Ikezoe Takayuki, Chen Sophie S, Yang Yang, Heber David, Taguchi Hirokuni, Koeffler H Phillip
Division of Hematology/Oncology, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
Int J Oncol. 2003 Nov;23(5):1461-70.
PC-SPES is an eight-herbal mixture which has activity against prostate cancer cells and can reduce the serum level of prostate specific antigen (PSA) in more than 80% of individuals with prostate cancer. We conducted this study to begin to clarify the molecular mechanism by which PC-SPES inhibited the growth of prostate cancer cells and down-regulated expression of PSA. Western blot analysis, luciferase reporter assay using a variety of promoters of the PSA gene and the isolated androgen receptor response elements (ARE), as well as electrophoretic mobility shift assay (EMSA) were employed to study the effect of PC-SPES on DHT-induced expression of PSA in LNCaP androgen-dependent human prostate cancer cells. Also, Western blot analysis and luciferase reporter assay using 12-0-tetradecanoylphorbol-13-acetate response elements were employed to study the ability of PC-SPES to activate the c-Jun NH2-terminal kinase (JNK)/c-Jun/AP-1 signal pathway in these cells. Reporter studies showed that PC-SPES inhibited DHT-induced PSA promoter/enhancer-luciferase activity via inhibition of ARE transcriptional activity. Western blot analysis showed that PC-SPES down-regulated DHT-induced expression of PSA without decreasing DHT-induced nuclear level of AR. EMSA demonstrated that PC-SPES inhibited the binding of DHT-activated AR to ARE. Moreover, we found that PC-SPES phosphorylated JNK, increased levels of phosphorylated and unphosphorylated forms of c-Jun, and enhanced AP-1 transcriptional activity in LNCaP cells. Interestingly, when LNCaP cells were stably tranfected with the dominant negative JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1), these cells no longer underwent apoptosis and growth inhibition in the presence of PC-SPES. But, PC-SPES still decreased levels of PSA in the LNCaP-JIP-1 cells. Taken together, PC-SPES inhibited binding of DHT-activated AR to AREs of PSA gene resulting in down-regulation of ARE transcriptional activity and expression of PSA, and this occurred independently of the JNK/c-Jun/AP-1 signal pathway. Also, PC-SPES activated the JNK/c-Jun/AP-1 signal pathway resulting in growth arrest and apoptosis of prostate cancer cells.
PC-SPES是一种由八种草药组成的混合物，对前列腺癌细胞具有活性，并且能使超过80%的前列腺癌患者的前列腺特异性抗原（PSA）血清水平降低。我们开展这项研究，旨在初步阐明PC-SPES抑制前列腺癌细胞生长及下调PSA表达的分子机制。采用蛋白质免疫印迹分析、利用PSA基因的多种启动子和分离的雄激素受体反应元件（ARE）进行荧光素酶报告基因检测，以及电泳迁移率变动分析（EMSA），来研究PC-SPES对双氢睾酮（DHT）诱导的LNCaP雄激素依赖性人前列腺癌细胞中PSA表达的影响。此外，采用蛋白质免疫印迹分析和利用12-氧-十四烷酰佛波醇-13-乙酸酯反应元件进行荧光素酶报告基因检测，来研究PC-SPES激活这些细胞中c-Jun氨基末端激酶（JNK）/c-Jun/活化蛋白-1（AP-1）信号通路的能力。报告基因研究表明，PC-SPES通过抑制ARE转录活性，抑制DHT诱导的PSA启动子/增强子荧光素酶活性。蛋白质免疫印迹分析表明，PC-SPES下调DHT诱导的PSA表达，但不降低DHT诱导的AR核水平。EMSA证明，PC-SPES抑制DHT激活的AR与ARE的结合。此外，我们发现PC-SPES使JNK磷酸化，增加c-Jun磷酸化和未磷酸化形式的水平，并增强LNCaP细胞中的AP-1转录活性。有趣的是，当LNCaP细胞用JNK相互作用蛋白-1（JIP-1）的显性负性JNK结合结构域（JBD）进行稳定转染时，在存在PC-SPES的情况下，这些细胞不再发生凋亡和生长抑制。但是，PC-SPES仍然降低LNCaP-JIP-1细胞中的PSA水平。综上所述，PC-SPES抑制DHT激活的AR与PSA基因的ARE结合，导致ARE转录活性和PSA表达下调，且这一过程独立于JNK/c-Jun/AP-1信号通路。此外，PC-SPES激活JNK/c-Jun/AP-1信号通路，导致前列腺癌细胞生长停滞和凋亡。
