidant, a high dose of NAC (5 mM) has previously been shown to completely suppress ROS in cells, which could induce cell protection (34). In the present study, low-dose (0.5 mM) NAC only neutralized the ROS that initiated cell proliferation, but did not entirely suppress ROS levels. Additionally, the inhibitory effect of NAC was identified in previous studies (35,36); therefore, NAC was used in the present study to definitively identify whether the enhancement of the sensitivity of OC cells to PTX was related to GA-induced ROS reduction.

An increasing number of studies have reported that ROS can have dual roles, with different doses having different effects. High ROS concentrations inhibit cell survival, whereas low ROS concentrations promote cell survival (37). ROS may benefit therapeutic resistance in cancer (38). In most tumours, the high level of ROS production caused by chemotherapy or radiotherapy has been considered an antitumour factor (39); however, a low level of ROS is a key driver of tumour initiation. Immortal proliferation of cancer cells can be initiated by a low ROS environment that can regulate the cell cycle, autophagy and metastasis. Our previous study also reported that low levels of ROS could attenuate the antitumor effect of Raddeanin on SKOV3 cells (40). The detection of ROS implied that the levels of ROS in the GA + PTX group were significantly decreased compared with those in the PTX group, and the expression levels of Nrf-2 and NQO1 were downregulated, indicating that SKOV3 and A2780 cells were subjected to a lower degree of oxidative stress. These results suggested that low-dose ROS induced by PTX is one of the mechanisms limiting the tumour suppressive effect of PTX. Combining PTX with GA or NAC intensified the antiproliferative effect of PTX, implying that the sensitivity of OC cells to PTX was heightened when the cells lost the irritation of low-level ROS, also suggesting that GA enhanced the sensitivity of A2780 and SKOV3 cells to PTX by inhibiting intracellular ROS generation.

To further explore the mechanism underlying the antitumour effect of GA combined with PTX on OC cells, apoptosis and proliferation were detected. Cell death via apoptosis is required for homeostatic turnover of cells under physiological conditions; however, it is usually inhibited in tumours. Thus, apoptosis has been regarded as an essential mechanism of antitumor effects in most treatment methods (41). In addition, the apoptosis-related signalling pathway, the PI3K/Akt pathway, was evaluated (42). The results of the present study are consistent with those of previous studies (43,44), showing that PTX can exert its antitumour effect by promoting cell apoptosis; however, there was no difference between PTX and the combined treatment in terms of cell apoptosis. PTX is a drug that inhibits mitosis (45). High doses of PTX have been reported to promote apoptosis (43,44). In the present study, low doses of PTX were used. Although the percentage of apoptotic cells detected in response to PTX was <10%, there was still a statistically significant difference between the PTX and control groups. In addition, low-dose ROS stimulation produced by PTX upregulated the expression of p-AKT, which was significantly downregulated following treatment with GA. This indicated that the PI3K/Akt pathway may be involved in the mechanism by which GA mediates the enhancement of sensitivity to PTX in OC cells. Subsequent studies should consider using a PI3K/Akt inhibitor as a positive control to further assess this.

In addition, the relationship between the PI3K/Akt pathway and the oxidative stress-related Keap1/Nrf-2 pathway has been extensively reported. Resveratrol has been shown to protect porcine intestinal epithelial cells from oxidative stress through the PI3K/Akt-mediated Nrf2 signalling pathway (46). The protective effect of Chrysanthemum morifolium on cell oxidative damage has also been reported to be related to the PI3K/Akt-mediated Nrf2/HO-1 signalling pathway (47). As an upstream signalling pathway, the PI3K/Akt pathway serves a key role in regulating Nrf-2/HO-1 protein expression. PI3K/Akt can dissociate Nrf-2 from Keap1 and promote subsequent signal transduction, thus inducing the activation of antioxidant enzymes.

It has previously been reported that ROS regulate tumours via multiple mechanisms, including apoptosis and the cell cycle (48). The cell cycle consists of mainly two phases, G1/S and G2/M, is essential for cell survival and is modulated by a stream of molecules. The two cell cycle phases are tightly controlled by cyclins, CDKs and CDK inhibitors. The present study revealed that PTX could inhibit A2780 and SKOV3 cells by inducing G2/M phase arrest. Notably, the production of low levels of ROS could promote the cell cycle process (20), which was in accordance with the present finding that the ROS inhibitor GA or NAC could prolong the G2/M phase arrest induced by PTX. CDKs are a group of serine/threonine protein kinases, which drive the cell cycle through the chemical action of serine/threonine proteins and act synergistically with cyclin, which is an important factor in cell cycle regulation. Among the CDK family members, CDK4 has an important role in regulating G1 cells (27). Moreover, the decrease in cyclin B1 has been related to inactivation of the G2/M checkpoint and G2/M phase arrest (28). As cyclin B1/CDK1 can modulate mitochondrial activities in cell cycle progression and proliferation, subsequent studies will fully consider evaluating the expression of CDK1 to illustrate whether GA enhances the therapeutic effect of PTX through its effects on mitochondrial function.

All of these findings suggested that low levels of ROS may promote OC cell proliferation via a cell cycle mechanism and that the sensitivity of OC cells to PTX could be enhanced by the GA-induced reduction in ROS, which may further block the cell cycle compared with PTX alone. This model provides a strong strategy to target ROS with GA in OC therapy.

In conclusion, the present study showed that GA enhanced the inhibition rate of PTX by reducing the ROS levels in human OC cells both in vivo and in vitro. Furthermore, it was revealed that the mechanism was not related to the apoptosis pathway. However, G2/M phase arrest was associated with the mechanism. The present findings suggested that the combination of GA and PTX may be a promising treatment for OC.

Acknowledgements

Not applicable.

Funding

This work was supported by grants from the Jilin Province of Department Finance (grant nos. 2019SCZT040 and 2019SCZT050) and the Jilin Province Science and Technology Department (grant no. 20200201589JC).

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

SHZ, JL and TWW conceived and designed the experiments. TWW, CQZ, MYS, JYC and ZYY performed the experiments. XMH, MMC and SW analysed the data. XMH, SW, MMC and ZYY wrote the manuscript, critically revised the study for important intellectual content, and performed language editing. JL and SHZ supervised the study and were involved in project management. SHZ and TWW confirm the authenticity of all the raw data. All authors read and approved the final manuscript, and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval and consent to participate

The animal experiments were approved by the Institutional Animal Care and Use Committee of Jilin University Second Hospital.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

References