The Inhibitory Effects Of Combined Bevacizumab With Chemotherapy In Different Sequence On Human Gastric Cancer

Objective:Gastric cancer is often diagnosed at an advanced stage. Although chemotherapy prolongs survival and improves quality of life, the survival of gastric cancer patients with advanced disease is short. Thanks to recent insights into the molecular pathways involved in gastric carcinogenesis, new targeted treatment options have become available for gastric cancer patients. Neo-vascularization is a vital process for tumor growth and development which involves the interaction between tumor cells and stromal endothelial cells through several growth factors and membranous receptors which ultimately activate pro-angiogenic intracellular signaling pathways. Inhibition of angiogenesis has become a standard treatment option for several tumor types. In gastric cancer, the therapeutic role of anti-angiogenic agents is more controversial. Bevacizumab which target vascular endothelial growth factor-A and vascular endothelial growth factor receptor-2, respectively, have been demonstrated antitumor activity in patients with tumors of the stomach or esophagogastric junction. To establish subcutaneous xenograft models of gastric cancer in nude mice and to evaluate the inhibitory effects of combined targeted treatment with chemotherapy in differen t sequence. Methods:1 MGC-803 Cells with stable expression of GFP andluciferase were harvested in single cell suspension at a concentration of 5 X 106 cells/ ml. A total of 5 X 106 cells(in 1ml of growth medium) were injected into the BALB/c nude mice.2 Subcutaneous xenograft models were established using MGC-803 gastric cancer cell line in 25 male 4-week old BALB/C-nu/nu nude mice and were randomly divided into five groups. control group as A group;bevacizumab group as B group(Bev:4mg/kg/d,d1、4、7)、Combined treatment of bevacizumab with FP 24 h later as C group(Bev:4mg/Kg d2、5、8 ip;5-FU:20mg/Kg DDP: 1 mg/kg d1、4、7, ip,three weeks);Combined treatment of bevacizumab with FP at the same time as D group(Bev:4mg/Kg 、5-FU:20mg/Kg, DDP: 1 mg/kg d1、4、7, ip,three weeks);Combined treatment of bevacizumab with FP 24 h before(bevacizumab :5 mg/kg, d1、4、7,5-FU:15 mg/kg, DDP:1 mg/kg d2、5、8), with 5 mice in each group. Bevacizumab,5-FU and DDP were administered intraperitoneally every three day for three weeks.3 Tumor size were measured by using Small animal in vivo imaging everyseven day. Tumor volumes were measured using a Vernier caliper and calculated with the ellipsoid formula V=1/2 ab2, where a and b are the long and short axes of the tumor, respectively. Tumor volumes were measured at baseline and at the different time points at the end of the experiment(every two days). the animals were imaged every 7days with an Night Owl ⅡLB 983 after i.p. injection of 150 mg/kg Dluciferin in sterile saline. Data wereanalyzed offline with Living Image Software v3.1(Caliper Life Science) by drawing regions of interest(ROI) around the tumor masses to derive total photon flux. The weights were monitored twice a week.Mices were sacrificed on day 22 and mice at the termination of the experiment in order to excise,measure, and weigh the tumors.4 Expression of CD34 was examined by immunohistochemistry for evaluation of microvascular density(MVD). To assess the angiogenic properties of Bev, vascular endothelial growth factor(VEGF) expression, tumor microvessel density and Bcl-2 Assaciated X protein(Bax) were analyzed in gastric carcinoma by immunohis to chemistry.5 Statistical methods: ANOVA, Repeated Measures Analysis of Variance Design Information. Result: 1 The mice were all observed tumor appearancing. The belly ribs on the left side of 25 nude can reach about 0.8cm tumor. Tumorigenic rate: 100%. 2 The volum and flux of tumor 2.1 on the day 1,the mean tumor xenografts volume of the groups were(0.122 ± 0.021)、(0.119 ± 0.020) 、(0.114 ± 0.027) 、(0.115 ± 0.011) 、(0.116±0.024)cm3(P=0.975). On the day d8,d15,d22, Our results showed that the tumor xenografts volume in the test group was significantly lower compared with that in the control group(P < 0.01). tumor xenografts volume of mice in chemotherapy and targeted treatment was significantly lower compared with that bevacizumab group(P< 0.001). the tumor xenografts volume in E group reduced significantly lower than C、D(P<0.001) and kept that trence until the day 21. No significant differences in tumor xenografts volume were observed between the C/D groups at the any time-points examined(P>0.05). 2.2 on the day 1, the mean tumor photon flux of the control and test groups was(57784±2145),(60354±2422),(58335±1956),(59425±2145),(58778±20116)p/sec(P=0.843). On the day d8,d15, d22,Our results showed that the photon flux in the test group was significantly lower compared with that in the control group(P< 0.001). Photon flux of mice in chemotherapy and targeted treatment was significantly lower compared with that bevacizumab group(P< 0.001). On the day 15-21, the photon flux in E group reduced significantly lower than C、D(P<0.0001) and kept that trence until the day 21. No significant differences in photon flux were observed between the C/D groups at the any time-points examined(P>0.05). 3 On the day 22, the xenografts volume weight was as follows:(1.840 ± 0.041)、(1.503 ± 0.042)、(1.207 ± 0.051)、(1.159± 0.212)and(0.986± 0.323)g. The inhibitory rate of tumor in group was 18.31 %,34.4 %, 37.01 % and 46%. Compared with control group, the inhibitory rate of tumor in treatment group was different from that in control(P < 0.01). The inhibito-ry rate of tumor in chemotherapy and targeted treatment was different from that in bevacizumab group(P < 0.001). The inhibitory rate of tumor in E group began to reduce and significantly lower than C/D(P<0.001). No significant differences the inhibitory rate of tumor were observed between the C/D groups at the any time-points examined(P=0.342). 4 After obtaining tumor spheres 4.1 Compared to the E group, the test group had a significantly lower MVD[13.8±1.6,8.0±1.0,6.8±1.2 and 6.5±1.2 vs 4.0±1.2, P<0.001]. 4.2 A lower expression of the marker VEGF-A [91.15±3.30; 60.19±4.30,48.15±3.20, 46.15±3.60 vs 33.15±3.81, P<0.05]. 4.3 Expression of cell surface markers Bax, By comparing bevacizumab with FP 24 h later group with the other groups, we found about 90.26%±2.53 cells in parental cells. Bax was increased in the tumor spheres. Conclusion:1 Combined treatment of bevacizumab with FP 24 h later is superior to other modes of Combined treatment。2 The inhibitory effects of combined Bevacizumab with FP in different sequence on human Gastric cancer is different,the expression of cell surface markers VEGFA、bax was decreased in the tumor spheres.3 In the application of anti-angiogenic drugs, tumor volume may not be the true performance of tumor response to drugs.