| Ovarian cancer is a gynecological malignant tumor with the highest mortality.Platinum resistance often occurs in the treatment of ovarian cancer,which remains a difficult problem to be solved.Recent studies have shown that the anticancer and sensitization effects of the traditional Chinese medicine on drug-resistant ovarian cancer,but the underlying mechanism is not clear.Triptolide,a major active component extracted from the traditional Chinese herb Tripterygium wilfordii Hook F,which received great attention against tumors in recent years.Over the last several years,our prior studies have revealed the antitumor and sensitization properties of TPL against cisplatin-resistant epithelial ovarian cancer in vitro and in vivo,and apoptosis has been revealed to mediate this process.With the in-depth study of its mechanism of action,recent studies have shown that TPL promotes tumor cell death by inducing damaged autophagy,thereby producing anticancer effects.However,the autophagic effects of TPL on cisplatin-resistant human epithelial ovarian cancer cells remain unclear.Studies have shown that autophagy,as automatic degradation process,which has dual roles in chemo-resistant cancer.On the one hand,moderate autophagy(protective autophagy)can provide nutrients and energy for tumor cells,which is responsible for tumor cell survival and chemotherapy resistance.Studies have suggested that increased level of autophagy is closely related to cisplatin resistance in ovarian cancer.On the other hand,excessive autophagy(damaged autophagy)can degrade normal protein molecules and organelles in tumor cells to induce tumor cell injury,and cause programmed non-apoptotic death.Accumulating evidence and studies have established the role of the aberrantly active JAK2/STAT3 pathway in autophagy regulation and ovarian cancer progression and chemoresistance.It is currently believed that Mcl-1 protein,which is an important autophagic target effector that can be transcriptionally regulated by JAK2/STAT3 pathway.Mcl-1 has been reported to negatively regulate the autophagy induction through binding to the BH3 domain of Beclin1 protein.In view of this,we consider: could TPL inhibit cisplatin-resistant ovarian cancer cell proliferation via the autophagic pathway? Could TPL regulate the autophagy of cisplatin-resistant ovarian cancer cells via the JAK2/STAT3/Mcl-1/Beclin1 pathway?Therefore,on the basis of our previous work,cisplatin-resistant epithelial ovarian cancer SKOV3/DDP cells were used as an experimental model in this study.Experiments such as the fluorescence “autophagy flow” dynamic detection,CCK8 test and ectopic subcutaneous ovarian tumor in nude mice were used to determine whether TPL inhibit cisplatin-resistant ovarian cancer cell proliferation via the damaged autophagic pathway.Moreover,molecular biology techniques such as RNA interference,gene transfection,immunoprecipitation were performed to explore the molecular signal mechanism of TPL-induced SKOV3/DDP cell autophagy via the JAK2/STAT3/Mcl-1/Beclin1 pathway.Our study has not only further understood the new mechanism of TPL inhibiting drug-resistant ovarian cancer,but also opened up new ideas for the treatment of cisplatin-resistant ovarian cancer.Part 1 Study on the effect of triptolide(TPL)-induced autophagy in cisplatin-resistant human epithelial ovarian cancer SKOV3/DDP cells in vitroObjective: To investigate the effect of triptolide(TPL)on the autophagy of cisplatinresistant human epithelial ovarian cancer SKOV3/DDP cells in vitro.Methods: 1.SKOV3/DDP cells were treated with 50 n M TPL for 24 h,the morphological features of cell autophagy were observed under transmission electron microscope.2.SKOV3/DDP cells transfected with m RFP-GFP-LC3 adenovirus were treated with the indicated doses of TPL(0、25、50、100 n M)for 12 h,and then autophagic flux was analyzed using confocal microscope.3.SKOV3/DDP cells treated with indicated concentrations of TPL(0、25、50、100 n M)for 24 h were subjected to immunoblotting for the levels of LC3,p62 and Beclin1.4.SKOV3/DDP cells were treated with 100 n M TPL for 24 h in the presence or absence of 10 m M 3-MA,the cytotoxicity of TPL was detected by CCK-8 assay,and the expression level of autophagy-related protein LC3,p62 and apoptotic-related protein caspase-3 were measured by Western Blot.5.SKOV3/DDP cells were treated with 100 n M TPL for 24 h in the presence or absence of 10 μM CQ,the cytotoxicity of TPL was detected by CCK-8 assay,and the expression level of autophagy-related protein LC3,p62 and apoptotic-related protein caspase-3 were measured by Western Blot.Results: 1.The results of transmission electron microscope showed that the number of autophagic vesicles in the cytoplasm of SKOV3/DDP cells increased significantly after 24 h treatment of 50 n M TPL,compared with the control group.2.The results of m RFP-GFP-LC3 adenovirus transfection showed that compared with untreated cells,the number of autophagosomes(yellow spots)and autolysoso-mes(red spots)in the TPL(50 n M and 100 n M)group significantly increased in a concentration-dependent manner(P<0.01).3.SKOV3/DDP cells treated with indicated concentrations of TPL(0、25、50、100 n M)for 24 h.Western Blot results showed that with the concentration of TPL increasing,the ration of LC3-II/LC3-I and the expression of Beclin1 increased,whereas p62 expression reduced dramatically in SKOV3/DDP cells.4.After TPL was used alone or in combination with 3-MA in the SKOV3/DDP cells for 24 h.The results of CCK8 showed that there was no significance between the cell viability of the 3-MA(10 m M)group and the control group(P>0.05).SKOV3/DDP cells were treated with 100 n M TPL for 24 h,the equal cell viability was 30.80 ±3.92%.Compared with the TPL group,the cell viability in the combination of 3-MA and TPL group was significantly increased to 63.30±4.93%,the difference was significant(P<0.01).The results of Western Blot showed that the ration of LC3-II/LC3-I and the expression of activated caspase-3 protein were significantly increased,accompanying a decreased level of p62 in the TPL group;the expression of cleaved caspase-3 was significantly suppressed when TPL-induced autophagy was inhibited by 3-MA.5.After TPL was used alone or in combination with CQ in the SKOV3/DDP cells for 24 h.The results of CCK8 showed that there was no significance between the cell viability of the CQ(10 μM)group and the control group(P>0.05).SKOV3/DDP cells were treated with 100 n M TPL for 24 h,the cell viability was 37.34±1.22%.Compared with the TPL group,the cell viability in the combination of CQ and TPL group was significantly increased to 57.75±5.61%,the difference was significant(P<0.01).The results of Western Blot showed that the ration of LC3-II/LC3-I and the expression of activated caspase-3 protein were significantly increased,accompanying a decreased level of p62 in the TPL group;the expression of cleaved caspase-3 was significantly suppressed when TPL-induced autophagy was inhibited by CQ.Conclusion: 1.TPL induced SKOV3/DDP cell autophagy in a dose-dependent manner.2.TPL suppresses cell proliferation through triggering lethal autophagy in SKOV3/DDP cells.Part 2 Study on the role of TPL-induced autophagy on its resensitization the growth-inhibiting effect of DDP on drug-resistant ovarian cancer in vivo.Objective: To investigate whether TPL treatment enhanced the growth-inhibiting effect of DDP on SKOV3/DDP xenografts through inducing autophagy.Methods: 1.Single-tumor cells of SKOV3/DDP suspensions were subcutaneously injected into the right armpit of each female athymic BALB/c-nu nude mice to establish tumor xenograft.2.Total of 25 BALB/c-nu nude mice bearing tumors were randomly separated into five groups: Vehicle,TPL,DDP,TPL+DDP,and TPL+DDP+CQ.3.The mice bearing tumors were randomized into five groups and treated as follows :(1)Vehicle group(50 ml/kg/d 0.9%Nacl every day,ip);(2)TPL group(0.15 mg/kg/d TPL every day,ip);(3)DDP group(4 mg/kg/d DDP on the 1st and 8th days,ip);(4)TPL+DDP group(0.15 mg/kg/d TPL every day,4 mg/kg/d DDP on the 1st and 8th days,ip);and(5)TPL+DDP+CQ group(0.15 mg/kg/d TPL every day,4 mg/kg/d DDP on the 1st and 8th days,25 mg/kg/d CQ every day,ip).4.The general condition of each group of tumor-bearing nude mice were observed every day and the body weight of mice and tumor volume were measured every two days during the administration period.After treatment for 10 days,tumors were removed and weighed.5.Morphological changes in tumor tissue sections isolated from the indicated groups of mice were observed by HE staining.6.The expression of Ki67,cleaved caspase-3,LC3 and p62 in tumor tissue sections isolated from the indicated groups of mice were measured by immunohistochemical analysis.Results: 1.During the period of drug treatment,all tumor-bearing nude mice tolerated well,and there was no significant change in activity,reaction ability,diet and drinking water.No mice died and no tumors ruptured.Compared with the Vehicle group,the body weight of tumor-bearing nude mice in each treatment group also fluctuated in the normal range,and there was no significant difference between them(P>0.05).2.No matter the volume or weight of the transplanted tumor,the subcutaneous transplanted tumor in the TPL group and DDP group was significantly smaller than that in the Vehicle group.The volume or weight of the transplanted tumor in the TPL+DDP combined group decreased significantly,as compared with the single drug treatment group.After adding autophagy inhibitor CQ,the volume or weight of the transplanted tumor in the TPL+DDP+CQ group was significantly heavier than that in TPL+DDP group(P<0.05).3.HE staining under light microscope showed that the transplanted tumor cells in the Vehicle group grew actively,and which were irregularly and tightly arranged,with obvious atypia,large nucleus,deep staining,more pathological mitosis and no obvious necrosis.Tumor tissues from drug treatment(TPL,or DDP,or TPL+DDP,or TPL+DDP+CQ)groups showed varying degrees of necrosis,with inflammatory cell infiltration,disordered arrangement of cancer cells,nuclear chromatin pyknosis,nuclear fragmentation and few pathological mitosis.Among them,the TPL+DDP group was the most obvious,followed by TPL group and TPL+DDP+CQ group,the DDP group was the lightest.4.Immunohistochemistry assay showed that:(1)Tumor tissues from drug treatment(TPL,or DDP,or TPL+DDP)groups showed lower expression of Ki67,compared with the Vehicle group.Among drug treatment groups,the expression of Ki67 protein in TPL+DDP group was the lowest.After adding autophagy inhibitor CQ,the expression of Ki67 protein in TPL+DDP +CQ group was significantly higher than that in TPL+DDP group(P<0.05).(2)Tumor tissues from drug treatment(TPL,or DDP,or TPL+DDP)groups showed higher expression of cleaved caspase-3,compared with the Vehicle group.Among drug treatment groups,the expression of cleaved caspase-3 protein in TPL+DDP group was the highest.After adding autophagy inhibitor CQ,the expression of cleaved caspase-3 in TPL+DDP+CQ group was significantly lower than that in TPL+DDP group(P<0.05).(3)Tumor tissues from drug treatment(TPL,or TPL+DDP,or TPL+DDP+CQ)groups showed higher expression of LC3,compared with the control group.And the expression of LC3 in TPL+DDP+CQ group was significantly higher than that in TPL+DDP group(P<0.05).(4)Compared with the control group,Tumor tissues from drug treatment(TPL,or TPL+DDP)showed lower expression of p62.While the expression of p62 in TPL+DDP+CQ group greatly increased,which was significantly higher than that in TPL+DDP group(P<0.05).Conclusion:TPL treatment effectively enhanced the growth-inhibiting effect of DDP by enhancing autophagic activity in the SKOV3/DDP xenograft model.Part 3 Molecular Mechanism of TPL-induced SKOV3/DDP cell autophagy in vitro.Objective: To investigate the molecular mechanism of TPL-induced SKOV3/DDP cell autophagy in vitro.Methods: 1.SKOV3/DDP cells transfected with m RFP-GFP-LC3 adenovirus were treated with or without 50 μM AG490 for 24 h,autophagic flux was analyzed using confocal microscope,and the expression level of JAK2,p-JAK2,STAT3,p-STAT3(Tyr705),LC3,p62 were measured by Western Blot.2.SKOV3/DDP cells treated with indicated concentrations of TPL(0、25、50、100 n M)for 24 h were subjected to immunoblotting for the levels of JAK2,p-JAK2,STAT3,p-STAT3(Tyr705),Mcl-1.3.SKOV3/DDP cells were treated with or without 100 n M TPL for 24 h,then the expression of p-STAT3(Tyr705)was examined using immunofluorescence staining.4.SKOV3/DDP cells were pre-incubated with IL-6(100 ng/ml)for 1 h,then treated with TPL(100 n M)for another 24 h,the expression level of JAK2,p-JAK2,STAT3,p-STAT3(Tyr705),Mcl-1,LC3,p62 were measured by Western Blot.5.SKOV3/DDP cells were pre-incubated with IL-6(100 ng/ml)for 1 h,then treated with TPL(100 n M)for another 24 h.Cell lysates were subjected to immunoprecipitation with anti-Beclin1 antibody and interaction with Mcl-1 was determined by Western Blotting.6.SKOV3/DDP cells transfected with Mcl-1 overexpression plasmid were treated with or without TPL(100 n M)for 24 h,followed by Western Blot analysis to detect the levels of Mcl-1,LC3 and p62.7.SKOV3/DDP cells transfected with the Beclin1 siRNA or negative controlsiRNA were left cultured with or without TPL(100 n M)for 24 h,and then the amount of Beclin1,LC3 and p62 were analyzed by Western Blotting.Results: 1.The results of m RFP-GFP-LC3 adenovirus transfection showed that compared with untreated cells,the number of autophagosomes(yellow spots)and autophagy lysosomes(red spots)in SKOV3/DDP cells increased significantly after 24 hours of AG490 treatment.The results of Western Blot showed that compared with untreated cells,the ratio of p-JAK2/JAK2 and p-STAT3/STAT3 and the expression of p62 protein were dramatically reduced,accompanying an increased level of LC3-II/LC3-I in the AG490 group(P<0.05).2.SKOV3/DDP cells treated with indicated concentrations of TPL(0、25、50、100 n M)for 24 h.Western Blot showed that with the increase of TPL concentration,the ration of p-JAK2/JAK2 and p-STAT3/STAT3 were decreased gradually(P<0.05),the Mcl-1 level was gradually decreased(P<0.05).3.Immunofluorescence assay revealed that the nuclear level of p-STAT3(Tyr705)was dramatically decreased after SKOV3/DDP were treated with 100 n M TPL for 24 h.4.SKOV3/DDP cells were pre-incubated with IL-6(100 ng/ml)for 1 h,then treated with TPL(100 n M)for another 24 h.Western Blot showed that the ration of p-JAK2/JAK2 and p-STAT3/STAT3 and the protein expression of Mcl-1 were dramatically decreased,the ration of LC3-II/LC3-I increased after TPL treatment,which was reversed by IL-6 pretreatment.5.SKOV3/DDP cells were pre-incubated with IL-6(100 ng/ml)for 1 h,then treated with TPL(100 n M)for another 24 h.Immunoprecipitation assay showed that Beclin1 and Mcl-1 immunoprecipitated with each other in SKOV3/DDP cells under basal conditions;whereas the interaction markedly decreased in the presence of 100 n M TPL treatment for 24 h,which was significantly prevented by IL-6 pretreatment.6.SKOV3/DDP cells transfected with Mcl-1 overexpression plasmid were treated with or without TPL(100 n M)for 24 h.Western Blot showed that transient overexpression of Mcl-1 significantly attenuated LC3-II accumulation and p62degradation induced by TPL.7.SKOV3/DDP cells transfected with the Beclin1 siRNA or negative control siRNA were left cultured with or without TPL(100 n M)for 24 h.Western Blot showed that siRNA-mediated Beclin1 downregulation significantly attenuated LC3-II accumulation and p62 degradation induced by TPL.Conclusion: TPL treatment can suppress the JAK2/STAT3 signaling cascade and reduce Mcl-1 expression,thereby alleviating Beclin1/Mcl-1 interaction,which may be part of the molecular mechanism of TPL-induced SKOV3/DDP cell autophagy. |
PDF Full Text Request