Bommareddy Ajay, Hahm Eun-Ryeong, Xiao Dong, Powolny Anna A, Fisher Alfred L, Jiang Yu, Singh Shivendra V
Department of Pharmacology and Chemical Biology, and University of Pittsburgh Cancer Institute, Pennsylvania, USA.
Cancer Res. 2009 Apr 15;69(8):3704-12. doi: 10.1158/0008-5472.CAN-08-4344. Epub 2009 Mar 31.
Phenethyl isothiocyanate (PEITC) is a promising cancer chemopreventive agent but the mechanism of its anticancer effect is not fully understood. We now show, for the first time, that PEITC treatment triggers Atg5-dependent autophagic and apoptotic cell death in human prostate cancer cells. Exposure of PC-3 (androgen independent, p53 null) and LNCaP (androgen responsive, wild-type p53) human prostate cancer cells to PEITC resulted in several specific features characteristic of autophagy, including appearance of membranous vacuoles, formation of acidic vesicular organelles, and cleavage and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes. A normal human prostate epithelial cell line (PrEC) was markedly more resistant toward PEITC-mediated cleavage and recruitment of LC3 compared with prostate cancer cells. Although PEITC treatment suppressed activating phosphorylations of Akt and mammalian target of rapamycin (mTOR), which are implicated in regulation of autophagy by different stimuli, processing and recruitment of LC3 was only partially/marginally reversed by ectopic expression of constitutively active Akt or overexpression of mTOR-positive regulator Rheb. The PEITC-mediated apoptotic DNA fragmentation was significantly attenuated in the presence of a pharmacologic inhibitor of autophagy (3-methyl adenine). Transient transfection of LNCaP and PC-3 cells with Atg5-specific small interfering RNA conferred significant protection against PEITC-mediated autophagy as well as apoptotic DNA fragmentation. A xenograft model using PC-3 cells and Caenorhabditis elegans expressing a lgg-1:GFP fusion protein provided evidence for occurrence of PEITC-induced autophagy in vivo. In conclusion, the present study indicates that Atg5 plays an important role in regulation of PEITC-induced autophagic and apoptotic cell death.
苯乙基异硫氰酸酯（PEITC）是一种很有前景的癌症化学预防剂，但其抗癌作用机制尚未完全明确。我们首次发现，PEITC处理可引发人前列腺癌细胞中依赖自噬相关蛋白5（Atg5）的自噬性和凋亡性细胞死亡。将PC-3（雄激素非依赖性、p53缺失）和LNCaP（雄激素反应性、野生型p53）人前列腺癌细胞暴露于PEITC后，出现了一些自噬特有的特征，包括膜性空泡的出现、酸性囊泡细胞器的形成，以及微管相关蛋白1轻链3（LC3）的切割和募集至自噬体。与前列腺癌细胞相比，正常人类前列腺上皮细胞系（PrEC）对PEITC介导的LC3切割和募集具有明显更高的抗性。尽管PEITC处理可抑制Akt和雷帕霉素哺乳动物靶蛋白（mTOR）的激活磷酸化，这两种蛋白参与不同刺激对自噬的调节，但组成型活性Akt的异位表达或mTOR阳性调节因子Rheb的过表达仅部分/轻微逆转了LC3的加工和募集。在存在自噬药理抑制剂（3-甲基腺嘌呤）的情况下，PEITC介导的凋亡性DNA片段化显著减弱。用Atg5特异性小干扰RNA瞬时转染LNCaP和PC-3细胞，可显著保护细胞免受PEITC介导的自噬以及凋亡性DNA片段化。使用PC-3细胞和表达lgg-1:GFP融合蛋白的秀丽隐杆线虫建立的异种移植模型，为PEITC诱导的体内自噬提供了证据。总之，本研究表明Atg5在调节PEITC诱导的自噬性和凋亡性细胞死亡中起重要作用。
