| Background: Colorectal cancer is one of the most common gastrointestinal cancers.According to the data,incidence of colorectal cancer become the third in the malignant tumor.There is a rising trend for the incidence of colorectal cancer in our country.The main treatment of colorectal cancer are surgery,radiotherapy,chemotherapy,supportive care.However,the treatment of the most of the colorectal cancer patients are failed,because of recurrence and metastasis after surgical treatment or side effects and drug resistences of chemotherapy.Therefore,it is necessary to search for new therapeutic strategies to deal with it.Targeted therapy has been widely used in clinical practice.However,there are not effective targeted drugs for colorectal cancer patients with BRAFV600 E mutations.In malignant melanoma,BRAF gene mutation account for about 80% of the gene mutantions.In colorectal cancer,the BRAFV600 E gene mutation in approximately 12-15%.Vemurafenib for malignant melanoma with BRAF gene mutation showed good clinical efficiency,and the drug was well tolerated,but the efficiency of Vemurafenib for colorectal cancer patients with BRAF gene mutation were poor.Related studies have found that a feedback regulation of EGFR in colorectal cancer.EGFR rapidly reaction after the application of BRAF inhibitor,and then it promote cell growth,proliferation,differentiation and other biological activities.In malignant melanoma,there is no EGFR feedback pathway and the expression levels of EGFR is low.Anti-EGFR targeted drugs can enhance the sensitivity of Vemurafenib for colorectal cancer,but often requires three targeted drugs.That is limits the application of targeted drugs in clinical practice.The high levels of expression of c-Met is associated with the resistence to chemical and targeted therapy.In colorectal cancer,the activation of c-Met can activate the RAS / RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathway.Therefore,there may be inter action between the c-Met and BRAF gene.However,the combination therapy of c-Met inhibitor PHA665752 and Vemurafenib in colorectal cancer with BRAFV600 E gene mutation have not been reported.Objective: To evaluate the proliferation effect of colorectal cancer cell lines with BRAF gene mutation treated by c-Met inhibitor PHA665752 in combination with Vemurafenib.Method:1 The proliferation efficacy of different concentrations of c-Met inhibitor PHA665752 on HT29 and RKO cell lines detected by MTT assay at 24 h,48h and 72 h.2 The proliferation efficacy of different concentrations of Vemurafenib on HT29 and RKO cell lines detected by MTT assay at 24 h,48h and 72 h.3 The proliferation efficacy of different concentrations of PHA665752 in combination with Vemurafenib on HT29 and RKO cell lines detected by MTT assay at 24 h,48h and 72 h.4 The influence of cell cycle of PHA665752 in combination with Vemurafenib in HT29 and RKO cell lines detected by Flow Cytometry.5 To obtain the HT29 cell lines with expressing BRAF-short hairpin RNA(shRNA)by lentivirus transfection method.6 The BRAF mRNA and protein expression of HT29 cell transfected with BRAF small hairpin RNA detected by RT-PCR and Western blot method.7 The proliferation efficacy of different concentrations of Vemurafenib on HT29 cell with transfected with BRAF small hairpin RNA detected by MTT assay at 24 h,48h and 72 h.Results:1 The proliferation efficacy of different concentrations of c-Met inhibitor PHA665752 on HT29 and RKO cell lines detected by MTT assay.(1)With the treatment of PHA665752,the higher the concentration of PHA665752,the inhibit effect for proliferation of HT29 cell were more significantly(P<0.01).(2)With the treatment of PHA665752,the higher the concentration of PHA665752,the inhibit effect for proliferation of RKO cell were more significantly(P<0.01).2 The proliferation efficacy of different concentrations of Vemurafenib on HT29 and RKO cell lines detected by MTT assay.(1)With the treatment of Vemurafenib,3,6,9 μM concentration of Vemurafenib inhibit the proliferation of HT29 cell at 24h(P<0.05);0.1,0.5μM concentration of Vemurafenib promote the proliferation of HT29 cell,and 9μM concentration of Vemurafenib inhibit the proliferation of HT29 cell at 48h(P<0.05);0.5,1,3,6,9μM concentration of Vemurafenib inhibit the proliferation of HT29 cell at 72h(P<0.05).(2)With the treatment of Vemurafenib,0.1,0.5μM concentration of Vemurafenib promote the proliferation of RKO cell,and 6,9μM concentration of Vemurafenib inhibit the proliferation of RKO cell at 24h(P<0.05);0.01,0.1μM concentration of Vemurafenib promote the proliferation of RKO cell,and 6,9μM concentration of Vemurafenib inhibit the proliferation of RKO cell at 48h(P<0.05);0.5,1,3,6,9μM concentration of Vemurafenib inhibit the proliferation of RKO cell at 72h(P<0.05).3 The proliferation efficacy of different concentrations of PHA665752 in combination with Vemurafenib in HT29 and RKO cell lines detected by MTT assay.(1)Treatment of PHA665752 in combination with Vemurafenib has the strongest inhibition of proliferation among control group and Vemurafenib or PHA665752 monotherapy treatment in HT29 and RKO cell lines(P<0.01).(2)To evaluate the effect of dual treatment of PHA665752 and Vemurafenib by Zhengjun Jin method.The inhibition of HT29 cells treated with 2 μM PHA665752 and each concentration of Vemurafenib at 24 h show a synergistic effect(Q>1.15);The inhibition of HT29 cells treated with 2 μM PHA665752 and 1,3,6 μM concentrations of Vemurafenib at 48 h show a synergistic effect(Q>1.15);The inhibition of HT29 cells treated with 2 μM PHA665752 and each concentration of Vemurafenib at 72 h show a additive effect(0.85<Q<1.15).The inhibition of RKO cells treated with 1 μM PHA665752 and 1 μM Vemurafenib at 24 h show a synergistic effect(Q>1.15);The inhibition of RKO cells treated with 1 μM PHA665752 and each concentration of Vemurafenib at 48 h show a synergistic effect(Q>1.15);The inhibition of RKO cells treated with 1 μM PHA665752 and each concentration of Vemurafenib at 72 h show a synergistic effect(Q>1.15).4 The influence of cell cycle of PHA665752 in combination with Vemurafenib in HT29 and RKO cell lines detected by Flow Cytometry.(1)Cell cycle of HT29:The percentage of G0 / G1 phase of control group is(52.33±5.13)%,The percentage of G0 / G1 phase of PHA665752 group is(55.33±6.66)%,The two groups compared with each other was not statistical significance(P>0.05);The percentage of G0 / G1 phase of Vemurafenib group is(67.00±6.93)%,compared with control group have statistical significance(P<0.05);The percentage of G0 / G1 phase of PHA665752 in combination with Vemurafenib group is(74.67±5.51)%,and more than control group and PHA665752 group(P<0.05).(2)Cell cycle of RKO:The percentage of G0 / G1 phase of control group is(39.31±2.73)%,The percentage of G0 / G1 phase of PHA665752 group is(45.84±1.41)%,The two groups compared with each other was statistical significance(P<0.05);The percentage of G0 / G1 phase of Vemurafenib group is(41.08±2.71)%,compared with control group was not statistical significance(P>0.05);The percentage of G0 / G1 phase of PHA665752 in combination with Vemurafenib group is(65.58±2.81)% and more than control group,PHA665752 group and Vemurafenib group(P<0.05).5 To obtain the HT29 cell lines with expressing BRAF-short hairpin RNA(shRNA)by lentivirus transfection method.The proportion of HT29 cell lines with fluoresent were about 70%,which meet the requirement of infection.6 The BRAF mRNA and protein expression of HT29 cell transfected with BRAF small hairpin RNA detected by RT-PCR and Western blot method.(1)Four groups recombinant lentivirus that could express BRAF-sh RNA were successfully constructed.One of the groups(the 4-S group)could significantly suppress BRAF gene expression in HT29 cells,the mRNA expression fell by 76%(P<0.05).(2)Four groups recombinant lentivirus that could express BRAF-sh RNA were successfully constructed.One of the groups(the 4-S group)could significantly suppress BRAF protein expression in HT29 cells,the protein expression fell by 69%(P<0.05).7 The proliferation efficacy of different concentrations of Vemurafenib on HT29 cell transfected with BRAF small hairpin RNA detected by MTT assay.With the treatment of Vemurafenib,3,6,9 μM concentrations of Vemurafenib inhibit the proliferation of HT29 cell transfected with BRAF small hairpin RNA at 24h(P<0.05);0.01μM concentration of Vemurafenib promote the proliferation of HT29 cell transfected with BRAF small hairpin RNA,and other concentrations of Vemurafenib inhibit the proliferation of HT29 cell transfected with BRAF small hairpin RNA cell at 48h(P<0.05);0.1,0.5,1,3,6,9μM concentrations of Vemurafenib inhibit the proliferation of HT29 cell transfected with BRAF small hairpin RNA cell at 72h(P<0.05).The inhibition rate of HT29 cell after the treatment with 9μM concentration of Vemurafenib were smaller compaired with HT29 cell transfected with BRAF small hairpin RNA at 24h(P<0.05);The inhibition rate of HT29 cell after the treatment with 0.1,0.5,1,3,6,9μM concentrations of Vemurafenib were smaller compaired with HT29 cell transfected with BRAF small hairpin RNA at 48 h and 72h(P<0.05).Conclusion:1 The inhibition of the proliferation of HT29 and RKO cell lines treated with PHA665752 and Vemurafenib show a synergistic effect.2 The resistence of Vemurafenib in colon cancer cells was induced by BRAFV600 E mutation. |
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