M2 Receptor Activation Counteracts the Glioblastoma Cancer Stem Cell Response to Hypoxia Condition.

Ilaria Cristofaro,Ada Maria Tata,Luciano Conti,Chiara Limongi,Annamaria Confaloni,Claudia Guerriero,Mario Fiore,Paola Piscopo,Alessio Crestini

doi:10.3390/ijms21051700

Abstract

Glioblastoma multiforme (GBM) is the most malignant brain tumor. Hypoxic condition is a predominant feature of the GBM contributing to tumor growth and resistance to conventional therapies. Hence, the identification of drugs able to impair GBM malignancy and aggressiveness is considered of great clinical relevance. Previously, we demonstrated that the activation of M2 muscarinic receptors, through the agonist arecaidine propargyl ester (Ape), arrests cell proliferation in GBM cancer stem cells (GSCs). In the present work, we have characterized the response of GSCs to hypoxic condition showing an upregulation of hypoxia-inducible factors and factors involved in the regulation of GSCs survival and proliferation. Ape treatment in hypoxic conditions is however able to inhibit cell cycle progression, causing a significant increase of aberrant mitosis with consequent decreased cell survival. Additionally, qRT-PCR analysis suggest that Ape downregulates the expression of stemness markers and miR-210 levels, one of the main regulators of the responses to hypoxic condition in different tumor types. Our data demonstrate that Ape impairs the GSCs proliferation and survival also in hypoxic condition, negatively modulating the adaptive response of GSCs to hypoxia.

Highlights

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain tumor
In order to evaluate the effects of hypoxia and arecaidine propargyl ester (Ape) on GBM cancer stem cells (GSCs) cell survival, we investigated by FACS analysis and propidium iodide staining, the fraction of cells in the sub-G1 phase both in normoxia and hypoxia and upon M2 agonist treatment
Hypoxia has been reported to increase the expression of the stem cell marker CD133 [12], suggesting that O2 deprivation increases the stability of cancer stem cells, the subpopulation involved in tumors progression and invasion

Summary

Introduction

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain tumor. Individuals affected by this tumor have a prognosis of 12–15 months of life after standard treatment. Given the intrinsic genetic and phenotypic heterogeneity of GBM, it is possible that current standard treatments eliminate only specific and more susceptible GBM subpopulations, while the more resistant ones survive and repopulate the tumor. This leads to a more aggressive recurrent tumor that does not respond to initial therapy and significantly compromises the patient’s prognosis

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