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

Alanah Varricchio; Sidra Khan; Zoe K Price; Rohan A Davis; Sunita A Ramesh; Andrea J Yool

doi:10.3390/cancers15041027

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

Cancers (Basel). 2023 Feb 6;15(4):1027. doi: 10.3390/cancers15041027.

Authors

Alanah Varricchio¹, Sidra Khan¹, Zoe K Price², Rohan A Davis³, Sunita A Ramesh⁴, Andrea J Yool⁵

Affiliations

¹ Discipline of Physiology, School of Biomedicine, University of Adelaide, Adelaide, SA 5005, Australia.
² Obstetrics and Gynaecology, Adelaide Medical School, University of Adelaide, Adelaide, SA 5005, Australia.
³ Griffith Institute for Drug Discovery, School of Environment and Science, Griffith University, Nathan, QLD 4111, Australia.
⁴ Biological Sciences, College of Science and Engineering, Flinders University, Bedford Park, SA 5042, Australia.
⁵ Discipline of Physiology, School of Biomedicine and the Institute for Photonics and Advanced Sensing, University of Adelaide, Adelaide, SA 5005, Australia.

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.

Keywords: aquaporin; brain cancer; caelestine C; glutamate receptor; ion channel; natural products; xanthurenic acid.

Grants and funding

This research was funded by the NeuroSurgical Research Foundation Grant 2022; the Australian Research Council (grant number 19ARC_DP190101745), and a University of Adelaide Research Training Program Scholarship to A.V.