Butoxy Mansonone G Inhibits STAT3 and Akt Signaling Pathways in Non-Small Cell Lung Cancers: Combined Experimental and Theoretical Investigations

Abstract

Epidermal growth factor receptor (EGFR) is the key molecular target for non-small cell lung cancer (NSCLC) due to its major contribution to complex signaling cascades modulating the survival of cancer cells. Targeting EGFR-mediated signaling pathways has been proved as a potential strategy for NSCLC treatment. In the present study, mansonone G (MG), a naturally occurring quinone-containing compound, and its semi-synthetic ether derivatives were subjected to investigate the anticancer effects on human NSCLC cell lines expressing wild-type EGFR (A549) and mutant EGFR (H1975). In vitro cytotoxicity screening results demonstrated that butoxy MG (MG3) exhibits the potent cytotoxic effect on both A549 (IC₅₀ of 8.54 μM) and H1975 (IC₅₀ of 4.21 μM) NSCLC cell lines with low toxicity against PCS201-010 normal fibroblast cells (IC₅₀ of 21.16 μM). Western blotting and flow cytometric analyses revealed that MG3 induces a caspase-dependent apoptosis mechanism through: (i) inhibition of p-STAT3 and p-Akt without affecting upstream p-EGFR and (ii) activation of p-Erk. The 500-ns molecular dynamics simulations and the molecular mechanics combined with generalized Born surface area (MM/GBSA)-based binding free energy calculations suggested that MG3 could possibly interact with STAT3 SH2 domain and ATP-binding pocket of Akt. According to principal component analysis, the binding of MG3 toward STAT3 and Akt dramatically altered the conformation of proteins, especially the residues in the active site, stabilizing MG3 mainly through van der Waals interactions.

Keywords: Akt; STAT3; apoptosis; mansonone G; molecular dynamics simulation; non-small cell lung cancer.

Figure 1

Two-dimensional (2D) chemical structures of (A) MG and its semi-synthetic ether derivatives MG1-MG10 [30] and (B) the known STAT3 (cryptotanshinone (CST) and S3I201) and Akt (uprosertib and H8) inhibitors. Three-dimensional (3D) structures of (C) STAT3 and (D) Akt1 signaling proteins. The SH2 domain of STAT3 and the ATP-binding pocket of Akt are shown by blue surface and black circle, respectively.

Figure 2

Cell viability of NSCLC (A–C) and PCS201-010 (D–F) cell lines after treatment with MG3, MG4, and CDDP for 48 h. The IC₅₀ (μM) and SI of three focused compounds against all studied cell lines are shown in (G) and (H), respectively. (I) The morphological changes of two NSCLC cell lines treated with MG3 at various concentrations for 48 h.

Figure 3

Flow cytometric analysis of Annexin V/PI stained cells after MG3 and CDDP treatments for 24 h on (A) A549 and (B) H1975 cells. Western blot analysis of apoptotic markers, caspase-3 and PARP, for (C) A549 and (D) H1975 cells. Inhibition of MG3-induced apoptosis by the pan caspase inhibitor Z-VAD(OMe)-FMK in (E) A549 and (F) H1975 cells. Data are expressed as mean ± SEM (n = 3). * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 vs. control. ^@ p ≤ 0.05, ^@@ p ≤ 0.01, and ^@@@ p ≤ 0.001 vs. MG3. ^# p ≤ 0.05, ^## p ≤ 0.01, and ^### p ≤ 0.001 vs. CDDP.

Figure 4

MG3 dose-dependently inhibits the phosphorylation of STAT3 (Tyr705) and Akt (Ser473) in (A) A549 and (B) H1975 cells at 24 h. The expression of p-Erk (Thr202/Tyr204) is found to be increased upon MG3 and CDDP treatments. The phosphorylation of EGFR (Y1068) for both (C) A549 and (D) H1975 cells is not significantly affected by MG3 and CDDP treatments. (E) Proposed mechanisms of MG3 against two studied NSCLC cell lines, in which MG3 promotes cell apoptosis through the inhibition of p-Akt and p-STAT3 as well as through the activation of MAPK signaling pathway. Data are expressed as mean ± SEM of three independent experiments. * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 vs. control.

Figure 5

$\Delta G_{\mathrm{bind}}^{\mathrm{residue}}$ (kcal/mol) of (A) STAT3 and (B) Akt. The amino acids involved in ligand binding are shaded according to their $\Delta G_{\mathrm{bind}}^{\mathrm{residue}}$, in which the highest and lowest energies are ranged from red to magenta, respectively.

Figure 6

Porcupine plot of apo and holo forms of (A) STAT3 and (B) Akt across the PC1. The residues within 5 Å of ligand are presented in circle where the MG3 molecule is shown in vdW model. The PCA scree plot of quantitative characters for each protein is given below, in which the columns in black and grey represent the data for apo and holo forms, respectively.