Antitumor Effect And Mechanism Of PAB On Cervical Cancer Cells In Vitro Through Regulating PAX2

Background and objectiveThe morbidity and mortality of cervical cancer rank fourth among all cancers in women.Although new curative treatments of cervical cancer have emerged in recent years,the curative effect on advanced or recurrent cases is still poor,so it is still desirable to develop novel and efficient treatment methods,as well as drugs.Pseudolaric acid B(PAB)is a biologically active diterpenoid isolated from Tujingpi,which has been proven to have antitumor effect.However,the therapeutic effect on cervical cancer and its molecular mechanism is still unclear.Paired Box 2(PAX2)is a member of the PAX family of transcription factors.Studies have shown that PAX2 is reactivated in a variety of tumor tissues and exhibits characteristics that promote cancer cells proliferation,survival and migration,and may mediate the antitumor effect of PAB.This study intends to confirm the antitumor effect of PAB on cervical cancer cells in vitro,and explore the regulatory relationship between PAB and PAX2,so as to reveal the molecular mechanism of PAB antitumor effect,and provide theoretical basis for the further development and clinical application of PAB.Methods1.Antitumor effect of PAB on cervical cancer cells in vitro: MTT assay was used to detect the effect of PAB on the proliferation activity of cervical cancer cells,Hoechst 33258 staining and flow cytometry were used to detect the effect of PAB on HeLa cells apoptosis,Transwell chamber migration experiment was used to detect the effect of PAB on the migration ability of HeLa cells,Real-time fluorescent quantitative PCR was used to detect the effect of PAB on the levels of apoptosis and metastasis-related mRNA in HeLa cells,Western blot assay was used to detect the effect of PAB on the levels of apoptosis and metastasis-related proteins in HeLa cells,Cell mitochondrial isolation kit was used to detect the effect of PAB on the release ofCytochrome C(CytC)from mitochondria in HeLa cells.2.Exploration of antitumor effect and its mechanism of PAX2 mediated PAB on cervical cancer cells in vitro: Lentiviral shRNA interference technology was used to construct the PAX2 low expression HeLa cells,Flow cytometry was used to detect the effect of PAB and low expression of PAX2 on HeLa cells apoptosis,Transwell chamber migration experiment was used to detect the effect of PAB and low expression of PAX2 on the migration ability of HeLa cells,Double luciferase reporter assay was used to detect the interaction of PAX2 and BAX gene,Chromatin immunoprecipitation assay was used to verify the interaction of PAX2 and BAX gene,TOPflash fluorescence activity assay was used to detect the effect of PAB on Wnt/?-catenin signaling pathway,Real-time fluorescent quantitative PCR was used to detect the levels of apoptosis and PAX family-related mRNA in HeLa cells,Western blot assay was used to detect the levels of apoptosis,metastasis,Wnt/?-catenin signaling pathway and PAX family-related proteins in HeLa cells.Results1.PAB can inhibit cervical cancer cells proliferation in a time-and concentration-dependent manner.PAB can induce apoptosis and inhibit migration of HeLa cells in a concentration-dependent manner.PAB has no significant effect on the levels of Fas,Caspase-8,NIK mRNA in the Fas pathway and GRP78,Caspase-12,Calpain mRNA in the ERS apoptosis pathway,but can promote the level of CHOP mRNA in the ERS apoptosis pathway.PAB can induce a significant increase in the levels of Caspase-3,Caspase-9,BAX,CytC,Apaf-1 mRNA and BAX,CytC,Apaf-1 proteins in HeLa cells in a concentration-dependent manner,and significantly inhibit the levels of BCL-2mRNA and its protein;the different concentrations of PAB does not significantly affect the precursor content of Caspase-3 and Caspase-9 at the protein level,but significantly increases the content of Cleaved Caspase-3 and Cleaved Caspase-9 in a concentration-dependent manner.PAB can induce the release of CytC from the mitochondria of HeLa cells to the cytoplasm in a time-dependent manner.Mdivi-1,an inhibitor of mitochondrial outer membrane permeability,can reverse the decrease of BCL-2 mRNA and the increase of Caspase-9 and BAX mRNA in HeLa cells induced by PAB.PAB can reduce the levels of MMP2 and MMP9 proteins in HeLa cells in a concentration-dependent manner.2.PAB can inhibit the levels of PAX2 and PAX8 mRNA in HeLa cells,and has nosignificant effect on the levels of PAX3,PAX5 and PAX7 mRNA.The level of PAX2 protein in all cervical cancer cells are HeLa,SiHa,CaSki,C33 A,and MS751 in descending order,and are negatively correlated with IC50 of PAB on all cervical cancer cells at 24,48,and 72 h.PAB can inhibit the level of PAX2 protein in HeLa cells,PAB and low expression of PAX2 can induce HeLa cells apoptosis,and decrease the levels of BCL-2,Cyclin D1,Survivin proteins and increase the level of BAX protein;PAB and low expression of PAX2 can inhibit HeLa cells migration,and reduce the levels of MMP2 and MMP9 proteins.Overexpression of PAX2 binds to the promoter of BAX,significantly reduces the luciferase activity regulated by the BAX promoter in HeLa cells;Overexpression of PAX2 can significantly inhibit the levels of BAX,Caspase-3 mRNA and promote the level of BCL-2 mRNA in HeLa cells,while overexpression of PAX2 in HeLa cells simultaneously overexpression of BAX can reverse the effect of overexpression of PAX2 on BAX,Caspase-3 and BCL-2 mRNA.PAB can inhibit the levels of Wnt2,Wnt10 b,Wnt13 and Wnt14 proteins in HeLa cells,and has no significant effect on the levels of Wnt4,Wnt5 a and Wnt11 proteins;PAB can significantly reduce ?-catenin transcriptional activity in HeLa cells;PAB can increase the ratio of Cleaved Caspase-3/Caspase-3 and p-?-catenin/?-catenin,and reduce the level of PAX2 protein and the ratio of p-GSK-3?/GSK-3? in HeLa cells;When Tideglusib activates the Wnt signaling pathway,the ratio of Cleaved Caspase-3/Caspase-3 and p-?-catenin/?-catenin decrease,and the level of PAX2 protein and the ratio of p-GSK-3?/GSK-3? increase in HeLa cells,and the addition of PAB can reverse the effect of Tideglusib on HeLa cells;When IWP-O1 inhibits the Wnt signaling pathway,the ratio of Cleaved Caspase-3/Caspase-3 and p-?-catenin/?-catenin increase,and the level of PAX2 protein and the ratio of p-GSK-3?/GSK-3?decrease in HeLa cells,which is consistent with the effect of PAB on HeLa cells.Conclusions1.PAB inhibits cervical cancer cells proliferation,and induces apoptosis in HeLa cells and inhibits its metastasis.2.PAB induces HeLa cells apoptosis through the mitochondrial apoptosis pathway.3.PAB inhibits the level of PAX2 in HeLa cells,and then induces apoptosis in HeLa cells and inhibits its metastasis.4.PAX2 binds to the promoter of BAX and negatively regulates the transcriptionof BAX,and thereby inhibiting HeLa cell apoptosis.5.PAB reduces the level of PAX2 by inhibiting the Wnt/?-catenin signaling pathway,relieves the inhibition of PAX2 on BAX expression,and then induces HeLa cells apoptosis.