Study On The Activity Of Rhein Mediates That Rhein Against Colon Cancer And Increases EGFR Inhibitor Sensitivity Via Inhibiting STAT3

ObjectiveTo investigate STAT3 inhibitors feedback activating EGFR,and explore the effects of the combination of Rhein and EGFR inhibitors in vivo and in vitro on the proliferation,apoptosis,and migration of colorectal cancer cells.Methods Parting One1.The effect of STAT3 small molecule inhibitor(LY5)on the expression of p-STAT3,p-EGFR(Y1068),p-ERK1/2,and p-AKT in colorectal cancer cells was detected by Western blot.Cell scratch assay was used to explore the effect of STAT3 small molecule inhibitor(LY5)and EGFR inhibitor(Erlotinib)used alone or in combination on the cell migration ability of colorectal cancer HCT-116 and DLD-1 cell lines.MTT assay was used to detect the inhibitory effect of STAT3 inhibitor(LY5)and EGFR inhibitor(Erlotinib)used alone or in combination on HCT-116 and DLD-1 cell survival.2.Rhein is similar to the four known STAT3 inhibitors on the structure,Napabucasin,STA-21,LLL12,and LY5.Using the molecular docking simulation experiments of the STAT3 SH2 domain,a computer-aided preliminary simulation of the docking results showed the relationship between Rhein and STAT3 protein.3.In thecolorectal cancer cell lines HCT116 and SW620,the experiments were divided into 4 groups,DMSO / Rhein / Erlotinib / Erlotinib+Rhein.Survival rate and IC50 were calculated by MTT assay,exploring the effect of Rhein and EGFR inhibitor(Erlotinib)used alone or in combination on the survival rate of colorectal cancer cells.And Western blot was used to investigate the effect of Rhein on the expression of p-STAT3 in colorectal cancer cells.4.Toinvestigate the effect of Rhein and EGFR inhibitor(Erlotinib)used alone or in combination on the cell cycle of colorectal cancer HCT-116 and SW-620 cell lines by flow cytometry,and expore the expression of cell cycle related proteins Clin B1 and CDC2 by Western blotting,aiming to point out the mechanism of cell cycle arrest effect of Rhein and Erlotinib used alone or in combination on the colorectal cancer cells.5.Flow cytometry experiments were performed on colorectal cancer cells to detect early and late apoptosis.The expression of apoptosis-related protein Bcl-2 in HCT-116 cells weredetected by Western blot,aiming toexpore the induction effect of Rhein and Erlotinib used alone or in combination on the apoptosis of colorectal cancer cells.6.To observe the change of morphology of colon cancer cells HCT-116 under a microscope and investigate the cell deformability and colony formation ability of HCT-116 colorectal cancer cells treated with Rhein and Erlotinib alone or in combination by Colony test.Parting Two1.Construct a nude mouse tumor model and randomly divide the experimental nude mice into four groups,control / Rhein / Erlotinib / Erlotinib+Rhein.During the administration period,take photoes and record the tumor volume and quality,draw the relevant graphs.2.The expression of p-STAT3,p-EGFR and pro-apoptosis protein Bax was detected by Western blotting.Explore the anti-tumor effect of single drug and combination drug in vivo.3.By comparing the changes in body weight of mice before and after administration in each group,and observing tissue analysis,we evaluate the safety of Rhein and Erlotinib in combination.Results1.Western blot indicated the detection of p-STAT3,p-EGFR(Y1068),p-ERK1/2,and p-AKT that in the colorectal cancer cells,LY5 as a STAT3 inhibitor can also induce the phosphorylation of EGFR,which further activates the EGFR signaling pathway.The cell scratch test demonstrated that the combined use of STAT3 inhibitor(LY5)and EGFR inhibitor(Erlotinib)significantly inhibited the cell migration ability of colorectal cancer HCT116 and DLD-1 cell lines(P<0.05).MTT assay showed that the combined use of STAT3 inhibitor(LY5)and EGFR inhibitor(Erlotinib)inhibited the survival of HCT-116 and DLD-1 cells with a statistically significant difference(P<0.05).2.Molecular docking simulation experiments showed that Rhein maycombine directly with STAT3 protein and affect its Y705 phosphorylation active site.3.From the molecular cell level,detection of p-STAT3(Y705)by Western blot showed that Rhein inhibited the phosphorylation of STAT3 in colorectal cancer cells and inhibited the proliferation and apoptosis of colorectal cancer cells.MTT experiments found that Rhein inhibited the growth of colorectal cancer cells in a dose-dependent manner.Erlotinib,as an EGFR inhibitor combined with Rhein,had significantly better inhibitory activity against colorectal cancer than either(P<0.05).Cell cycle experiments showed that the two drugs synergistically enhanced of the cell G2/M phase arrest.Down-regulation of Clin B1 and CDC2 cell cycle arrest-related protein expression further confirmed that the combined use of Rhein and Erlotinib enhanced the cycle arrest effecton colorectal cancer cells more than single use(P<0.05).Apoptosis results showed that Rhein could promote the apoptosis of HCT-116 and SW-620 cell lines.The combined use of drugs significantly increasing the apoptosis rate and down-regulated expression of Bcl-2indicated that the combined use could synergistically induce apoptosis of colorectal cancer cells(P<0.05).Rhein can inhibit the colony formation and promote cell morphology changes of HCT-116 cells,which demonstrated that the combined use of EGFR inhibitors(Erlotinib)and Rhein in colorectal cancer cell lines significantly attenuated cell viability than use alone.4.The vivo experiments in nude mice showed that the volume and quality in the combined administration group were significantly lower than that of the single drug group,suggesting that combination therapy can significantly inhibit tumor growth.Western blot analysis confirmed that the combination therapy up-regulated the expression of pro-apoptotic protein Bax,and significantly inhibited the expression of p-STAT3 and p-EGFR comparing with the use of drugs alone.The results of histological analysis indicated that the combined administration had no obvious toxicity to the normal tissues of mice.Conclusions1.At the molecular and cellular levels,STAT3 inhibitors have been shown to activate EGFR signaling pathways in a feedback manner,and the combined use of STAT3 and EGFR inhibitiors have synergistic anti-tumor effect.2.The new STAT3 inhibitor Rhein has better anti-tumor activity.Based on the simulation docking of Rhein and STAT3,STAT3 may be the target of Rhein.3.At the cytological level,Rhein exerted an anti-tumor effect through inhibiting STAT3 and increased the Antitumor activity of EGFR inhibitors.And the combined use of Rhein and EGFR inhibitors can synergistically promote apoptosis and inhibit cell proliferation.4.At the animal level,Rhein and EGFR inhibitors also showed a good synergistic anti-tumor effect.Rhein can effectively reverse the resistance of EGFR inhibitors and has a good safety.