School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur 721302, West Bengal, India.
Life Sci. 2013 Nov 13;93(21):783-90. doi: 10.1016/j.lfs.2013.09.009. Epub 2013 Sep 15.
Thymoquinone (TQ), the predominant bioactive constituent of black seed oil (Nigella Sativa), has been shown to possess antineoplastic activity against multifarious tumors. However, the meticulous mechanism of TQ on Akt mediated survival pathway is still unrevealed in breast cancer. Here, we investigated TQ's mechanism of action against PI3K/Akt signaling and its downstream targets by modulating proteins translational machinery, leading to apoptosis in cancer cells.
MDA-MB-468 and T-47D cells were treated with TQ and evaluated for its anticancer activity through phase distribution and western blot. Modulatory effects of TQ on Akt were affirmed through kinase and drug potential studies.
Studies revealed G1 phase arrest till 24h incubation with TQ while extended exposure showed phase shift to subG1 indicating apoptosis, supported by suppression of cyclin D1, cyclin E and cyclin dependent kinase inhibitor p27 expression. Immunoblot and membrane potential studies revealed mitochondrial impairment behind apoptotic process with upregulation of Bax, cytoplasmic cytochrome c and procaspase-3, PARP cleavage along with Bcl-2, Bcl-xL and survivin downregulation. Moreover, we construed the rationale behind mitochondrial dysfunction by examining the phosphorylation status of PDK1, PTEN, Akt, c-raf, GSK-3β and Bad in TQ treated cells, thus ratifying the involvement of Akt in apoptosis. Further, the consequential effect of Akt inhibition by TQ is proven by translational repression through deregulated phosphorylation of 4E-BP1, eIF4E, S6R and p70S6K.
Our observations for the first time may provide a new insight for the development of novel therapies for Akt overexpressed breast cancer by TQ.
黑种草籽油（Nigella Sativa）中的主要生物活性成分百里醌（TQ）已被证明对多种肿瘤具有抗肿瘤活性。然而，TQ 在 Akt 介导的存活途径中的细致机制在乳腺癌中仍未被揭示。在这里，我们通过调节蛋白质翻译机制来研究 TQ 对 PI3K/Akt 信号及其下游靶标的作用，从而导致癌细胞凋亡。
用 TQ 处理 MDA-MB-468 和 T-47D 细胞，并通过相位分布和 Western blot 评估其抗癌活性。通过激酶和药物潜力研究证实了 TQ 对 Akt 的调节作用。
研究表明，用 TQ 孵育 24 小时可导致 G1 期停滞，而延长暴露则显示出向 subG1 的相位转移，表明凋亡，这得到了细胞周期蛋白 D1、细胞周期蛋白 E 和细胞周期蛋白依赖性激酶抑制剂 p27 表达抑制的支持。免疫印迹和膜电位研究显示，凋亡过程背后的线粒体损伤伴随着 Bax、细胞质细胞色素 c 和 procaspase-3 的上调，PARP 裂解以及 Bcl-2、Bcl-xL 和 survivin 的下调。此外，我们通过检查 TQ 处理细胞中 PDK1、PTEN、Akt、c-raf、GSK-3β 和 Bad 的磷酸化状态来推断线粒体功能障碍的原理，从而证实 Akt 在凋亡中的参与。此外，通过 TQ 对 Akt 的抑制作用来证明翻译抑制作用，通过对 4E-BP1、eIF4E、S6R 和 p70S6K 的去调节磷酸化来证明。
我们的观察结果首次可能为 TQ 对 Akt 过表达乳腺癌的新型治疗方法的开发提供新的见解。
