Xiao Dong, Johnson Candace S, Trump Donald L, Singh Shivendra V
Department of Pharmacology and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.
Mol Cancer Ther. 2004 May;3(5):567-75.
Phenethyl isothiocyanate (PEITC), a constituent of many cruciferous vegetables, offers significant protection against cancer in animals induced by a variety of carcinogens. The present study demonstrates that PEITC suppresses proliferation of PC-3 cells in a dose-dependent manner by causing G(2)-M-phase cell cycle arrest and apoptosis. Interestingly, phenyl isothiocyanate (PITC), which is a structural analogue of PEITC but lacks the -CH(2) spacers that link the aromatic ring to the -N=C=S group, neither inhibited PC-3 cell viability nor caused cell cycle arrest or apoptosis. These results indicated that even a subtle change in isothiocyanate (ITC) structure could have a significant impact on its biological activity. The PEITC-induced cell cycle arrest was associated with a >80% reduction in the protein levels of cyclin-dependent kinase 1 (Cdk1) and cell division cycle 25C (Cdc25C; 24 h after treatment with 10 micro M PEITC), which led to an accumulation of Tyr(15) phosphorylated (inactive) Cdk1. On the other hand, PITC treatment neither reduced protein levels of Cdk1 or Cdc25C nor affected Cdk1 phosphorylation. The PEITC-induced decline in Cdk1 and Cdc25C protein levels and cell cycle arrest were significantly blocked on pretreatment of PC-3 cells with proteasome inhibitor lactacystin. A 24 h exposure of PC-3 cells to 10 micro M PEITC, but not PITC, resulted in about 56% and 44% decrease in the levels of antiapoptotic proteins Bcl-2 and Bcl-X(L), respectively. However, ectopic expression of Bcl-2 failed to alter sensitivity of PC-3 cells to growth inhibition or apoptosis induction by PEITC. Treatment of cells with PEITC, but not PITC, also resulted in cleavage of procaspase-3, procaspase-9, and procaspase-8. Moreover, the PEITC-induced apoptosis was significantly attenuated in the presence of general caspase inhibitor and specific inhibitors of caspase-8 and caspase-9. In conclusion, our data indicate that PEITC-induced cell cycle arrest in PC-3 cells is likely due to proteasome-mediated degradation of Cdc25C and Cdk1, and ectopic expression of Bcl-2 fails to confer resistance to PEITC-induced apoptosis. Furthermore, the results of the present study point toward involvement of both caspase-8- and caspase-9-mediated pathways in apoptosis induction by PEITC.
异硫氰酸苯乙酯（PEITC）是许多十字花科蔬菜的一种成分，对多种致癌物诱导的动物癌症具有显著的保护作用。本研究表明，PEITC通过引起G(2)-M期细胞周期阻滞和凋亡，以剂量依赖的方式抑制PC-3细胞的增殖。有趣的是，异硫氰酸苯酯（PITC）是PEITC的结构类似物，但缺少将芳香环与-N=C=S基团连接的-CH(2)间隔基团，它既不抑制PC-3细胞活力，也不引起细胞周期阻滞或凋亡。这些结果表明，即使异硫氰酸酯（ITC）结构发生细微变化也可能对其生物活性产生重大影响。PEITC诱导的细胞周期阻滞与细胞周期蛋白依赖性激酶1（Cdk1）和细胞分裂周期25C（Cdc25C；用10 μM PEITC处理24小时后）的蛋白质水平降低80%以上有关，这导致酪氨酸（Tyr(15)）磷酸化（无活性）的Cdk1积累。另一方面，PITC处理既不降低Cdk1或Cdc25C的蛋白质水平，也不影响Cdk1磷酸化。用蛋白酶体抑制剂乳胞素预处理PC-3细胞后，PEITC诱导的Cdk1和Cdc25C蛋白质水平下降及细胞周期阻滞被显著阻断。PC-3细胞暴露于10 μM PEITC 24小时，但不暴露于PITC，分别导致抗凋亡蛋白Bcl-2和Bcl-X(L)水平下降约56%和44%。然而，Bcl-2的异位表达未能改变PC-3细胞对PEITC诱导的生长抑制或凋亡的敏感性。用PEITC而非PITC处理细胞也导致procaspase-3、procaspase-9和procaspase-8的裂解。此外，在存在通用半胱天冬酶抑制剂以及半胱天冬酶-8和半胱天冬酶-9的特异性抑制剂时，PEITC诱导的凋亡显著减弱。总之，我们的数据表明，PEITC诱导PC-3细胞的细胞周期阻滞可能是由于蛋白酶体介导的Cdc25C和Cdk1降解，并且Bcl-2的异位表达未能赋予对PEITC诱导的凋亡的抗性。此外，本研究结果表明半胱天冬酶-8和半胱天冬酶-9介导的途径均参与了PEITC诱导的凋亡。
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