Yin Ming-Biao, Li Zhan-Rong, Cao Shousong, Durrani Farukh A, Azrak Rami G, Frank Cheryl, Rustum Youcef M
Department of Pharmacology and Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.
Mol Pharmacol. 2004 Jul;66(1):153-60. doi: 10.1124/mol.66.1.153.
Methylselenocysteine (MSC) is an organic selenium compound in preventative clinical trials involving prostate, lung, and colon carcinoma. We found that methioninase-activated MSC potentiates 7-ethyl-10-hydroxycamptothecin (SN-38)-induced cell lethality in vitro in the p53-defective human head and neck carcinoma A253 cells. Activated MSC increases chk2 phosphorylation at threonine-68 induced by SN-38, with no significant effect on chk1 phosphorylation. Cell cycle arrest induced by SN-38, however, was not abrogated or potentiated by MSC. These results suggest that the enhanced cellular lethality of SN-38 by MSC was not associated with cell cycle regulation pathways. Because chk2, in addition to its role in cell cycle arrest, can induce apoptosis by phosphorylation/activation, we examined whether increased chk2 phosphorylation could induce preapoptotic DNA fragmentation. DNA damage analysis showed that megabase DNA fragmentation is decreased, accompanied by the increased 30 to 300 kilobase pairs of DNA fragmentation after exposure to SN-38 with MSC, compared with SN-38 alone. No significant changes in the amount of DNA fragments were observed in cells treated with SN-38 or MSC alone. Moreover, proteolytic destruction of DNA replication-associated proteins cdc6, MCM2, and cdc25A may induce a DNA damage checkpoint response. The observed down-regulation of DNA replication proteins cdc6, MCM2, and cdc25A after exposure to SN-38 with MSC further indicates a relationship between drug response and DNA damage. Exposure to SN-38 with MSC resulted in a significant increase of poly(ADP-ribose) polymerasecleavage and caspase 3 activation. All together, the data support the hypothesis that enhanced lethality of this combination is associated with increased chk2 phosphorylation at Thr68 and down-regulation of specific DNA replication-associated proteins, which result in poly(ADP-ribose) polymerase cleavage, caspase 3 activation, and the induction of 30 to 300 kilobase pairs of DNA fragmentation.
甲基硒代半胱氨酸（MSC）是一种有机硒化合物，正用于前列腺癌、肺癌和结肠癌的预防性临床试验。我们发现，甲硫氨酸酶激活的MSC在体外可增强7-乙基-10-羟基喜树碱（SN-38）对p53缺陷型人头颈部癌A253细胞的致死作用。激活的MSC可增加由SN-38诱导的苏氨酸68位点的chk2磷酸化，而对chk1磷酸化无显著影响。然而，MSC并未消除或增强SN-38诱导的细胞周期阻滞。这些结果表明，MSC增强SN-38的细胞致死作用与细胞周期调控途径无关。由于chk2除了在细胞周期阻滞中发挥作用外，还可通过磷酸化/激活诱导凋亡，因此我们研究了chk2磷酸化增加是否能诱导凋亡前DNA片段化。DNA损伤分析表明，与单独使用SN-38相比，暴露于SN-38和MSC后，兆碱基DNA片段化减少，同时30至300千碱基对的DNA片段化增加。单独用SN-38或MSC处理的细胞中未观察到DNA片段数量的显著变化。此外，DNA复制相关蛋白cdc6、MCM2和cdc25A的蛋白水解破坏可能诱导DNA损伤检查点反应。暴露于SN-38和MSC后观察到的DNA复制蛋白cdc6、MCM2和cdc25A的下调进一步表明了药物反应与DNA损伤之间的关系。暴露于SN-38和MSC导致聚（ADP-核糖）聚合酶裂解和半胱天冬酶3激活显著增加。总之，这些数据支持以下假设：这种联合用药增强的致死作用与苏氨酸68位点chk2磷酸化增加以及特定DNA复制相关蛋白的下调有关，这导致聚（ADP-核糖）聚合酶裂解、半胱天冬酶3激活以及30至300千碱基对DNA片段化的诱导。
