Pharmacological Inhibition of Membrane Signaling Mechanisms Reduces the Invasiveness of U87-MG and U251-MG Glioblastoma Cells In Vitro

Abstract

Impairing the motility of glioblastoma multiforme (GBM) cells is a compelling goal for new approaches to manage this highly invasive and rapidly lethal human brain cancer. Work here characterized an array of pharmacological inhibitors of membrane ion and water channels, alone and in combination, as tools for restraining glioblastoma spread in human GBM cell lines U87-MG and U251-MG. Aquaporins, AMPA glutamate receptors, and ion channel classes (shown to be upregulated in human GBM at the transcript level and linked to mechanisms of motility in other cell types) were selected as pharmacological targets for analyses. Effective compounds reduced the transwell invasiveness of U87-MG and U251-MG glioblastoma cells by 20–80% as compared with controls, without cytotoxicity. The compounds and doses used were: AqB013 (14 μM); nifedipine (25 µM); amiloride (10 µM); apamin (10 µM); 4-aminopyridine (250 µM); and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; 30 µM). Invasiveness was quantified in vitro across transwell filter chambers layered with extracellular matrix. Co-application of each of the ion channel agents with the water channel inhibitor AqB013 augmented the inhibition of invasion (20 to 50% greater than either agent alone). The motility impairment achieved by co-application of pharmacological agents differed between the GBM proneural-like subtype U87-MG and classical-like subtype U251-MG, showing patterns consistent with relative levels of target channel expression (Human Protein Atlas database). In addition, two compounds, xanthurenic acid and caelestine C (from the Davis Open Access Natural Product-based Library, Griffith University QLD), were discovered to block invasion at micromolar doses in both GBM lines (IC50 values from 0.03 to 1 µM), without cytotoxicity, as measured by full mitochondrial activity under conditions matching those in transwell assays and by normal growth in spheroid assays. Mechanisms of action of these agents based on published work are likely to involve modulation of glutamatergic receptor signaling. Treating glioblastoma by the concurrent inhibition of multiple channel targets could be a powerful approach for slowing invasive cell spread without cytotoxic side effects, potentially enhancing the effectiveness of clinical interventions focused on eradicating primary tumors.

Multiomic screening of invasive GBM cells reveals targetable transsulfuration pathway alterations

“…GBM43/CTHkd cells expanded over 5 days in culture at varying seeding densities to the same degree as control GBM43 cells ( P = 0.1–0.9; Supplemental Figure 4D ). Similarly, to confirm that the antiinvasive effects of CSE-γ-IN did not reflect effects on cell survival, we assessed the concentration window for which CSE- γ-IN inhibited GBM spheroid invasion without cytotoxicity and found that the invasion inhibitory effect of CSE-γ-IN on spheroids derived from GBM43 cells, which are of the proneural GBM subtype ( 37 ), began at 40 μM ( P < 0.0001; Figure 5D ) with 40 μM CSE-γ-IN also inhibiting spheroid invasion in classical subtype U251 cells ( 38 ) ( P < 0.001; Supplemental Figure 4E ). Concentrations of CSE-γ-IN above 100 μM began to affect the viability of GBM43 or U251 cells ( Supplemental Figure 4F ).…”

Chemical and Antiplasmodial Investigations on Eremophila-Derived Alkaloids and Semisynthetic Ether Analogues

“…None of the new ether derivatives displayed significantly improved activity in the current study, suggesting that further chemical modifications at the phenol moiety of 1 are not warranted for antiplasmodial investigations. All new chemistry produced during this project will be added to the Davis Open Access Natural Product-Based Library , and screened in other bioassays in the future.…”

Synthesis and Antimalarial Evaluation of Halogenated Analogues of Thiaplakortone A

“…The outcomes from these proof-of-concept late-stage functionalisation studies suggest that with respect to antimalaria potential, further halogenation efforts at C-2 and C-7 are not warranted for this particular scaffold. All new chemistry produced during this project will be added to the Davis Open Access Natural Product-based Library [ 31 , 32 ] and screened in other bioassays in the future. Thus, while compounds 3 – 11 will not directly impact antimalarial drug discovery and development, they may provide future leads for other biomedical applications or be the source of new probes for chemical biology research.…”