The Experimental Study of Antiangiogenic and Induction of Apoptosis by Maximum Tolerated Dose Combined with Low-dose Cyclophosphamide in Breast Cancer
|School||Dalian Medical University|
|Keywords||Cyclophosphamide Angiogenesis Breast cancer low-dose metronomic chemotherapy Maximum tolerated dose|
Objective:Observe the effects of angiogenesis and tumor cell’s apoptosis by maximum tolerated dose combined with low-dose cyclophosphamide on transplanted human breast cancer on nude mice.Method:1. Established nude mice models of transplanted human breast cancer:Human breast cancer cells line MCF-7 were inoculated in IMDM medium which contained 10% fetal bovine serum. In the logarithmic phase, the concentration of MCF-7 cells was adjusted to 6×107 /ml . A nude mice was injected 0.2ml cells suspension under the right breast pad. When the tumor volume was about 150-200mm3,nude mice were divided into groups to start medication.2.Divided into experimental groups and drug treatment: The 20 tumor-bearing mice were randomly (random number table) divided into 4 groups. Every group contained 5 nude mice. Group A (low-dose cyclophosphamide, LDM) CTX20mg / kg, qd, ip; Group B (maximum tolerated dose combined with low-dose cyclophosphamide) CTX150mg / kg, qod, ip (3 times) CTX20mg / kg, qd, ip; Group C (maximum tolerated dose, MTD) CTX150mg / kg, qod, ip (3 times); Group D (control group) NS same volume, qd, ip.3.Volumes of tumor were measured every 2 days and white blood cells of nude mice were count in each group every week.4. Tumor dissection and detection of pathology: All nude mice were killed by cervical dislocation after treatments, then took the tumors complete under sterile conditions. All tumors were soaked by 10% formaldehyde solution respectively in 15 minutes and fixed 8 ~ 24h at room temperature. After that we deal with the tumors with paraffin-embedded and serial sections of 2um. Immunohistochemistry was used to detect the expressions of microvessel density (MVD) and thrombosponain-1 (TSP-1) in different tumor tissues. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) was used to observed tumor cells apoptosis.Results:1.Inhibit tumor’s growth: The experiment showed that tumor volume began to appear difference after 14 days. Compared with group NS , the trend growth of treatment groups slowed.Tumor volume was significantly smaller than the low dose of rhythm group(PAB=0.008)and maximum tolerated dose(PBC=0.038) after 21 days.Tumor weight of combined treatment group was the smallest,and inhibition ratios of the tumor weight was 69.15%.2. Side effects: Mice were at good conditions after drug treatment. Group NS ate less with the weight loss badly.The dietary and action were nomal without diarrhea and ecchymosis.The statistical analysis of the leukocyte count showed there were not different among every experimental group.3. The results of immunohistochemistry:(1) Microvessel density :The expression of the control’s MVD was significantly higher than other treatment groups.The most obvious group is the combination group.There was statistically significant between the MTD and combined group(PBC=0.012),while the LDM and combined group(PAB=0.525), MTD and the control (PCD=0.208)had no statistically significant.(2) Thrombosponain-1:The positive expression of TSP-1 in combined group was the highest,while the control was the lowest.The combined group was significant than the MTD and NS by statistical analysis(PBC=0.004,PBD=0.001). Group LDM was the same as combined group.There was no statistically significant between LDM and combined group.4.The results of apoptosis: The number of combined group’s apoptotic cells were significantly more than the other groups. The statistical analysis showed that there were differences between the control and all the treatments. Combined group was more significant than the other two methods(MTD、LDM).There was not difference between maximum tolerated dose and low-dose cyclophosphamide.Conclusion : Maximum tolerated dose combined with low dose cyclophosphamide could inhibit tumor growth, tumor angiogenesis and promote tumor cells apoptosis, but did not increase side effects.