Ying Li, Li Gang, Wei Si-si, Wang Hong, An Pei, Wang Xun, Guo Kai, Luo Xian-jin, Gao Ji-min, Zhou Qing, Li Wei, Yu Ying, Li Yi-gang, Duan Jun-li, Wang Yue-peng
1] Department of Cardiology, Affiliated Xinhua Hospital, Shanghai Jiaotong University (SJTU) School of Medicine, Shanghai 200092, China [2] Gerontology, Affiliated Xinhua Hospital, Shanghai Jiaotong University (SJTU) School of Medicine, Shanghai 200092, China.
Pediatrics, Affiliated Xinhua Hospital, Shanghai Jiaotong University (SJTU) School of Medicine, Shanghai 200092, China.
Acta Pharmacol Sin. 2015 Feb;36(2):229-40. doi: 10.1038/aps.2014.115. Epub 2014 Dec 29.
Small GTPase Rac1 is a member of the Ras superfamily, which plays important roles in regulation of cytoskeleton reorganization, cell growth, proliferation, migration, etc. The aim of this study was to determine how a constitutively active Rac1b regulated cell proliferation and to investigate the effects of the Rac1b inhibitor sanguinarine.
Three HEK293T cell lines stably overexpressing GFP, Rac1-GFP or Rac1b-GFP were constructed by lentiviral infection. The cells were treated with sanguinarine (1 μmol/L) or its analogue berberine (1 μmol/L) for 4 d. Cell proliferation was evaluated by counting cell numbers and with a BrdU incorporation assay. The levels of cleaved PARP-89 (an apoptosis marker) and cyclin-D1 (a proliferative index) were measured using Western blotting.
In 10% serum-containing media, overexpressing either Rac1 or Rac1b did not significantly change the cell proliferation. In the serum-starved media, however, the survival rate of Rac1b cells was significantly increased, whereas that of Rac1 cells was moderately increased. The level of cleaved PARP-89 was significantly increased in serum-starved Rac1 cells, but markedly reduced in serum-starved Rac1b cells. The level of cyclin-D1 was significantly increased in both serum-starved Rac1 and Rac1b cells. Treatment with sanguinarine, but not berberine, inhibited the proliferation of Rac1b cells, which was accompanied by significantly increased the level of PARP-89, and decreased both the level of cyclin-D1 and the percentage of BrdU positive cells.
Rac1b enhances the cell proliferation under a growth-limiting condition via both anti-apoptotic and pro-proliferative mechanisms. Sanguinarine, as the specific inhibitor of Rac1b, is a potential therapeutic agent for malignant tumors with up-regulated Rac1b.
小GTP酶Rac1是Ras超家族的成员，在细胞骨架重组、细胞生长、增殖、迁移等的调控中发挥重要作用。本研究的目的是确定组成型激活的Rac1b如何调节细胞增殖，并研究Rac1b抑制剂血根碱的作用。
通过慢病毒感染构建了三个稳定过表达绿色荧光蛋白（GFP）、Rac1-GFP或Rac1b-GFP的HEK293T细胞系。用1 μmol/L血根碱或其类似物黄连素处理细胞4天。通过细胞计数和BrdU掺入试验评估细胞增殖。使用蛋白质免疫印迹法检测裂解的PARP-89（一种凋亡标志物）和细胞周期蛋白D1（一种增殖指数）的水平。
在含10%血清的培养基中，过表达Rac1或Rac1b均未显著改变细胞增殖。然而，在血清饥饿培养基中，Rac1b细胞的存活率显著增加，而Rac1细胞的存活率适度增加。血清饥饿的Rac1细胞中裂解的PARP-89水平显著增加，但血清饥饿的Rac1b细胞中明显降低。血清饥饿的Rac1和Rac1b细胞中细胞周期蛋白D1水平均显著增加。用血根碱而非黄连素处理可抑制Rac1b细胞的增殖，同时伴随着PARP-89水平显著增加，细胞周期蛋白D1水平和BrdU阳性细胞百分比降低。
Rac1b通过抗凋亡和促增殖机制在生长限制条件下增强细胞增殖。血根碱作为Rac1b的特异性抑制剂，是Rac1b上调的恶性肿瘤的潜在治疗药物。
