Academy of Scientific and Innovative Research, New Delhi, India.
Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, J&K, 180001, India.
Breast Cancer. 2019 May;26(3):378-385. doi: 10.1007/s12282-018-0933-x. Epub 2018 Nov 29.
Phosphatidylinositol-3 kinases (PI3Ks) are involved in regulating cell growth, proliferation, differentiation, apoptosis and survival. p110α and p110β, two ubiquitously expressed isoforms of PI3K signalling, are involved in growth factor mediated signaling and survival by generating second messengers. Earlier, we have generated GFP-fusion proteins of p110α and p110β and expressed them in normal and cancer cell-lines to investigate their subcellular localization and their role in various activities. Here, we sought to examine the role of p110α and p110β isoforms in protecting MCF-7 breast cancer cells against oxidative stress.
We performed cytotoxicity assays, DNA transfection, Plasmid DNA preparation, western blotting, flourscence microscopy and statistical analysis.
To know whether p110α and p110β are involved in protecting MCF-7 breast cancer cells against oxidative stress, we subjected MCF-7 cells to H2O2 treatment and observed a dose dependent decrease in cell viability and a marked increase in the levels of pro-apoptotic markers which include PARP, Bcl-2, Bax and procaspase-9. We then over-expressed recombinant GFP-fusion p110α and p110β proteins in MCF-7 cells and observed a significant decrease in apoptosis and a concomitant increase in pAkt levels.
We report the involvement of p110α and p110β isoforms of Class 1A PI3K signalling in rescue from oxidative stress-induced apoptosis in MCF-7 cells in Akt dependent manner.
磷脂酰肌醇-3 激酶(PI3Ks)参与调节细胞生长、增殖、分化、凋亡和存活。p110α 和 p110β 是 PI3K 信号的两种广泛表达的同工型,通过生成第二信使参与生长因子介导的信号转导和存活。此前,我们生成了 p110α 和 p110β 的 GFP 融合蛋白,并在正常和癌细胞系中表达它们,以研究它们的亚细胞定位及其在各种活动中的作用。在这里,我们试图研究 p110α 和 p110β 同工型在保护 MCF-7 乳腺癌细胞免受氧化应激中的作用。
我们进行了细胞毒性测定、DNA 转染、质粒 DNA 制备、Western 印迹、荧光显微镜和统计分析。
为了了解 p110α 和 p110β 是否参与保护 MCF-7 乳腺癌细胞免受氧化应激,我们用 H2O2 处理 MCF-7 细胞,观察到细胞活力呈剂量依赖性下降,促凋亡标志物(包括 PARP、Bcl-2、Bax 和 procaspase-9)水平显著增加。然后,我们在 MCF-7 细胞中转染重组 GFP 融合 p110α 和 p110β 蛋白,观察到凋亡明显减少,pAkt 水平同时增加。
我们报告 Class 1A PI3K 信号的 p110α 和 p110β 同工型参与了 MCF-7 细胞中依赖 Akt 的氧化应激诱导凋亡的挽救。