The Inhibitory Effect Of Endostatin Combined With Ifosfamide Chemoththerapy In Treatment Of Transplant Sarcoma In Mice

Part one BackgroundTumor is a serious harm to human health,Currently,there are surgery, chemotherapy and radiotherapy, biological therapy etc.Various treatments,but curative effect is not ideal, the mortality rate remains high. in the 21st century totally conquering the disease,looking for more effective treatment methods and new no-side-effects drug treatment, have become a hot spots of general medical researchers.Many studies show that tumor formation and growth, the key is invasion and metastasis of tumor vascularization. Tumor did ont enter the invasion of the avascular, when tumor diameter general less than 2-3mm,cell count in 1 x 107, tumor cells are mainly supplied nutrition by dispersion. When the tumor diameter is more than 2-3mm,Tumor will need new blood vessels to supply nutrition,.discharge metabolites and provide transfer channel. The formation of a tumor blood essels is complicated process, many kinds of cell factors participate the process, and influences the formation of new blood vessels, Herefore, the research development of the antiangiogenesis drugs is to inhibit tumor growth, and prevent tumor metastasis effective strategy. Since the 20th century 70's, Folkman puts forward the growth of tumor relys on new angiogenesis, antiangiogenic can treat the substantive tumor. Many preclinical experimental study attempt to deal with a certain angiogenesis link by antiangiogenic drugs in situ tumor of animal or subcutaneous transplantation tumor, which provide some clinical application of the theory and experiment. Currently, antiangiogenic methods mainly have three directions:The first is to neutralize or inhibit the factor, which can promote the formation of blood vessels; The sceond is to give the factor, which can inhibit the formation of blood vessels; The third is to provide paranormal effect of toxic material or antibody make tumor infarction, Many antiangiogenic drugs already have been into clinical trials stage, and hav already obtained encouraging results, Especially vascular statin and endothelial statin. Molecular biology research shows that ES can by induce endothelial cell apoptosis, block activity of the vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP), thereby inhibit tumor growth and metastasis, ES is currently accepted the strongest inhibit angiogenesis factor, has the very strong antitumor activity, can significantly reduce tumor microvascular density within the tumor, increase tumor cell apoptosis. ES can strongly inhibit tumor endothelial cells, and can effectively restrain the growth of the tumor above 60, can adjust 12% of the human genome, and then cut the express pathological angiogenesis,and no side effects, also no resistance and side effects. Several studies have also reported that ES has the good anti-tumor effect in the liver, thyroid cancer, breast cancer, colorectal, cervical cancer and lung adenocarcinoma, and can prevent the formation of metastases. Currently ES are III period clinical trials, many experiments showed that ES has specific inhibition of vascular endothelial cells, has a stronger inhibitory effect for the clinical treatment of tumor, and break a new path. Recombinant human endothelial statin injection is our country experts and scholars research a new human blood endothelial statin, which can specific strongly inhibit endothelial cell proliferation and growth, is a kind of brand-new inhibit angiogenesis inhibitors, with the chemotherapy drug joint act synergistically. With the deepening of the research of molecular biology, chemotherapy combined molecular target therapy has become a hotspot of tumor treatment research. Part two The inhibitory effect of recombinant human endostatin on angiogenesis in tumorsObjective1. To evaluate the inhibitory effect of endostatin on angiogenesis and sarcoma growth in mice.2. To evaluate the concentration-response relationship of endostatin on antiangiogenesis and inhibit sarcoma growth in mice.Material and methods1. Establish animal modelS180 cells was provided form abdomen of mice by the department of biology of Nanjing University. The cells were collected and centrifuged at 1000r/min,and then prepared in DEME into 5×107/ml cell suspension.0.1ml cell suspension was inoculated subcutaneouly into the right axillary of mice with sterile syringe, thus the animal model of sarcomas were set up.Observe mice subcutaneous transplant tumors, 75 mice have tumor after S180 cells inoculation only 5 days, all tumor diameter of biggest (a) and the least(b) are measured, calculation tumor size:V=ab2/2, tumor size too big and too small is excluded, There are 60mice only enter laboratory,tumor size about 90mm3.2. Grouping and drug treatmentsThe sixty mice implanted with sarcoma were randomly divided into the control, ES low dose(5mg/kg.d)groups,ES middle dose(10mg/kg.d)groups and ES high dose(15mg/kg.d) groups.ES and NS was injected around the tumor,once a day for 14 days.3. Testing indexThe tumor growth situation are observed, the tumor diameter of the biggest (a) and the least (b) are measured with vernier caliper every 3 days until over drug injection, Calculating volume, Taking average, drawing tumor growth curve, The mice in the 4 groups were sacrificed by cervical dislocation at 15 days after initiation of therapy. This DiOC7 was injected into the tail vein 1 min before the mice were sacrificed, at a concentration of 1.0 mg/kg. the tumors were complete removed, Tumor volume was calculated by the formula:l/2×a×b2.where a and b are the major and minor diameters of the tumor as measured using vernier caliper every 3 days after administration. The tumor inhibition rate (TIR) was calculated by the formula:TIR= [(A-B)/A]×100%, where A and B are mean volume of the tumors of the control and treatment groups, respectively. The tumors were then removed and split. Half of the tissue was made frozen sections (5μm thick) for DiOC7 perfusion staining immediately and then the perfusion vessels was observed using fluorescence microscopy,and the average distance of perfusion blood vessels are calculated, and the other half was fixed in formalin and embedded in paraffin, Then, Paraffin sections (5μm thick) were maked for immunohistochemistry. The MVD was quantified in each of the five 'hotspot' areas on a slide at high at×200 magnification,Tumor MVD was calculated as the mean of vessel counts recorded at 5 representative locations.4. Data and StatisticalAll the data were processed by Spss 13.0.All the data were noted in mean±standard deviation (mean±SD); To determine the statistically significant difference in parameters among in the four groups, the one-way ANOVA test was employed, and the LSD or Dunnett's was used in multiple comparison. Statistical significance was set at P< 0.05.ResultsNo one mice in the 4 groups died of their tumor burdens before scheduled sacrifice, All mice were killed at 15 days after initiation of therapy. All animals in each group developed tumors (100% tumorigenicity). Five days later, subcutaneous tumor nodules can be seen in 60 mice. The curves showed that tumors size increased gradually in the four groups. All treatment groups decreased tumor volume and weight relative to the control group (P< 0.05), Tumor growth inhibition was greater. Tumor growth was significantly inhibited in the 3 treatment groups(P<0.05), The TIR in the low dose,the middle dose and the high dose groups were 24.91%,45.83%and65.3%,respectively,and showed a marked inhibition of Tumor angiogenesis, The value of MVD and perfusion vessel average distance in Control groups,ES the low dose groups,ES the middle dose groups and ES the high dose groups were 43.13±9.86,30.27±9.95,17.87±7.69,9.60±4.31 and 43.33±8.21,66.06±14.98,84.94±6.99,108.16±18.10, respectively; while Compared with the control group, and among ES significantly (P<0.01).The effect of endostatin on tumor perfused vessels were evaluated using DiOC7 staining.endostatin-treated tumors had markedly increase in mean distances to the nearest perfused vessel compared with control group (P< 0.01),The numbers of perfused vessels was decrease. Mean distance to the perfused vessel was markedly lower in control group.in high dose therapy group, the distance to the perfused vessel was markedly increased than the low dose group and the middle dose group(P< 0.01), indicating a pronounced vascular reduce. The MVD was significantly less in tumors treated with high dose therapy compared to control tumors and tumors treated with low dose and the middle dose esdostatin (P< 0.01).Conclusion1.The results show that ES markedly inhibits tumor angiogenesis and tumor growth.2.The results show that ES have the obvious curative effect depends on the doses. Part three The inhibitory effect of endostatin combined with Ifosfamide chemoththerapy in treatment of transplant sarcoma in miceObjectiveTo evaluate the inhibitory effect of recombinant human endostatin combined with ifosfamide on angiogenesis and transplant sarcoma growth in mice.Material and methods1. Establish animal modelS180 cells was provided form abdomen of mice by the department of biology of Nanjing University. The cells were collected and centrifuged at 1000r/min,and then prepared in DEME into 5×107/ml cell suspension.0.1ml cell suspension was inoculated subcutaneouly into the right axillary of mice with sterile syringe, thus the animal model of sarcomas were set up.Observe mice subcutaneous transplant tumors, 75 mice have tumor after S180 cells inoculation only 5 days, all tumor diameter of biggest (a) and the least (b) are measured, calculation tumor size:V=ab2/2, tumor size too big and too small is excluded, There are 60 mices only enter laboratory,tumor size about 90mm3.2. Grouping and drug treatments60 nodules with unanimous size were randomized into one control group, two single agent groups and one combination group, fifteen for each group. In the ifosfamide (IFO) group, ifosfamide, 50mg.kg-1, was given by intraperitoneal injection, once a day for 5 days. In the endostatin (ES) group, endostatin,10mg.kg-1, was injected around the tumors, once a day for 14 days. In the combination(ES+IFO) group, endostatin, 10mg.kg-1, was injected around the tumor, once a day for 14 days, meanwhile the ifosfamide,50mg.kg-1,was given by intraperitoneal injection, once a day for 5 days. In the control (NS) group, the same volume of normal saline was given by intraperitoneal injection. 3. Testing indexThe tumor growth situation are observed, the tumor diameter of the biggest (a) and the least (b) are measured with vernier caliper every 3 days until over drug injection, Calculating volume, Taking average, drawing tumor growth curve, The mice in the 4 groups were sacrificed by cervical dislocation at 15 days after initiation of therapy. This DiOC? was injected into the tail vein 1 min before the mice were sacrificed, at a concentration of 1.0 mg/kg. the tumors were complete removed, Tumor volume was calculated by the formula:1/2×a×b2.where a and b are the major and minor diameters of the tumor as measured using vernier caliper every 3 days after administration. The tumor inhibition rate (TIR) was calculated by the formula:TIR= [(A-B)/A]×100%, where A and B are mean volume of the tumors of the control and treatment groups, respectively. The tumors were then removed and split. Half of the tissue was made frozen sections (5μn thick) for DiOC7 perfusion staining immediately and then the perfusion vessels was observed using fluorescence microscopy,and the average distance of perfusion blood vessels are calculated, and the other half was fixed in formalin and embedded in paraffin, Then, Paraffin sections (5μm thick) were maked for immunohistochemistry. The MVD was quantified in each of the five 'hotspot' areas on a slide at high at×200 magnification,Tumor MVD was calculated as the mean of vessel counts recorded at 5 representative locations.4. Data and StatisticalAll the data were processed by Spss 13.0.All the data were noted in mean±standard deviation (mean±SD); To determine the statistically significant difference in parameters among in the three groups, the one-way ANOVA test was employed, and the LSD or Dunnett's was used in multiple comparison. Statistical significance was set at P< 0.05.Results No one mice in the 4 groups died of their tumor burdens before scheduled sacrifice, All mice were killed at 5 days after initiation of therapy All animals in each group developed tumors (100% tumorigenicity),. Five days later, subcutaneous tumor nodules can be seen in 60 mice. The curves showed that tumors size increased gradually in the four groups.All treatment groups decreased tumor volume and weight relative to the control group (P< 0.01), Tumor growth inhibition was greater. Two single agent groups resulted in a significant reduction of the mean tumor volume and weight compared to control group(P< 0.01), The combined treatment group showed the smallest mean tumor volume and weight relative to the single treatment groups(P<0.01).the growth rate of tumor in combination group was significantly lower than that in other three groups. The tumor-inhibiting rate was 45.83%,76.57% and 84.66% respectively in endostatin group,ifosfamide group and combination group in comparison to control group. The value of MVD and perfusion vessel average distance in Control groups,ES groups,IFO groups and ES+IFO groups were 43.13±9.86,17.87±7.69,29.20±9.28,7.60±3.18 and 43.33±8.21,84.94±6.99,68.96±10.90,121.08±22.75, respectively.The effects of endostatin combined with ifosfamide on tumor perfused vessels were evaluated using DiOC7 staining.endostatin-treated tumors and combination-treated tumors had markedly increase in mean distances to the nearest perfused vessel compared with control group (P<0.01),The numbers of perfused vessels was decrease. Mean distance to the perfused vessel was markedly lower in control group.in combination therapy group, the distance to the perfused vessel was markedly increased (P<0.01) than endostatin group, indicating a pronounced vascular reduce. In summary, in therapy groups The distance to the perfused vessel remained significantly increased compared to control group (P<0.01).The MVD were markedly differences among the four groups.The MVD had a significant reduction in three treatment groups. The MVD was significantly less in tumors treated with endostatin or combination therapy compared to control tumors and tumors treated with ifosfamide alone (P<0.01).In addition, there was a further significant decrease in MVD in tumors treated with endostatin plus ifosfamide compared to tumors treated with either agent alone (P<0.01).ConclusionRecombinant human endostatin combined with ifosfamide has synergistic effect on inhibitory activity against the growth of transplanted sarcoma in mice and reduce the formation of capillary than either endostatin or ifosfamide used alone.