Stolfi Carmine, Fina Daniele, Caruso Roberta, Caprioli Flavio, Fantini Massimo Claudio, Rizzo Angelamaria, Sarra Massimiliano, Pallone Francesco, Monteleone Giovanni
Dipartimento di Medicina Interna, Università Tor Vergata, Via Montpellier 1, 00133 Rome, Italy.
Carcinogenesis. 2008 Jun;29(6):1258-66. doi: 10.1093/carcin/bgn122. Epub 2008 May 20.
Regular consumption of mesalazine has been associated with a reduced risk of colorectal cancer (CRC) in patients with inflammatory bowel disease. The molecular mechanisms underlying the antineoplastic effect of 5-aminosalicylic acid remain, however, poorly characterized. In this study, we examined whether mesalazine affects cell cycle progression and analyzed specific checkpoint pathways in experimental models of CRC. Mesalazine inhibited the growth of HCT-116 and HT-29 cells, two CRC cell lines that express either a wild-type or mutated p53. Cell cycle analysis revealed that mesalazine induced cells to accumulate in S phase. This effect was associated with a sustained phosphorylation of the cyclin-dependent kinase (CDK)2 at threonine 14 and tyrosine 15 residues, an event that inactivates the CDK2-cyclin complex and blocks S-G(2) phase cell cycle transition. Consistently, mesalazine reduced the protein content of CDC25A, a phosphatase that regulates CDK2 phosphorylation status. Analysis of upstream kinases that negatively control CDC25A expression showed that mesalazine enhanced the activation of CHK1 and CHK2. However, silencing of CHK1 and CHK2 did not prevent the mesalazine-induced CDC25A protein downregulation. In contrast, CDC25A protein ubiquitination and degradation and accumulation of cells in S phase following mesalazine exposure were reverted by proteasome inhibitors. Notably, mesalazine also inhibited CDC25A in human CRC explants. Finally, we showed that mesalazine downregulated CDC25A in CT26, a murine CRC cell line, and prevented the formation of CT26-derived tumors in mice. Data show that mesalazine negatively regulates CDC25A protein expression, thus delaying CRC cell progression.
规律服用美沙拉嗪与炎症性肠病患者结直肠癌（CRC）风险降低相关。然而，5-氨基水杨酸抗肿瘤作用的分子机制仍不清楚。在本研究中，我们检测了美沙拉嗪是否影响细胞周期进程，并在CRC实验模型中分析了特定的检查点途径。美沙拉嗪抑制了HCT-116和HT-29细胞的生长，这两种CRC细胞系分别表达野生型或突变型p53。细胞周期分析显示，美沙拉嗪诱导细胞在S期积累。这种效应与细胞周期蛋白依赖性激酶（CDK）2苏氨酸14和酪氨酸15残基的持续磷酸化有关，该事件使CDK2-细胞周期蛋白复合物失活并阻断S-G(2)期细胞周期转换。一致地，美沙拉嗪降低了CDC25A的蛋白质含量，CDC25A是一种调节CDK2磷酸化状态的磷酸酶。对负调控CDC25A表达的上游激酶的分析表明，美沙拉嗪增强了CHK1和CHK2的激活。然而，CHK1和CHK2的沉默并不能阻止美沙拉嗪诱导的CDC25A蛋白下调。相反，蛋白酶体抑制剂可逆转美沙拉嗪暴露后CDC25A蛋白的泛素化和降解以及细胞在S期的积累。值得注意的是，美沙拉嗪在人CRC外植体中也抑制了CDC25A。最后，我们表明美沙拉嗪在小鼠CRC细胞系CT26中下调了CDC25A，并阻止了CT26衍生肿瘤在小鼠中的形成。数据表明，美沙拉嗪负调控CDC25A蛋白表达，从而延缓CRC细胞进程。
