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

目的：p53诱导p21表达水平升高是早衰的分子靶点之一。活性氧（ROS）参与了这一过程。在本研究中，我们利用致癌基因K-ras转化NIH3T3细胞模型，研究了ROS如何介导细胞生长和抑制内质网介导细胞衰老的机制。材料与方法：1)建立2株仅表达致癌K-ras （K12V）和共表达显性负ER （K12VDNER）的NIH3T3细胞株，分析细胞生长情况。2)通过β-gal染色评价衰老诱导情况。使用氨基苯基荧光素（APF）用流式细胞仪测定细胞内ROS水平。3)将活性氧抑制剂n -乙酰基- l -半胱氨酸（NAC）加入细胞系，观察其抗衰老作用。4)免疫印迹法检测ras信号相关蛋白。结果：1)与模拟细胞或K12V细胞相比，K12VDNER细胞的生长明显受到抑制，出现多核扁平细胞，β-gal阳性染色明显增加，并伴有衰老。3) K12VDNER细胞随着衰老的诱导，胞内ROS水平升高约2倍。3)在K12VDNER细胞中，NAC可使β-gal阳性染色细胞减少，恢复细胞生长。4)与模拟细胞或K12V细胞相比，K12VDNER细胞中p21的表达水平升高。结论：细胞衰老是由致癌的K-ras和与ROS相关的ER介导的。我们认为衰老途径是通过ROS，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.