Cerquetti L, Bucci B, Marchese R, Misiti S, De Paula U, Miceli R, Muleti A, Amendola D, Piergrossi P, Brunetti E, Toscano V, Stigliano A
Endocrinology II Faculty of Medicine, University La Sapienza, Rome, Italy.
Endocr Relat Cancer. 2008 Jun;15(2):623-34. doi: 10.1677/erc.1.1315.
Mitotane, 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane (o,p'-DDD) is an agent with adrenotoxic effect, which is able to block cortisol synthesis. This drug and radiotherapy are used also in adrenal cancer treatment even if their biological action in this neoplasia remains unknown. We investigated the effects of o,p'-DDD and ionizing radiations (IR) on cell growth inhibition and cell cycle perturbation in H295R and SW13 adrenocortical cancer cells. Both cell lines were irradiated at a 6 Gy dose and were treated with o,p'-DDD 10(-5) M separately and with IR/o,p'-DDD in combination. This combination treatment induced an irreversible inhibition of cell growth in both adrenocortical cancer cells. Cell cycle analysis showed that IR alone and IR/o,p'-DDD in combination induced the cell accumulation in the G2 phase. At 120 h after IR, the cells were able to recover the IR-induced G2 block while cells treated with IR/o,p'-DDD were still arrested in G2 phase. In order to study the molecular mechanism involved in the G2 irreversible arrest, we have considered the H295R cell line showing the highest inhibition of cell proliferation associated with a noteworthy G2 arrest. In these cells, cyclin B1 and Cdk2 proteins were examined by western blot and Cdk2 kinase activity measured by assay kit. The H295R cells treated with IR/o,p'-DDD shared an increase in cyclin B1 amount as the coimmunoprecipitation of Cdc2-cyclin B1 complex. The kinase activity also shows an increase in the treated cells with combination therapy. Moreover, in these cells, sequence analysis of p53 revealed a large deletion of exons 8 and 9. The same irreversible block on G2 phase, induced by IR/o,p'-DDD treatment, happened in H295R cells with restored wild-type p53 suggesting that this mechanism is not mediated by p53 pathway.
米托坦，即1,1-二氯-2-(邻氯苯基)-2-(对氯苯基)乙烷（邻，对'-滴滴滴），是一种具有肾上腺毒性作用的药物，能够阻断皮质醇的合成。即便这种药物和放疗在肾上腺癌治疗中的生物学作用尚不清楚，但它们仍被用于肾上腺癌的治疗。我们研究了邻，对'-滴滴滴和电离辐射（IR）对H295R和SW13肾上腺皮质癌细胞生长抑制和细胞周期扰动的影响。两种细胞系均接受6 Gy剂量的照射，并分别用10(-5) M的邻，对'-滴滴滴处理，以及接受IR/邻，对'-滴滴滴联合处理。这种联合治疗在两种肾上腺皮质癌细胞中均诱导了不可逆的细胞生长抑制。细胞周期分析表明，单独的IR以及IR/邻，对'-滴滴滴联合处理均诱导细胞在G2期积累。IR照射后120小时，细胞能够恢复IR诱导的G2期阻滞，而接受IR/邻，对'-滴滴滴处理的细胞仍停滞在G2期。为了研究G2期不可逆阻滞所涉及的分子机制，我们选用了H295R细胞系，该细胞系显示出最高的细胞增殖抑制率，并伴有明显的G2期阻滞。在这些细胞中，通过蛋白质免疫印迹法检测细胞周期蛋白B1和细胞周期蛋白依赖性激酶2（Cdk2）蛋白，并使用检测试剂盒测量Cdk2激酶活性。接受IR/邻，对'-滴滴滴处理的H295R细胞中，细胞周期蛋白B1的量增加，这与Cdc2-细胞周期蛋白B1复合物的共免疫沉淀有关。联合治疗的处理细胞中，激酶活性也有所增加。此外，在这些细胞中，p53的序列分析显示外显子8和9有大片段缺失。在恢复野生型p53的H295R细胞中，IR/邻，对'-滴滴滴处理同样诱导了G2期的不可逆阻滞，这表明该机制并非由p53途径介导。
