| Purpose Selenium(Se) has been widely reported to possess anti-tumor effects. Angiogenesis is required to supply oxygen, nutrients, and growth factors for tumor growth. Angiopoietin-2(Ang-2), vascular endothelial growth factor(VEGF), and platelet-derived growth factor(PDGF) are the important cytokines for the regulation of angiogenesis and tumor growth. To explore whether the anti-tumor effect of Se was associated with angiogenesis in vivo, we studied the effects of sodium selenite(Sel) and methylseleninic acid(MSA) on tumors induced by canine mammary tumor cells(CMT1211) in mice; cyclophosphamide(CTX) served as a positive control.Method(1) The establishment of the model of mice transplanted tumor. To implant the tumors, the CMT1211 cells were subcutaneously injected into the right flank of each mouse. After 7 days, there has visible tumor on every mouse. Then the animals were randomly divided into four groups: A,B,C,D(10 mice/group).(2) Transplanted tumor mice were treated with drugs. Group A: untreated controls(oral gavage of normal saline), Group B: cyclophosphamide(CTX) injected by the intraperitoneal route at a dose of 40 mg/kg weekly, Group C: sodium selenite(Sel) was given by gavage at a dose of 3 mg/kg/d, and Group D: methylseleninic acid(MSA) was given by gavage at a dose of 3 mg/kg/d. Treatment continued for 21 days.(3) Tumor growth inhibition rate was calculated. The animals received intraperitoneal anesthesia with pentobarbital natrium and were sacrificed 24 hours after the last administration. Collect the tumor, calculate the tumor weights, volumes anti-tumor rate.(4) Determination of Se content in tumor tissues. The Se content in the tumor tissue was estimated by fluorescent atomic absorption spectrometry.(5) The tumor tissue were performed H&E staining for histopathological evaluation.(6) To determine the intratumoral microvessel density(MVD) and vascular maturation index(VMI) of the xenografts, we performed immunofluorescent double staining with anti CD31 and anti-α-SMA antibodies specific for the endothelial cells and pericytes of microvessels, respectively.(7) The assessment of the production of cytokines which are important for the regulation of angiogenesis and tumor growth by the mouse tumors, including angiopoietin-2(Ang-2), vascular endothelial growth factor(VEGF), and platelet-derived growth factor(PDGF), by IHC, western blotting, and RT-QPCR.Result(1) The CMT1211 cells(2×106 cells per mouse) were subcutaneously injected into the right flank of each mouse. After 7 days, there has visible tumor on every mouse. The formation rate of transplanted tumor was 100%.(2) Compared with the control group, administration of Sel, MSA, and CTX to the mice with xenografts significantly reduced the growth of tumors induced by the CMT1211 cells. The average tumor weights of the CTX, MSA, and Sel group decreased by approximately 51, 40, and 32 %, respectively, and the mean tumor volume decreased by approximately 56, 46, and 37 %, respectively.(3) Compared with the control group and CTX group, the Se content was significantly increased in the Sel and MSA groups.(4) H&E staining showed that there was a larger necrotic area in the treatment group compared with the control group. Cell morphological changes, including the protoplasm concentration, nucleus pyknosis, karyorrhexis.(5) Treatment with CTX, MSA, and Sel resulted in a reduction in MVD and a increase in VMI, respectively.(6) The changes of m RNA and protein expression of Ang-2, PDGF, and VEGF shared similar pattern and were lower(p<0.05) in MSA, Sel, and CTX groups compared with the control group.Conclusion Selenium compounds inhibit the growth of tumor in vivo, the mechanism of anti-tumor effect may be selenium compounds induces an anti-angiogenesis effect via the inhibition of angiogenetic factors. |
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