Muscarinic receptor drug trihexyphenidyl can alter growth of mesenchymal glioblastoma in vivo

Abstract

Glioblastoma (GBM) is the most commonly occurring and most aggressive primary brain tumor. Transcriptomics-based tumor subtype classification has established the mesenchymal lineage of GBM (MES-GBM) as cancers with particular aggressive behavior and high levels of therapy resistance. Previously it was show that Trihexyphenidyl (THP), a market approved M1 muscarinic receptor-targeting oral drug can suppress proliferation and survival of GBM stem cells from the classical transcriptomic subtype. In a series of in vitro experiments, this study confirms the therapeutic potential of THP, by effectively suppressing the growth, proliferation and survival of MES-GBM cells with limited effects on non-tumor cells. Transcriptomic profiling of treated cancer cells identified genes and associated metabolic signaling pathways as possible underlying molecular mechanisms responsible for THP-induced effects. In vivo trials of THP in immunocompromised mice carry orthotopic MES-GBMs showed moderate response to the drug. This study further highlights the potential of THP repurposing as an anti-cancer treatment regimen but mode of action and d optimal treatment procedures for in vivo regimens need to be investigated further.

Keywords: cystathionine beta-synthase; drug repurposing; glioblastoma; mesenchymal transformation; trihexyphenidyl.

FIGURE 1

Cell viability of all the cell lines (JHH520, BTSC233, NCH644, GBM1, U87 and HUVEC) using ATP consumption assay treated with five different concentrations of THP. Control samples were treated by MeOH without THP. Each experiment executed at least n = 3 independent repetitions (A–F).

FIGURE 2

Proliferation results using FACS based quantification for 10 µM THP treatment of cells for 48 and 72 h. Cell proliferation was expressed as percentage of Ki67-positive cells to total amount of gated events recorded. Control samples were without THP treatment (A,B). Each experiment executed at least for 3 independent repetitions. Significant findings (*) with a p value < 0.05 and highly significant findings (**) with a p value < 0.01 or (***) with a p value < 0.001.

FIGURE 3

Cell cycle distribution in GBM1, JHH520, NCH644 and BTSC233 cells following 10 µM THP treatment for 48 h. Control samples were without THP treatment. Each experiment executed at least for 3 independent repetitions (A–D).

FIGURE 4

Apoptosis analysis results using Annexin V/PI staining and FACS based quantification for 10 µM THP treatment of cells for 48 and 72 h. Viable cells (A), early apoptotic cells (B), total apoptotic cells (C) and late apoptotic cells (D). Control samples were without THP treatment. Each experiment executed at least for three independent repetitions. Significant findings (*) with a p-value <0.05 and highly significant findings (**) with a p-value <0.01 or (***) with a p-value <0.001.

FIGURE 5

RNA sequencing of cell lines with or without THP treatment for 48 h. Shared regulatory genes were processed and represented via Venn diagram and subsequent survival analysis of five identified gene signals using TSCG dataset (A), differential expression of CBS-L (B), and GSEA result by KEGG for the related pathways (C).

FIGURE 6

Detection of persulfidated proteins in NCH644 48 h post THP treatment (A). Oxyblot THP 48 h -GBM1/NCH644/JHH520 (B). Oxyblot THP 72 h -GBM1/NCH644/JHH520 (C).

FIGURE 7

Bioluminescence based assessment of orthotopic tumor growth in immunocompromised mice for mesenchymal tumor subtype cell models BTC233, NCH421k and JHH520. (A–C) Exemplary images of animals in treatment groups at latest time point when both groups were still complete. (D–F) Mean relative signal intensities (day x to day 12) until measurement when individual groups were still complete. n = 5 or 6.