重组转化生长因子α-绿脓杆菌外毒素融合蛋白对人膀胱癌细胞生长的抑制作用

目的 探讨重组转化生长因子α-绿脓杆菌外毒素融合蛋白(TP40)对膀胱癌细胞生长的抑制作用。方法 采用蛋白印迹(Western blot)法检测人膀胱癌124细胞表皮生长因子受体(EGFR)的表达。用浓度为5～1000μg／L的TP40处理T24细胞,甲基噻唑基四唑(MTY)法检测细胞生长24、48、72和96h时的生长情况。氚-胸腺嘧啶核苷(^3H]-TdR)掺入法检测TP40对T24细胞蛋白质合成的抑制作用。用浓度为1～7500μg／L的表皮生长因子(EGF)竞争拮抗TP40的细胞毒作用。结果 T24细胞表达EGFR强阳性。浓度为5、50、100、250、500、750和1000μg／L的TP40处理T24细胞96h,细胞生长抑制率分别为10％、19％、27％、41％、47％、53％和6l％。用750μg／L的TP40处理T24细胞2,4、48、72和96h,^3H]-TdR掺入率分别为80％,69％,48％和51％。浓度为1～7500μg／L的EGF对TP40有竞争抑制作用。结论 人膀胱癌T24细胞能高水平地表达EGFR;重组TP40对T24细胞的生长具有剂量、时间依赖性抑制作用,该作用是通过EGFR进行的。

Inhibitory effect of recombinant transforming growth factor alpha-pseudomonas exotoxin 40 on human bladder cancer cell proliferation

OBJECTIVE: To study inhibitory effect of recombinant transforming growth factor alpha-Pseudomonas exotoxin fusion protein (TP40) on proliferation of the human bladder cancer T24 cells. METHODS: Expression of epidermal growth factor receptor (EGFR) in cultured T24 cells was analyzed with Western blot assay. Human bladder cancer T24 cells were exposed to TP40 at 5 - 1 000 microg/L. Methyl thiazolyl tetrazolium assay was applied to evaluate the cell proliferation by measuring the absorbance (A) at 570 nm with a microplate reader. Tritium labeled thymine deoxyriboside ((3)H]-TdR) uptake was measured to observe DNA synthesis. Competition assays were performed by the EGF at 1 - 7 500 microg/L. RESULTS: Expression of EGFR was high in human bladder cancer T24 cells. Cell growth was suppressed by 10%, 19%, 27%, 41%, 47%, 53% and 61% after 96 h treatment with TP40 at 5, 50, 100, 250, 500, 750 and 1 000 microg/L, respectively. (3)H]-TdR incorporation was 80%, 69%, 48% and 51% after 24 h, 48 h, 72 h, 96 h treatment with TP40 at 750 microg/L, respectively. When the concentration was 1 - 7 500 microg/L, EGF could block the inhibitory effect of TP40 to some extent. CONCLUSIONS: Human bladder cancer T24 cells express EGFR at a high level. TP40 could inhibit the growth of T24 cells effectively in a dose- and time-dependent manner. The cytotoxic effects of TP40 were specifically mediated by EGFR.