Hajiahmadi Sima, Panjehpour Mojtaba, Aghaei Mahmoud, Shabani Mahdi
a Department of Clinical Biochemistry, School of Pharmacy and Isfahan Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Isfahan, Iran.
b Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.
Biochem Cell Biol. 2015 Aug;93(4):321-9. doi: 10.1139/bcb-2014-0117. Epub 2015 Mar 16.
A2b adenosine receptor (A2bAR) acts as a potent regulator of cell growth in various cell lines. The present study was designed to understand the controlling mechanism of A2bAR agonist (NECA)-induced apoptosis in ovarian cancer cells. Real-time PCR and western blotting assays were used to evaluate the gene and protein expression profiles of A2bAR, respectively. MTT assay was used to study the cell proliferation effect of A2bAR agonist (NECA). Detection of apoptosis was conducted using annexin V-FITC/PI staining, caspase-3 activation assay, and the expression of Bax and Bcl-2 proteins analysis. The mitochondrial membrane potential (ΔΨM) was analyzed by employing JC-1 prob. The mRNA and protein expression levels of A2bAR in ovarian cancer cells were detected. NECA significantly reduced cell viability in a dose-dependent manner in OVCAR-3 and Caov-4 cell lines. The growth inhibition effect of NECA was related to the induction of cell apoptosis, which was manifested by annexin V-FITC staining, activation of caspase-3, and loss of mitochondrial membrane potentials (ΔΨm). In addition, downregulation of the regulatory protein Bcl-2 and upregulation of Bax protein by NECA were also observed. These findings demonstrated that NECA induces apoptosis via the mitochondrial signaling pathway. Thus, A2bAR agonists may be a potential agent for induction of apoptosis in ovarian cancer cells.
A2b 腺苷受体（A2bAR）在多种细胞系中作为细胞生长的有效调节因子发挥作用。本研究旨在了解A2bAR激动剂（NECA）诱导卵巢癌细胞凋亡的调控机制。分别使用实时聚合酶链反应（PCR）和蛋白质免疫印迹分析来评估A2bAR的基因和蛋白质表达谱。采用MTT法研究A2bAR激动剂（NECA）的细胞增殖作用。使用膜联蛋白V-异硫氰酸荧光素/碘化丙啶（annexin V-FITC/PI）染色、半胱天冬酶-3（caspase-3）激活检测以及Bax和Bcl-2蛋白表达分析来检测细胞凋亡。采用JC-1探针分析线粒体膜电位（ΔΨM）。检测了卵巢癌细胞中A2bAR的mRNA和蛋白质表达水平。NECA在OVCAR-3和Caov-4细胞系中以剂量依赖性方式显著降低细胞活力。NECA的生长抑制作用与细胞凋亡的诱导有关，这通过annexin V-FITC染色、caspase-3激活以及线粒体膜电位（ΔΨm）的丧失得以体现。此外，还观察到NECA使调节蛋白Bcl-2下调以及Bax蛋白上调。这些发现表明NECA通过线粒体信号通路诱导细胞凋亡。因此，A2bAR激动剂可能是诱导卵巢癌细胞凋亡的潜在药物。
