Hou Ming-Feng, Kuo Ho-Chang, Li Jih-Heng, Wang Yu-Shiuan, Chang Chen-Chia, Chen Ku-Chung, Chen Wei-Chiao, Chiu Chien-Chih, Yang Shengyu, Chang Wei-Chiao
Cancer Center, Kaohsiung Medical University Hospital, Taiwan.
Biochim Biophys Acta. 2011 Dec;1810(12):1278-84. doi: 10.1016/j.bbagen.2011.07.001. Epub 2011 Jul 14.
Orai1/CRACM1 is a principal component of the store-operated calcium channels. Store-operated calcium influx is highly correlated with inflammatory reactions, immunological regulation, and cell proliferation. Epidermal growth factor (EGF), which plays an important role in the regulation of cell proliferation, can activate store-operated calcium channels. However, the consequences of Orai1/CRACM1 overexpression in EGF-mediated lung cancer cells growth are not known.
To investigate the role of Orai1/CRACM1 in EGF-mediated lung cancer cell proliferation, Orai1/CRACM1 plasmids were transfected into cells by lipofection. A cell proliferation assay, immunofluorescence staining, flow cytometry, and real-time polymerase chain reaction were employed to monitor cell proliferation. The calcium influx signals were investigated using a fluorescent-based calcium assay.
Transfection of Orai1/CRACM1 plasmids resulted in the inhibition of EGF-mediated cell proliferation. ERK1/2 and Akt phosphorylation were inhibited by Orai1/CRACM1 overexpression. Expression of the cell cycle modulator p21 was induced in the Orai1/CRACM1-overexpressing cells, whereas the expression of cyclin D3 was reduced. Flow cytometry revealed that overexpression of Orai1/CRACM1 resulted in G0/G1 cell cycle arrest. Importantly, Orai1/CRACM1 overexpression significantly attenuated EGF-mediated store-operated calcium influx. In addition, application of 2-APB, a store-operated calcium channel inhibitor, resulted in the inhibition of EGF-mediated cancer cell proliferation.
We conclude that Orai1/CRACM1 overexpression attenuates store-operated Ca(2+) influx that in turn blocks EGF-mediated proliferative signaling and drives cell cycle arrest.
Orai1/CRACM1是储存式钙通道的主要组成部分。储存式钙内流与炎症反应、免疫调节和细胞增殖高度相关。在细胞增殖调节中起重要作用的表皮生长因子（EGF）可激活储存式钙通道。然而，Orai1/CRACM1过表达在EGF介导的肺癌细胞生长中的后果尚不清楚。
为研究Orai1/CRACM1在EGF介导的肺癌细胞增殖中的作用，通过脂质体转染将Orai1/CRACM1质粒转染到细胞中。采用细胞增殖试验、免疫荧光染色、流式细胞术和实时聚合酶链反应来监测细胞增殖。使用基于荧光的钙测定法研究钙内流信号。
转染Orai1/CRACM1质粒导致EGF介导的细胞增殖受到抑制。Orai1/CRACM1过表达抑制了ERK1/2和Akt磷酸化。在过表达Orai1/CRACM1的细胞中诱导了细胞周期调节因子p21的表达，而细胞周期蛋白D3的表达降低。流式细胞术显示，Orai1/CRACM1过表达导致G0/G1期细胞周期停滞。重要的是，Orai1/CRACM1过表达显著减弱了EGF介导的储存式钙内流。此外，应用储存式钙通道抑制剂2-APB导致EGF介导的癌细胞增殖受到抑制。
我们得出结论，Orai1/CRACM1过表达减弱了储存式Ca(2+)内流，进而阻断了EGF介导的增殖信号并导致细胞周期停滞。
