Development of novel molecular-target therapy focus on type I, type II uterine endometrial cancer

目的：P53诱导的p21表达水平升高是早衰的分子靶点之一。活性氧物种(ROS)参与了这个过程。本研究利用K-ras基因转化NIH3T3细胞的模型，研究了ROS是如何通过抑制ER来调节细胞生长和衰老的。材料和方法：1)建立了两个单独表达致癌K-ras(K12V)或共表达显性负性ER(K12VDNER)的NIH3T3细胞株，并对细胞生长进行了分析。2)β-GAL染色检测衰老诱导作用。用氨基苯基荧光素(APF)流式细胞仪测定细胞内ROS水平。3)在细胞中加入ROS抑制剂N-乙酰-L-半胱氨酸(NAC)，观察其抗衰老作用。4)免疫印迹法检测ras信号转导相关蛋白的表达。结果：1)K12VDNER细胞生长受到明显抑制，出现多核扁平细胞，β-GAL染色阳性细胞明显增多。3)诱导衰老的K12VDNER细胞内ROS水平增加约一倍。3)在K12VDNER细胞中，加入NaC后，β-Gal染色阳性细胞减少，细胞生长恢复。4)K12VDNER细胞中p21的表达水平明显高于模型细胞和K12V细胞。结论：K-ras和ER参与了ROS介导的细胞衰老。我们提出了通过ROS进行衰老的途径，因为抑制ROS挽救了衰老的诱导。

Objectives : Increased expression level of p21 induced by p53 is one of the molecular target of premature senescence. Reactive oxygen species (ROS) is involved in this process. In this study, we investigated how ROS is mediated with the cell growth and mechanism of cellular senescence by suppressed ER using the model of NIH3T3 cell transformed by oncogenic K-ras.Materials and Methods : 1) We established two NIH3T3 cell lines that expressed only oncogenic K-ras (K12V) or co-expressed dominant negative ER (K12VDNER) and analyed the cell growth. 2) Induction of senescence was evaluated by β-gal staining. Intracellular levels of ROS were measured by FACS using Aminophenyl fluorescin (APF). 3) Inhibitor of ROS, N-acetyl -L-cystein (NAC), was added onto the cell lines, and effect against the induction of senescence was evaluated. 4) Several proteins related with ras signaling was evaluated with immunoblots.Results : 1) Cell growth of K12VDNER cells were markedly suppressed compared to mock or K12V cells accompanied with senescence by the appearance of multi-nucleated flat cells and significant increase of positive β-gal staining. 3) Intracellular levels of ROS were increased about two times in K12VDNER cells accompanying with induction of senescence. 3) In K12VDNER cell, positive β-gal staining cells were decreased and cell growth was recovered by the addition of NAC. 4) Expression level of p21 was increased in K12VDNER cell compared to mock or K12V cell.Conclusion : Cellular senescence mediated with oncogenic K-ras and ER related with ROS. We suggested the pathway of senescence via ROS because inhibition of ROS rescued the induction of senescence.