Ravizza Raffaella, Gariboldi Marzia B, Passarelli Laura, Monti Elena
Dept. of Structural and Functional Biology, Section of Pharmacology, University of Insubria, Via A, da Giussano 10, 21052 Busto Arsizio (VA), Italy.
BMC Cancer. 2004 Dec 15;4:92. doi: 10.1186/1471-2407-4-92.
Colon adenocarcinomas are refractory to a number of widely used anticancer agents. Multifactorial mechanisms have been implicated in this intrinsically resistant phenotype, including deregulation of cell death pathways. In this regard, the p53 protein has a well established role in the control of tumor cell response to DNA damaging agents; however, the relationship between p53-driven genes and drug sensitivity remains controversial. The present study investigates the role of the p53/p21 system in the response of human colon carcinoma cells to treatment with the cytotoxic agent doxorubicin (DOX) and the possibility to modify the therapeutic index of DOX by modulation of p53 and/or p21 protein levels.
The relationship between p53 and p21 protein levels and the cytotoxic effect of DOX was investigated, by MTT assay and western blot analysis, in HCT116 (p53-positive) and HT29 (p53-negative) colon cancer cells. We then assessed the effects of DOX in two isogenic cell lines derived from HCT116 by abrogating the expression and/or function of p53 and p21 (HCT116-E6 and HCT116 p21-/-, respectively). Finally, we evaluated the effect of pre-treatment with the piperidine nitroxide Tempol (TPL), an agent that was reported to induce p21 expression irrespective of p53 status, on the cytotoxicity of DOX in the four cell lines. Comparisons of IC50 values and apoptotic cell percentages were performed by ANOVA and Bonferroni's test for independent samples. C.I. calculations were performed by the combination Index method.
Our results indicate that, in the colon carcinoma cell lines tested, sensitivity to DOX is associated with p21 upregulation upon drug exposure, and DOX cytotoxicity is potentiated by pre-treatment with TPL, but only in those cell lines in which p21 can be upregulated.
p21 induction may significantly contribute to the response of colon adenocarcinomas cells to DOX treatment; and small molecules that can exploit p53-independent pathways for p21 induction, such as TPL, may find a place in chemotherapeutic protocols for the clinical management of colorectal cancer, where p53 function is often lost, due to genetic or epigenetic defects or to post-transcriptional inactivating mechanisms.
结肠腺癌对多种广泛使用的抗癌药物具有耐药性。多种机制与这种内在的耐药表型有关，包括细胞死亡途径的失调。在这方面，p53蛋白在控制肿瘤细胞对DNA损伤剂的反应中具有公认的作用；然而，p53驱动的基因与药物敏感性之间的关系仍存在争议。本研究调查了p53/p21系统在人结肠癌细胞对细胞毒性药物阿霉素（DOX）治疗反应中的作用，以及通过调节p53和/或p21蛋白水平来改变DOX治疗指数的可能性。
通过MTT法和蛋白质印迹分析，研究了HCT116（p53阳性）和HT29（p53阴性）结肠癌细胞中p53和p21蛋白水平与DOX细胞毒性作用之间的关系。然后，我们通过消除p53和p21的表达和/或功能（分别为HCT116-E6和HCT116 p21-/-），评估了DOX对源自HCT116的两种同基因细胞系的影响。最后，我们评估了用哌啶氮氧化物Tempol（TPL）预处理的效果，据报道TPL是一种无论p53状态如何均可诱导p21表达的药物，对四种细胞系中DOX细胞毒性的影响。通过ANOVA和独立样本的Bonferroni检验对IC50值和凋亡细胞百分比进行比较。通过联合指数法进行CI计算。
我们的结果表明，在所测试的结肠癌细胞系中，对DOX的敏感性与药物暴露后p21上调有关，并且TPL预处理可增强DOX的细胞毒性，但仅在那些p21可上调的细胞系中如此。
p21的诱导可能显著有助于结肠腺癌细胞对DOX治疗的反应；并且能够利用不依赖p53的途径诱导p21的小分子，如TPL，可能在结直肠癌临床管理的化疗方案中占有一席之地，在结直肠癌中，由于遗传或表观遗传缺陷或转录后失活机制，p53功能常常丧失。
