Methuosis Contributes to Jaspine-B-Induced Cell Death

Abstract

Methuosis is a type of programmed cell death in which the cytoplasm is occupied by fluid-filled vacuoles that originate from macropinosomes (cytoplasmic vacuolation). A few molecules have been reported to behave as methuosis inducers in cancer cell lines. Jaspine B (JB) is a natural anhydrous sphingolipid (SL) derivative reported to induce cytoplasmic vacuolation and cytotoxicity in several cancer cell lines. Here, we have investigated the mechanism and signalling pathways involved in the cytotoxicity induced by the natural sphingolipid Jaspine B (JB) in lung adenocarcinoma A549 cells, which harbor the G12S K-Ras mutant. The effect of JB on inducing cytoplasmic vacuolation and modifying cell viability was determined in A549 cells, as well as in mouse embryonic fibroblasts (MEF) lacking either the autophagy-related gene ATG5 or BAX/BAK genes. Apoptosis was analyzed by flow cytometry after annexin V/propidium iodide staining, in the presence and absence of z-VAD. Autophagy was monitored by LC3-II/GFP-LC3-II analysis, and autophagic flux experiments using protease inhibitors. Phase contrast, confocal, and transmission electron microscopy were used to monitor cytoplasmic vacuolation and the uptake of Lucifer yellow to assess macropinocyosis. We present evidence that cytoplasmic vacuolation and methuosis are involved in Jaspine B cytotoxicity over A549 cells and that activation of 5' AMP-activated protein kinase (AMPK) could be involved in Jaspine-B-induced vacuolation, independently of the phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin complex 1 (PI3K/Akt/mTORC1) axis.

Kinase inhibitor-induced cell-type specific vacuole formation in the absence of canonical ATG5-dependent autophagy

“…Further studies using VE-821, PIKfyve inhibitors, SB202190 and SB220025 could reveal novel mechanisms and targets responsible for this interesting phenotype relevant to anti-cancer therapy. Methuosis, another micropinocytosis mediated vacuolation process also shows RAB7+ vacuoles and crosstalk with autophagy/lysosomal transport pathways (23, 69). Micropinocytosis inhibitors (70) like dynasore and dyngo-4a does not inhibit SB202190-induced vacuolation in A549 cells (Figure 4A & Supplementary Table S1).…”

“…Paraptosis, a caspase-independent cell death pathway with accumulation of cytoplasmic vacuoles originating from endoplasmic reticulum and swollen mitochondria has been targeted for cancer therapy (20, 21). Methuosis is another vacuolation dependent mode of programmed cell death where vacuoles originate from macropinosomes and swollen endosomes (22, 23). SB202190 was originally shown to induce vacuoles specifically in colorectal cancer cells (24, 25).…”

Nutlin-3a inducesKRASmutant lung cancer specific methuosis-like cell death that is dependent on GFPT2

“…The underlying mechanism or pathway of methuosis or methuosis-like cell death remains unclear, but the involvement of Ras (Bhanot et al, 2010), Rac1 (Bhanot et al, 2010;Chi et al, 1999), Akt-mTOR (Liu et al, 2021), and AMPK (Bielsa et al, 2022) in a caspaseindependent manner has been reported. Consistent with these reports, nutlin-3a downregulated KRAS activity as well as the KRAS downstream signal, KRAS-PI3K/Akt-mTOR, but not MEK/ERK.…”

Progress in the discovery and development of small molecule methuosis inducers

“…Interestingly, the vacuoles of normal HEK293T cells caused by the stimulation of jaspine B would gradually disappear as time went on (after 24 h), indicating that its induced methuosis has a certain selectivity toward the tested tumor cells. In a recent report by Bielsa et al , 35 they further proved that the cavitation induced by jaspine B was related to the activation of adenosine 5′-monophosphate activated protein kinase.…”