Ghiasi Naghmeh, Habibagahi Mojtaba, Rosli Rozita, Ghaderi Abbas, Yusoff Khatijah, Hosseini Ahmad, Abdullah Syahrilnizam, Jaberipour Mansooreh
Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran E-mail :
Asian Pac J Cancer Prev. 2014 Jan;14(11):6605-11. doi: 10.7314/apjcp.2013.14.11.6605.
WEE1 is a G2/M checkpoint regulator protein. Various studies have indicated that WEE1 could be a good target for cancer therapy. The main aim of this study was to asssess the tumor suppressive potential of WEE1 silencing in two different breast cancer cell lines, MCF7 which carries the wild-type p53 and MDA-MB468 which contains a mutant type.
After WEE1 knockdown with specific shRNAs downstream effects on cell viability and cell cycle progression were determined using MTT and flow cytometry analyses, respectively. Real-time PCR and Western blotting were conducted to assess the effect of WEE1 inhibition on the expression of apoptotic (p53) and anti-apoptotic (Bcl2) factors and also a growth marker (VEGF).
The results showed that WEE1 inhibition could cause a significant decrease in the viability of both MCF7 and MDA-MB-468 breast cancer cell lines by more than 50%. Interestingly, DNA content assays showed a significant increase in apoptotic cells following WEE1 silencing. WEE1 inhibition also induced up- regulation of the apoptotic marker, p53, in breast cancer cells. A significant decrease in the expression of VEGF and Bcl-2 was observed following WEE1 inhibition in both cell lines.
In concordance with previous studies, our data showed that WEE1 inhibition could induce G2 arrest abrogation and consequent cell death in breast cancer cells. Moreover, in this study, the observed interactions between the pro- and anti-apoptotic proteins and decrease in the angiogenesis marker expression confirm the susceptibility to apoptosis and validate the tumor suppressive effect of WEE1 inhibition in breast cancer cells. Interestingly, the levels of the sensitivity to WEE1 silencing in breast cancer cells, MCF7 and MDA-MB468, seem to be in concordance with the level of p53 expression.
WEE1是一种G2/M期检查点调节蛋白。多项研究表明，WEE1可能是癌症治疗的一个良好靶点。本研究的主要目的是评估在两种不同的乳腺癌细胞系中，即携带野生型p53的MCF7细胞系和含有突变型p53的MDA-MB468细胞系中，WEE1沉默的肿瘤抑制潜力。
用特异性短发夹RNA（shRNA）敲低WEE1后，分别使用MTT和流式细胞术分析确定其对细胞活力和细胞周期进程的下游影响。进行实时聚合酶链反应（PCR）和蛋白质印迹法以评估WEE1抑制对凋亡因子（p53）和抗凋亡因子（Bcl2）表达以及生长标志物（血管内皮生长因子，VEGF）表达的影响。
结果显示，WEE1抑制可导致MCF7和MDA-MB-468两种乳腺癌细胞系的活力显著降低超过50%。有趣的是，DNA含量检测显示WEE1沉默后凋亡细胞显著增加。WEE1抑制还诱导乳腺癌细胞中凋亡标志物p53的上调。在两种细胞系中，WEE1抑制后均观察到VEGF和Bcl-2表达显著降低。
与先前的研究一致，我们的数据表明WEE1抑制可诱导乳腺癌细胞中的G2期阻滞解除并导致细胞死亡。此外，在本研究中，观察到的促凋亡蛋白和抗凋亡蛋白之间的相互作用以及血管生成标志物表达的降低证实了对凋亡的敏感性，并验证了WEE1抑制在乳腺癌细胞中的肿瘤抑制作用。有趣的是，乳腺癌细胞MCF7和MDA-MB468对WEE1沉默的敏感程度似乎与p53表达水平一致。
