雄激素非依赖性前列腺癌DU145细胞中TRPM8和TRPA1的表达及活性研究

Study on the expression and activity of TRPM8 and TRPA1 in androgen-independent prostate cancer cells DU145

目的探讨TRPM8和TRPA1在雄激素非依赖性前列腺癌DU145细胞中是否表达,以及薄荷醇是否通过作用于TRPM8通道来影响DU145细胞的生物学行为。方法采用Nested RT-PCR实验、Western Blot实验和免疫组织化学法从不同水平(mRNA水平、蛋白水平)以及直观观察层面检测TRPM8和TRPA1在DU145细胞中的表达;采用Ca^(2+)成像实验观察薄荷醇能否诱导DU145细胞内Ca^(2+)浓度的变化;采用MTT法检测梯度浓度的薄荷醇对DU145细胞增殖能力的影响。结果 Nested RT-PCR实验、Western Blot实验和免疫组织化学法结果证明,在DU145细胞中检测到TRPM8表达,而未检测到TRPA1表达。Ca^(2+)成像实验结果显示,在37℃环境下细胞荧光非常弱,加入薄荷醇后细胞内Ca^(2+)荧光强度急剧升高。MTT法检测结果显示,与未经薄荷醇处理的对照组相比不同浓度(25、50、75、100μmol/L)薄荷醇处理组的细胞增殖率均下降(P<0.05)。经BCTC预处理20 min,薄荷醇所致的细胞增殖抑制被部分恢复(P<0.05)。结论雄激素非依赖性前列腺癌DU145细胞中有大量的具有生物活性的TRPM8通道蛋白表达,而可同被薄荷醇激活的TRPA1却未被检测到表达。薄荷醇可激活DU145细胞中的TRPM8通道诱导Ca^(2+)荧光强度急剧升高,抑制DU145细胞的增殖。

Objective To investigate the expression of TRPM8 and TRPA1 in androgen-independent prostate cancer （AIPC） cells DU145 and the mechanism of menthol mediating the biological behaviors of DU145 cells through TRPM8 channel. Method RT-PCR, Western Blot and immunohistochemical assay were performed to investigate the expression of TRPM8 and TRPA1 on mRNA and protein levels, and the expression of TRPM8 and TRPA1 in DU145 cells were ob-served visually;Ca2＋imaging was performed to investigate whether menthol could induce the Ca2＋concentration changes in DU145 cells;MTT assay was utilized to investigate the effect of menthol on the proliferation of DU145 cells. Result The results of RT-PCR, Western Blot and immunohistochemical assay suggested that TRPM8 expressed in DU145 cells while there was no TRPA1 expression. Ca2＋imaging experiments showed a weak cell fluorescence in 37℃environment, and then increased sharply after adding menthol. The results of MTT assay indicated that, compared with the non-treated cells, the cell increment rate of cells treated with menthol of different concentrations （25, 50, 75, 100 μmol/L） consistent-ly decreased. But the decrease was partly corrected by BCTC treatment for 20 min （P〈0.05）. Conclusion There are a large number of bioactive TRPM8 pathway proteins in the AIPC DU145 cells, and TRPA1, which can be activated by menthol, has not been detected. Menthol may activate the TRPM8 channel in the DU145 cells to induce a sharp increase in Ca2＋fluorescence intensity and inhibit the proliferation of DU145 cells.