Antibodies to phosphorylated (p)-JAK2 (product no. 3776; 1:1,000), JAK2 (product no. 3230; 1:1,000), p-STAT3 (Y705) (product no. 9145; used at 1:1,000 for western blotting and 1:100 for immunofluorescence staining), STAT3 (product no. 12640; 1:1,000), myeloid cell leukemia-1 (Mcl-1; product no. 94296; 1:1,000), LC3 (product no. 4599; used at 1:1,000 for western blotting and 1:6,400 for immunohistochemistry), p62 (product no. 88588; used at 1:1,000 for western blotting and 1:1,600 for immunohistochemistry), Beclin1 (product no. 3495; 1:1,000), caspase-3 (product no. 9662; used at 1:1,000 for western blotting and immunohistochemistry), β-actin (product no. 3700; 1:1,000) and Ki67 (product no. 12202; 1:800) were purchased from Cell Signaling Technology, Inc. HRP-labeled anti-rabbit (cat. no. SA00001-2; 1:5,000) and anti-mouse IgG (cat. no. SA00001-1; 1:5,000) were obtained from ProteinTech, Group, Inc. Chemicals, including triptolide (TPL), chloroquine (CQ), 3-methyladenine (3-MA), N-acetyl-L-cysteine (NAC), cisplatin (DDP), interleukin (IL)-6 and AG490 were purchased from Sigma-Aldrich; Merck KGaA. Cell Counting Kit-8 (CCK-8) assay and Reactive Oxygen Species (ROS) Assay Kit were obtained from Beyotime Institute of Biotechnology.

Cisplatin-resistant SKOV3/DDP and parental SKOV3 cells were purchased from China Center for Type Culture Collection and preserved at the Key Laboratory of Molecular Center of Jiangxi Province. The cells were routinely cultured in RPMI-1640 medium containing fetal bovine serum (FBS; 10%), penicillin/streptomycin (100 U/ml) in a 5% humidified CO₂ atmosphere at 37°C. Cisplatin (0.3 µg/ml) was added to the SKOV3/DDP culture media to maintain its acquired resistance.

To track autophagic flux, SKOV3/DDP cells were transfected with mRFP-GFP-LC3 adenovirus (Hanbio Biotechnology Co., Ltd.) following the manufacturer's recommendations and performed as previously described (23). Briefly, SKOV3/DDP cells (1×10⁶ cells/ml) were treated with TPL (0, 25, 50 and 100 nM) for 12 h, or treated with AG490 (50 µM) for 24 h at 37°C, respectively. After drug treatment, the formation of autophagosomes (yellow puncta) and autolysosomes (red puncta) were detected using a confocal laser scanning microscope under an ×400 magnification.

Cell viability was measured using Cell Counting Kit-8 (CCK-8) assay. Briefly, SKOV3/DDP cells (1×10⁴ cells/well) were treated with TPL (100 nM), CQ (10 µM), 3-MA (10 mM) or a combination of these compounds for 24 h at 37°C. Following treatment, CCK-8 solution was added and incubated at 37°C for 4 h. The optical density (OD) value of each well was measured at 450 nm using a Microplate Reader (Molecular Devices, LLC), and the cell viability was then calculated.

Immunoblotting analyses were performed as previously described (12). The whole cell protein extracts were prepared using the RIPA lysis buffer (Solarbio Life Science), and the protein concentrations were quantified with BCA Protein Assay Kit. Equal amounts of protein (30 µg/lane) were separated on 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific, Inc.). Following blocking for 2 h using 5% non-fat milk at room temperature, the membrane was incubated overnight with the specific primary antibodies against p-JAK2, JAK2, p-STAT3 (Y705), STAT3, Mcl-1, LC3, p62, Beclin1, caspase-3, and the internal control β-actin at 4°C. After washing with PBS, the membranes were incubated with the corresponding horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or anti-mouse IgG at room temperature for 1 h. The blot bands were detected using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, Inc.), and imaged and quantified using the ChemiDoc XRS System (Bio-Rad Laboratories, Inc.).

Reactive Oxygen Species Assay Kit was applied to measure the level of intracellular ROS in SKOV3/DDP cells. Briefly, SKOV3/DDP cells (1×10⁶ cells/ml) were treated with TPL (100 nM) alone or in presence of NAC (5 mM) at 37°C for 12 h, then washed three times in PBS and incubated with DCFH-DA at 37°C for 20 min in the dark. The DCF fluorescence intensity was examined and analyzed by cell flow cytometry (FACSCalibur; BD Biosciences).

Immunofluorescence staining was carried out to analyze the expression of p-STAT3 (Y705) in SKOV3/DDP cells. Briefly, SKOV3/DDP cells (1×10⁶ cells/ml) were treated with or without 100 nM TPL at 37°C for 24 h, and then fixed with 4% paraformaldehyde for 20 min on ice and permeablized with 0.5% Triton X-100 for 10 min at 37°C. Then, the cells were incubated with anti-p-STAT3 (Y705) antibody at 4°C overnight and subsequently with CoraLite 488-conjugated Affinipure goat anti-rabbit IgG secondary antibody (cat. no. SA00013-2; 1:500; ProteinTech Group, Inc.) for 1 h at room temperature in the dark. Finally, the cells were incubated with DAPI at room temperature for 10 min to stain nuclei, and the and the images were obtained using a confocal microscope (Olympus FV1000; Olympus Corporation) at a magnification of ×400.

Co-IP assay was performed as previously reported (24). Briefly, 1,000 µg of whole cell lysates were prepared with 1 ml of IP lysis buffer (cat. no. 87787; Pierce IP Lysis Buffer; Thermo Fisher Scientific, Inc.) after drug treatment. Then the lysate was incubated with the antibody against Beclin1 and 100 µl of Protein A/G magnetic beads (cat. no. 88803; Pierce; Thermo Fisher Scientific, Inc.) at 4°C overnight on a rocking platform. The immunoprecipitated pellets were collected by centrifugation at 12,000 × g for 5 min at 4°C. Then the pellets were washed three times with the cold lysis buffer, boiled in 2X SDS loading buffer and analyzed by western blot analysis with anti-Mcl-1.

After SKOV3/DDP cells were grown into 6-well plates and reached 50% confluence, cells were transiently transfected with the Beclin1 small interfering (si)RNA or negative control siRNA (100 pmol) using Lipofectamine 2000 ((Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Briefly, 5 µl (100 pmol) Beclin1 siRNA and 5 µl Lipofectamine 2000 were diluted in 50 µl RPMI-1640 medium, respectively, and were incubated for 5 min at room temperature. Then, the diluted Beclin1 siRNA and Lipofectamine 2000 were mixed and incubated for 15 min at room temperature. Next, the complexes were added to cells and incubated for 6 h at 37°C in a 5% CO₂ incubator. Thereafter, the cells were washed with PBS and suspended in the RPMI-1640 medium plus 10% FBS. After 48 h of transfection, cells were treated or untreated with 100 nM TPL for an additional 24 h and collected for western blot analysis. The Beclin1 siRNA (5′-GGATGACAGTGAACAGTTA-3′) and negative control siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′) were designed and synthesized by Guangzhou Ribobio Co., Ltd.

Proliferating SKOV3/DDP cells in a 6-well plate were transiently transfected with the pcDNA3.1-3×FLAG vector (GV141; Shanghai Genechem, Co., Ltd.) encoding Mcl-1 or the empty vector (1 µg/µl) at 50% confluence using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Briefly, 5 µl (1 µg/µl) Mcl-1-vector and 10 µl Lipofectamine 3000 were diluted in 125 µl RPMI-1640 medium, respectively, and were incubated for 5 min at room temperature. Then, the diluted Mcl-1-vector and Lipofectamine 3000 mixture were maintained at room temperature for 15 min. Next, the complexes were added to each well and incubated for 6 h at 37°C. Subsequently, the cells were washed with PBS and replaced with RPMI-1640 medium containing 10% FBS. After 48 h of transfection, cells were treated or untreated with 100 nM TPL for an additional 24 h and harvested for western blot analysis.

The female athymic BALB/CA-u nude mice (4-6 weeks old; 18±2 g; n=25) were purchased from Hunan SJA Laboratory Animal Co., Ltd. and housed at the SPF-level laboratory animal center in Nanchang University (26-28°C; 55% humidity; 10-h light/14-h dark cycle). All mice had free access to sterilized food and water. All animal experiments were approved by the Ethics Committee of Nanchang University and performed in compliance with the approved guidelines. A tumor xenograft was established in a murine model as described in our previous study (11,13). Briefly, single-tumor cells of SKOV3/DDP suspensions (5×10⁶) were subcutaneously injected into the right armpit of each BALB/CA-u nude mice. Seven days after cell implantation, the mice bearing tumors were randomized into five groups (n=5/group) and initially treated as follows: i) Vehicle group (50 ml/kg/day PBS every day, i.p.); ii) TPL group (0.15 mg/kg/day TPL every day, i.p.); iii) DDP group (4 mg/kg/day DDP on the 1st and 8th days, i.p.); iv) TPL + DDP group (0.15 mg/kg/day TPL every day, 4 mg/kg/day DDP on the 1st and 8th days, i.p.); and v) TPL + DDP + CQ group (0.15 mg/kg/day TPL every day, 4 mg/kg/day DDP on the 1st and 8th days, 25 mg/kg/day CQ every day, i.p.). All drugs were intraperitoneally (i.p.) injected into the mice as aforementioned (day 0 was considered as the beginning day of drug treatment). Tumor volumes and body weight were verified every other day. The tumor volume was calculated using the standard formula: Volume (mm³) = width² (mm²) × length (mm) ×0.5. After treatment for 10 days, all the mice were euthanized. The tumors were harvested, weighed and subjected to further immunohistochemical analysis as previously described (11,13).

All experiments were performed at least three times, and the results are presented as the mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism version 6.0 software (GraphPad Software, Inc.).

Unpaired Student's t-test was employed for comparisons of means between two groups, and one-way ANOVA with Tukey's post hoc test were employed for the comparison of multiple groups. The criterion for statistical significance was indicated as P<0.05.

TPL has been revealed to stimulate autophagy in some cancer cells (25–28), while the effect of TPL on autophagy regulation in drug-resistant ovarian cancer cells has not been reported. Therefore, mRFP-GFP tandem fluorescent-tagged LC3 was first used to track autophagic flux in SKOV3/DDP cells. As revealed in Fig. 1A, a significant increase in the number of autophagosomes (yellow puncta) and autolysosomes (red puncta) were observed following TPL treatment under a confocal microscope. Next, the expression of two specific autophagy markers, the proteins LC3 and p62 were assessed. Western blot analysis revealed that TPL treatment markedly increased the ratio of LC3-II/LC3-I but reduced p62 expression in a dose-dependent manner in SKOV3/DDP cells (Fig. 1B). These results clearly revealed that TPL treatment enhanced autophagic flux in ovarian cancer cells, which was consistent with previous studies (25–28).

Our group previously revealed that TPL induced apoptosis-mediated cell death in DDP-resistant ovarian cancer cells (9,10,12,13). Given the dual role of autophagy in cancer cell death, widely used autophagy inhibitors (3-MA and CQ) were applied to further confirm whether this autophagic response mediates the cytotoxic effects of TPL. As revealed in Fig. 1C, cell viability was significantly restored following treatment with TPL in the presence of 3-MA or CQ. Moreover, the expression of cleaved caspase-3 was also significantly suppressed when TPL-induced autophagy was inhibited by 3-MA or CQ (Fig. 1D and E). These data indicated that autophagy contributed to TPL-induced SKOV3/DDP cell death. The autophagic effect of TPL on parental SKOV3 cells was then evaluated. Consistent with the effect observed in SKOV3/DDP cells, TPL also prompted autophagy of SKOV3 cells (Fig. S1A). In addition, it was also revealed that the cytotoxic effect of TPL on SKOV3 cells was significantly reduced by pretreatment with 3-MA (Fig. S1B). Thus, the data aforementioned suggested that the autophagy induced by TPL was cytotoxic to ovarian cancer cells, rather than cytoprotective.

Our group previously demonstrated that TPL could enhance the suppressive effects of DDP on tumor growth in a SKOV3/DDP xenograft model (11,13). To further evaluate the effects of TPL-induced autophagy on chemosensitization in vivo, the impact of the combination of TPL + DDP + CQ on the growth of SKOV3/DDP xenografts was determined. Mice-bearing SKOV3/DDP cells were randomly separated into five groups: Vehicle, TPL, DDP, TPL + DDP, and TPL + DDP + CQ. All animals tolerated TPL treatment well. The mice did not exhibit any obvious change in body weight (Fig. 2A). The tumor volume and tumor weight in the TPL + DDP group were significantly reduced compared to those in the TPL + DDP + CQ group or any other single treatment group (Fig. 2B and C). In agreement with this observation, proliferation-related Ki-67 (the brown color area) was greatly downregulated in the TPL + DDP group (Fig. 2D). Additionally, the immunohistochemical staining results also revealed that autophagy inhibition significantly reduced the chemosensitization effect of TPL on SKOV3/DDP cells in vivo (Fig. 2D). It was concluded from these results that the chemosensitization effect of TPL was dependent on autophagy.

Then, systematic research on the underlying mechanisms involved in TPL-induced autophagy against SKOV3/DDP cells was performed in vitro. ROS have been documented as common mediators of cell apoptosis and autophagy (29). In a previous study by our group, it was found that TPL induced ROS-related apoptosis in DDP-resistant ovarian cancer cells (12). Therefore, DCFH-DA was first used as a probe to examine the intracellular ROS levels. As revealed in Fig. 3A and B, the mean DCF fluorescence intensity of SKOV3/DDP cells was significantly increased in response to 100 nM of TPL treatment, as compared with the control group. Additionally, this increase was significantly attenuated when the cells were pretreated with the well-known antioxidant NAC (Fig. 3A and B). These data indicated that TPL promoted the generation of cellular ROS in SKOV3/DDP cells. To evaluate the role of ROS in TPL-induced autophagy, SKOV3/DDP cells were incubated with TPL in the presence or absence of NAC. Notably, pretreatment with NAC strongly reduced the number of autophagosomes and autolysosomes compared with TPL treatment alone (Fig. 3C). Consistently, NAC strongly blocked the TPL-induced upregulation of LC3-II/LC3-I expression levels while enhancing p62 expression (Fig. 3D). Overall, these findings suggested that the autophagic inducing effect of TPL on SKOV3/DDP cells was associated with ROS generation.

The JAK2/STAT3 pathway is constitutively activated in ovarian cancer (21,22), has a well-established role in autophagy regulation and can be influenced by ROS (30). First, to evaluate the association between activation of the JAK2/STAT3 pathway and autophagy in SKOV3/DDP cells, AG490 (a JAK2/STAT3 signaling inhibitor) was used. As revealed in Fig. 4A and B, the level of autophagy significantly increased after treatment with AG490, suggesting that inhibition of the JAK2/STAT3 pathway may play an important role in autophagy induction.

The effect of TPL treatment on the JAK2/STAT3 signaling pathway was next examined. Western blot analysis revealed that TPL treatment led to noticeable inhibition of p-JAK2 and p-STAT3 (Y705) (Fig. 4C). Additionally, immunofluorescence analysis revealed that TPL incubation significantly decreased the nuclear level of p-STAT3 (Y705) (Fig. 4D). These results indicated that TPL treatment inhibited the JAK2/STAT3 pathway in SKOV3/DDP cells.

To further reveal the importance of the JAK2/STAT3 pathway in modulating TPL-induced autophagy, SKOV3/DDP cells were pretreated with IL-6 (a JAK2/STAT3 signaling activator). It was revealed that IL-6 strongly attenuated the LC3 conversion and p62 degradation accompanied by the upregulation of p-JAK2 and p-STAT3 (Y705) compared to those of the TPL group (Fig. 4E). These data indicated that TPL triggered autophagy in SKOV3/DDP cells at least partially by modulating JAK2/STAT3 signaling. Notably, the TPL-induced reductions in p-JAK2 and p-STAT3 (Y705) could be restored in the presence of NAC (Fig. 4F). Thus, ROS-mediated inhibition of the JAK2/STAT3 signaling pathway was involved in TPL-induced autophagy.

Next, the downstream signaling molecules of STAT3 involved in the regulation of TPL-induced autophagy were investigated. It has been reported that Mcl-1, a well-known antiapoptotic protein that is transcriptionally regulated by STAT3, negatively regulates autophagy by binding to Beclin1 (24,31). Therefore, the protein levels of Mcl-1 and Beclin1 were analyzed and it was revealed that TPL efficiently decreased Mcl-1 levels and increased Beclin1 protein levels in a dose-dependent manner (Fig. 4C). Next, an immunoprecipitation assay was conducted to monitor the interaction between Beclin1 and Mcl-1 in SKOV3/DDP cells. It was revealed that Mcl-1 and Beclin1 immunoprecipitated with each other in SKOV3/DDP cells under basal conditions (Fig. 5A), whereas the interaction markedly decreased in the presence of TPL (Fig. 5B).

Notably, IL-6 significantly prevented TPL-mediated Mcl-1 downregulation (Fig. 4E) and the Beclin1/Mcl-1 interaction (Fig. 5B). Furthermore, transient overexpression of Mcl-1 (Fig. 5C) and siRNA-mediated Beclin1 downregulation (Fig. 5D) significantly attenuated LC3-II accumulation and p62 degradation induced by TPL. Collectively, these results indicated that inhibition of the JAK2/STAT3/Mcl-1 pathway and the subsequent disruption of the Beclin1/Mcl-1 interaction are vital to TPL-induced autophagy.