Epimagnolin targeting on an active pocket of mammalian target of rapamycin suppressed cell transformation and colony growth of lung cancer cells

Abstract

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.

Fargesin Inhibits EGF-Induced Cell Transformation and Colon Cancer Cell Growth by Suppression of CDK2/Cyclin E Signaling Pathway

“…Moreover, aschantin did not inhibit protein kinase activities of PI3K, PDK-1, or AKT [11,12]. Recently, our research group reported that epimagnolin, a stereochemical epimer of magnolin, targets the active pocket of the mTOR kinase, but not that of ERK1 and ERK2, contrasting with our expectation that epimagnolin could inhibit the activity of ERK1 and ERK2 [13]. These results demonstrated that slight differences in chemical structure might produce large differences in the molecular targeting of proteins that are involved in human diseases including cancers.…”

“…Additionally, we did not observe the cytotoxic effects of fargesin at 60 μM by 48 h in JB6 Cl41 cells (Supplemental Figure S2) as well as fargesin-induced apoptosis in JB6 Cl41 and HaCaT cells by 48 h and 72 h, respectively (Supplemental Figure S3). The cell cycle distribution of JB6 Cl41 and HaCaT cells in normal cell culture conditions was similar to the previous literature [11,13,22]. To examine whether the G1-phase cell cycle accumulation of JB6 Cl41 and HaCaT cells treated by fargesin was mediated by a growth factor-induced G1/S cell cycle transition or not, we starved the cells and co-treated them with EGF and fargesin as indicated.…”

“…Previously, our research demonstrated that magnolin targeted the active pockets of ERK1 and ERK2 by forming hydrogen bonds with Lys168 of ERK1 and Met108 and Lys54 of ERK2, resulting in a 68 nM IC50 for ERK1 and a 16.5 nM IC50 for ERK2 [11]. Moreover, our research interest was expanded to include identifying the molecular targets of aschantin and epimagnolin [12][13][14]. The results demonstrated that those two compounds targeted the active pocket of the mTOR kinase with a less than 400 nM IC50 [11][12][13].…”

“…Moreover, our research interest was expanded to include identifying the molecular targets of aschantin and epimagnolin [12][13][14]. The results demonstrated that those two compounds targeted the active pocket of the mTOR kinase with a less than 400 nM IC50 [11][12][13]. However, other Shin-Yi compounds that might have biological activities in diverse human diseases have not been elucidated.…”

WNT7A Promotes EGF-Induced Migration of Oral Squamous Cell Carcinoma Cells by Activating β-Catenin/MMP9-Mediated Signaling

“…EGF Induces Cell Migration via MEK/ERK and PI3K/AKT Signalings, but Only p-AKT is Involved in the Wnt/b-Catenin Pathway ERK and PI3K/AKT are critical components of EGF-activated signaling, which has been associated with human cancer EMT (Wudtiwai et al, 2018;Jiang et al, 2019;Park et al, 2019;Yang et al, 2019;Yoo et al, 2019). To verify whether ERK and PI3K/ AKT could affect EGF-mediated migration of CAL27 cells, MEK inhibitor U0126 and PI3K/AKT inhibitor LY294002 were applied in observing the cell migration in the absence or presence of EGF (20 ng/mL) for 24 h. The results showed that pre-treatment with U0126 and LY294002 could reverse the EGFinduced migration (Figures 4A, B).…”