The autophagy inducer trehalose stimulates macropinocytosis in NF1-deficient glioblastoma cells

Abstract

Background Glioblastoma is a highly aggressive brain tumor. A big effort is required to find novel molecules which can cross the blood–brain barrier and efficiently kill these tumor cells. In this perspective, trehalose (α-glucopyranosyl‐[1→1]‐α‐d‐glucopyranoside), found in various dietary sources and used as a safe nutrient supplement, attracted our attention for its pleiotropic effects against tumor cells. Methods Human glioblastoma cell lines U373-MG and T98G were exposed to trehalose and analyzed at different time points. Cell proliferation was evaluated at medium term, and clonogenic capacity and cell morphology were evaluated at long term. Western blot was used to evaluate biochemical markers of autophagy (also measured in cells co-treated with EIPA or chloroquine), and mTOR, AMPK and ERK 1/2 signalling. Macropinocytosis was evaluated morphologically by bright-field microscopy; in cells loaded with the fluorescein-conjugated fluid-phase tracer dextran, macropinocytic vacuoles were also visualized by fluorescence microscopy, and the extent of macropinocytosis was quantified by flow cytometry. Results The long-term effect of trehalose on U373-MG and T98G cell lines was impressive, as indicated by a dramatic reduction in clonogenic efficiency. Mechanistically, trehalose proved to be an efficient autophagy inducer in macropinocytosis-deficient T98G cells and an efficient inducer of macropinocytosis and eventual cell death by methuosis in U373-MG glioblastoma cells, proved to be poorly responsive to induction of autophagy. These two processes appeared to act in a mutually exclusive manner; indeed, co-treatment of U373-MG cells with the macropinocytosis inhibitor, EIPA, significantly increased the autophagic response. mTOR activation and AMPK inhibition occurred in a similar way in the two trehalose-treated cell lines. Interestingly, ERK 1/2 was activated only in macropinocytosis-proficient U373-MG cells harbouring loss-of-function mutations in the negative RAS regulator, NF1, suggesting a key role of RAS signalling. Conclusions Our results indicate that trehalose is worthy of further study as a candidate molecule for glioblastoma therapy, due to its capacity to induce a sustained autophagic response, ultimately leading to loss of clonogenic potential, and more interestingly, to force macropinocytosis, eventually leading to cell death by methuosis, particularly in tumor cells with RAS hyperactivity. As a further anticancer strategy, stimulation of macropinocytosis may be exploited to increase intracellular delivery of anticancer drugs.

Progress in the discovery and development of small molecule methuosis inducers

“…52 Researchers from Maellaro's team recently discovered that trehalose could induce methuosis but not autophagy in U373-MG glioblastoma cells. 53 In contrast, it was found to only induce autophagy in another macropinocytosis-deficient glioblastoma cell line, T98G, indicating that these two processes (methuosis and autophagy) appeared to act in a mutually exclusive manner. This may be related to the coincident AMPK–mTOR1 signaling pathway in both methuosis and autophagy.…”

Pleiotropic physiological functions of Piezo1 in human body and its effect on malignant behavior of tumors

“…To adapt to this extreme environment, tumor cells often reprogram their cellular metabolism to take up essential nutrients from the nutritionally deprived environment and adjust intracellular metabolic pathways to supply the more important biological processes ( Pavlova Natalya and Thompson, 2016 ). Macropinocytosis is an important non-selective extracellular amino acid uptake pathway, which has been reported in tumor cells ( Del Bello et al, 2022 ; Xia et al, 2023 ) and even tumor stromal cells ( Zhang et al, 2021 ). Macropinocytosis is dependent on the formation of peripheral folds, whose formation and migration are regulated by actin ( Isogai et al, 2015 ).…”

Amyloids and brain cancer: molecular linkages and crossovers

“…In C6 glioma cells, Aβ42 treatment induces cyclooxygenase-2 and inducible nitric oxide synthase (iNOS) overexpression followed by nuclear factor kappa B (NF-κB), which could be reversed by an anti-aggregatory compound resveratrol [ 86 ]. Several other anti-aggregatory natural molecules, for example trehalose, quercetin, and myricetin, have shown anti-neoplastic and apoptosis-inducing potential in glioma cells [ 87–90 ]. Another highly effective anti-amyloid agent epigallocatechin-3-gallate (EGCG) leads to cell death, reduced cellular proliferation, and suppression of invasion in multiple glioma cell lines and is suggested to be used as an adjuvant in anti-glioma therapies [ 91 ].…”