| ObjectiveIn United States, cryoablation therapy has become one of the first-line treatments for prostate cancer. While in local treatment of metastatic prostate cancer, there exist a contradiction between the the destruction of the tumor and the protection of normal tissue. So there will exist a district called sublethal district for some paatients.Residual tumor cells and tumor microenvironment is closely related to tumor recurrence and metastasis. TGF-β is closely related with invasion, metastasis, the transformation of epithelial mesenchymal and the tumor immune. In our previous study, we found TGF-β in plasma changed after cryoablation. We think the TGF-β in the microenvironment may play a role in prostate cancer.Therefore, we simulated state of cells in different microenvironment after cryoablation invitro and at the cellular level,we explored the change of apoptosis,TGF-β1 concentration and the expression leval of TGF-β1, smad2,smad3,smad4 in CWR- 22 RV1 prostate cancer cells following cryoablation. And on this basis,we further explored the influence of TGF-β block on migration and the aggressivity of prostate cancer cells after cryoablation.ContentTGF-β is closely related with invasion, metastasis, the transformation of epithelial mesenchymal and the tumor immune. We simulated the model of prostate cancer treated with cryoablation in cells level to explore the releasing rules of the cells in different districts in the prostate cancer treated with cryoablation and in which the variation of the proteins in the TGF-β pathway like TGF-β, smad2, smad3, smad4.Then we explored the influence of TGF-β block on migration and the aggressivity of prostate cancer cells after cryoablation by transwell test.MethodsAccording to the district in the tumor following cryoablation, the CWR-22 RV prostate cancer cells were divided into four groups to simulate the freezing model of prostate cancer in cell level :Group A, control cells( the untreated prostate cancer cell).Group B, treated with low temperature(put the cell suspension in the-80℃ icebox for 7min, then rewarm it in 37℃ water).Group C, normal cells cultured with1640 and necrosis supernatant(put the cell suspension in the liquid nitrogen for4 min,then rewarm it in 37℃ water,got the supernate after centrifuge).Group D, cells treated with low temperature were cultured with1640 and necrosis supernatant. The cell apoptosis was observed by Flow Cytometry Analysis at 10 h.Then supernatant in four groups was extracted to test the concentration of TGF-β by ELISA at 5h, 10 h,20h, 36 h, 48 h to explore the releasing rules of the cells in different districts in the prostate cancer treated with cryoablation, At the same time, the level of intracellular TGF-β, smad2,smad3, smad4 of four groups were detected by Western Blot at10 h.Mean while,we use transwell test to observe the influence in the ability of migration and invasiveness of prostate cancer cell.Results1. The apoptosis rate in group B is higher than that in group A(21.83±1.03 vs 11.2±1.57, P<0.05).The apoptosis rate in group D is higher than that in group B(29.50±0.52 vs 21.83±1.03,P<0.05).2. According to the result of Enzyme-linked immunosorbent assay, the changing tendency of TGF-β1 concentration in four groups is roughly consistent. TGF-β1concentration in four groups has been increasing until 36 hours, then the TGF-βconcentration decreased. The concentration of TGF-β1 in group D is higher than that in group A(P<0.05). The concentration of TGF-β1 in group B is lower than that in group A(P<0.05).3. In Western Blot test, the expression level of TGF-β1,smad2,smad3 and smad 4 in group D is higher than that in group A, the expression level of TGF-β1,smad2,smad3 and smad 4 in group B is lower than that in group A.4. The transwell test showed that the number of cells traversing gale tin in Group A、B、C、D、E was 100±3.2 vs 60±4.2 vs 126±3.8 vs 158±3.3vs 30±5.1,P<0.05. The number of cells traversing the role of the small room was 89±1.2 vs65±2.8 vs 115±4.6 vs 132±3.1 vs 48±4,P<0.05。The ability of invasiveness in group D is stronger than that in group A, The ability of invasiveness in group B is weaker than that in group A. The block of TGF-βcan inhibit the invasiveness of sublethal cells.5. ConclusionIn our research, we simulated a model of prostate cancer treated with cryoablation in cells level. And we found that the apoptosis rate is higher in sublethal cells and the sublethal cells in necrosis liqhid have higher apoptosis rate than that in sublethal without necrosis.TGF-β concentration in four groups is roughly consistent. It has been increasing until 36 hours, then the TGF-β concentration decreased. At the same time. TGF-β1 concentration in the sublethal cells in necrosis district close to necrosis liquid(group D) and in district close to necrosis liquid out of ice ball(group C) were higher than that in district away from necrosis liquid out of ice ball(group A)which had higher TGF-β1 concentration in district away from necrosis liquid(group B). As time going, TGF-β1 concentration in the sub-lethal district close to necrosis liquid(group D) became higher than that in district close to necrosis liquid out of ice ball(group C). Compared with the district away from necrosis liquid out of ice ball(group A), the activity of TGF-β pathway and the ability of invasiveness in the sub-lethal district away from necrosis liquid was subdued.While the activity of TGF-β pathway and the ability of invasiveness in the sub-lethal district close to necrosis liquid was enhanced. The TGF-β inhibitor could inhibit the invasiveness of prostate cancer cell treated with cryoablation. |
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