| Objectives:To observe and analyze the effect of antitumor and anti-angiogenesis of endostar combined with docetaxel under different sequences. According to the changes of biological markers which reflect invasion and angiogenesis of tumor, we want to investigate the mechanism of the two different administration sequences in order to put it in clinical work.Method:Nude mice with xenograft tumor were randomized into 3 groups:concurrent administration group (endostar 400μg·mouse-1·d-1, dl-d35, docetaxel 10mg·kg-1·3-d, d1-d19), endo-first group(endostar 400μg·mouse-1·d-1, dl-d35, docetaxel 10mg·kg-1·3d-1, d16-d34), model group (positive control, mice burdened tumor without treatment) and blank control group (negative control). The volume of tumor and the weight of mouse were measured during treatment. Activated circulating endothelial cells (aCECs) were detected by flowcytometry, and the expression of MMP-2, MMP-9, TIMP-1, TIMP-2, EMMPRIN,CD34, a-SMA were determined by immunohistochemistry. To capture images with professional digital camera, and batch measure them with the macro (Pathology6) of the image analysis soft named "image pro plus 6.0". After the integrated optical density (IOD) of each image was given, we use the mean value of IOD of each slice as the reflection of protein expression. Quantitative calculation and statistic analysis were made at the endpoint.Results:1. The results of animal experiment:The tumor growth of concurrent administration group(39.94mm3)is less than the endo-first group(99.57mm3)during the treatment, both of them were smaller than the positive control groups(P=0.003, P=0.015). The concurrent administration group had slightly decreased the weight of mouse, similar to the blank control group. And the mouse weight in the endo-first group and model group were lighter than the blank control group(P=0.061,P=0.073)2. The results of flowcytometry:The amount of aCECs in both treatment groups were more than the control, and it is greater in the endo-first group than the concurrent administration group(P<0.001).3. The results of immunohistochemistry staining:Compared with model group, the expression of MMP-2 and MMP-9 in treatment groups were obviously down-regulated(P＜0.05). The expression of TIMP-1 in the endo-first group and TIMP-2 in the concurrent administration group were up-regulated (P=0.048, P=0.002). And the EMMPRIN expression was significantly down-regulated in the concurrent administration group (P=0.021).The expression of a-SMA in both of the administration groups were less than model group(P=0.009, P=0.014).4. The comparison of MVD:The MVD of the administration groups were less than model group(P=0.022, P=0.036).No significant difference was seen between the administration groups.5. The correlation analysis:A significant positive correlation was seen between MMP-2 expression and tumor volume(P=0.020), TIMP-1 expression and aCECs(P=.041),MMP-2 expression and MVD(P=0.031),α-SMA expression and MVD(P=0.002), MMP-2 expression and MMP-9 expression(P=0.008), EMMPRIN expression and MMP-9 expression(P=0.001). A negative correlation was seen between TIMP-2 expression and tumor volume(P=0.027),MMP-2 expression and aCECs9(P=0.025),α-SMA expression and aCECs(P=0.044).Conclusion:In comparison with the endo-first group, the anti-tumor effect and survival quality of the concurrent administration group is better. Both of endostar and docetaxel may have "vascular normalization effect" by down-regulating MMPs expression through different point. Both of endostar and docetaxel inhibit the stromal reaction induced by cancer, and restrain cancer progress to a certain extent. The change of aCECs should be a dynamic process with rise in early-stage suggesting the decrease of vascular bed and sequent decline ascribed to apoptosis of aCECs and the tumor-regression after combined therapy. Investigation of its dynamic change may be helpful to know the change of tumor burden and vascular bed and predict the antitumor effect. |
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