Pharmaceutical Biochemistry, Basic Research Laboratory & BK21 PLUS Team for Creative Leader Program for Pharmacomics-based Future Pharmacy, College of Pharmacy, The Catholic University of Korea, Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do, Republic of Korea.
Division of Applied Life Science (BK21 Plus), PMBBRC, Gyeongsang National University, Jinju-si, Gyeongsangnam-do, Republic of Korea.
Mol Carcinog. 2019 Jul;58(7):1221-1233. doi: 10.1002/mc.23005. Epub 2019 Mar 18.
Mammalian target of rapamycin (mTOR) has a pivotal role in carcinogenesis and cancer cell proliferation in diverse human cancers. In this study, we observed that epimagnolin, a natural compound abundantly found in Shin-Yi, suppressed cell proliferation by inhibition of epidermal growth factor (EGF)-induced G1/S cell-cycle phase transition in JB6 Cl41 cells. Interestingly, epimagnolin suppressed EGF-induced Akt phosphorylation strongly at Ser473 and weakly at Thr308 without alteration of phosphorylation of MAPK/ERK kinases (MEKs), extracellular signal-regulated kinase (ERKs), and RSK1, resulting in abrogation of the phosphorylation of GSK3β at Ser9 and p70S6K at Thr389. Moreover, we found that epimagnolin suppressed c-Jun phosphorylation at Ser63/73, resulting in the inhibition of activator protein 1 (AP-1) transactivation activity. Computational docking indicated that epimagnolin targeted an active pocket of the mTOR kinase domain by forming three hydrogen bonds and three hydrophobic interactions. The prediction was confirmed by using in vitro kinase and adenosine triphosphate-bead competition assays. The inhibition of mTOR kinase activity resulted in the suppression of anchorage-independent cell transformation. Importantly, epimagnolin efficiently suppressed cell proliferation and anchorage-independent colony growth of H1650 rather than H460 lung cancer cells with dependency of total and phosphorylated protein levels of mTOR and Akt. Inhibitory signaling of epimagnolin on cell proliferation of lung cancer cells was observed mainly in mTOR-Akt-p70S6K and mTOR-Akt-GSK3β-AP-1, which was similar to that shown in JB6 Cl41 cells. Taken together, our results indicate that epimagnolin potentiates as chemopreventive or therapeutic agents by direct active pocket targeting of mTOR kinase, resulting in sensitizing cancer cells harboring enhanced phosphorylation of the mTORC2-Akt-p70S6k signaling pathway.
哺乳动物雷帕霉素靶蛋白（mTOR）在多种人类癌症的致癌作用和癌细胞增殖中起关键作用。在这项研究中，我们观察到，大量存在于新沂的表木兰醇通过抑制表皮生长因子（EGF）诱导的 JB6 Cl41 细胞 G1/S 细胞周期相转变来抑制细胞增殖。有趣的是，表木兰醇强烈抑制 EGF 诱导的 Akt 在 Ser473 处的磷酸化，在 Thr308 处弱磷酸化，而不改变丝裂原活化蛋白激酶/细胞外信号调节激酶（MEKs）、细胞外信号调节激酶（ERK）和 RSK1 的磷酸化，导致 GSK3β 在 Ser9 处和 p70S6K 在 Thr389 处的磷酸化被阻断。此外，我们发现表木兰醇抑制 c-Jun 在 Ser63/73 处的磷酸化，从而抑制激活蛋白 1（AP-1）的转录激活活性。计算对接表明，表木兰醇通过形成三个氢键和三个疏水相互作用靶向 mTOR 激酶结构域的活性口袋。该预测通过使用体外激酶和三磷酸腺苷-珠竞争测定得到证实。mTOR 激酶活性的抑制导致锚定非依赖性细胞转化的抑制。重要的是，表木兰醇有效地抑制了 H1650 而非 H460 肺癌细胞的增殖和锚定非依赖性集落生长，这依赖于 mTOR 和 Akt 的总蛋白和磷酸化蛋白水平。在肺癌细胞中，表木兰醇对细胞增殖的抑制作用主要在 mTOR-Akt-p70S6K 和 mTOR-Akt-GSK3β-AP-1 信号通路中观察到，这与在 JB6 Cl41 细胞中观察到的相似。综上所述，我们的研究结果表明，表木兰醇通过直接靶向 mTOR 激酶的活性口袋，增强了对 mTORC2-Akt-p70S6k 信号通路磷酸化增强的癌症细胞的敏感性，从而作为化学预防或治疗剂发挥作用。
