| Objectives1. To evaluate the effect and the mechnism of genistein (Gen) on proliferation, migration and adhesion to endothelium of human gastric adenocarcinoma SGC7901 cell.2. To examine the feasibility of using combination of adenovirus-mediated gene delivery of TIMP-1 plus endostatin and cell transplantation technique to inhibit tumor growth and metastasis in mouse melanoma. To demonstrate the possibility that the implantation of TIMP-1- and endostatin-producing fibroblasts at a site in vivo where direct secretion of TIMP-1 and endostatin into the blood is a promising approach for the development of cancer therapy.Methods1. Cell counting assay and MTT assay were performed to study the effect of Gen on SGC7901 cell proliferation.2. Wound-healing assay was used to test the effect of Gen on SGC7901 cell migration. Wound-healing assay and transwell invasion assay were also used to assess the effect of the supernatants of cultured mouse primary fibroblasts after infection with indicated adenovirus on mouse melanoma B16BL6 cell migration and invasion.3. Tumor cell adhesion to endothelium assay was used to explore the ability of SGC7901 cells pretreated with Gen, and the the ability of mouse melanoma B16BL6 cells pre-incubated with the supernatants of cultured mouse primary fibroblasts after infection with Ad-TIMP-1 and Ad-End, to adhere to the monolayer of HUVECs.4. Immunofluorescence assay was used to evaluate the effect of Gen on the distribution of E-cadherin,β-catenin, and f-actin, and western blotting assay was used to test the expression of FAK,p-FAK,E-cadherin andβ-catenin in SGC7901 cell.5. Indirect enzyme-linked immunosorbent assay (ELISA) was used to detect the level of TIMP-1 and endostatin secretion in supernatants from the culture of indicated adenovirus-infected primary fibroblasts, and in sera collected from indicated adenovirus-infected primary fibroblasts- treated mice.6. Tumor inoculation assay in animal experiments was used to explore the therapeutic effect of in vivo production of human TIMP-1 and endostatin after implantation of primary fibroblasts infected with indicated adenovirus into tumor-bearing mice and the cytochemical method was used to obseve the tumor histopathological changes.7. Experimental lung metastasis model was established by injection with B16BL6 cells into mice tail vein, and indicated adenovirus-infected primary fibroblasts after 24 h were also subcutaneously implanted into mice. Twenty one days after tumor cells injection, mice were sacrificed to examine the effect on nodules visible as black forms on the surface of the lungs in B16BL6 cells.Results1. Results from cell counting and MTT assays demonstrated that Gen suppressed SGC7901 cell proliferation, especially in the dose of 7.5μmol/L (P<0.01).2. Results from wound-healing assay showed that Gen significantly inhibited the migration of SGC7901 cell in the dose-dependent manner (with the range of concentration from 5.0μmol/L to 20.0μmol/L) (P<0.01). Wound-healing assay and transwell invasion assay showed that conditioned medium either from Ad-End- or Ad-TIMP-1-infected primary fibroblasts significantly suppressed B16BL6 cell migration and invasiveness (P<0.05), while the conditioned medium from primary fibroblasts infected with Ad-TIMP-1 plus Ad-End dramatically inhibited the migration and invasiveness of B16BL6 cell (P<0.01).3. Findings from SGC7901 cell adhesion to HUVECs assay showed that Gen in the concentration from 5.0μmol/L to 10.0μmol/L significantly decreased the ability of SGC7901 cells adhesion to monolayer of HUVECs (P<0.01), and no inhibition was shown in the dose of 2.5μmol/L of Gen treated group (P>0.05), when compared with control. Interestingly, it is also no difference between Gen treated group in the dose of 20.0μmol/L and control (P>0.05). Data from adhesion of B16BL6 cell to the monolayer of HUVECs assay demonstrated that conditioned medium from Ad-TIMP-1 alone, Ad-End alone, or Ad-TIMP-1 plus Ad-End- infected primary fibroblasts can markedly inhibit the adhesion of B16BL6 cell to HUVECs, especially in both Ad-TIMP-1- and Ad-TIMP-1 plus Ad-End- treated group (P<0.01), when compared with mock- and Ad-GFP- infected groups.4. Immunofluorescence assay showed that Gen in the dose of 7.5μmol/L led to re-distribution of E-cadherin,β-catenin, and actin cytoskeleton in SGC7901 cells, and the number of the stress fibers in SGC7901 cells treated with Gen markedly decreased relative to control. Results from western blotting assay showed that Gen down-regulated the expression of p-FAK and E-cadherin (except in the dose of 10.0μmol/L), but no inhibition to the expression of FAK andβ-catenin in SGC7901 cell. Surprisingly, when the dose of Gen is 10.0μmol/L, obvious up-regulation of E-cadherin in Gen treated cells was observed.5. Secretion of TIMP-1 or/and End was shown in supernatants from the culture of indicated adenoviruses infected mouse primary fibroblasts, and in sera of mice after implantation of fibroblasts infected with indicated adenoviruses. It was shown from the ELISA that the levels of TIMP-1 or endostatin secretion into supernatants in Ad-TIMP-1- or Ad-End- infected primary fibroblasts increased in a time-dependent manner, suggesting that primary fibroblasts infected with Ad-TIMP-1 or Ad-End are suited for the production of active TIMP-1 or endostatin. Secretion of TIMP-1 or endostatin in sera was detectable within 24 h and remained elevated until the final measurement at 20 days in mice after implantation of primary fibroblasts infected with Ad-TIMP-1 and Ad-End, demonstrating that implantation of TIMP-1 and endostatin -producing fibroblasts at a site in vivo where direct secretion of TIMP-1 and endostatin into the blood.6. Subcutaneously injection with either Ad-TIMP-1 or Ad-End treatment fibroblasts into tumor-bearing mice showed a significant delay of tumor growth compared with that of mock and Ad-GFP groups. It was also shown that injection with fibroblasts combinational treatment of Ad-TIMP-1 with Ad-End could synergistically inhibit tumor growth, and resulted in more effective tumor regression than those injections with either Ad-TIMP-1 alone or Ad-End alone infected fibroblasts. Tumor samples, examined histologically using H&E staining, demonstrated that the B16Bl6 tumors displayed significant lymphocyte infiltration and many wide areas of necrosis in the groups treated with indicated adenovirus-infected fibroblasts, especially, in the group of combinational treatment of Ad-TIMP-1 with Ad-End infected fibroblasts.7. A statistically significant decrease in the number of the surface of lung metastases of B16BL6 cells was observed in mice injected with Ad-TIMP-1- or Ad-End -infected primary fibroblasts, as compared with that in mice implanted with mock and Ad-GFP treated groups. Interestingly, lung metastasis by B16BL6 cells in mice injection with fibroblasts combinational treatment of Ad-TIMP-1 with Ad-End demonstrated synergistic and more effective regression than those in either Ad-TIMP-1- or Ad-End- infected primary fibroblasts therapeutic mice.Conclusion1. Gen inhibits cell proliferation, migration, and adhesion on endothelium presumably through its regulating the redistribution of E-cadherin,β-catenin, and actin cytoskeleton, and down-regulating the expression of p-FAK and E-cadherin in SGC7901 cells.2. Secretion of TIMP-1 and endostatin in supernatants of cultured mouse primary fibroblasts after infection with Ad-TIMP-1 and Ad-End might significantly inhibit B16BL6 cell migration, invasion and adhesion onto the monolayer of HUVECs.3. Implantation of fibroblasts, infected with Ad-TIMP-1 alone, Ad-End alone, or Ad-TIMP-1 plus Ad-End, producing high amounts of TIMP-1 or/and endostatin in vivo, resulted in detectable in sera levels which may clearly inhibit the tumor growth and lung metastasis in murine melanoma model. Meanwhile, implantation of fibroblasts cotransduction with TIMP-1 and endostatin genes demonstrated the synergistic antitumor growth and metastasis.4. Findings present here suggest that adenovirus-mediated gene-infected primary fibroblasts in vivo provided a new cellular gene therapy strategy and had potential clinical applications in the treatment of tumors. Moreover, the synergistic and more effective anti-tumor growth and metastasis were shown in combinational treatment of Ad-TIMP-1 with Ad-End fibroblasts. The results also demonstrat that the combination gene therapy with cellular therapy is possible represented a promising approach for the development of cancer therapy.
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