Inhibition Effects Of AMD3100,PD98059 And Anti-CXCR7 On The Growth Of Endometrial Cancer Subcutaneous Xenografts In Nude Mice

Objective:To explore the therapeutic effects and possible mechanism of blocking CXCL12-specific receptors CXCR4,CXCR7 and intracellular ERK signaling pathway on subcutaneous xenografts of human endometrial cancer Ishikawa cells in nude mice.Method:1.Mixed the Ishikawa cell suspension of endometrial cancer with Matrigel 1:1 to adjust the cell density to 7.5×10~7/ml with a cell counting plate and injected 200ul into subcutaneous of the right back of female BALB/c nude mouse with a 1ml disposable sterile syringe to establish transplanted tumor animal model.2.A successful modeling was established when the diameter of subcutaneous xenografts in nude mice were more than 5 mm.All tumor-bearing nude mice were randomly divided into 5 groups(n=6):AMD3100 group(trade name:Plerixafo,CXCR4specific antagonist),PD98059 group(ERK signaling pathway blocker),Anti-CXCR7 group(CXCR7 neutralizing antibody),AMD3100+Anti-CXCR7 group,Nacl group,once every 3 days for 3 weeks;all drugs were injected intraperitoneally into the body.3.During the entire treatment cycle,closely observed the mental state,diet of tumor-bearing nude mice and the growth of subcutaneous xenografts.The long diameter(a)and short diameter(b)of xenografts were measured with a vernier caliper before each administration.Calculated the volume according to the formula(V=a×b~2/2,mm~3)and drew the tumor growth curves of each group.4.After the end of the treatment period,all the tumor-bearing nude mice were removed from the neck and tumors were completely removed at the super clean bench.Weighed(g)after removing fat tissue and blood stains and calculated tumor inhibition rates(%)according to the formula:inhibition rate(%)=(tumor weight of the control group-tumor weight of the experimental group)/tumor weight of the control group×100%.5.Calculated the q value according to the Golden's formula:q=E(A+B)/(EA+EB-EA·EB).The E(A+B)was the tumor inhibition rate when the two drugs were used together;EA,EB is the tumor inhibition rate when used as a single drug;q<0.85 described that the combination drug had antagonistic effect;q>1.15,synergistic effect;q=0.85~1.15,additive effect.6.Detected the expression of apoptosis-inhibiting gene Survivin mRNA and protein in subcutaneous xenografts of each group by Real-time quantitative PCR and Western Blotting.Result:1.All nude mice were successfully modeled.During the whole experiment,there was no accidental death and the skin of the inoculation site was not ulcerated.At the end of the treatment period,the tumor growth rate and tumor mass of the experimental group AMD3100,PD98059,Anti-CXCR7,AMD3100+Anti-CXCR7 were significantly lower than those of the control group(P<0.01).2.The tumor inhibition rates of the experimental groups were AMD3100(53.09±6.04)%,PD98059(50.39±4.58)%,Anti-CXCR7(48.42±14.99)%,AMD3100+Anti-CXCR7(45.24±8.08)%and the above experimental groups were statistically significant compared with the control group(P<0.01).3.According to the inhibition rate of each group,q=0.60<0.85 was calculated by the formula,indicating that the combination did not produce synergistic anti-tumor effect.4.Western Blotting and Real-time quantitative PCR results showed that the expression levels of Survivin protein and mRNA in AMD3100 group,PD98059 group,Anti-CXCR7 group and AMD3100+Anti-CXCR7 group were significantly lower than those in the control group(P<0.01).But there were no significant differences in tumor growth curve,tumor weight,tumor inhibition rate,and expression levels of Survivin protein and mRNA between the experimental groups(P>0.05).Conclusion:Animal experiments showed that blocking CXCL12-specific receptors CXCR4,CXCR7 and intracellular ERK signaling pathway could inhibit the growth of subcutaneous xenografts of endometrial cancer Ishikawa cells in nude mice,which may be related to the down-regulation of apoptosis inhibitory gene Survivin in tumor tissues.The CXCR4,CXCR7 and ERK signaling pathways are expected to be important targets for the treatment of endometrial cancer and their corresponding antagonists may provide a new strategy for targeted therapy of endometrial cancer.