Knockdown TIGAR And Inhibition Of Autophagy Enhance The Anticancer Effect Of Aescinate Sodium

Recent studies have found that aescinate sodium has anti-tumor effect in vitro and in vivo.TP53-induced glycolysis and apoptosis regulator(TIGAR)plays multiple roles in regulating tumor cell metabolism,oxidative stress,autophagy and apoptosis.However,the roles of TIGAR-regulated oxidative stress and autophagy in the anti-tumor effect of aescinate sodium are still unknown.The aims of this study are to investigate the effects of TIGAR and autophagy on the anti-tumor effect of aescinate sodium and its related mechanisms.Part I: Aescinate sodium inhibits cell proliferation and induces DNA damage,cell cycle arrest and apoptosis in colorectal cancer cells.Aims:Aescinate sodium is mainly used to treat vascular diseases in clinic.Recent studies have found that aescinate sodium has a good anti-tumor effect.The effects of TIGAR and regulated autophagy,which maybe influence the antitumor activity of aescinate sodium,were studied.Therefore,we first examined the regulatory effects of aescinate sodium on the proliferation,DNA damage,cell cycle and apoptosis of HCT116 and HCT-8 colorectal cancer cells,and preliminarily discussed the anti-tumor effects of aescinate sodium and its working concentration and the time course.Methods:CCK-8 and clonogenesis assay were performed to determine the effects of aescinate sodium on the short-term and long-term proliferation of colorectal cancer cells.Western blot(WB)was used to detect the levels of DNA damage-and apoptosis-related proteins.Annexin v-FITC /PI double staining flow cytometry was performed to detect cell cycle and apoptosis in response to aescinate sodium in colorectal cancer cells.Hoechst staining was used to detect DNA damage that was induced by aescinate sodium.Results:1.Aescinate sodium inhibited the short-term and long-term proliferation in HCT116 and HCT-8 colorectal cancer cells.CCK-8 results showed that aescinate sodium inhibited the short-term proliferation of tumor cells in concentration-and time-dependent methods.The proliferation of normal colon epithelial cells(NCM460)was inhibited when the cells were treated with high concentration of aescinate sodium(80 or 100 μg/mL)for 24 or 48 hours.However,low concentrations of aescinate sodium(40 or 60 μg/mL)for 12 hours treatment had little effect on proliferation of NCM460 cells.So,40 or 60 μg/mL aescinate sodium to treat cells for 12 hours was seclected to do the following experiments.In addition,the results of cloning formation experiment showed that aescinate sodium inhibited the formation of cell cloning in a concentration dependent method,and the higher concentration of aescinate sodium,the more obvious inhibitory effect.2.Aescinate sodium induced DNA damage,cell cycle inhibition and apoptosis in colorectal cancer cells.The expression levels of activated PARP(act-PARP)and gamma H2 AX were up-regulated in response to aescinate sodium in HCT116 and HCT-8 cells in concentrationand time-dependent methods,which indicated the DNA damage induction.Similarly,40 and 60 μg/mL of aescinate sodium induced more severe chromosome aggregation and breakage than control group by Hoechst staining.In addition,high concentration of aescinate sodium can induce more G1 phase arrest and apoptosis than low concentration of aescinte sodium.Conclusion: Low concentration aescinate sodium(40 or 60 μg/mL)had no significant inhibitory effect on normal colon epithelial cells.However,it can inhibit the proliferation of colorectal cancer cells in long-or short-time proliferation,and induce cell DNA damage,cell cycle arrest and apoptosis.Aescinate sodium possessed the anti-tumor effect in vitro.Part Ⅱ: TIGAR influence the anti-tumor activity of aescinate sodium in vitro and in vivoAims:TIGAR increases the levels of intracellular NADPH and ribose by inhibiting the flux of glycolysis and increasing the flux of PPP,and plays a key role in regulating cellular stress response,DNA damage,autophagy and apoptosis.Our previous results showed that aescinate sodium had significant anti-tumor effects.In addition,it has been reported to induce oxidative stress in tumor cells.So,is TIGAR involved in regulating the oxidative stress and autophagy,which may affect the anti-tumor effect of aescinate sodium? The purpose of this study is to investigate the effect of TIGAR on the anti-tumor activity of aescinate sodium and to explore whether interfering TIGAR expression could be a new target in increasing the anti-tumor activity of aescinate sodium.Methods:The expression of p53 and TIGAR in colorectal cancer cells were detected by WB in response to aescinate sodium.The apoptosis of colorectal cancer cells that were treated with TIGAR knockdown and/or aescinate sodium was detected by flow cytometry.Meanwhile,the expression of apoptosis-related proteins was detected by WB.TIGAR overexpression plasmid was constructed in HCT116 cells.The expression of TIGAR and related proteins was detected by WB,and the apoptosis was detected by flow cytometry.In the case of TIGAR knockdown,the downstream products of TIGAR including nicoamide adenine dinucleotide phosphate(NADPH)and ribose were supplemented.The effects of the NADPH and ribose on DNA damage and apoptosis induced by aesinate sodium were detected by WB and flow cytometry.TIGAR-shRNA lentivirus was used to construct TIGAR knockdown cell line,which was inoculated subcutaneously in nude mice to do the in vivo experiment.The expression of TIGAR and related proteins in tumor tissues were detected by WB and immunohistochemistry.Results:1.TIGAR was upregulated by aescinate sodium,and TIGAR knockdown increased aesinate sodium-induced apoptosis.Aescinate sodium increased the expression of TIGAR in HCT116 and HCT-8 cells.TIGAR knockdown significantly increased the apoptosis induced by aescinate sodium.Moreover,TIGAR knockdown increased the expression of act-PARP and gamma-H2 AX in response to aescinate sodium.The levels of cleaved caspase-9 and cleaved caspase-3 were also increased,which indicated that TIGAR knockdown augmented the DNA damage and apoptosis in response to aescinate sodium.2.TIGAR overexpression or supplement NADPH and ribose,the downstream products of TIGAR,can partly rescue aescinate sodium-induced DNA damage and apoptosis.The expression of related proteins and apoptosis induced by aescinate sodium were partially inhibited by TIGAR overexpression.Similarly,NADPH and ribose had the same protective functions on partially rescuing the DNA damage and apoptosis.These results indicated that overexpression of TIGAR and supplement of TIGAR downstream products could partially save cell DNA damage and apoptosis,which were induced by aescinate sodium.3.TIGAR knockdown enhanced the anti-tumor activity of aescinate sodium.In addition,TIGAR knockdown could induce more DNA damage in tumor cells than related control groups and significantly inhibit tumor growth,which indicated that TIGAR knockdown enhanced the anti-tumor activity of aescinate sodium in vivo.Conclusion:Aescinate sodium can induce the up-regulation of TIGAR to antagonize the stress-induced injury of tumor cells.TIGAR knockdown removed the protective stress response and enhanced the anti-tumor activity of aescinate sodium.Part Ⅲ: TIGAR knockdown and autophagy inhibition synergistically increased the apoptosis induced by aescinate sodium.Aims:Aescinate sodium induced oxidative stress in tumor cells.A large number of literatures have proved that ROS,as an upstream signal,can activate autophagy of tumor cells.The levels of ROS and autophagy can affect the apoptosis of tumor cells.Our previous study found that TIGAR,as a stress regulating protein,can affect the sensitivity of chemotherapy drugs by regulating intracellular ROS and autophagy.So,does aescinate sodium induce oxidative stress that activates autophagy? Does TIGAR regulate oxidative stress and autophagy,which maybe affect tumor cell apoptosis? The aims of this study were to investigate the effects of TIGAR-regulated ROS and autophagy on the apoptosis in response to aescinate sodium in tumor cells.Methods:The expression of autophagy-related proteins induced by aescinate sodium in HepG2 and HCT116 cells was detected by WB.The autophagy flux in tumor cells was detected by immunofluorescence.ATG5 was knocked down with RNAi,and the effect of ATG5 knockdown was detected by WB.The apoptosis induced by autophagy inhibition and/or aescinate sodium treatment was detected by flow cytometry.ATG5-shRNA knockdown was used to construct stable cell line,and the effect of ATG5 knockdown on the anti-tumor activity of sodium aescinate was observed in vivo.2 ’,7 ’-dichlorodifluorescein diacetate(DCFH2-DA)labeled flow cytometry was used to detect the ROS.Finally,apoptosis-related proteins were detected by WB to explore the effect of TIGAR and autophagy in increasing the anti-tumor effect of aescinate sodium in tumor cells.Results:1.Aescinate sodium induced oxidative stress in HepG2 cells.HepG2 cells were treated with aescinate sodium at different concentrations for 12 hours or with 60 μg/mL of aescinate sodium for 0,3,6,9,12 or 18 hours.The results showed that aescinate sodium induced concentration-dependent ROS up-regulation.Moreover,ROS levels in cells reached a rapid peak after 60μg/mL of aescinate sodium treatment for 3 hours.2.Aescinate sodium activated autophagy and promoted the formation of autophagosomes in HepG2 and HCT116 cells.Apoptosis was induced by aescinate sodium in HepG2 and HCT116 cells,which was consistent with the previous results.At the same time,we found that aescinate sodium can obviously increase the levels of autophagy-related proteins ATG5 and LC3-Ⅱ,which indicated the activation of cell autophagy.As the immunofluorescence results shown,aescinate sodium promoted the formation of autophagosomes in tumor cells.However,the activation of autophagy by aescinate sodium was not dependent on the m TOR signaling pathway.3.ATG5 knockdown or the inhibition of autophagy by 3-MA increased apoptosis induced by aescinate sodium.In HepG2 cells,ATG5 knockdown significantly increased the apoptosis induced by aescinate sodium.The expression levels of act-PARP and cleaved caspase-3 were futher increased by ATG5 knockdown in response to aescinate sodium.In HCT116 cells,the apoptosis was further induced by 3-methyladenine(3-MA),an inhibition of autophagy,in response to aescinate sodium.4.Inhibition of autophagy enhanced the anti-tumor activity of aescinate sodium in vivo.After inhibition of autophagy by ATG5 lentivirus(LV-shRNA ATG5),the growth of tumor cells in nude mice was significantly inhibited,and the anti-tumor activity of aescinate sodium was enhanced in vivo.5.TIGAR knockdown increased the levels of autophagy and ROS induced by aescinate sodium.In order to further study the effects of TIGAR on ROS and autophagy induced by aescinate sodium,we tested the levels of ROS and autophagy,which were induced by aescinate sodium after TIGAR knockdown.Reduction of TIGAR can obviously increase of the levels of ROS and LC3-Ⅱ,and activate autophagy in tumor cells.6.TIGAR knockdown and autophagy inhibition synergistically increased aescinate sodium-induced apoptosis.The expression of TIGAR and ATG5 was reduced by TIGAR si RNA1 and ATG5 si RNA2.Cells were treated with aescinate sodium for 12 hours.Knockdown of TIGAR and ATG5 futher increased the expression of act-PARP,gamma-H2 AX and act-caspase-9 in response to aescinate sodium,which indicated the increase of DNA damage and apoptosis.Similarly,inhibition of autophagy by 3-MA and combination with TIGAR knockdown could also increase the expression of these proteins.TIGAR knockdown combined with autophagy inhibition synergistically increased DNA damage and apoptosis induced by aescinate sodium.Conclusion:Oxidative stress and protective autophagy were induced by aescinate sodium in tumor cells.Inhibition autophagy enhanced the anti-tumor activity of aescinate sodium.TIGAR knockdown enhanced ROS and autophagy in response to aescinate sodium.TIGAR knockdown combined with the inhibition of autophagy futher increased aescinate sodium-induced apoptosis.Conclusions for the full text: In this paper,we discussed the internal and external functions of TIGAR and autophagy on the anti-tumor effect of aescinate sodium via TIGAR knockdown and overexpression.We can draw the following conclusions:(1)Aescinate sodium possesses anti-tumor effect.At the same time,it activates a strong protective response including the increase of TIGAR expression and activation of autophagy.However,TIGAR upregulation and autophagy activation have an intrinsic relationship with the anti-tumor effect of aescinate sodium.(2)TIGAR inhibits oxidative stress,DNA damage and autophagy by increasing intracellular NADPH and Ribose in tumor cells.TIGAR knockdown auguments oxidative stress and DNA damage in response to aescinate sodium and further activates intracellular protective autophagy.Inhibition of autophagy and TIGAR knockdown enhance the anti-tumor activity of aescinate sodium in vitro.(3)While targeting TIGAR to enhance the anti-tumor activity of aescinate sodium is a good therapeutic strategy,it is necessary to inhibit the protective autophagy of tumor cells to maximize the anti-tumor activity of aescinate sodium.