The Antitumor Activities Of Stat3 Inhibitor Napabucasin On Ovarian Cancer Cell SKOV-3 And The Related Molecular Mechanism

Objective:The purpose of this study was to investigate the in vitro inhibitory effect of the STAT3 inhibitor Napabucasin?Napa?on human ovarian cancer SKOV-3 cells and the related molecular mechanism,and to further study the antic-ancer effect of combination use of Napa and proteasome inhibitor MG132 on SKOV-3 cells and the synergistic anticancer mechanism,so as to provide theoretical basis for the application of STAT3 inhibitor in the treatment of ovarian cancer.Methods:1.The inhibitory effect of Napa on SKOV-3 cells proliferation was analyzed by MTT assay.2.The effect of Napa on SKOV-3 cell cycle distribution,the expression of cell cycle-related proteins p21,cyclin B1 and cdc2,and the mRNA expression level of p21 were analyzed by flow cytometry,Western Blot and qRT-PCR,respectively.3.Flow cytometry was used to evaluate whether Napa can induce SKOV-3 cells apoptosis.4.Multiple analytical methods such as MDC staining,Western Blot assay and mRFP-GFP-LC3 plasmid transfection method were adopted to examine whether Napa induced autophagy in SKOV-3 cells.5.Anti-tumor activity of Napa after blocking its autophagy-inducing effect by a autophagy inhibitor Baf-A1 was tested by MTT assay.6.Wound healing assay,Transwell migration assay and Transwell invasion assay were used to analyze the effects of Napa on SKOV-3 cells migration and invasion.Then Western Blot method was used to analyze the phosphorylation of Integrin ?1 and expression level of uPA.7.We performed synergisitc assay by using Chou and Talalay's method to examine the anti-cancer effect of combination use of Napa and MG132.Fa-CI data were analyzed by CalcuSyn software to determine drug combination effeciency.8.The effect of the combination use of Napa and MG132 on cell cycle distribution,apoptosis induction and the related mechanism were analyzed by flow cytometry and Western Blot.Multiple analytical methods such as MDC staining,Western Blot assay and mRFP-GFP-LC3 plasmid transfection method were adopted to examine whether Napa combined with MG132 induced autophagy in SKOV-3 cells.Results:1.Napa showed significantly growth inhibitory effect on SKOV-3 cells.MTT assay showed that Napa reduced SKOV-3 cells viability in a dose-dependent manner.The IC₅₀ value was calculated to be 0.649?M.2.Napa induced cell cycle arrest at G2/M phase in SKOV-3 cells.Napa treatment increased the expression of p21 but had no influence in the expression of cyclin B1 and cdc-2.qRT-PCR showed that the level of p21 mRNA also increased after Napa treatment.3.Napa did not induce cells apoptosis of SKOV-3 cells.4.Napa could induce autophagy in SKOV-3 cells.MDC staining assay showed that the amount of the bright green fluorescence near the nuclear was increased in Napa-treated SKOV-3 cells;the expression of autophagy marker proteins including LC3-II and Atg5 increased,p62 protein decreased;a smooth process from forming autophagosome to the degradation of lysosome was observed.5.The anti-cancer activity of Napa on SKOV-3 cells was increased after inhibition of autohpagy by autophagy inhibitor Baf-A1,indicating that Napa-induced autophagy was beneficial to SKOV-3 cells survival.6.Napa inhibited SKOV-3 cells migration and invasion.Wound healing experiment and Transwell migration experiment showed that the mobility of SKOV-3 cells reduced after Napa treatment.Similarly,Transwell invasion assay showed that the invasion rate of SKOV-3 cells also reduced.Western Blot showed that Napa inhibited cells migration and invasion by reducing the phosphorylation of Integrin?1 and the expression of uPA.7.Synergisitc assay showed that combination use of Napa with MG132 at the ratio as IC_{50 Napa}?2×IC_{50 MG132} led to synergistic effect.Combination indexes?CI? were then calculated,with CI<1,suggesting Napa and MG132 synergistic inhibited SKOV-3 cells proliferation under the concentration ratio.8.Studies on the mechanism of drug synergism showed that compared with the single drug group,Napa combined with MG132 exhibited a stronger apoptosis-inducing effect,and the apoptosis-related proteins including caspase-9,caspase-3 and PARP also increased.The autophagy inducing effect by the combination of two drugs was more obvious than using either drug alone.Also,the two drugs combination-induced autophagy showed a protective effect to cells viability.However,the cell cycle distribution in combination group and in the single drug group was very close.Conclusions:1.STAT3 inhibitor Napa significantly inhibited the proliferation of ovarian cancer SKOV-3 cells.The anti-cancer effect can be attributed to the fact that Napa induced cell cycle arrest in G2/M phase by upregulation of the cycle-blocking protein p21.2.Napa induced autophagy in SKOV-3 cells,which reduced the anti-cancer activity of Napa.3.Napa significantly inhibited SKOV-3 cells migration and invasion,indicating that it might be a good candidate to resist ovarian cancer metastasis.4.Napa combined with proteasome inhibitor MG132 showed synergistic anti-tumor effect on SKOV-3 cells,and the synergistic mechanism is mainly reflected in inducing cells apoptosis.