Enhanced Anti-tumor And Anti-angiogenic Effects Of Metronomic Vinorelbine Combined With Endostar On Lung Carcinoma Research

Objective:whether an angiogenesis inhibitor?Endostar?combined with metronomic chemotherapy?MET?Vinorelbine have synergistic anti-tumor and anti-angiogenic responses or lighter adverse events on lung carcinoma model;and discussed the potential mechanisms in the anti-angiogenesis aspect.Methods:To establish a mouse xenograft model,C57BL/6J mice were inoculated subcutaneously with lewis lung carcinoma cells?LLC?and when the tumors reached a size of approximately 150²00 mm³,tumor-bearing mice were randomized into six groups?n=15 mice per group?and treated for 14consecutive days as follows:i)Control group?negative control?,ii)Endostar group?10 mg/kg?,iii)MET NVB group?3 mg/kg body weight?bw?of Vinorelbine every other day?,i.v.)MET NVB+Endo group,v)maximum tolerated dose?MTD?NVB group,?10 mg/kg bw of Vinorelbine i.p.on days 1and 8?;and vi)MTD NVB+Endo group.During the treatment period,tumors were measured every other day and the tumor volumes were calculated by the following formula:tumor volume?cm³?=length×width²×0.5.After treatment,the tumor growth curve was plotted and the tumor growth inhibition rate was calculated.Six of animals in each group were observed using micro fluorine-18-deoxyglucose PET/computed tomography(¹⁸F-FDG PET/CT)to identify changes by comparing SUVmax values.The animals were sacrificed by orbital puncture at second days after treatment,and tumor tissue and peripheral blood were harvested for futher examination.Circulating endothelial progenitor cells?CEPs?stained with CD133⁺CD34⁺Flk-1⁺,apoptosis rate,expression of CD31,vascular endothelial growth factor?VEGF?,hypoxia inducible factor-1?HIF-1??were determined using flow cytometry,western blot analysis,immunofluorescence staining and Enzyme-linked immunosorbent assay?ELISA?analysis.In addition,white blood cell?WBC?counts and H&E-stained sections of liver,lungs,kidney,and heart were performed in order to monitor toxicity assessments.Results:We found that the tumors grew rapidly in the control group,but the tumor growth was significantly decreased in all treatment groups?P<0.001?.Treatment with MET NVB and administration with MTD NVB group showed effective in inhibiting tumor growth and inhibited tumor weights by 43.57%and 39.65%,respectively.Furthermore,the inhibition rate of the MET NVB+Endo group and MTD NVB+Endo group were 70.82%and 65.89%,respectively.And the combined groups showed the greatest efficacy in tumor growth inhibition compared with other treatment groups?P<0.001 in all cases?.Flow cytometry analysis revealed that the frequency of CEPs in the MET NVB combined with Endostar group was 0.023±0.012%,and was significantly lower compared to the control group and the MTD NVB+Endo group?P<0.001?.Immunofluorescence staining analysis demonstrated that a lower MVD value was observed in the MET NVB group and Endo group.The MET NVB+Endo group had the least MVD value,and was significantly lower compared to the MTD NVB+Endo group?P<0.001?.The expression of HIF-1?and VEGF were decreased in the MET NVB group and Endo group by immunofluorescence staining analysis,ELISA analysis and western blot analysis,and were significantly further decreased in the MET NVB+Endo group compared to the MTD NVB+Endo group?P<0.001?.Flow cytometry analysis also showed that treatment with MET NVB+Endo and MTD NVB+Endo groups led to higher apoptosis rate compared with all other groups?P<0.001?,however no statistically significant differences were observed between groups?P>0.05?.Western blot analysis also indicated that the amount of Bax and Caspase-3 protein was increased,but the Bcl-2 protein level was decreased in the MET NVB+Endo group and MTD NVB+Endo group.Representative ¹⁸F-FDG PET/CT images and the SUVmax values of mice in the control group were 2.72±0.17,when compared with other groups?P<0.001 in all cases?.The SUVmax values of mice in the Endo group,the MTD NVB group and the MET NVB group were 2.34±0.16,1.72±0.12 and1.61±0.13 respectively.Moreover,the SUVmax values of mice in the MET NVB+Endo and MTD NVB+Endo groups were 1.21±0.11 and 1.32±0.13?P<0.001 vs all other groups?,indicating that the combination of drugs resulted in decrease in the metabolic activity of the tumor and showing that the efficacy of combined treatment groups was better than that of single drug groups,however no statistically significant differences were observed between groups?P>0.05?.To investigate the side effects induced by the different therapeutic regimens,these results showed WBC counts were reduced in the MTD NVB group and the MTD NVB+Endo group and there was a bone marrow suppression,while in the MET NVB group and the MET NVB+Endo group WBC counts were within the normal range.H&E-stained sections of liver,lungs,kidney,and heart also indicated that metronomic chemotherapy had little damage to internal organs,while conventional chemotherapy had severe damage to internal organs.Conclusion:?1?Treatment with NVB had anti-tumor effect on lung carcinoma,and combined with Endostar had a synergistic anti-tumor response.?2?Administration with MET NVB combined with Endostar had synergistic anti-tumor and anti-angiogenic responses,and the potential mechanisms of combined groups were to reduce the frequency of CEPs,to decrease the MVD values and to inhibit the expression of HIF-1?and VEGF.?3?Treatment with MET NVB or combined with Endostar showed lighter adverse events.