白消安通过调节雄激素受体磷酸化诱导前列腺癌细胞凋亡的分子机制

Molecular mechanism of prostate cancer cell apoptosis induced by busulfan via adjustment of androgen receptor phosphatization.

作者信息

Liu Jun, Jiang Guojun, Yang Aiping, Yang Guohui, Yang Wenjuan, Fang Yi

机构信息

Department of Clinical Laboratory, Zhejiang Xiaoshan Hospital Hangzhou, Zhejiang 311201, China.

Department of Pharmacy, Zhejiang Xiaoshan Hospital Hangzhou, Zhejiang 311201, China.

出版信息

Am J Transl Res. 2016 Nov 15;8(11):4881-4891. eCollection 2016.

PMID:27904688

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5126330/

Abstract

OBJECTIVE

To probe killing effect of busulfan to prostate cancer cell without androgen and the influence of androgen receptor phosphatization and analyze its molecular mechanism.

METHODS

prostate cancer cell line 22RV1, LAPC4 and LNCaP treated with busulfan under androgen-free condition underwent CCK-8 examination to probe killing ability of the medicine. Flow cytometry was used to check the influence of busulfan on apoptosis rate of prostate cancer cell line LAPC4. Expression level of androgen receptor (AR), Src and Ack1 and change in phosphatization of AR after busulfan treatment were measured by RT-PCR and Western blotting. Finally, influence o proliferation ability and apoptosis of LAPC4 were measured using EGF-busulfan co-processing.

RESULTS

Significant dose-dependency was observed as killing ability rises with higher busulfan concentration (p<0.05). Significant improvement in prostate cancer cell inhibition ability of busulfan was also observed with prolonging of time (p<0.05). Then we discovered, as indicated by flow cytometry, that busulfan inhibits prostate cancer cell LAPC4 proliferation by strengthening its apoptosis (p<0.05), which showed significant dose- and time-dependency. Detection of AR expression and phosphatization level showed no significant influence on mRNA and protein expression level of AR made by busulfan. However, decline of phosphatization level at AR Y534 site was positively related to busulfan treatment time. Busulfan was found to be inhibitory to Src kinase induced by EGF and level of resulting AR phosphatization in our further probe into the mechanism of busulfan influence on phosphatization level at AR Y534 site. Nude mice experiment indicated that busulfan was inhibitory to protein expression of AR downstream target gene prostate specific antigen (PSA) and human tissue kallikrein2 (hk-2), thus inhibited in vivo tumorigenic ability of prostate cancer cells.

CONCLUSION

Busulfan was significantly inhibitory to prostate cancer cell proliferation by inhibiting phosphatization of Src kinase at AR Y534 site.

摘要

目的

探讨白消安对去雄激素状态下前列腺癌细胞的杀伤作用及对雄激素受体磷酸化的影响，并分析其分子机制。

方法

在无雄激素条件下，用白消安处理前列腺癌细胞系22RV1、LAPC4和LNCaP，进行CCK-8检测以探究药物的杀伤能力。采用流式细胞术检测白消安对前列腺癌细胞系LAPC4凋亡率的影响。通过RT-PCR和蛋白质免疫印迹法检测白消安处理后雄激素受体（AR）、Src和Ack1的表达水平以及AR磷酸化的变化。最后，用表皮生长因子（EGF）与白消安共同处理，检测对LAPC4增殖能力和凋亡的影响。

结果

随着白消安浓度升高，杀伤能力增强，呈现显著的剂量依赖性（p<0.05）。随着时间延长，白消安对前列腺癌细胞的抑制能力也显著提高（p<0.05）。然后我们通过流式细胞术发现，白消安通过增强前列腺癌细胞LAPC4的凋亡来抑制其增殖（p<0.05），呈现显著的剂量和时间依赖性。AR表达和磷酸化水平检测显示，白消安对AR的mRNA和蛋白表达水平无显著影响。然而，AR Y534位点的磷酸化水平下降与白消安处理时间呈正相关。在进一步探究白消安对AR Y534位点磷酸化水平影响的机制时，发现白消安可抑制EGF诱导的Src激酶及由此导致的AR磷酸化水平。裸鼠实验表明，白消安可抑制AR下游靶基因前列腺特异性抗原（PSA）和人组织激肽释放酶2（hk-2）的蛋白表达，从而抑制前列腺癌细胞的体内致瘤能力。