| Objective: To explore the effect of the c-Met inhibitor PHA-665752 combined with vemurafenib on the growth of colon cancer xenograft tumor with BRAFV600 E mutation.Methods:1 The establishment of animal model of nude miceThe male nude mice aged 4~5 weeks were raised in SPF environment.Preparation of cell suspension from human colon cancer HT-29 cell line(5.5×107/ml).Extract 0.15 ml cell suspension(7~8×106)and inoculate in nude mice’s left ribs abdominal subcutaneous.Generally,the tumors were visible after four days.The models were established successfully.2 The experimental groupsWhen subcutaneous tumors grew to a diametrical length of about 0.6 cm,the nude mice were randomly divided into four groups(7 mice in each group): DMSO(control),PHA-665752,vemurafenib,the combination group of PHA-665752 and vemurafenib.The PHA-665752 and DMSO were administered intraperitoneally every two days,and vemurafenib was intragastriced twice daily.The mice were treated for three weeks.3 Testing of the growth of transplanted tumorThe mice living conditions and tumors were daily observed.When the subcutaneous tumors of nude mice grew to a length of about 0.6cm,the long diameter and short diameter of the tumors and the mice weight were measured every three days.4 After three weeks of treatment,the mice were euthanized at application of anesthetics chloral hydrate.The tumors were dissected out and fixed in 10% formalin.5 Pathological changes were detected by HE staining.The expression levels of HGF,p-c-Met,p-Akt and p-ERK were detected by immunohistochemistry.Results: 1 The influence on the general condition of the nude miceThere was no evident difference in diet,vigor,hair and body weight ofnude mice between each group.The mice weight continued to grow until theend of the experiment.During the course of the experiment,one nude mouse in the control group died from lymph node metastasis,two mice in vemurafenib group and three mice in the combined group died due to improper administration of intragastric administration.2 The growth of colon cancer xenograft tumorThe tumor growth curves were drawn according to the volume of the tumor(long diameter×short diameter2/2).The tumor changes were observed.At the end of the experiment,we found that the tumor volume of the combined group was smaller than other groups(P<0.05).There were no significant differences between the other three groups.3 The pathological detection in the colon xenograft tumorLots of tumor necrosis was observed in the combined group,associated with inflammatory cells invasion.4 The expression of HGF,p-c-Met,p-Akt and p-ERK in the xenograft tumor 4.1 The expression of HGF in the xenograft tumorThe immunohistochemical scores of the DMSO group,PHA-665752 group,vemurafenib group and the combined group were: 7.00±1.38,8.48±1.50,6.80±1.05,7.32±1.62.There was no statistical difference between the groups(P>0.05).4.2 The expression of p-c-Met in the xenograft tumorThe immunohistochemical scores of the DMSO group,PHA-665752 group,vemurafenib group and the combined group were: 6.56±2.23,3.76±1.34,7.52±1.24,1.20±1.17.The expression of p-c-Met was downregulated in PHA-665752 group and the combined group(P<0.05).There were significant differences between the combined group and the other groups(P<0.01).The expression of p-c-Met was upregulated in vemurafenib group.4.3 The expression of p-Akt in the xenograft tumorThe immunohistochemical scores of the DMSO group,PHA-665752 group,vemurafenib group and the combined group were: 6.80±2.47,4.24±0.78,6.96±1.60,2.24±1.11.The expression of p-Akt was downregulated in PHA-665752 group and the combined group(P<0.05).There were significant differences between the combined group and the other groups(P<0.01).The expression of p-Akt in group vemurafenib was slightly decreased,but there was no significant difference compared the DMSO group.4.4 The expression of p-ERK in the xenograft tumorThe immunohistochemical scores of the DMSO group,PHA-665752 group,vemurafenib group and PHA-665752 combined with vemurafenid group were: 8.16±1.71,3.40±2.41,7.40±2.10,0.64±0.83.The expression of p-ERK was downregulated in PHA-665752 group and the combined group(P<0.05).There were more significant differences between the combined group and the other groups(P<0.01).Vemurafenib had no significant inhibitory effect on p-ERK.Conclusions:1 PHA-665752 combined with vemurafenib has synergistic inhibitory effect on BRAFV600 E mutant colon cancer xenografts.2 PHA-665752 combined with vemurafenib may play a synergistic role in the inhibition of p-c-Met,PI3 K and MAPK signaling pathways. |
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