| Objective1. To discover and research the acting machenism of anti-tumor metastasis of basil polysaccharide through external experiment.2. To study the relationship between the anti-tumor metastasis of basil polysaccharide and epithelial-mesenchymal transition (EMT) through internal experiment.Methods1. In external experiments, we researched effect of basil polysaccharide on ability of cell proliferation, transfer, invasion ability, and expression level, expression of protain by MTT assay, wound healing assay, transwell, fluorescent quatititive PCR (RT-PCR), and western-blot assay.2. In internal experiments, tumor-bearing model under skin of nude mouse were constructed and divided into five groups stochastically. During experiments, we observed the variation of tumors in different groups, calculated the effect of anti-tumor, and used fluorescent quatititive PCR (RT-PCR) and western-blot assay to test the effect of basil polysaccharide on expression level of related components in tumor organization.3. Wistar model of transplating liver cancer was built in livers of rats. After model constructed successfully, hepatic artery ligation was undertook on the model. Then the rats were separated into 5 groups and forcedly feed with different solutions with different concentration of basil polysaccharide. After this experiment, we measured the gross tumor volume, calculated the number of tumor metastases in enterocoelia, and tested the the effect of basil polysaccharide on expression level of related components in tumor organization by fluorescent quatititive PCR (RT-PCR) and western-blot assay.Results1. MHCC97H and MHCC97L cells were processed by COCl2 solution with concentration of 200μmol·l-1. After 24 hours, the mRNA expression level of HIF-1 α in MHCC97H and MHCC97L cells rose apparently and have significant difference compared with regular group (P<0.01). Under the condition of COCl2 solution with concentration of 200 μ mol·l-1, the survival rate of MHCC97H and MHCC97L cells are high —— 77.76% and 87.76%, respectively. As a consequence, the condition of hypoxia model would be under the function of COCl2 solution with concentration of 200μmol·l-1 for 24 hours.In normoxic state, there was few influence of basil polysaccharide on multiplication capability of MHCC97H and MHCC97L cells. The survival rate of both of the cells is nearly 100%. The difference compared to regular groups has no statistical significance. Solution with medium concentration of basil polysaccharide has inhibiting effect on transfer ability of MHCC97H cells. The result shows significant difference compared with regular group (P<0.05). Basil polysaccharide has no apparent effect on invasion ability of MHCC97H cells or the transfer and invasion ability of MHCC97L cells. The result has no statistical significance compared to regular group (P>0.05). Basil polysaccharide has up-regulated effect on the mRNA and protein expression level of E-cadherin in MHCC97H and MHCC97L cells, and protein expression level of VMP1 and ZO-1 in MHCC97H cells. Basil polysaccharide also has down-regulated effect on the mRNA expression level of Vimentin in MHCC97H cells. Compared to regular group, the results of the up and down regulation of basil polysaccharide have significant difference (P<0.01, P<0.05).Under anoxic condition, basil polysaccharide has inhibiting effect on cell multiplication and transfer of MHCC97H and MHCC97L cells, and invasion ability of MHCC97H cells. The result has apparent difference compared to the group of hypoxia model (P<0.01, P<0.05). In the same condition, in MHCC97H and MHCC97L cells, mRNA and protein expression level of HIF-1α、N-cadherin and Vimentin will raise, the protein expression of β-catenin will raise, and the mRNA and protein expression level of E-cadherin and VMP1 will decline. In these case, basil polysaccharide could down-regulate the mRNA and protein expression level of HIF-1α、 N-cadherin and Vimentin and the protein expression of β-catenin, and up-regulate the mRNA and protein expression level of E-cadherin and VMP1 in MHCC97H cells. While in MHCC97L cells, basil polysaccharide could up-regulate the mRNA and protein expression level of E-cadherin and down-regulate the mRNA and protein expression level of HIF-1α.2. The experiment of tumor-burdened nude mouse model with MHCC97H cells shows:low, medium and high dose of basil polysaccharide and solution of sorafenib have different anti-tumor rate —— 25.15%,14.72%,46.63% and 55.58% respectively to subcutaneous tumors of nude mouse, which, compared to model group, have no significant difference. Also, in tumor tissue, basil polysaccharide could down-regulate the mRNA and protein expression of HIF-1 a, Vimentin and N-cadherin and protein expression of -catenin, and up-regulate the mRNA and protein expression of E-cadherin, VMP1 and ZO-1, which, compared to model group, have significant difference (P<0.05, P<0.01). What’s more, in tumor tissue, Sorafenib could up-regulate the mRNA and protein expression of E-cadherin, down-regulate the protein expression of β-catenin, the mRNA expression of N-cadherin, and the mRNA and protein expression of Vimentin, hich also shows great difference (P<0.01), compared to model group.3. The result of wistar model of transplating liver cancer in rats shows: HAL could diminish the volume of liver tumor apparently, which, compared to model group without HAL, has significant difference (p<0.05). While, after HAL operation, the amount of metastasis (36±8) increased obviously, which shows great difference to the one (11+3) of model group. After HAL operation, in tumor tissue, the mRNA and protein expression level of HIF-1 a, N-cadherin and Vimentin rised, the protein expression level of β-catenin rised, and the mRNA and protein’expression level of E-cadherin and VMP1 reduced. Compared with the HAL group, medium and high dose of basil polysaccharide groups have much less transferring amount of tumor to abdominal cavity —— 22±4 and 19 ±6 respectively, and have significant difference —— P<0.05 and P<0.01. In tumor tissue, basil polysaccharide could down-regulate the mRNA and protein expression level of HIF-α , N-cadherin and Vimentin, down-regulate the protein expression level of β-catenin, and up-regulate the mRNA and protein expression level of E-cadherin and VMP1, which also have great difference compared with HAL groups (P<0.05, P<0.01).Conclusion1. Oxygen deficit could promote EMT of MHCC97 and MHCC97L. In anoxic state, in MHCC97 and MHCC97L cells, the expression of HIF-1a could raise, and the expression of E-cadherin and VMP1 related to EMT could decline, and the expression of β-catenin、N-cadherin and Vimentin could raise, which will induce the occurrence of EMT, and enhance the capacity of sports and invasion of cells. As a result, the invasion and transfer of tumor was sped up.2. Basil polysaccharide could inhibit the EMT in MHCC97H and MHCC97L. This function has close relationship with hypoxic microenvironment of tumor, shows difference in different cells, but works very well to MHCC97H. The research shows that, basil polysaccharide could control the sports and invasion capacity of MHCC97H by regulating the expression of HIF-1 a and related E-cadherin, β-catenin, N-cadherin, Vimentin and VMP1 to influent the occurring of EMT in anoxic state. While, basil polysaccharide could only control the sports ability of MHCC97L by regulating the expression of HIF-1 a and related E-cadherin, but has nothing to do with invasion ability.3. Both basil polysaccharide and sorafenib could treat tumor by control EMT, but with different principle. Basil polysaccharide could improve the hypoxic microenvironment, and down-regulate the expression of HIF-1α, then regulate the expression of related E-cadherin, β-catenin, N-cadherin, Vimentin, VMP1 and ZO-1, and control the occurring of EMT. While, sorafenib could only inhibit the EMT by control the protein expression of E-cadherin, β-catenin and Vimentin.4. Basil Polysaccharide could resist tumor metastasis by influence the tumor in many aspects. The results of this experiment and previous research found that Basil Polysaccharide could improve the hypoxic microenvironment, induce the occurrence of tumor cell EMT, strengthen the immunity of the organism, and improve the survival state. In conclusion, Basil Polysaccharide has influence on the tumor with multi-target and in multiple pathways, which, as the result let Basil Polysaccharide having the function of anti-tumor metastasis. |
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