Molecular Mechanism Of Curcumin On Ovarian Cancer Cells

Introduction:Ovarian cancer is the most lethal malignancy in gynecological field due to difficulty to early detect.Ovarian cancer is estimated to die nearly 13,980 women in the United States in 2019.Standard treatments for ovarian cancer include cytoreductive surgery and chemotherapy drugs.However,the recurrence rate of ovarian cancer still exceeded 85%.The emergence of drug resistance affects the PFS and OS of EOC.Therefore,the molecular mechanism to reverse drug resistance of ovarian cancer has attracted much attention.Curcumin,a common Chinese medicine,a yellow-colored dietary flavour from the plant(Curcuma longa),has been reported to be effective in the treatment of ovarian cancer and put into several clinical trials owing to its apoptotic effect.Although several studies have reported that curcumin can induce apoptotic cell death and play an anti-cancer role,the drug resistance of curcumin limits its clinical utilization in the treatment of ovarian cancer.Recent studies have shown that drug resistance induced by curcumin is closely related to autophagy.Autophagy mainly maintains intracellular homeostasis.More and more evidence shows that curcumin-induced autophagy is not only a pro-death signal,but also an adaptation to stress to avoid cell death and apoptosis,leading to chemotherapy resistance.Most studies have shown that drug resistance on ovarian cancer is associated with protective autophagy.In addition,curcumin has been identified as an autophagy inducer in various cancer studies,such as melanoma,glioma,breast cancer and oral cancer.Therefore,inhibition of autophagy may become an effective way to overcome the resistance of curcumin on ovarian cancer.Autophagy inhibitors,such as chloroquine,in combination with chemotherapy drugs and radiotherapy,have enhanced anti-tumor effect,suggesting an important role in anti-cancer treatment.Thus,autophagy inhibitors are expected to address the resistance of curcumin on ovarian cancer.This study is to investigate the molecular mechanism of curcumin in the treatment of ovarian cancer cell resistance,the key role of protective autophagy in curcumin-induced resistance and the mechanism of autophagy inhibitors to reverse the drug resistance of curcumin-induced ovarian cancer.Objective:The purpose of this paper is to investigate the molecular mechanism of curcumin in the treatment of ovarian cancer cell resistance,the key role of protective autophagy in curcumin-induced resistance and the mechanism of autophagy inhibitors to reverse the drug resistance of curcumin-induced ovarian cancer.Materials:In this study,SK-OV-3 and A2780 human EOC cell lines were purchased from American typical culture collection(ATCC).Human EOC cell line HO-8910 and human embryonic kidney cell line HEK-293T were purchased from the Chinese academy of sciences(Shanghai).SK-OV-3 and A2780 human EOC cell lines were transfected with shRNA and selected by puromycin to construct stable SK-OV-3-pLKO.1-shLC3B and A2780-pLKO.1-shLC3B cell lines,and the corresponding control cell lines SK-OV-3-pLKO.1-NC and A2780-pLKO.1-NC were established.Methods:MTT assay,EdU incorporation assay,colony forming assay and flow cytometric analysis of apoptosis were used to detect the effect of different concentrations of curcumin on SK-OV-3 and A2780 cell survival rate in different time.Western blot was used to analyze the expression of apoptosis-related proteins on curcumin-treated SK-OV-3 and A2780 cells.Autophagy of SK-OV-3 and A2780 cell lines was verified by electron microscopy,which was used to observe autophagosome directly,and fluorescence microscopy,which was used to detect LC3-labeled immunofluorescence of autophagosome.Western blot and immunofluorescence staining assay were used to detect the expression of autophagy-related proteins on curcumin-treated SK-OV-3 and A2780 cells and the target proteins of autophagy-related AKT/mTOR/p70S6K signaling pathway.LY294002,a PI3K inhibitor,and the plasmids to construct AKT-overexpressed SK-OV-3 cell line were used to verily the correlation between the protective autophagy induced by curcumin in ovarian cancer cell lines and the target proteins in the autophagy-related AKT/mTOR/p70S6K signaling pathway by western blot.LC3B,a key autophagy target,was stably knocked down by chloroquine and shRNA.The autophagy induction effect of curcumin on SK-OV-3 and A2780 cell lines was demonstrated by MTT assay,EdU incorporation assay,flow cytometric analysis of apoptosis and western blot.SPSS 23.0 statistical software was used to analyze the experimental data.P<0.05 was statistically significant.Three independent experiments were performed for statistical analysis.The results were shown in the form of mean and SD.Results:1.Effect of curcumin on cell viability of SK-OV-3,A2780 and HO-8910 cell lines.Effect of curcumin on cell viability of SK-OV-3,A2780 and HO-8910 cells were detected by MTT assay.We examined the effect of curcumin on cell viability in three ovarian cancer cell lines(SK-OV-3,A2780 and HO-8910 cell lines).The cells were respectively treated with different concentrations(0,5,10,20,40,80 μM)of curcumin for 24,48 and 72h.The results showed that curcumin repressed proliferation of all three types of ovarian cancer cells by time-dependent and dose-dependent manner.The results above were statistically significant(P<0.05).The IC50 values of curcumin were 41.05,31.84 and 30.29 μM for SK-OV-3 cell line at 24,48 and 72h,respectively;18.37,13.26 and 13.83 μM for A2780 cell line at 24,48 and 72h,respectively;and 18.93,14.46,and 13.92μM for HO-8910 cell line at 24,48 and 72h,respectively.We utilize the colony formation assay to observe the long-term inhibitory effect of curcumin on EOC cell proliferation.Over 10μM,curcumin suppressed colony-forming ability of EOC cells significantly for SK-OV-3 cell line.To test whether curcumin had a direct impact on cell proliferation,we used EdU staining assay to find that treatment with curcumin for 48 h significantly inhibited EdU uptake rate in a dose-dependent manner in SK-OV-3 cells.Collectively,these results suggested that curcumin has a killing effect on all three ovarian cancer cell lines.The results above were statistically significant(P<0.05).2.Effect of curcumin on apoptosis of SK-OV-3 and A2780 ovarian cancer cells.To study the underlying mechanism by which curcumin inhibited cell growth in ovarian cancer cells,apoptosis assay was performed by flow cytometry.We detected that 40μM curcumin could significantly induce apoptosis in SK-OV-3 cell line while 30 μM curcumin triggered apoptosis in A2780 cell line.The results above were statistically significant(P<0.05).We analyzed the effects of curcumin on activity of caspases and PARP.Western blotting assay showed that curcumin activated Caspase-9 and PARP,suggesting that curcumin-induced cell death might be dependent of apoptosis by caspases activation in EOC cells.3.The mechanism of curcumin-induced autophagy in SK-OV-3 and A2780 ovarian cancer cells.We used electron microscopy to observe the ultrastructure of A2780 cells.The results indicated the presence of a large number of autophagic vesicles in curcumin-treated A2780 cells as compared to the control A2780 cells.In addition,increased formation/expression of LC3B-Ⅰ/Ⅱ in response to curcumin was confirmed by immunofluorescent staining of SK-OV-3 cells in culture.SK-OV-3 cells treated with 40μM curcumin exhibited increasing puncta formation and fluorescence intensity of LC3B compared with the control group significantly,suggesting that treatment of curcumin resulted in autophagy of SK-OV-3 cells.The results above were statistically significant(P<0.05).We utilized western blot analysis to evaluate expression levels of certain proteins related to autophagy.The cells were respectively treated with different concentrations of curcumin for 48h.Proteins from SK-OV-3 and A2780 ovarian cancer cell lines were extracted to detect the expressions of autophagy-related target molecules such as LC3B,Atg3,beclin-1 and P62 by western blot.Exposure to curcumin dramatically increased the expression levels of LC3B-II in a dose-dependent manner,compared with the control group.Besides,curcumin increased the expression of Atg3,Beclin1 and decreased the expression of P62 in a concentration-dependent manner.These results indicated that curcumin induced autophagy in SK-OV-3 and A2780 ovarian cancer cells.4.Autophagy inhibitors increased the sensitivity of curcumin against SK-OV-3 and A2780 ovarian cancer cells.The role of curcumin-induced autophagy in SK-OV-3 and A2780 ovarian cancer cells was investigated by using autophagy inhibitors.The cell viability examined by using the MTT assay was significantly decreased in the combined treatment of CQ and curcumin,compared with curcumin treatment alone in SK-OV-3 and A2780 ovarian cancer cell lines.Moreover,the apoptosis assay confirmed that CQ-curcumin combined group had more apoptotic cell death than curcumin group.To determine whether CQ-curcumin combination has a direct impact on cell proliferation,we found that the combined treatment of CQ and curcumin significantly inhibited EdU uptake rate,compared with curcumin treatment alone in SK-OV-3 ovarian cancer cell line.Taken together,these results indicated that blockage of autophagy aggravated curcumin-induced apoptosis and CQ could be used in combination with curcumin drug to treat ovarian cancer.The results above were statistically significant(P<0.05).5.Role of LC3B in curcumin-induced autophagy of SK-OV-3 and A2780 ovarian cancer cells.We silenced the expression of LC3B and examined the modulation of LC3B expression by curcumin treatment combined with shRNA-induced LC3B knockdown in SK-OV-3 and A2780 ovarian cancer cells.SK-OV-3 and A2780 human EOC cell lines were transfected with shRNA and selected by puromycin to construct stable SK-OV-3-pLKO.1-shLC3B and A2780-pLKO.1-shLC3B cell lines,and the corresponding control cell lines SK-OV-3-pLKO.1-NC and A2780-pLKO.1-NC were established.Western blot assay showed that transfection with shLC3B blocked the autophagic effect of curcumim-treated SK-OV-3 and A2780 cells.LC3B protein expression levels were significantly increased in SK-OV-3 and A2780 cells treated with LC3B shRNAs and curcumin in comparison to cells treated with LC3B shRNAs alone.The results above were statistically significant(P<0.05).Additionally,to assess the effects of LC3B shRNAs on curcumin-mediated cell apoptosis,we found that the protein expression of cleaved Caspase-9 and cleaved PARP increased consistently,demonstrating that combination group of LC3B shRNAs and curcumin had more apoptotic cell death than curcumin group.Collectively,these findings suggested that downregulation of LC3B enhanced the sensitivity of SK-OV-3 and A2780 cells toward curcumin treatment.6.The mechanism of protective autophagy of curcumin in SK-OV-3 and A2780 ovarian cancer cells.The phosphorylation levels of AKT,mTOR as well as downstream factors of mTOR,such as p70S6K and 4E-BPI,were markedly decreased in the ovarian cancer cells treated with different concentrations(0,10,20,40μM for SK-OV-3 cell line and 0,7.5,15,30μM for A2780 cell line)of curcumin respectively.The results above were statistically significant(P<0.05).These data suggested that the curcumin-induced protective effects on ovarian cancer cells might depend on inhibition of AKT/mTOR pathway.We transiently transfected SK-OV-3 and A2780 ovarian cancer cells with an AKT overexpression plasmid,which is a vector constitutively expressing the active form of AKT1.The overexpression of AKT1 reduced expression of LC3B-Ⅱ,leading to the reversal of curcumin-induced cell death.The results above were statistically significant(P<0.05).Given the high level of activated AKT in SK-OV-3 and A2780 ovarian cancer cells,the pretreatment of cells with LY294002,an inhibitor of PI3K,suppressed the function of AKT,as demonstrated by higher expression of LC3B-II,contributing to curcumin-induced cell death markedly.These results indicated that the downregulation of AKT signaling pathway markedly contributed to curcumin-induced cell death and autophagy in SK-OV-3 and A2780 ovarian cancer cells.The results above were statistically significant(P<0.05).Conclusion:1.Curcumin plays an anti-cancer role on ovarian cancer cells by inducing apoptosis and inhibiting the proliferation of ovarian cancer cells.2.Curcumin induces protective autophagy by inhibiting AKT/mTOR/p70S6K pathway in human ovarian cancer cells resulting in drug resistance.3.Chloroquine as an autophagy inhibitor sensitized ovarian cancer cells to curcumin.Innovation:1.It was the first time to find curcumin combined with autophagy inhibitors exerted synergistic anti-ovarian cancer effect;2.It was proposed that autophagy inhibitors could enhance curcumin’s anti-ovarian cancer effect by inhibiting curcumin-induced protective autophagy.3.It provides a new strategy to solve apoptosis resistance and chemotherapy resistance on ovarian cancer cells.Limitations:1.It was not studied in vivo and clinical trials about curcumin-treatment of ovarian cancer.2.It failed to explore the main target molecules of curcumin.