| Background: Gastric cancer remains the most malignant tumor with high morbidity and mortality throughout China and worldwide.In China,more than 60% of patients diagnosed gastric cancer were in advanced period.Side effects of conventional chemotherapy drugs limit the treatment of advanced gastric cancer.Therefore,molecular targeted therapy has gradually become a research focus of anti-tumor therapy.However,the presence of primary drug-resistant or secondary drug-resistant of targeted therapy has become a clinical problem.Studying the regulatory mechanism of gastric cancer targeted therapy,finding ways to increase its sensitivity will provide new ideas and methods for clinical treatment of gastric cancer.Receptor tyrosine kinases(RTKs)are the largest enzyme-linked receptors,which are both receptors and enzymes.After binding to ligands,phosphorylating the tyrosine residues of target proteins,they activate downstream signal pathways.Abnormal expressions of RTKs are associated with many tumor occurrence and development.Drugs targeted receptor tyrosine kinases have been a hot spot for advanced cancer treatment.Cetuximab,an Ig G1 anti-EGFR m Ab,can bind specially to the extracellular domain of EGFR on both normal and tumor cells,thus competitively inhibiting the binding of EGF.Cetuximab is the first anti-EGFR antibody to be approved by the US FDA since 2004 for treating squamous cell carcinomas and colorectal cancer,which can inhibit the proliferation of cancer cells,induce apoptosis of cancer cells and reduce the matrix metalloproteinases and vascular endothelial growth factor.Gastric cancer is the third most prevalent malignancy and the second most common cause of cancer-related deaths worldwide.Although combination of cetuximab with chemotherapy for advanced gastric cancer(GC)show an active result in phase 2 trials,in phase 3 trial,addition of cetuximab don't provided additional benefit to chemotherapy alone.So we conducted this study to explore more effective drugs,increasing cetuximab sensitivity by durg combination.Berberine is a traditional Chinese medicine widely used in the treatment of intestinal infections,and now scientists have found that berberine had great potential in cancer treatment.In recent years,some studies have suggested that berberine can inhibit tumor cell proliferation,promote tumor cell apoptosis,inhibit tumor cell metastasis and enhance the sensitivity of tumor cells to chemotherapy drugs.In addition,inhibition of EGFR by berberine has recently been reported in human cancers.In this study,we hypothesized that berberine targeted EGFR signaling and enhanced efficacy of EGFR inhibitors in gastric cancer.This study sought to determine the impact of berberine on human gastric cancer cell sensitivity to EGFR inhibitors in vitro and in vivo through inhibition of EGFR/STAT3 signaling and inducing apoptosis and cell cycle arrest.This study is helpful to reveal the new target of berberine on gastric cancer,and to elucidate the molecular mechanism of berberine to increase the sensitivity of EGFR inhibitors,and to provide a theoretical basis for the clinical application of berberine in the treatment of gastric cancer.Part? Effect of berberine on the sensitivity of EGFR inhibitors in gastric cancer cell linesPurposes: To study the effect of berberine on the sensitivity of EGFR inhibitors in gastric cancer cells and its possible target.Methods:The gastric cancer cells were treated with different concentrations of berberine,and the inhibition of cell proliferation was detected by MTT assay.Erlotinib or cetuximab combined with different concentrations of berberine on gastric cancer cells,MTT assay to determine cell proliferation inhibition,to find whether berberine can increase erlotinib / cetuximab sensitivity to gastric cancer cells.Flow cytometry was used to analyze the changes of apoptosis and cell cycle after treated with berberine alone,erlotinib alone and berberine combined with erlotinib on gastric cancer cells.Results:1.Effects of berberine on cell proliferation of human gastric cancer cells The proliferation of gastric cancer cells under different concentrations of berberine was detected by MTT.It was found that the inhibitory effect on the proliferation of gastric cancer cells was enhanced with the increase of the concentration of berberine and the prolongation of the time.The apoptotic rate significanty increased after treatment of different concentrations of berberine in gastric cancer cells(P <0.05).2.Effects of berberine on apoptosis and cycle arrest of gastric cancer cells The percentage of G0 / G1 phase cells increased after berberine intervention in gastric cancer cells,and the difference was statistically significant(P <0.05).3.Effects of berberine combined with EGFR inhibitors on proliferation inhibition of gastric cancer cellsGastric cancer cells were treated with berberine alone,EGFR inhibitors alone,and berberine combined with EGFR inhibitors for 48 hours.MTT was used to observe the proliferation inhibition of gastric cancer cells in different treatment groups.One-way ANOVA test showed that the inhibitory effect of combination therapy on gastric cancer cell proliferation was significantly stronger than that of single drug group,and the synergistic effect was dose-dependent(P <0.05).Therefore,It suggest that berberine combined with EGFR inhibitors synergistically inhibit the proliferation of gastric cancer cells.Calcu Syn software was used to calculate the combined index of combination treatment.With the increasing concentration of berberine,the inhibitory effect of proliferation was gradually increased,CI value was less than 1.It was confirmed that the combination of berberine and EGFR INHIBITORS had synergistic effect on the inhibition of gastric cancer cell proliferation.4.Effects of berberine on apoptosis and cell cycle arrest induced by erlotinib in gastric cancer cells.The effects of berberine and erlotinib,berberine and erlotinib alone on the apoptosis and cell cycle of gastric cancer cells were detected by flow cytometry.The results showed that the apoptotic rate of gastric cancer cells treated by berberine and erlotinib alone increased,apoptosis rates were(20.5 ± 4.5)% and(10.6 ± 2.3)% respectively,which were higher than those of untreated group(P <0.05).After combination treatment,apoptosis significantly increased,was(55.6 ± 8.8)%(combination vs.erlotinib,P=0.000),have statistically difference,It indicated that berberine combined with erlotinib possesses synergistic effect on gastric cancer cell apoptosis.Flow cytometry results showed that berberine induced cell cycle arrest,cells treated with single drug berberine or erlotinib stagnated in the G0 / G1 phase.Compared with the single drug treatment group,the number of G0 / G1 phase cells in the two drug combination groups was significantly increased(P <0.05),suggesting that combination of berberine and erlotinib to promote cell cycle arrest in gastric cancer cell lines.5.Berberine inhibited the phosphorylation of EGFR in gastric cancer cellsWestern blot showed that EGFR was highly expressed in gastric cancer cell lines MKN45,BGC823 and SGC7901.Gastric cancer cells were treated with different concentrations of berberine or treated at different times to observe the changes of p EGFR and EGFR expression.The results showed that the expression level of total EGFR was not decreased after treated with berberine,but the phosphorylation level of p EGFR was decreased and the phosphorylation level of p EGFR was correlated with the time and drug concentration of berberine.Conclusion:1.Berberine can inhibit the proliferation of human gastric cancer cells,with concentration and time-dependent.2.The apoptosis rate and G0 / G1 phase cells increased after treatment with berberine in gastric cancer cells.3.Berberine enhances the sensitivity of gastric cancer cells to EGFR inhibitors.4.Berberine combined with EGFR inhibitors promotes gastric cancer cell cycle arrest,apoptosis.5.Berberine inhibits the phosphorylation of EGFR time and concentration dependently.Part? Molecular Mechanism of Berberine Increasing the Sensitivity of EGFR-TKIs in Gastric Cancer CellsPurpose: To study the mechanism of berberine increasing the sensitivity of gastric cancer cells to EGFR INHIBITORS.Methods: 1.EGFR high-expressive gastric cancer cell lines were treated with different concentrations of berberine,and the protein was extracted at 24 h,48 h and 72 h after treatment.The phosphorylation of EGFR,AKT,ERK,NF?B,and STAT3 were detected by immunofluorescence and Western blotblot.Western blotting was used to detect the expression of Bcl-x L,PARP and cyclin D1.2.To observe the effect of berberine on EGFR pathway on the basis of EGF-activated EGFR pathway or erlotinib inhibition of EGFR pathway,Western blot was used to detect the phosphorylation of EGFR,AKT,ERK,NF?B,STAT3 and expression of Bcl-x L,PARP and cyclin D1.Si RNA was used to knockdown STAT3 and U0126 was used to inhibit phosphorylation of ERK.The phosphorylation of ERK,STAT3 and the expression of Bcl-x L,PARP and Cyclin D1 were detected by Western blot.Results: 1.Berberine significantly inhibited EGFR downstream signaling pathway and target gene Cyclin D1,Bcl-x LWestern blot analysis showed that the expression of EGFR,AKT,ERK,NF?B and STAT3 did not decrease after treatment with berberine,but the phosphorylation levels of EGFR,AKT,ERK,NF?B and STAT3 were lowered,Bcl-Xl and Cyclin D1 expression were decreased and Cleaved-PARP was increased.With increasing concentrations of berberine,the phosphorylation level of EGFR pathway was more strongly inhibited.2.Berberine can counteract EGF activation of the EGFR pathway and amplify the inhibitory effect of erlotinib on the EGFR pathwayBerberine regulates apoptosis and cell cycle by inhibiting EGFR downstream molecules such as STAT3,AKT,ERK,NF?B,and Bcl-x L and cyclin D1.These data suggest that berberine regulates downstream target proteins Bcl-xL and cyclin D1 by inhibiting the phosphorylation of EGFR and downstream AKT,ERK,STAT3.Then,when pretreated with EGF,the inhibition of EGFR pathway was weakened by berberine.When combination with erlotinib,the inhibitory effect was amplified.3.To observe the effect of berberine on target gene after inhibition of ERK or STAT3Furthermore,When STAT3 si RNA inhibited STAT3,the expression of Bcl-xL and cyclin D1 protein was decreased in BGC823 cells,and cleaved PARP was increased.The expression of cyclin D1 protein was decreased,but the apoptotic protein was not changed when U0126 specifically inhibited ERK.Conclusion: 1.Berberine inhibits EGFR pathway activation and has a time and concentration dependency.2.Berberine combined with erlotinib can amplify the inhibition of EGFR pathway.3.Berberine can inhibit the phosphorylation of EGFR,and can regulate the expression of Bcl-x L and cyclin D1 through STAT3 pathway.Part? The mechanism of berberine combined with cetuximab in inhibiting gastric cancer in vivoPurposes: To study the molecular mechanism of berberine combined with cetuximab in inhibiting tumor in vivo.Methods: Subcutaneous xenograft model of gastric cancer was established using BALB / c female nude mice.Nude mice were randomly divided into four groups: control group,cetuximab group,berberine group,cetuximab and berberine group.The body weight and tumor volume of nude mice were measured every week.After 4 weeks,the nude mice were sacrificed and the tumor was removed.The tumor volume,weight and were measured.Neutral formaldehyde solution was used to fixtumor.Ki67,p-EGFR and p-STAT3 were analyzed by immunohistochemistry.The apoptotic index was calculated by TUNEL staining.The expression of STAT3,Bcl-x L,PARP,cyclin D1 and the phosphorylation of STAT3 were detected using western blot.Results: 1.Inhibitory effect of berberine combined with cetuximab on the growth of gastric cancer xenografts in vivo.The tumor volume of each group of nude mice was measured dynamically.The tumor weight was measured at the end.The average tumor volume of cetuximab group,berberine group and berberine and cetuximab group were significantly lower than those of control group group.The tumor volume and weight of the combination group were significantly lower than those of the cetuximab group(P <0.001).The combination of the two drugs has a synergistic effect on the inhibition of tumor growth.2.Effects of berberine combined with cetuximab on survival time of gastric cancer xenograft model.Gastric cancer xenografts models were randomly divided into four groups: control group,berberine group,cetuximab group and berberine combined with cetuximab group.The death time of each nude mouse was recorded until the last nude mice died.The survival curves of nude mice were drawn after 90 days.Compare to other groups,the survival time of berberine combined with cetuximab group was the longest(P <0.05).3.Berberine combined with cetuximab inhibits proliferation of gastric cancer xenografts through EGFR pathwayThe expression of Ki67 and p-EGFR was detected by immunohistochemistry.The results showed that cetuximab group,berberine,berberine and cetuximab group positive cells were less than the control group.The number of positive staining cells in the combination group was significantly lower than that in the cetuximab group(P<0.05).The results of Western blot showed that the phosphorylation level of STAT3 was decreased and the cell cycle related protein Cyclin D1 decreased.These results suggest that the combination of berberine and cetuximab has a synergistic effect on the inhibition of EGFR / STAT3 pathway and tumor proliferation.4.Berberine combined with cetuximab promotes apoptosis of gastric cancer xenografts.The apoptotic cells were observed after TUNEL staining.The results showed that cetuximab group,berberine and berberine joint cetuximab group number of apoptotic cells was significantly higher than the control group.The apoptotic rate of the combination group was significantly higher than that of the cetuximab group(P <0.05).It suggests that berberine combined with cetuximab has synergistic effect on gastric cancer cell apoptosis.5.Effects of berberine combined with cetuximab on p-EGFR in gastric cancer xenografts.Immunohistochemistry showed high expression of p-EGFR in the control group.Berberine treatment alone or in combination with cetuximab decreased p-EGFR expressionCompared with berberine or cetuximab group,the expression of p-EGFR was lower than that of berberine combined with cetuximab group(P <0.05).6.Effects of berberine combined with cetuximab on expression of key protein in EGFR pathway.The expression of p-STAT3,STAT3,Bcl-xL,CyclinD1 and Cleaved-PARP were detected by Western blot.The results showed that the expression of phosphorylated STAT3,Bcl-x L and cyclin D1 were decreased in mice treated with cetuximab combined with berberine compared to the control and cetuximab alone.Conclusion:1.Berberine or cetuximab alone can inhibit the growth of gastric cancer xenografts and prolong the survival time of nude mice.The combination treatment group was more effective in the inhibition of tumor growth,the longest survival time of nude mice.2.Berberine combined with cetuximab inhibited the proliferation and induced apoptosis of gastric cancer by EGFR / STAT3 pathway.Part? Berberine Simultaneously Inhibits Phosphorylation of Multiple RTKs in Gastric Cancer CellsPurposes: To study the inhibitory effect of berberine on the activation of multiple tyrosine kinase receptors.Methods: The inhibition effect of berberine on phosphorylation of RTKs was examined using the P-Tyr-1000 antibody and the RTK Signaling Antibody Array Kit.Results: 1.Berberine inhibited the phosphorylation of multiple proteins in gastric cancer cells.MKN45,BGC823 and SGC7901 gastric cancer cell lines were treated with berberine,or treated with DMSO as negative control.Western blot was used to detect the phosphorylation of protein in gastric cancer cells.The results showed that the phosphorylation level of proteins at different molecular weights was significantly decreased after normalization.2.Berberine can inhibit phosphorylation of multiple tyrosine kinase receptors.We found that phosphorylation levels of most tyrosine kinase receptors such as EGFR,ERBB2,FGFR1,MET,VEGFR2 decreased after berberine.At the same time,ERK,SRC and STAT3 phosphorylation signals also reduced.Conclusion: Berberine can inhibit the activation of multiple tyrosine kinase receptors,including EGFR,ERBB2,FGFR1,MET and VEGFR2. |
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