Department of Biochemistry, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia in Katowice, Jedności 8, 41-200 Sosnowiec, Poland.
Department of Medical Genetics, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia in Katowice, Jedności 8, 41-200 Sosnowiec, Poland.
Molecules. 2017 Oct 3;22(10):1657. doi: 10.3390/molecules22101657.
AKT, a serine/threonine protein kinase and mammalian target of rapamycin (mTOR) plays a critical role in the proliferation and resistance to apoptosis that are essential to the development and progression of colon cancer. Therefore, AKT/mTOR signaling pathway has been recognized as an attractive target for anticancer therapy. Inositol hexaphosphate (InsP6), a natural occurring phytochemical, has been shown to have both preventive and therapeutic effects against various cancers, however, its exact molecular mechanisms of action are not fully understood. The aim of the in vitro study was to investigate the anticancer activity of InsP6 on colon cancer with the focus on inhibiting the AKT1 kinase and p70S6K1 as mTOR effector, in relation to proliferation and apoptosis of cells. The colon cancer Caco-2 cells were cultured using standard techniques and exposed to InsP6 at different concentrations (1 mM, 2.5 mM and 5 mM). Cellular proliferative activity was monitored by 5-bromo-2'-deoxyuridine (BrdU) incorporation into cellular DNA. Flow cytometric analysis was performed for cell cycle progression and apoptosis studies. Real-time RT-qPCR was used to validate mRNA levels of CDNK1A, CDNK1B, CASP3, CASP9, AKT1 and S6K1 genes. The concentration of p21 protein as well as the activities of caspase 3, AKT1 and p70S6K1 were determined by the ELISA method. The results revealed that IP6 inhibited proliferation and stimulated apoptosis of colon cancer cells. This effect was mediated by an increase in the expression of genes encoding p21, p27, caspase 3, caspase 9 as well a decrease in transcription of AKT1 and S6K1. InsP6 suppressed phosphorylation of AKT1 and p70S6K1, downstream effector of mTOR. Based on these studies it may be concluded that InsP6 can reduce proliferation and induce apoptosis through inhibition of the AKT/mTOR pathway and mTOR effector followed by modulation of the expression and activity of several key components of these pathways in colon cancer cells.
AKT 是一种丝氨酸/苏氨酸蛋白激酶和哺乳动物雷帕霉素靶蛋白 (mTOR)，在增殖和抗凋亡方面发挥着关键作用，这对于结肠癌的发生和发展至关重要。因此，AKT/mTOR 信号通路已被认为是一种有吸引力的抗癌治疗靶点。肌醇六磷酸 (InsP6) 是一种天然存在的植物化学物质，已被证明对多种癌症具有预防和治疗作用，但其确切的作用机制尚不完全清楚。本体外研究旨在研究 InsP6 对结肠癌的抗癌活性，重点是抑制 AKT1 激酶和 mTOR 效应物 p70S6K1，以研究细胞增殖和凋亡。采用标准技术培养结肠癌 Caco-2 细胞，并将其暴露于不同浓度的 InsP6（1 mM、2.5 mM 和 5 mM）中。通过 BrdU 掺入细胞 DNA 来监测细胞增殖活性。进行流式细胞术分析以研究细胞周期进展和细胞凋亡。实时 RT-qPCR 用于验证 CDKNA1、CDKNB1、CASP3、CASP9、AKT1 和 S6K1 基因的 mRNA 水平。通过 ELISA 法测定 p21 蛋白浓度以及 caspase 3、AKT1 和 p70S6K1 的活性。结果表明，InsP6 抑制结肠癌细胞的增殖并刺激其凋亡。这种作用是通过增加编码 p21、p27、caspase 3、caspase 9 的基因的表达以及降低 AKT1 和 S6K1 的转录来介导的。InsP6 抑制 AKT1 和 p70S6K1 的磷酸化，后者是 mTOR 的下游效应物。基于这些研究，可以得出结论，InsP6 可以通过抑制 AKT/mTOR 通路和 mTOR 效应物，以及随后调节这些通路中的几个关键成分的表达和活性，减少结肠癌细胞的增殖并诱导其凋亡。
