International Journal of Molecular Sciences Article Methuosis Contributes to Jaspine-B-Induced Cell Death Núria Bielsa 1 , Mireia Casasampere 1 , Jose Luis Abad 1 , Carlos Enrich 2,3 , Antonio Delgado 1,4,† , Gemma Fabriàs 1,5 , Jose M. Lizcano 6,7, * and Josefina Casas 1,5, * 1 Research Unit on BioActive Molecules, Department of Biological Chemistry, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), 08034 Barcelona, Spain; nubielsa@gmail.com (N.B.); mireia.casasampere@iqac.csic.es (M.C.); joseluis.abad@iqac.csic.es (J.L.A.); gemma.fabrias@iqac.csic.es (G.F.) 2 Unitat de Biologia Cel·lular, Departament de Biomedicina, Facultat de Medicina i Ciències de la Salut, Universitat de Barcelona, 08036 Barcelona, Spain; enrich@ub.edu 3 Centre de Recerca Biomèdica CELLEX, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain 4 Unit of Pharmaceutical Chemistry (Associated Unit to CSIC), Department of Pharmacology, Toxicology and Medicinal Chemistry, Faculty of Pharmacy and Food Sciences, University of Barcelona, 08028 Barcelona, Spain 5 Liver and Digestive Diseases Networking Biomedical Research Centre (CIBEREHD), ISCIII, 28029 Madrid, Spain 6 Protein Kinases and Signal Transduction Laboratory, Departament de Bioquímica i Biologia Molecular and Institut de Neurociències, Universitat Autònoma de Barcelona (UAB), 08193 Barcelona, Spain 7 Protein Kinases in Cancer Research, Vall Hebron Institut de Recerca (VHIR), 08193 Barcelona, Spain * Correspondence: josemiguel.lizcano@uab.es (J.M.L.); fina.casas@iqac.csic.es (J.C.) † Deceased author. 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 Citation: Bielsa, N.; Casasampere, M.; involved in the cytotoxicity induced by the natural sphingolipid Jaspine B (JB) in lung adenocarcinoma Abad, J.L.; Enrich, C.; Delgado, A.; A549 cells, which harbor the G12S K-Ras mutant. The effect of JB on inducing cytoplasmic vacuolation Fabriàs, G.; Lizcano, J.M.; Casas, J. and modifying cell viability was determined in A549 cells, as well as in mouse embryonic fibroblasts Methuosis Contributes to (MEF) lacking either the autophagy-related gene ATG5 or BAX/BAK genes. Apoptosis was analyzed Jaspine-B-Induced Cell Death. Int. J. by flow cytometry after annexin V/propidium iodide staining, in the presence and absence of z-VAD. Mol. Sci. 2022, 23, 7257. https:// Autophagy was monitored by LC3-II/GFP-LC3-II analysis, and autophagic flux experiments using doi.org/10.3390/ijms23137257 protease inhibitors. Phase contrast, confocal, and transmission electron microscopy were used to Academic Editor: Paola Giussani 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 Received: 3 June 2022 Accepted: 27 June 2022 over A549 cells and that activation of 50 AMP-activated protein kinase (AMPK) could be involved in Published: 29 June 2022 Jaspine-B-induced vacuolation, independently of the phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin complex 1 (PI3K/Akt/mTORC1) axis. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: autophagy; methuosis; apoptosis; sphingolipids; cytoplasmic vacuolization published maps and institutional affil- iations. 1. Introduction Copyright: © 2022 by the authors. In living organisms, there is a regulated equilibrium between the generation of new Licensee MDPI, Basel, Switzerland. cells by cell division and the elimination of the damaged ones. The most well-known mech- This article is an open access article anism of programmed cell death is apoptosis [1]. In recent decades, however, new types of distributed under the terms and cell death independent of apoptosis have gained interest for their potential importance in conditions of the Creative Commons pathological processes, toxicology, and cancer therapy [2]. One of the latest additions to Attribution (CC BY) license (https:// the list of phenotypes of cell death is methuosis [3], first described by Overmeyer et al. in creativecommons.org/licenses/by/ 2008 [4]. The name derives from the Greek methuo, which means “to drink to intoxication”, 4.0/). Int. J. Mol. Sci. 2022, 23, 7257. https://doi.org/10.3390/ijms23137257 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022, 23, 7257 2 of 15 because in this type of cell death, the cytoplasm is occupied by fluid-filled vacuoles that originate from macropinosomes. Macropinocytosis is a clathrin-independent endocytic process by which cells internal- ize extracellular fluid, nutrients, and proteins in vesicles (macropinosomes) generated from protrusion of the plasma membrane [2]. Macropinosomes enter the endocytic pathway and are recycled to the plasma membrane, or mature and become late endosomes that eventually fuse with lysosomes [5]. In methuosis, endosomal trafficking is not functional, and the vacuoles that are originated from macropinocytic activity are accumulated in the cy- toplasm and they fuse with each other, producing larger vacuoles that finally occupy most of the cytoplasm. Cytoplasmic vacuolization can be transient or irreversible. Irreversible vacuolization produces different types of caspase-independent cell death, including methu- osis, paraptosis, oncosis, and necroptosis [6], which occur without cellular shrinkage and nuclear fragmentation, typical of apoptosis [7]. The appearance of vacuoles can resemble autophagy but, in contrast to autophagosomes that have double membranes surrounding luminal cytoplasmic contents, methuosis vacuoles have a single membrane [7]. Paraptosis and oncosis proceed with cytoplasmic vacuoles, but in these cases, vacuoles are originated from endoplasmic reticulum or mitochondria [2] instead of macropinocytosis. Methuosis was originally described in glioblastoma cells after the ectopic expression of activated Ras (rat sarcoma virus) [4] and small molecules that induce methuosis in numerous cancer cell types have been described, including apoptosis-resistant cancer cells. Among these molecules, indol-based chalcones have been explored in U251 human glioblastoma cells [7–9], as well as the CK2 (casein kinase II) inhibitor silmitasertib in colorectal cancer cells [10]. Jaspine B (JB) is a natural anhydrous sphingolipid (SL) derivative isolated from the marine sponge Jaspis sp. reported to induce cytoplasmic vacuolation [11] and cytotoxicity in several cancer cell lines [11–13]. SL metabolism is a complex network of regulated reactions. SLs are important constitutive elements of the cellular membranes, and also display roles as bioactive molecules and intervene in different cellular-signalling pathways. JB interferes in SL metabolism acting as a competitive inhibitor of the CerS (ceramide synthase) enzymes [11]. Alterations in the SL metabolism have been associated with cell death by apoptosis and autophagy [12,13]. However, JB induces methuosis independently of the alterations it induces in the SL metabolism [11]. Importantly, JB capacity for inducing vacuolation is not cell-type-dependent, since it has been demonstrated in several cell lines with different genetic profiles [13]. Previously, we reported that JB induced cytoplasmic vacuolization in the human gastric cancer HGC27 cell line [11], which expresses wild-type K-RAS [14]. Since it has been reported that active K-Ras promotes methuosis [4,15], we investigated the activity of JB in lung adenocarcinoma A549 cells, which express the active G12S K-Ras mutant. Here, we describe that JB induces methuosis in these cells thorough activation of the 5’ AMP-activated protein kinase (AMPK). 2. Results 2.1. JB Induces Cytoplasmic Vacuolization in A549 and MCF7 Cells Lung adenocarcinoma A549 cells were treated with different concentrations of JB (Figure 1A) (CC50 /24 h = 2.05 µM) (Figure S1A) and analyzed for vacuolization at different times. In agreement with the results previously reported for human gastric cancer HGC27 cells [11], phase contrast microscopy showed that 5 µM JB induced the formation of cytosolic vacuoles (4 h) in A549 cells (Figure 1B), with a cellular viability of ~80%. These conditions were selected for vacuolization induction in further experiments. JB interferes with SLs’ metabolism by inhibiting CerS [11]. To establish the role of CerS in cellular vacuolation induced by JB, we used Fumonisin B1 (FB1), a canonical CerS inhibitor [16]. As shown in Figure 1B, no vacuoles were observed in A549 cells treated with FB1, indicating that inhibition of CerS by JB is not implicated in its capacity to induce cytoplasmic vacuolation. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 15 inhibitor [16]. As shown in Figure 1B, no vacuoles were observed in A549 cells treated Int. J. Mol. Sci. 2022, 23, 7257 with FB1, indicating that inhibition of CerS by JB is not implicated in its capacity to induce 3 of 15 cytoplasmic vacuolation. Figure 1. Induction Inductionofofcell vacuolization cell vacuolization by by Jaspine B (JB) Jaspine in A549 B (JB) cells.cells. in A549 (A) Chemical structure (A) Chemical of struc- JB. (B) ture of Phase JB. (B)contrast images of Phase contrast A549 cells images treated of A549 cellswith 0.05% treated ethanol with 0.05%for 24 h, 5for ethanol µM24JBh,for 4 h, JB 5 µM or 50 µM for 4 h,Fumonisin or 50 µMB1 (FB1) for 24 Fumonisin B1h.(FB1) The images for 24 h.are The representative images areofrepresentative three experiments performed of three exper- in triplicate. iments Scale bar: performed 50 µm. (C) Scale in triplicate. TEM images bar: 50ofµm.A549(C)cells treated TEM withof images 0.05% A549ethanol for 4 h or cells treated 10 with µM JB ethanol 0.05% for 5 h and forMCF7-LC3GFP 4 h or 10 µM JBcells for 5treated h andwith 0.05% ethanol MCF7-LC3GFP fortreated cells 6 h or 10with µM 0.05% JB for 6ethanol h. Arrows for point to membrane extensions at the surface of A549 or MCF7 cells and asterisks show endolyso- 6 h or 10 µM JB for 6 h. Arrows point to membrane extensions at the surface of A549 or MCF7 cells some accumulation indicating defective endocytic flux. Scale bars: 2 µm. (D) Incorporation of Luci- and asterisks show endolysosome accumulation indicating defective endocytic flux. Scale bars: 2 µm. fer yellow (LY) in A549 cells incubated with 5 µM JB and 0.5 mg/mL LY for 4 h. Scale bar: 50 µm. (D) Incorporation (E) Phase contrastofimages Luciferofyellow A549 (LY) cellsin A549 cellswith pretreated incubated 25 µMwith 5 µM JB and 0.5 mg/mL 5-[N-ethyl-N-isopropyl] LY for amiloride 4(EIPA) h. Scale(or methanol) for 1 h and then treated with 5 µM JB (or ethanol) for 24 h. The images are bar: 50 µm. (E) Phase contrast images of A549 cells pretreated with 25 µM 5-[N-ethyl-N- representative isopropyl] of two(EIPA) amiloride independent experiments (or methanol) for 1 hperformed in triplicate. and then treated with 5Scale µM JB bar: (or50 µm. (F)for ethanol) Viabil- 24 h. ity ofimages The A549 cells pretreated with are representative of 25 two µM EIPA (or methanol) independent experimentsfor 1performed h and thenintreated withScale triplicate. 5 µMbar: JB (or µm. 50 ethanol) (F) for 24 h, asofassessed Viability withpretreated A549 cells 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl with 25 µM EIPA (or methanol) tetrazolium for 1 h andbromide then (MTT). with treated Control cells 5 µM JBwere treated for (or ethanol) with24vehicles (without h, as assessed EIPA with and without JB). Results are the mean 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Control cells were treated with vehicles (without EIPA and without JB). Results are the mean ± SD of two experiments in triplicate. * p < 0.0005. (G) A549 cells were 3 treated with 5 µM JB and 0.5 mg/mL LY for 4 h. An amount of 75 nM LT was added 0.5 h prior to visualization. Scale bar: 10 µm. Int. J. Mol. Sci. 2022, 23, 7257 4 of 15 Transmission electron microscopy (TEM) revealed that JB-induced vesicles are mul- tisized and surrounded by a single membrane (Figure 1C), in contrast to the double- membrane structures that characterize autophagosomes (see below). Interestingly, TEM showed the presence of lamellipodial membrane projections, suggesting that the vacuoles were originated from macropinocytic activity (Figure 1C). To investigate this possibility, we incubated cells with JB in the presence of the fluorescent dye Lucifer yellow (LY), a fluid-phase tracer that is incorporated in cells during macropinocytosis [4,17]. Fluorescence microscopy analysis showed round-shaped vacuoles filled with the fluorescent tracer LY, which matched with those observed by contrast phase imaging (Figure 1D). This indicates that vacuoles exhibited characteristics of macropinosomes, as reported for other cancer cell lines [11]. To confirm that JB-induced vacuolization occurred via macropinocytosis, we used EIPA (5-[N-ethyl-N-isopropyl]amiloride), a Na+ /H+ exchanger inhibitor that blocks macropinocy- tosis [18,19]. The incubation of A549 cells with EIPA inhibited vacuolization induced by JB (Figure 1E). Furthermore, EIPA significantly rescued the viability of cells treated with JB (Figure 1F), indicating that vacuolization by macropinocytosis was implicated in JB cytotoxicity. When the endocytic pathway is functional, vacuoles internalized by macropinocy- tosis are either recycled to the plasma membrane, or mature and become late endo- somes that eventually fuse with lysosomes [20,21]. Despite the rise in the macropinocytic activity, methuosis induces a dysfunctional macropinocytotic process, so the nascent macropinosomes do not recycle or fuse with lysosomes [4]. To investigate whether the cytoplasmic vacuolization induced by JB was related to methuosis, cells were treated with JB, and the fusion of vacuoles with lysosomes was examined by confocal microscopy. As illustrated in Figure 1G, vacuoles originated by JB treatment (stained with LY) did not colocalize with the lysosomal marker lysotracker. This suggests that JB disrupts the endosome–lysosome fusion, which is indicative of methuosis [4]. In order to examine further the vacuolization induced by JB, we incubated cells with the JB fluorescent analogue JB-Bodipy (Figure S2), which induced a dose-dependent de- crease in cell viability with an IC50 of 16 µM (24 h) (Figure S1B). A549 cells were treated with JB-Bodipy for a short time (2 h), and examined by phase contrast microscopy. JB-Bodipy induced cellular vacuolization but vacuoles did not contain JB-Bodipy (Figure S3), whose transport across the plasma membrane, as a long-chain base analog, is likely mediated by acyl CoA synthetases [22]. In turn, JB-Bodipy stain was localized in distinct vesicular structures (Figure S2). These results agree with those reported by Rozié et al. [23], who found JB to localize in cytoplasmatic aggregates. 2.2. JB-Induced Cytotoxicity Involves Methuosis JB induced cytotoxicity in A549 cells, with an IC50 of 2.05 µM (Figure S1A). This result is similar to those reported for other human cancer cell lines, such as HGC27 [10], U2OS [23], HT29 [24], MCF7 [25], A-375 [26], and HeLa [27]. In all the cases, reported IC50 values were in the micromolar to submicromolar range. Some of these cell lines are not mutated in the K-RAS/MAPK pathway, which means that Jaspine B toxicity does not necessarily require hyperactivation of this route. Next, we investigated the type of cell death induced by JB. As reported with other cell death inducers [2,4,10,11,15,28,29], we observed that the necroptosis inhibitor necrostatin-1 failed to recover the cytotoxicity induced by JB in A459 cells (Figure S4). Therefore, we next investigated the role of apoptosis. JB has been reported to induce apoptosis in different cancer cell lines [11–13]. In our case, we evaluated whether JB induced apoptosis in A459 cells using flow cytometry analysis. While JB showed a poor proapoptotic effect at short (4 h) incubation time, discrete late apoptosis was detected at 24 h treatment (Figure 2A). To investigate the role of apoptosis in JB-induced cytotoxicity, we used the pancaspase inhibitor carbobenzoxy- valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (z-VAD). As shown in Figure 2B, Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 15 Int. J. Mol. Sci. 2022, 23, 7257 late apoptosis was detected at 24 h treatment (Figure 2A). To investigate the role of apop- 5 of 15 tosis in JB-induced cytotoxicity, we used the pancaspase inhibitor carbobenzoxy-valyl- alanyl-aspartyl-[O-methyl]-fluoromethylketone (z-VAD). As shown in Figure 2B, z-VAD recovered z-VAD cell death recovered cellinduced by H2Oby death induced 2 (aH known 2 O2 (a apoptotic inducer), known apoptotic but it did inducer), butnot recover it did not the viability recover of cells treated the viability of cells with JB. with treated This JB. result Thissuggests that JB isthat result suggests a poor JB isinducer a poor of apop- inducer tosis, of and thatand apoptosis, apoptosis is not theismain that apoptosis not thecause of cell main death cause observed of cell death upon observedJB treatment. upon JB To further To treatment. confirm furtherthis observation, confirm we used a genetic this observation, we usedapproach, a genetic using mouse approach, embryonic using mouse fibroblasts fibroblasts embryonic (MEF) double (MEF)knockout doublefor both Bcl-2-associated knockout X protein (BAX) for both Bcl-2-associated and(BAX) X protein Bcl-2- antagonist/killer 1 (BAK). MEF lacking Bax and Bak cannot undergo apoptosis, as these and Bcl-2-antagonist/killer 1 (BAK). MEF lacking Bax and Bak cannot undergo apoptosis, proteins as are essential these proteins regulators are essential of the of regulators apoptotic signaling the apoptotic [30]. Firstly, signaling we observed [30]. Firstly, we observedthat JB induced that JB inducedmicropinocytosis-mediated micropinocytosis-mediated vacuolization vacuolizationin both in bothBAX BAX+/+/BAK +/+ (wild +/+ /BAK type) +/+ (wild −/− /BAK−/− (knockout) MEF (Figure S5). Interestingly, no difference and BAX type) /BAK-/- (knockout) and-/-BAX MEF (Figure S5). Interestingly, no difference in cell viability was in observed cell viabilitywhen wild-typewhen was observed and knockout wild-typeMEF andwere treated knockout MEFwithwere JB (Figure treated2D),withsug- JB gesting that apoptosis does not mediate JB cytotoxicity. Our results agree with previous (Figure 2D), suggesting that apoptosis does not mediate JB cytotoxicity. Our results agree works with showing previous that cytotoxicity works showing thatinduced by methuosis cytotoxicity induced by is not prevented methuosis byprevented is not apoptosis by in- hibitors [2,4,7,11,31]. apoptosis inhibitors [2,4,7,11,31]. Figure2. Figure 2. Analysis Analysis of of apoptosis apoptosis induced inducedbybyJaspine JaspineBB(JB) (JB)in in A549 A549cells. cells. (A) (A) A549 A549 cells cells were were incubated incubated with 0.05% EtOH for 24 h (control), 5 µM JB for 4 h or 1 µM JB for 24 h and with 0.05% EtOH for 24 h (control), 5 µM JB for 4 h or 1 µM JB for 24 h and then stained then stained with annexin with V (AV)/propidium iodide (PI). Fluorescence was analyzed by flow cytometry. annexin V (AV)/propidium iodide (PI). Fluorescence was analyzed by flow cytometry. Data were Data were obtained from 2 experiments in triplicate. (B) Determination of cell viability by MTT assay in A549 cells obtained from 2 experiments in triplicate. (B) Determination of cell viability by MTT assay in treated for 24 h with 1 µM JB in the presence or absence of 100 µM carbobenzoxy-valyl-alanyl-as- A549 cells treated for 24 h with 1 µM JB in the presence or absence of 100 µM carbobenzoxy-valyl- partyl-[O-methyl]-fluoromethylketone (z-VAD) or 10 µM H2O2 in the presence or absence of 100 alanyl-aspartyl-[O-methyl]-fluoromethylketone µM z-VAD. Results are the mean ± SD for two experiments(z-VAD) orperformed 10 µM H2 O in the presence in2 triplicate and areorexpressed absence as the percentage of the viability compared to the control. *p < 0.005. (C) Determination of and of 100 µM z-VAD. Results are the mean ± SD for two experiments performed in triplicate cell via- are bility by MTT expressed assay as the of mouse percentage ofembryonic fibroblasts the viability compared(MEF)to thedouble control.knockouts * p < 0.005. (Bax/Bak KO) for Bcl- (C) Determination 2-associated of X protein cell viability by MTT(Bax) assayand Bcl-2-antagonist/killer of mouse 1 (Bak)(MEF) embryonic fibroblasts and MEF doublewild-type knockouts(Bax/Bak WT) (Bax/Bak incubated with different concentrations of JB for 24 h. Results are the mean ± SD KO) for Bcl-2-associated X protein (Bax) and Bcl-2-antagonist/killer 1 (Bak) and MEF wild-type of two experiments in triplicate. (Bax/Bak WT) incubated with different concentrations of JB for 24 h. Results are the mean ± SD of two experiments in triplicate. Macroautophagy, hereafter referred to as autophagy, is a highly conserved cellular process characterized by Macroautophagy, self-degradation hereafter referred toofasintracellular autophagy, components that are included is a highly conserved cellular in double-membrane process characterized vesicles known as autophagosomes by self-degradation [32]. In somethat of intracellular components cases, areautophagy included can in trigger cell death, vesicles double-membrane a processknown called as autophagy-mediated autophagosomes [32]. cell In death someor cases, cytotoxic autoph- autophagy agy trigger can [33]. Interestingly, cell death, aautophagy has also process called been described to occur autophagy-mediated alongor cell death with methuosis cytotoxic au- [11]. Therefore, tophagy we next studied [33]. Interestingly, whether autophagy JB induced has also autophagy been described in our to occur cells.with along Onemethu- of the hallmarks osis of autophagy [11]. Therefore, is conjugation we next of the soluble studied whether form of JB induced the microtubule-associated autophagy in our cells. One of the hallmarks of autophagy is conjugation of the soluble form of the microtubule- associated protein 1 light chain 3 (LC3) with phosphatidylethanolamine and conversion 5 to autophagosomal membrane-associated form (LC3-II), which can be monitored by im- Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 15 Int. J. Mol. Sci. 2022, 23, 7257 6 of 15 protein 1 light chain 3 (LC3) with phosphatidylethanolamine and conversion to autopha- gosomal membrane-associated form (LC3-II), which can be monitored by immunoblot analysis. munoblotAsanalysis. shown inAs(Figure shown3A), treatment in (Figure 3A),oftreatment A549 cellsofwith A549JB cells resulted withinJBrapidly (4 resulted h) in elevated rapidly (4LC3-II levels. LC3-II h) elevated To demonstrate levels. Tothat JB induced demonstrate autophagic that JB induced flux, cells wereflux, autophagic in- cubated cells werewith the lysosomal incubated with the inhibitors lysosomal pepstatin inhibitors A and E64d, pepstatin to block A and E64d, the finalthe to block step of final autolysosomal degradation. step of autolysosomal Incubation degradation. with protease Incubation inhibitors with protease and E64d inhibitors further and E64d in- further creased increasedLC3-II LC3-IIlevels (Figure levels 3B,3B, (Figure 1 vs. 2, 52,vs. 1 vs. 6), indicating 5 vs. thatthat 6), indicating JB induces dynamic JB induces au- dynamic tophagy in A549 cells. autophagy in A549 cells. Figure Figure 3.3. Analysis Analysisof ofautophagy autophagyinducedinduced by byJaspine JaspineBB(JB) (JB)ininA549 A549cells. cells.(A) (A)Analysis Analysisofofmicrotubule- microtubule- associated associated protein protein11light lightchain chainIIII(LC3II) (LC3II)expression expressionby byWestern WesternblotblotininA549 A549cells cellstreated treated with JBJB with 5 µM 5 µM atat different differenttimes timesforforupuptoto1212h.h.(B) (B)Analysis AnalysisofofLC3II LC3IIexpression expressionby byWestern Western blotblot in in A549 A549 cells cells pretreated pretreated oror not not for for 22 h h with with protease protease inhibitors inhibitors (PI) (PI) (10 (10 µg/mL µg/mL E-64-D E-64-D and and 55 µg/mL µg/mL pepstatin pepstatin A)A) and/or for 0.5 h with 5 mM 3-methyladenine (3MA) and then treated with JB and/or for 0.5 h with 5 mM 3-methyladenine (3MA) and then treated with JB (5 µM, 4 h) or vehicle (5 µM, 4 h) or vehicle (0.05% (0.05% EtOH). EtOH). Results Results areare representative representative of of three experiments. (C) three experiments. (C) Determination Determination of of cell cell viability viability with 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) in A549 preincubated or with 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) in A549 preincubated not with 5 mM 3-methyladenine for 0.5 h and treated with JB at different concentrations for 4 h. or not with 5 mM 3-methyladenine for 0.5 h and treated with JB at different concentrations for Results are the mean ± SD of two experiments in triplicate. (D) Determination of cell viability by 4 h. Results MTT assay inare the mean ± SD of autophagy-related two5 experiments gene inKO) (Atg5)−/− (Atg5 triplicate. and Atg5(D)+/+Determination (Atg5 WT) mouse of cell viability embryonic − / − +/+ fibroblasts by MTT assay (MEF)in incubated with different autophagy-related gene concentrations 5 (Atg5) of JBKO) (Atg5 for 24 andh. Atg5 Data (+/-SD) (Atg5 correspond WT) mouse to three experiments embryonic in triplicate. fibroblasts (MEF) incubated with different concentrations of JB for 24 h. Data (+/−SD) correspond to three experiments in triplicate. We used a pharmacological approach to block the formation of autophagosomes, by inhibiting vacuolar We used protein sortingapproach a pharmacological 34 class III to phosphoinositide block the formation 3-kinase (class III PI3K of autophagosomes, Vps34), an essential protein for cellular autophagy. The incubation of cells with 3-methyl- by inhibiting vacuolar protein sorting 34 class III phosphoinositide 3-kinase (class III PI3K Vps34), an essential protein for cellular autophagy. The incubation adenine resulted in a decrease in JB-induced LC3-II expression levels (Figure 3B, column of cells with 3-methyladenine resulted in a decrease in JB-induced LC3-II expression levels 5 vs. 7, column 6 vs. 8). However, 3-methyladenine did not recover the loss of cell viability (Figure 3B, column 5 vs. 7, column 6 vs. 8). However, 3-methyladenine did not recover in response to JB (Figure 3C). In order to obtain genetic evidence of whether the JB induces the loss of cell viability in response to JB (Figure 3C). In order to obtain genetic evidence cytotoxic autophagy, we used autophagy-related gene 5 (ATG5) knockout MEF (ATG5-/-). of whether the JBprotein Atg5 induces cytotoxic plays autophagy, an essential role inwe theused autophagy-related elongation gene 5and of the phagophore (ATG5) knockout its maturation MEFthe (ATG5 − / − ). Atg5 protein playstherefore, an essential into complete autophagosome; cellsrole in theATG5 lacking elongation of the phagophore are autophagy-deficient and its maturation into the complete autophagosome; therefore, cells lacking ATG5 are autophagy-deficient [34]. JB induced macropinocytic vacuoles in both wild-type (ATG5+/+ ) 6 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 15 Int. J. Mol. Sci. 2022, 23, 7257 7 of 15 [34]. JB induced macropinocytic vacuoles in both wild-type (ATG5+/+) and ATG5−/− MEF (Figure and ATG53A), −/− butMEFATG5 gene deletion (Figure 3A), buthad ATG5no effect on JB-induced gene deletion had nocytotoxicity (Figure 3D). effect on JB-induced Finally, to investigate cytotoxicity (Figure 3D). whether vacuoles induced by JB fuse with autophagosomes, we performed fluorescence Finally, to investigate microscopy whether colocalization vacuoles induced by analysis, using JB fuse with a MCF7 cancer we autophagosomes, cell line that stably overexpresses GFP-tagged LC3-II. TEM analysis revealed performed fluorescence microscopy colocalization analysis, using a MCF7 cancer cell line the formation of vacuoles that stablyby macropinocytosis overexpresses (Figure GFP-tagged 1C) inTEM LC3-II. these cells inrevealed analysis responsethetoformation JB treatment. As of vac- shown uoles byin Figure 4A, vacuoles macropinocytosis and (Figure autophagosomes 1C) generatedtoinJBresponse in these cells in response treatment. to As JB shown did not incolocalize, Figure 4A,indicating vacuoles that and they are different generated autophagosomes subcellularinstructures. response toThis wasnot JB did confirmed by colocalize, confocal microscopy of cells expressing GFP-tagged LC3 and stained with indicating that they are different subcellular structures. This was confirmed by confocal the fluorescent tracer LY. As microscopy shown of cells in FigureGFP-tagged expressing 4B, most JB-induced vacuoles LC3 and stained did with not the colocalizetracer fluorescent with LY. au- Astophagosomes shown in Figure (Mander’s 4B, mosttest: LY on GFP JB-induced 0.048;did vacuoles GFP noton LY 0.469). colocalize withOverall, our results autophagosomes suggest that (Mander’s JBLY test: does on not GFPinduce autophagy-mediated 0.048; GFP cell death, on LY 0.469). Overall, in agreement our results suggest thatwith JBprevi- does ousinduce not reportsautophagy-mediated [4,11,15,28]. cell death, in agreement with previous reports [4,11,15,28]. Figure4.4.Analysis Figure Analysisof ofvacuoles vacuolesand andautophagosomes autophagosomesininMCF7 MCF7cells. cells.(A) (A)MCF7 MCF7cells cellsstably stablytransfected transfected withGFP-tagged with GFP-tagged microtubule-associated microtubule-associated protein protein 11light lightchain chainIIII(LC3II) (LC3II)were wereincubated incubated with JB JB with (10 µM, 2 h). Vacuoles were observed under a phase microscopy and autophagosomes, (10 µM, 2 h). Vacuoles were observed under a phase microscopy and autophagosomes, labeled withlabeled with GFP,were GFP, wereanalyzed analyzedbybyfluorescence fluorescencemicroscopy. microscopy.Scale Scalebar: bar:5050µm. µm.(B)(B)MCF7 MCF7cells cellsstably stablytransfected transfected with GFP-tagged LC3II were incubated with 10 µM JB and 0.5 mg/ml Lucifer yellow with GFP-tagged LC3II were incubated with 10 µM JB and 0.5 mg/mL Lucifer yellow (LY) for (LY) for22hh and analyzed by confocal microscopy. Images are representative of at least two experiments. Scale and analyzed by confocal microscopy. Images are representative of at least two experiments. Scale bar: 10 µm. bar: 10 µm. Allin All inall, all,our ourfindings findingsstrongly stronglysuggest suggestthat thatvacuoles vacuolesformed formedininresponse responsetotoJBJBdo donot not merge with lysosomes or autophagosomes, and that JB cytotoxic activity merge with lysosomes or autophagosomes, and that JB cytotoxic activity occurs throughoccurs through methuosis.Thus, methuosis. Thus,wewepropose propose JBJB asas a methuosis-inducing a methuosis-inducing agent, agent, as itashas it has beenbeen proposed proposed for for other small molecules, such as 3-(5-methox-y-2-methyl-1H-indol-3-yl)- other small molecules, such as 3-(5-methox-y-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2- 1-(4-pyridi- nyl)-2-propene-1-one propene-1-one (MOMIPP) (MOMIPP) [35] and[35] and silmitasertib silmitasertib (CX-4945) (CX-4945) [36]. [36]. 2.3.JB 2.3. JBActivates ActivatesAMPK AMPK Recentstudies Recent studieshave haveshown shownthat thatmacropinocytosis macropinocytosiscontributes contributestotocell cellgrowth growthbyby stim- stimu- ulating lating mTORC1 mTORC1 activity activity [37].[37]. Although Although inmodel in our our model macropinocytosis macropinocytosis inducesinduces cell cell death death than rather rather than contributing contributing to cell growth, to cell growth, we addressed we addressed the mTORC1the mTORC1 pathway.pathway. To this end,To this end, protein protein kinase kinase B (Akt) B (Akt) and and ribosomal ribosomal protein S6protein S6 (S6) phosphorylation (S6) phosphorylation was ana- was analyzed by lyzed by immunoblot. immunoblot. JB treatmentJB treatment of A549 cellsof A549 cells resulted inresulted in a rapidin a rapid reduction reduction in phos- phosphorylated phorylated Akt (30 min) Akt and (30 min) S6 (at and 8 h) S6 (at levels 8 h) levels (Figure (Figure 5A, left 5A,These panels). left panels). Theseconfirmed results were results werein HGC27 human gastric cancer cells, where JB inhibited Akt and S6 phosphorylation after 7 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 8 of 15 Int. J. Mol. Sci. 2022, 23, 7257 8 of 15 confirmed in HGC27 human gastric cancer cells, where JB inhibited Akt and S6 phosphor- ylation after 1 h and 2 h incubation, respectively (Figure 5A, right panels). To investigate the 1 hrole andof2h the Akt/mTORC1 incubation, pathway(Figure respectively in JB-induced cellpanels). 5A, right vacuolation, we investigated To investigate the rolethe of effect of everolimus the Akt/mTORC1 (mTORC1 pathway inhibitor),cell in JB-induced MK-2206 (Akt we vacuolation, inhibitor), and the investigated AZD-2014 effect of (mTORC1/2 everolimus inhibitor) (mTORC1ininhibitor), A549 cells. None of(Akt MK-2206 Akt or mTOR inhibitors inhibitor), induced and AZD-2014 vacuoliza- (mTORC1/2 tion (Figure inhibitor) in5B), A549incells. spiteNone of inhibition of Akt orofmTOR mTORC1 (Figure inhibitors 5C). These induced results indicate vacuolization (Figurethat 5B), the Akt/mTORC1 in spite signaling of inhibition pathway of mTORC1 is not (Figure involved 5C). in theindicate These results induction thatofthe vacuolization Akt/mTORC1 in our cells. signaling pathway is not involved in the induction of vacuolization in our cells. Figure Figure 5.5. (A) (A)A549 A549andandHGC27 HGC27cells cellstreated treatedwith withvehicle vehicle(0, (0,0.05% 0.05%EtOH, EtOH,2424h)h)or or55µM µMJBJBat atthe the indicated times (h) were lysed, and the levels of total and phosphorylated Akt and S6 proteins indicated times (h) were lysed, and the levels of total and phosphorylated Akt and S6 proteins were were analyzed analyzedby byimmunoblotting. immunoblotting.β-Actin was β-Actin used was as loading used control. as loading Results control. are representative Results of two are representative of to three independent experiments. (B) A549 cells were treated for 24 h with 1 µM everolimus, two to three independent experiments. (B) A549 cells were treated for 24 h with 1 µM everolimus, 10 µM MK-2206, or 100 nM AZD-2014 and compared to 4 h with 5 µM JB. Phase contrast images are 10 µM MK-2206, or 100 nM AZD-2014 and compared to 4 h with 5 µM JB. Phase contrast images representative of two experiments performed in triplicate. Scale bar: 50 µm. (C) Protein extracts are representative of two experiments performed in triplicate. Scale bar: 50 µm. (C) Protein extracts were analyzed by immunoblotting for total ribosomal protein S6 and phospho-S6. Numbers denote were analyzedofby quantification immunoblotting levels of phospho-S6fornormalized total ribosomal by S6protein protein,S6expressed and phospho-S6. Numbers as percentage denote of vehicle. quantification of levels of phospho-S6 normalized by S6 protein, Results are representative of two experiments. * p < 0.01, **p < 0.0001. expressed as percentage of vehicle. Results are representative of two experiments. * p < 0.01, ** p < 0.0001. Recent work has proposed the AMP-dependent kinase (AMPK) as an upstream in- Recent work has proposed the AMP-dependent kinase (AMPK) as an upstream in- ducer of autophagy. AMPK activates the master regulator of autophagy, Atg1/Unc-51- ducer of autophagy. AMPK activates the master regulator of autophagy, Atg1/Unc-51-Like Like Autophagy Activating Kinase 1 (ULK), by two different mechanisms: inhibition of Autophagy Activating Kinase 1 (ULK), by two different mechanisms: inhibition of mTORC1 mTORC1 by phosphorylating tuberous sclerosis complex 2 (TSC2) and Raptor proteins, by phosphorylating tuberous sclerosis complex 2 (TSC2) and Raptor proteins, and by direct and by direct phosphorylation of Atg1/ULK [38]. Therefore, we first studied whether JB phosphorylation of Atg1/ULK [38]. Therefore, we first studied whether JB induced AMPK induced AMPK activity in A549 cells. Immunoblot analysis showed that JB induced activity in A549 cells. Immunoblot analysis showed that JB induced AMPK phospho- AMPK phosphorylation (Thr172) as early as upon 1h incubation (Figure 6A). Since JB in- rylation (Thr172) as early as upon 1h incubation (Figure 6A). Since JB induced cellular duced cellular vacuoles within 1-2 h treatment, and mTORC1 activity was unaffected at vacuoles within 1–2 h treatment, and mTORC1 activity was unaffected at this time, we next this time, we next investigated the role of active AMPK in JB-mediated cellular vacuoliza- investigated the role of active AMPK in JB-mediated cellular vacuolization. Interestingly, tion. Interestingly, treatment of A459 cells with the AMPK activator phenformin resulted treatment of A459 cells with the AMPK activator phenformin resulted in the induction in ofthe induction vacuoles of vacuoles (Figure (Figure 6B), 6B), implicating implicating active AMPK as active AMPK a player in as a player cellular in cellular vacuolization. Furthermore, phenformin-induced vacuoles were formed by macropinocytosis (Figure 6B) and did not merge with lysosomes (Figure 6C), as it happened for the vacuoles formed in re- 8 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 9 of 15 Int. J. Mol. Sci. 2022, 23, 7257 9 of 15 vacuolization. Furthermore, phenformin-induced vacuoles were formed by macropinocy- tosis (Figure 6B) and did not merge with lysosomes (Figure 6C), as it happened for the vacuoles sponse toformed in response JB. Besides to JB. Besides the formation the formation of vacuoles, of vacuoles, phenformin induced aphenformin induced clear ER dilation, in aagreement clear ER dilation, with the in agreement reported with the reported phenformin-induced phenformin-induced UPR UPR activation activation in an AMPK-dependent in an AMPK-dependent manner [39]. manner [39]. Figure Figure 6.6. (A) (A)A549 A549andandHeLa HeLa cells treated cells with treated withvehicle (0, 0.05% vehicle EtOH (0, 0.05% 24 h)24orh)5 or EtOH µM5 JB µMat JB theatindi- the cated times indicated (h) (h) times were lysed, were lysed,and andthethe levels ofoftotal levels totaland andphosphorylated phosphorylated5'5 AMP-activated 0 AMP-activated protein protein kinase kinase (AMPK) (AMPK)were wereanalyzed analyzed bybyimmunoblotting. immunoblotting. β-Actin was used β-Actin as loading was used control.control. as loading Results are Re- representative of two toofthree sults are representative two toindependent experiments. three independent (B) A549(B)cells experiments. A549were cellstreated with 0.05% were treated with ethanol or 10 mM 0.05% ethanol or 10phenformin mM phenforminfor 4 h.forArrows showshow 4 h. Arrows membrane extensions, membrane the asterisk extensions, shows the asterisk en- shows dolysosome accumulation indicating defective endocytic flux, and the arrowhead, dilated ER. Scale endolysosome accumulation indicating defective endocytic flux, and the arrowhead, dilated ER. Scale bars: 2 µm. (C) A549 cells treated with 10 mM phenformin and 0.5 mg/ml of LY for 4 h. 75 nM bars: 2 µm. (C) A549 cells treated with 10 mM phenformin and 0.5 mg/mL of LY for 4 h. 75 nM lysotracker was added 0.5 h prior to visualization. Cells were observed under confocal microscopy. lysotracker Scale bar: 10was µm.added Images0.5are h prior to visualization. representative Cells were observed of two experiments under confocal microscopy. in duplicate. Scale bar: 10 µm. Images are representative of two experiments in duplicate. 3. Discussion 3. Discussion Methuosis Methuosis isis an an emergent emergenttypetypeof ofcell celldeath deaththat thathashasbeen beenrecently recentlyproposed proposed asas a tar- a target get for designing new antitumoral therapies [40]. In this regard, very for designing new antitumoral therapies [40]. In this regard, very few small moleculesfew small molecules have have been been reported reported as as methuosis methuosis inducers inducers [7–11]. [7–11]. However, However, mostmost of of the the protein protein targets targets of of these compounds remain to be identified. Among them, the inhibition these compounds remain to be identified. Among them, the inhibition of class III phospho-of class III phos- phoinositide kinase inositide kinase PIKFYVE PIKFYVE [8] triggers [8] triggers vacuolization, vacuolization, a hallmark a hallmark of methuosis. of methuosis. Addi- Additionally, tionally, CK2 inhibition by silmitasertib also induces methuosis [10]. Here, CK2 inhibition by silmitasertib also induces methuosis [10]. Here, we described the natural we described the natural SLJaspine SL derivative derivative B as Jaspine a compoundB as athat compound that triggers vacuolization triggers vacuolization by mi- by micropinocytosis cropinocytosis and induces methuosis in cancer cells. Additionally, we and induces methuosis in cancer cells. Additionally, we provide evidence that JB induces provide evidence that JB induces autophagy and autophagy apoptosis in and apoptosis cancer in cancer cells, but cells, but these cellular these cellular processes are notprocesses involvedare in not involved inphenotype the methuosis the methuosis phenotype induced by JB. induced by JB. Although Although the the exact exact signaling signaling pathways pathways by by which which small small compounds compounds induce induce methu- methu- osis osis are not fully described, implication of Ras (rat sarcoma virus)/Rac (Ras-related C3 are not fully described, implication of Ras (rat sarcoma virus)/Rac (Ras-related C3 botulinum toxin substrate 1) [4,15], and Akt-mTOR [10] pathways have been suggested. 9 Int. J. Mol. Sci. 2022, 23, 7257 10 of 15 Deregulation of macropinocytosis in cancer cells correlates with oncogenic RAS, as RAS- dependent signaling pathways hyperstimulate macropinocytosis in glioblastoma [4], and CD99 triggering induces methuosis of Ewing sarcoma cells through the insulin-like growth factor type 1 receptor (IGF-1R)/RAS/Rac1 signalling [15]. On the other hand, 3-(5-methoxy- 2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one (MOMIPP) induces methuosis in glioblastoma and other cancer cell lines by modulating the MAPK/Jun N-Terminal Kinase (JNK) signalling pathway [29]. Interestingly, the CK2 inhibitor silmitasertib promotes methuosis-like cell death associated with massive catastrophic vacuolization, most likely due to the inhibition of the Akt/mTORC1 axis (Akt is a known CK2 target) [10]. In our study, we found that treatment of cancer cells with JB resulted in inhibition of Akt and mTORC1. However, specific inhibition of Akt or mTORC1/mammalian target of rapamycin complex 2 (mTORC2) did not result in vacuolization, discarding the Akt/mTOR pathway as a player in the methuosis phenotype induced by JB in cancer cells. Additionally, here we described that JB induced autophagic flux in cancer cells, but provided genetic evidence that autophagy is not involved in JB-induced macropinocytic vacuoles in these cells. AMP-activated protein kinase (AMPK) plays an essential role as sensor of energy imbalance in cells, thereby being pivotal for cell survival under various environmental stressors [41]. AMPK is involved in both tumor suppression and tumor-promoting ac- tivity depending on distinct cellular contexts. Since AMPK has been shown to display tumor-suppressive roles in certain conditions, several small molecules that activate AMPK have been tested in preclinical models of cancer [42]. Our results suggest that AMPK activation is involved in methuosis induced by JB, in agreement with previous reports. Thus, Kim et al. reported that phosphatase and tensin homolog (PTEN)-deficient prostate cancer cells use macropinocytosis to survive and proliferate under nutrient stress, and that AMPK activation is a general requirement for macropinocytosis of extracellular material induced by PTEN loss [43]. On the other hand, AMPK has been reported to be required for the macropinocytic internalization of ebolavirus [44]. Furthermore, methuosis can be induced by activation of the MAPK/JNK signalling pathway [26]. Our results provide further evidence suggesting that AMPK mediates methuosis in lung cancer cells carrying KRAS mutation. In conclusion, JB induces cancer cell vacuolation and death through a mechanism that cannot be prevented by inhibiting apoptosis, autophagy, or necrosis and it is most likely due to the activation of methuosis. Although more research is needed, we propose that the activation of AMPK would play a critical role in JB-induced cell vacuolization, independently of the PI3K/Akt/mTORC1 axis, which in turn is involved in the autophagic response of cells to JB (Figure 7). olization, most likely due to the inhibition of the Akt/mTORC1 axis (Akt is a known CK2 target) [10]. In our study, we found that treatment of cancer cells with JB resulted in inhi- bition of Akt and mTORC1. However, specific inhibition of Akt or mTORC1/mammalian target of rapamycin complex 2 (mTORC2) did not result in vacuolization, discarding the Akt/mTOR pathway as a player in the methuosis phenotype induced by JB in cancer cells. Int. J. Mol. Sci. 2022, 23, 7257 Additionally, here we described that JB induced autophagic flux in cancer cells, but 11 pro- of 15 vided genetic evidence that autophagy is not involved in JB-induced macropinocytic vac- uoles in these cells. Figure 7. Figure 7. Proposed Proposed mode mode of of action action of of Jaspine Jaspine BB in in lung lung adenocarcinoma adenocarcinoma A549 A549 and and gastric gastric cancer cancer HGC-27 cells. HGC-27 cells. AMP-activated 4. Materials protein kinase (AMPK) plays an essential role as sensor of energy and Methods imbalance inand 4.1. Reagents cells, thereby being pivotal for cell survival under various environmental Antibodies stressors MEM[41]. AMPK isessential (minimum involved in both tumor medium), DMEM suppression (Dulbecco’sand tumor-promoting Modified activ- Eagle Medium), ity depending FBS on distinct (Fetal bovine serum),cellular contexts. Since nonessential AMPK amino has trypsin-EDTA, acids, been shown to display tumor- MTT (3-(4,5- suppressive roles in certain conditions, dimethylthiazol-2-yl)-2,5-diphenyl several bromide), tetrazolium small molecules that activate BSA (bovine serum AMPK albumin),have5- been tested (N-ethyl- in preclinical models(EIPA), N-isopropyl)amiloride of cancer ECL™[42].Prime Our results Westernsuggest thatDetection Blotting AMPK activation Reagent, is involved and in methuosis 3-methyladenine wereinduced by JB, in agreement with from Sigma-Aldrich/Merck. previous DMEM reports. Thus, FluoroBrite, Kim et lysotracker al. reported red, that yellow and Lucifer phosphatase and tensin (LY) were homolog (PTEN)-deficient from ThermoFisher prostate Scientific. z-VAD andcancer cells fumonisin use(FB1) B1 macropinocytosis were from Enzo to survive and proliferate Life Sciences. Annexinunder V-FITCnutrient stress, anddetection early apoptosis that AMPK kit activation is a general requirement for macropinocytosis of extracellular material induced was from Cell Signaling. Laemmli buffer 4x and 30% acrylamide/Bis 37.5:1 were from by PTENLysotracker Bio-Rad. loss [43]. OnRedtheandother hand, PDVF AMPK haswere membranes beenfrom reported Roche. toAntibodies: be required β-actin for the macropinocytic internalization of ebolavirus [44]. Furthermore, methuosis (mouse) was from Sigma; LC3II (rabbit) from MBL; Caspase 3 (rabbit), Aktcan be induced (rabbit), pAkt (rabbit), AMPK (rabbit), pAMPK (rabbit), S6 (rabbit), and pS6 (rabbit) were from Cell Sig- naling. HRP-secondary antibody goat anti-Mouse IgG was from Thermo Fisher Scientific (Barcelona, Spain). HRP-secondary antibody goat anti-Rabbit was from Sigma (Fontenay- sous-Bois, France). 10 4.2. Jaspine B and Jaspine B-Bodipy Synthesis Synthetic pachastrissamine (Jaspine B) was obtained from phytosphingosine using the protocol described by Overkleef et al. [45]. Jaspine B-Bodipy was synthesized by a sequence of reactions, starting with a cross metathesis of two advanced synthones, prepared by procedures previously described. Jaspine B alkene functional moiety was carried out following the methodology described by Jana and Panda [27], and the C10-Bodipy alkene moiety was prepared as described by Saba et al. [46]. The unsaturated isopropylidene-Boc- protected Jaspine B-Bodipy obtained was reduced to the saturated intermediate, and finally deprotected using acidic conditions to afford the wanted fluorophore (Figure S2). 4.3. Cell Lines HGC27 cells (human gastric cancer cells, provided by Prof. Riccardo Ghidoni, Univer- sity of Milan, Italy) were maintained in MEM supplemented with 10% FBS and 1% nonessential amino acids. Cells were grown without reaching confluence. A549 cells Int. J. Mol. Sci. 2022, 23, 7257 12 of 15 (human lung carcinoma, ATCC). Atg5− /− and Atg5+ /+ T-large antigen-transformed MEF cells (provided by Dr. Noboru Mizushima, Tokyo Medical University, Tokyo, Japan), Bax− /− /Bak− /− and Bax+ /+ /Bak+ /+ T-large antigen-transformed MEF cells [47] (pro- vided by Stanley J. Korsmeyer, Dana-Farber Cancer Institute, Boston, MA, USA), and MCF7-LC3GFP cells (provided by Dr. Yaowen Wu, Umeå University, Umeå, Sweden) were cultured in DMEM supplemented with 10% FBS. All cells were maintained at 37 ◦ C in 5% CO2 . 4.4. Cell Viability 1 × 105 cells were seeded in 96-well plates and grown for 24 h. Cell viability was examined in triplicate by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- mide) assay. Absorbance was measured at 570 nm using the multi-detection microplate reader BioTek Synergy 2. 4.5. Annexin V-FITC Staining 1 × 105 cells were plated in 6-well plates and allowed to grow overnight. After treatment, cells and medium were collected and centrifuged and cellular pellets washed with 50 mM PBS-EDTA 1% BSA. For cell staining, Annexin V-FITC kit (Cell Signaling) was used according to the manufacturer’s instructions. Stained cells were analyzed in a Guava EasyCyte™ flow cytometer (Merck Millipore, Billerica, MA, USA). Data analysis was performed using the Multicycle AV program (Phoenix Flow Systems, San Diego, CA, USA). 4.6. Uptake of Fluid-Phase Tracer LY 1 × 105 cells were seeded in 6-well plates or 35 mm glass-bottom dishes and grown overnight. Medium was replaced with 1 mL of fresh medium containing LY (0.5 mg/mL) and cells were incubated with 5 µM JB or 0.05% ethanol (vehicle) for 4 h at 37 ◦ C. Then, Lysotracker (75 nM) was added to each well and left 30 min prior to observation. Medium was removed, cells were washed three times with PBS, and fresh DMEM FluoroBrite was added. Fluorescent images of live cells were taken using a Digital Sight DS-2Mv camera, acquired with Nis Element F 3.0 software or a Zeiss 880 laser scanning confocal microscope and analyzed using Fiji-ImageJ software. 4.7. Western Blotting Cells were lysed in Laemmli sample buffer. Equal amounts of protein (15–30 µg) were loaded and separated on a polyacrylamide/SDS gel and transferred onto a PVDF mem- brane. Membranes were blocked in 5% milk or 5% BSA in 0.1% TBS-Tween and incubated with the corresponding primary antibody. After washing with 0.1% TBS-Tween, mem- branes were probed with the correspondent horseradish-peroxidase-conjugated secondary antibody. Finally, protein detection was carried out using ECL reactive and visualized in a LI-COR C-DiGit® blot scanner. Alternatively, AMPK and pAMPK proteins were detected using photographic films (Fuji Medical X-ray film, Fijifilm, Greenwood, SC, USA) in a FUJI PHOTO FPM-100A. Band intensity was quantified by LI-COR Image Studio Lite software. 4.8. Phase Contrast and Confocal Microscopy Cells (0.3 × 106 /mL) were seeded in 6-well plates. Phase contrast pictures were taken using a Nikon Eclipse TS100 inverted microscope, connected to a Digital Sight DS-2Mv camera, and acquired with Nis Element F 3.0 software. Confocal microscopy images were obtained in a Zeiss 880 confocal microscope, and analyzed by Fiji-Image J program. 4.9. Transmission Electron Microscopy 1 × 105 cells were seeded in 60 mm dishes and grown overnight. Medium was replaced with fresh medium containing JB, phenformin, or ethanol (0.05%). After 4–6 h of treatment, cells were washed 3 times with PBS. Then, cells were fixed in 3% glutaraldehyde in Int. J. Mol. Sci. 2022, 23, 7257 13 of 15 0.1 M phosphate buffer for 1 h at room temperature. Fixing buffer was removed and fresh fixative buffer was added. Cells were collected by gently scrapping from the Petri dish. Samples were centrifuged (200× g for 5 min) and resuspended in the same fixation buffer. Fixing buffer was removed, new fixation buffer was added, and samples were centrifuged again (200× g for 5 min) and kept in fixative at 4 ◦ C. Pellets were washed in PB and incubated with 1% OsO4 for 90 min at 4 ◦ C. Then, samples were dehydrated, embedded in Spurr, and sectioned using Leica ultramicrotome (EM VC7, Leica Microsystems, Wetzlar, Germany). Ultrathin sections (50–70 nm) were stained with 2% uranyl acetate for 10 min, a lead-staining solution for 5 min and observed using a transmission electron microscope, JEOL JEM-1010, fitted with a Gatan Orius SC1000 (model 832) digital camera. Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms23137257/s1. Author Contributions: Conceptualization, J.M.L. and J.C.; Data curation, N.B. and M.C.; Formal analysis, N.B., M.C., C.E., G.F. and J.C.; Funding acquisition, G.F., A.D., J.M.L. and J.C.; Investigation, N.B., J.L.A., G.F., A.D., J.M.L. and J.C.; Methodology, N.B., J.L.A. and J.M.L.; Supervision, G.F., A.D., J.M.L. and J.C.; Validation, N.B. and G.F.; Visualization, N.B. and J.M.L.; Writing—original draft, N.B. and G.F.; Writing—review and editing, J.M.L. and J.C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by MCIN/AEI/10.13039/501100011033 and by the European Union (“ERDF A way of making Europe”), grants number CTQ2017-85378-R and PID2020-113813RB- I00 (to G.F.) and PID2019-107561RB-I00 (to J.M.L.). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data supporting reported results can be provided upon request. Acknowledgments: We thank Noboru Mizushima, Stanley J. Korsmeyer, and Yaowen Wu for pro- viding the cells used in this study and Eva Dalmau (deceased) for her excellent technical assistance. This manuscript is dedicated to her. Conflicts of Interest: The authors declare no conflict of interest. References 1. Pistritto, G.; Trisciuoglio, D.; Ceci, C.; Garufi, A.; D’Orazi, G. Apoptosis as anticancer mechanism: Function and dysfunction of its modulators and targeted therapeutic strategies. Aging 2016, 8, 603–619. [CrossRef] [PubMed] 2. Maltese, W.A.; Overmeyer, J.H. Methuosis: Nonapoptotic cell death associated with vacuolization of macropinosome and endosome compartments. Am. J. Pathol. 2014, 184, 1630–1642. [CrossRef] 3. Kornienko, A.; Mathieu, V.; Rastogi, S.K.; Lefranc, F.; Kiss, R. Therapeutic agents triggering nonapoptotic cancer cell death. J. Med. Chem. 2013, 56, 4823–4839. [CrossRef] [PubMed] 4. Overmeyer, J.H.; Kaul, A.; Johnson, E.E.; Maltese, W.A. 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