Glioblastoma Multiforme Stem Cell Cycle Arrest by Alkylaminophenol Through the Modulation of EGFR and CSC Signaling Pathways

Abstract

Cancer stem cells (CSCs), a small subpopulation of cells existing in the tumor microenvironment promoting cell proliferation and growth. Targeting the stemness of the CSC population would offer a vital therapeutic opportunity. 3,4-Dihydroquinolin-1(2H)-yl)(p-tolyl)methyl)phenol (THTMP), a small synthetic phenol compound, is proposed to play a significant role in controlling the CSC proliferation and survival. We assessed the potential therapeutic effects of THTMP on glioblastoma multiforme (GBM) and its underlying mechanism in various signaling pathways. To fully comprehend the effect of THTMP on the CSCs, CD133⁺ GBM stem cell (GSC) and CD133^- GBM Non-stem cancer cells (NSCC) population from LN229 and SNB19 cell lines was used. Cell cycle arrest, apoptosis assay and transcriptome analysis were performed for individual cell population. THTMP strongly inhibited NSCC and in a subtle way for GSC in a time-dependent manner and inhibit the resistance variants better than that of temozolomide (TMZ). THTMP arrest the CSC cell population at both G1/S and G2/M phase and induce ROS-mediated apoptosis. Gene expression profiling characterize THTMP as an inhibitor of the p53 signaling pathway causing DNA damage and cell cycle arrest in CSC population. We show that the THTMP majorly affects the EGFR and CSC signaling pathways. Specifically, modulation of key genes involved in Wnt, Notch and Hedgehog, revealed the significant role of THTMP in disrupting the CSCs' stemness and functions. Moreover, THTMP inhibited cell growth, proliferation and metastasis of multiple mesenchymal patient-tissue derived GBM-cell lines. THTMP arrests GBM stem cell cycle through the modulation of EGFR and CSC signaling pathways.

Keywords: GBM stem cells; alkylaminophenol and cell death; cell cycle arrest; non-stem cancer cells; resistance population.

Figure 1

Effect of 3,4-Dihydroquinolin-1(2H)-yl)(p-tolyl)methyl)phenol (THTMP) treatment on cell survival in GBM cells. (A) Molecular structure of three tested alkylaminophenols (HNPMI, THMPP and THTMP) and % cell viability of those compounds on LN229 and SNB19 cell lines at 100 µM concentration. (B) Percentage of cell viability for LN229 and SNB19 cell lines upon treatment with THTMP/TMZ in the dilution series from 10 µM to 100 µM. (C) Representative image and intensity of CD133 on GSC, NSCC and mixed population of LN229 and SNB19. (D) Time-dependent effect of THTMP on GSC and NSCC cells. The results were normalized to DMSO control. One-way ANOVA was conducted (P < 0.05) to determine the statistical significance in all the conditions compared to DMSO control. (E) The top DEGs involved in DNA damage on GSC-LN229, GSC-SNB19, NSCC-LN229 and NSCC-SNB19. The DEGs were color coded corresponding to the up- and down- expressed genes. (F) Relative cytotoxicity of resistant variants, LR1, LR2 (derived from GSC-LN229) and SR1, SR2 (derived from GSC-SNB19) to THTMP and/or TMZ. The results were normalized to DMSO control. One-way ANOVA was done (P < 0.05) to determine the statistical significance in all the conditions compared to DMSO control. All experiments were performed with N = 5. * p < 0.05, ns—non significant.

Figure 2

THTMP triggers GSC and NSCC cell cycle arrest. (A) Graphs represented the distribution of cells in different phases of cell cycle (B) The bar diagram representing the percentage of total cells in different cell cycle phases treated with THTMP/TMZ. (C) The top DEGs involved in the cell cycle in GSC and NSCC population. The DEGs were color coded corresponding to the up- and down- expressed genes. Five biological and technical repeats were used in cell cycle and gene expression analysis. All experiments were performed with N = 5.

Figure 3

THTMP induces apoptosis in NSCCs and GSCs. (A) Percentage of apoptotic cells and necrotic cells upon THTMP/TMZ treatment at 24 h, stained with Annexin V-FITC/PI. (B) The top DEGs which are involved in apoptosis induction on GSCs and NSCCs. The DEGs were color coded that corresponds to the up- and down-expressed genes. (C) Effect of THTMP/TMZ on NSCCs and GSCs intracellular ROS production. (D) Activity of caspases 3/7 on NSCCs and GSCs in THTMP/TMZ treatment. Fold increase in ROS and caspases 3/7 activity of cells was calculated with triplicates for each condition. The data were normalized against DMSO control (C,D). * p < 0.05, ** p < 0.01.

Figure 4

Effect of THTMP on GSC, NSCC and their progenitors gene expression patterns (A) Overlapping DEGs in GSC, NSCC and mixed populations of LN229 and SNB19 cell lines. (B) Circos plot of overlapping up- and down-regulated genes detected in GSC, NSCC and progenitor lines. (C) Common KEGG biological process in GSC, NSCC and progenitor lines. (D) Common signaling pathways in GSC, NSCC and progenitor lines.

Figure 5

Effect of THTMP on multiple signaling pathways (A) Significant targeted signaling pathways upon THTMP treatment. (B) Comparison of DEGs fold change in EGF, PDGF, JAK/STAT, and TGF-β signaling pathways of GSC, NSCC and mixed populations. (C) Comparison of DEGs fold change in CSC signaling pathways in GSC, NSCC and progenitor lines.

Figure 6

THTMP inhibited cell growth and cell migration of patient-derived mesenchymal subtype of GBM cells. (A) Demonstrated images of morphological changes in patient-derived GBM cells at 24 h after THTMP and DMSO treatment. (B). Growth inhibitory effect of THTMP on different cell lines, MMK1, RN1 and PB1 at 10 and 100 µM after 24 h post treatment. (C) Example images of scratch assay shows the closing/widening of the scratched area over the time. All images were taken using a light microscope with 10× objective. (D) Quantification of the percentage of invading area of MMK1 and RN1 cells for every 2 h over the period of 10 h after the scratch. * p < 0.05, ** p < 0.01, ns—non significant.