The Mechanism Of Luteolin Increasing Cisplatin Sensitivity Of Ovarian Carcinoma Cells

Part one.Luteolin enhances the sensitivity of ovarian carcinoma cells to cisplatin by inhibiting autophagyBackground and Objective:Ovarian cancer is one of the most lethal gynecologic malignancies which is considered to be a silent killer because of its insidious onset and nonspecific early symptoms.Currently,cisplatin(DDP)-based chemotherapy combined with cytoreduction is the main clinical strategy for ovarian cancer,but is limited by high recurrence and chemoresistance.Luteolin is a natural four hydroxyl flavone compound widely distributed in the plant kingdom.Previous studies reported that higher concentration of luteolin could suppress the proliferation and metastasis of various cancer cells.Lower concentration of luteolin exhibits hypotoxicity to cancer,but it can boost the sensitivity of multiple malignant tumor cells to chemotherapeutic drugs.In ovarian cancer,luteolin reduced the migration and invasion of ovarian cancer cells.However,the effect of luteolin on chemosensitivity of ovarian cancer cells and the mechanism have rarely been reported.Autophagy is an important survival mechanism in response to several types of stress.Previous studies have demonstrated that autophagy plays key role in various types of cancer cells in response to anticancer therapies.Microtubule-associated protein 1 light chain 3(LC3)-Ⅱ is a marker of autophagy and increased LC3-Ⅱ levels indicate that the autophagy has been initiated.Elucidating the mechanisms involved in autophagy may provide effective strategies for cancer therapyIn the present study,we explored whether luteolin could confer cisplatin-sensitivity to SKOV-3 cells and preliminarily reveal the potential mechanism.Methods:1.SKOV3 cells were exposed to an increased concentration of luteolin(0,5,10,20,40,80 and 160 μM)for CCK-8 to identify the IC50 of luteolin.Then luteolin(0,20 and 40 μM)were co-cultured with the gradient increased of cisplatin for 24 h respectively before CCK-8 to evaluate cell viability and calculate IC50 of cisplatin.2.Flow cytometry and Western blot were used to detect the apoptosis rate and the expressions of caspase-3 in SKOV3 cells upon luteolin combined with cisplatin administration.3.Electron microscopy,immunofluorescence and Western blot analysis were performed to observe the autophagic corpuscle formation and the expression of LC3-II in SKOV3 cells upon luteolin combined with cisplatin administration.Results:1.Higher concentration of luteolin could inhibit the proliferation of SKOV3 cells.The IC50 of luteolin was 65 μM,so 20 μM and 40 μM luteolin,which concentration was below the IC50,was chose to compare the inhibitory effect in SKOV3 cells co-cultured with various concentrations of cisplatin.Further studies found that compared with cisplatin-treated SKOV3 cells(cisplatin IC50=21 μM),the proliferation of SKOV3 cells was decreased significantly upon 20 μM or 40 μM luteolin combined with cisplatin administration(IC50 was 14 μM and 10 μM respectively).Therefore,we selected 40 μM luteolin and the corresponding 10 μM cisplatin for subsequent research.2.Compared with control,the apoptosis rate of SKOV3 cells was no significant change with luteolin treatment alone,but was elevated significantly with cisplatin treatment alone.The apoptosis rate of SKOV3 cells upon luteolin and cisplatin co-incubation were 2.25-fold higher than those with cisplatin treatment alone(p<0.001).Similarly,compared with control,the expression of caspase-3 in SKOV3 cells was no significant change under luteolin treatment alone,but was increased significantly under cisplatin treatment alone.The expression of caspase-3 in SKOV3 cells with luteolin and cisplatin co-incubation were 2.15-fold higher than those with cisplatin treatment alone(p<0.001).3.The number of autophagy bodies in SKOV3 cells were increased obviously after cisplatin treatment under electron microscopy,which were apparent decline after luteolin and cisplatin co-incubation.Correspondingly,immunofluorescence and Western blot results showed that the expression of LC3-II in SKOV3 cells with cisplatin treatment alone was significantly upregulated compared with control,which was downregulated by 3.16-fold after luteolin and cisplatin co-incubation compared with cisplatin treatment alone(p<0.001).Conclusions:1.Luteolin increases the sensitivity to cisplatin-mediated suppression of proliferation in SKOV-3 cells.2.Luteolin increases the sensitivity to cisplatin-induced apoptosis in SKOV-3 cells.3.Luteolin enhances the cisplatin-sensitivity of ovarian carcinoma cells by inhibiting autophagy.In summary,our data preliminarily revealed the mechanism by which luteolin enhance the chemosensitivity of ovarian cancer cells.Luteolin enhances the sensitivity of ovarian cancer cells to cisplatin by inhibiting autophagy,so that low concentration of cisplatin exhibit effective cytostatic effect in SKOV3 cells.Therefore,luteolin is expected to be a potential chemosensitizer for ovarian cancer.Part two.Luteolin inhibits autophagy by repressing the expression of PARP1 to confer cisplatin-sensitivity in ovarian carcinoma cells Background and Objective:PARP1,a member of the ribozyme family poly(ADP-ribose)(PAR)polymerases(PARPs),is capable of repairing the damage DNA via base excision repair to maintain DNA stability.Clinical researches reported that PARP1 is upregulated in various malignant tumors,and PARP-1 inhibitors could enhance the therapeutic effects of radiotherapy and chemotherapy.In vitro studies demonstrated that PARP-1 deficient mice were more sensitive to genotoxic drugs.Previous studies found that PARP inhibitors can be used to potentiate chemotherapy and regulate cancer proliferation and apoptosis through inducing autophagy in different type of cancers such as nasopharyngeal carcinoma cells.Moreover,it is reported that PARP1 inhibition could permit a substantial lowering of cisplatin concentrations without diminishing treatment efficacy,potentially reducing systemic side effects.In addition,PARP-1 participates in mammalian rapamycin target protein(mTOR)-mediated autophagy pathway.As our previous data indicated that.luteolin enhanced the sensitivity of ovarian cancer cells to cisplatin by inhibiting autophagy.we further presume that luteolin may inhibit the autophagy of ovarian cancer cells by inhibiting PARP1.Hence,this study investigate the expression of PARP 1 in luteolin and cisplatin co-treated SKOV3 cells.Then we further assessed the molecular mechanism in cisplatin sensitized ovarian cancer cells by which luteolin inhibits the autophagy through silencing and over expressing PARP1 respectively.Methods:1.Real-time PCR(RT-PCR)and Western blot were employed to identify the expression of PARP1 in SKOV3 cells incubated with different concentrations of luteolin(0,10,20 and 40 μM)for different intervals(0,12,24 and 48 h),as well as the expression of PARP1 in SKOV3 cells co-treated with luteolin and cisplatin.2.CCK-8,flow cytometry and Western blot were conducted to measure the proliferation and apoptosis of SKOV3 cells upon luteolin and cisplatin administration after silencing PARP 1 by RNAi.3.Western blot and immunofluorescence assay were used to evaluate autophagy in SKOV3 cells under luteolin and cisplatin co-treatment after silencing PARP1 by RNAi.4.CCK-8,flow cytometry and Western blot were conducted to measure the proliferation and apoptosis of SKOV3 cells upon luteolin and cisplatin administration after overexpressing PARP1.5.Western blot and immunofluorescence assay were used to evaluate autophagy in SKOV3 cells under luteolin and cisplatin co-treatment after overexpressing PARP1.Results:1.Luteolin significantly inhibited the expression of PARP1 at both mRNA and protein levels in dose-dependent and time-dependent manners(p<0.01).Cisplatin notably increased the expression of PARP1,while the mRNA and protein levels of PARP1 in SKOV3 cells with luteolin and cisplatin co-treatment were 2.69 folds and 2.44 folds lower than that with cisplatin treatment alone(p<0.001).2.Both silencing PARP1 in cisplatin treated-SKOV3 cells and administrating luteolin and cisplatin to SKOV3 cells could dose dependently suppress cell proliferation,and the cisplatin IC50s of the two groups are close(≈10 μM).However,the IC50 was 5 μM when silencing PARP1 in luteolin and cisplatin co-treated-SKOV3 cells.Therefore,5 μM cisplatin was used for subsequent experiments.Flow cytometry showed that the apoptosis rate of SKOV3 cells under luteolin and cisplatin co-treatment with siPARPl tranfection was 1.3-fold higher than that without siPARPl tranfection(p<0.001).Similarly,under the same luteolin and cisplatin co-treatment,the expression of caspase-3 in SKOV3 cells with siPARPl transfection was significantly upregulated compared with non siPARPl transfected SKOV3 cells p<0.001),3.Under the same luteolin and cisplatin co-treatment,the expression of LC3-Ⅱ in SKOV3 cells with siPARPl transfection was downregulated by 9 folds compared with non siPARP1 transfected SKOV3 cells(p<0.001).4.Cisplatin IC50 was 8 μM in luteolin and cisplatin co-treated SKOV3 cells,which was elevated to 20 μM when overexpressing PARP1 in SKOV3 cells under the same drugs treatment.Therefore,we chose 5 μM cisplatin for subsequent experiments.Flow cytometry showed that the apoptosis rate of SKOV3 cells with luteolin and cisplatin co-treatment was 1.7 folds higher than that with cisplatin treated NC group,overexpressing PARP1 decreased the apoptosis rate by 1.4 folds under the same drugs treatment(p<0.001).Similarly,compared with NC,the expression of caspase-3 was upregulated in SKOV3 cells with luteolin and cisplatin co-treatment,while overexpressing PARP1 could significantly reverse the expression of caspase-3(p<0.01).5.Compared with NC,the expression of LC3-Ⅱ remarkably downregulated in SKOV3 cells with luteolin and cisplatin co-treatment,overexpressing PARP1 reversed LC3-Ⅱ expression in SKOV3 cells with same drugs treatment.Conclusions:1.Luteolin inhibits autophagy by suppressing the expression of PARP 1.2.Silencing PARP1 further promotes cisplatin sensitivity of ovarian cancer cells in coordination with luteolin.3.Silencing PARP1 further reduced the autophagy of ovarian cancer cells in coordination with luteolin.4.Overexpressing PARP 1 reverses luteolin-increased cisplatin sensitivity of ovarian cancer cells.5.Overexpressing PARP 1 reverses luteolin-reduced autophagy of ovarian cancer cells.Taken together,our study proved that luteolin inhibited the autophagy by decreasing the expression of PARP1,thereby enhancing the sensitivity of ovarian cancer cells to cisplatin.Our data identified the molecular mechanism on luteolin-stimulated chemosensitivity in ovarian cancer cells.Overall,luteolin may become a promising candidate for treating cisplatin-resistant ovarian cancer to increase the duration of survival in ovarian cancer patients.