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

目的：p53诱导的p21蛋白表达水平升高是细胞早衰的分子靶点之一。活性氧（ROS）参与了这一过程。本研究以K-ras癌基因转化的NIH 3 T3细胞为模型，探讨ROS在细胞生长中的作用以及ER抑制对细胞衰老的影响。材料与方法：1）建立了两株仅表达K-ras癌基因（K12 V）和共表达显性负性ER（K12 VDNER）的NIH 3 T3细胞系，并对细胞生长进行了分析。2)通过β-gal染色评价衰老的诱导。使用氨基苯基荧光素（APF）通过FACS测量细胞内ROS水平。3)将ROS的抑制剂N-乙酰基-L-半胱氨酸（NAC）加入到细胞系中，并评估对衰老诱导的影响。4)结果：1）K12 VDNER细胞的生长明显受到抑制，细胞出现多核扁平细胞，β-gal染色阳性细胞明显增多，细胞衰老; 3)K12 VDNER细胞衰老诱导过程中，细胞内ROS水平增加约2倍。3)在K12 VDNER细胞中，加入NAC后，β-gal染色阳性细胞减少，细胞生长恢复。4)结论：K-ras和ER介导的细胞衰老与ROS有关。我们认为衰老的途径是通过活性氧，因为活性氧的抑制挽救了衰老的诱导。

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.