Naja Atra Cardiotoxin 3 Elicits Autophagy and Apoptosis in U937 Human Leukemia Cells Through the Ca2+/PP2A/AMPK Axis

toxins Article Naja atra Cardiotoxin 3 Elicits Autophagy and Apoptosis in U937 Human Leukemia Cells through the Ca2+/PP2A/AMPK Axis Jing-Ting Chiou 1, Yi-Jun Shi 1, Liang-Jun Wang 1, Chia-Hui Huang 1, Yuan-Chin Lee 1 and Long-Sen Chang 1,2,* 1 Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan 2 Department of Biotechnology, Kaohsiung Medical University, Kaohsiung 807, Taiwan * Correspondence: [email protected]; Tel.: +886-7-5252-000-5813 Received: 23 August 2019; Accepted: 10 September 2019; Published: 12 September 2019 Abstract: Cardiotoxins (CTXs) are suggested to exert their cytotoxicity through cell membrane damage. Other studies show that penetration of CTXs into cells elicits mitochondrial fragmentation or lysosome disruption, leading to cell death. Considering the role of AMPK-activated protein kinase (AMPK) in mitochondrial biogenesis and lysosomal biogenesis, we aimed to investigate whether the AMPK-mediated pathway modulated Naja atra (Taiwan cobra) CTX3 cytotoxicity in U937 human leukemia cells. Our results showed that CTX3 induced autophagy and apoptosis in U937 cells, whereas autophagic inhibitors suppressed CTX3-induced apoptosis. CTX3 treatment elicited Ca2+-dependent degradation of the protein phosphatase 2A (PP2A) catalytic subunit (PP2Acα) and phosphorylation of AMPKα. Overexpression of PP2Acα mitigated the CTX3-induced AMPKα phosphorylation. CTX3-induced autophagy was via AMPK-mediated suppression of the Akt/mTOR pathway. Removal of Ca2+ or suppression of AMPKα phosphorylation inhibited the CTX3-induced cell death. CTX3 was unable to induce autophagy and apoptosis in U937 cells expressing constitutively active Akt. Met-modified CTX3 retained its membrane-perturbing activity, however, it did not induce AMPK activation and death of U937 cells. These results conclusively indicate that CTX3 induces autophagy and apoptosis in U937 cells via the Ca2+/PP2A/AMPK axis, and suggest that the membrane-perturbing activity of CTX3 is not crucial for the cell death signaling pathway induction. Keywords: cardiotoxin; Ca2+; PP2A; AMPK; autophagy; apoptosis Key Contribution: The cytotoxicity of N. atra CTX3 on U937 cells is related to the Ca2+/PP2A/AMPK axis-elicited autophagy and apoptosis; Our data suggest a dissociation of CTX3-induced death signaling from its effect on cell membrane damage. 1. Introduction Cobra cardiotoxins (CTXs) show a wide range of biological activities including hemolysis, cardiotoxicity, and disruption of muscular integrity [1–3]. Moreover, accumulated evidence has shown that CTXs induce apoptosis in cancer cells through the mitochondria-mediated apoptotic pathway, lysosome-mediated death pathway, or membrane-damaging effects [4–8]. It is widely believed that the plasma membrane is a target of CTXs, but the membrane pore formed by CTXs may have a limited lifetime on the cell surface due to membrane reorganization [9]. Therefore, some studies indicate that the cytotoxicity of CTXs is related to their capability of cell penetration [4,7,10,11]. The internalized CTXs are suggested to elicit mitochondrial fragmentation [10,11] or lysosome disruption [4,7], and thereby induce the death of the myoblast cells, primary cortical neurons, or cancer cells. Notably, Langone Toxins 2019, 11, 527; doi:10.3390/toxins11090527 www.mdpi.com/journal/toxins Toxins 2019, 11, x FOR PEER REVIEW 2 of 12 Toxinslysosome2019, 11 disruption, 527 [4,7], and thereby induce the death of the myoblast cells, primary cortical2 of 12 neurons, or cancer cells. Notably, Langone et al. [12] found that activated AMPK can attenuate the etcytotoxicity al. [12] found of Naja that activatedpallida CTX AMPK on myoblast can attenuate cells. AMPK the cytotoxicity has been ofdemonstratedNaja pallida CTX to be on involved myoblast in cells.mitochondrial AMPK has biogenesis been demonstrated [13,14] and to belysosomal involved biogenesis in mitochondrial [15]. Some biogenesis studies [13 have,14] and revealed lysosomal that biogenesisAMPK acts [15 as]. a Sometumor studies suppressor have or revealed oncogene that in AMPKdifferent acts cancer as a tumorcells [16]. suppressor Other studies or oncogene indicate inthat di ffAMPK-mediatederent cancer cells signaling [16]. Other elicits studies death indicate in cancer that AMPK-mediatedcells via autophagy signaling and/or elicitsapoptosis death [17]. in cancerTherefore, cells it via is autophagyintriguing to and explore/or apoptosis the role [17 of]. the Therefore, AMPK-mediated it is intriguing pathway to explore in the thecytotoxicity role of the of AMPK-mediatedCTXs. Prior studies pathway have inshown the cytotoxicity that human of myeloid CTXs. Prior leukemia studies cells have are shown highly that susceptible human myeloid to the leukemiacytotoxicity cells of are CTXs highly [4,7,18]. susceptible Therefore, to the we cytotoxicity investigated of whether CTXs [4 ,7the,18 AMPK-associated]. Therefore, we investigated pathway is whetheran important the AMPK-associated mediator in N. atra pathway CTX3-induced is an important death ofmediator human leukemia in N. atra U937CTX3-induced cells. death of human leukemia U937 cells. 2. Results 2. ResultsTreatment with CTX3 reduced the U937 survival of cells in a concentration- and time-dependent mannerTreatment with an with IC50 CTX3 value reduced of approximately the U937survival 150 nM for of cells a 4-h in treatment a concentration- (Figure and1A). time-dependent Hence, we used mannerthis dose with of CTX3 an IC 50tovalue study of the approximately mechanism of 150 itsnM cytotoxicity. for a 4-htreatment CTX3 treatment (Figure 1increasedA). Hence, the we number used thisof annexin dose of CTX3V-FITC to staining study the U937 mechanism cells (Figure of its 1B). cytotoxicity. In line with CTX3 this, treatment the CTX-treated increased cells the numbershowed ofdegradation annexin V-FITC of procaspase-3 staining U937 and PARP cells (Figure (Figure1 B).1C), In whereas line with the this, caspase the CTX-treated inhibitors inhibited cells showed the cell degradationdeath induced of procaspase-3by CTX3 (Figure and PARP1D). These (Figure results1C), whereas indicated the that caspase CTX3 inhibitors induces apoptosis inhibited thein U937 cell deathcells. induced by CTX3 (Figure1D). These results indicated that CTX3 induces apoptosis in U937 cells. Figure 1. Cobra cardiotoxin (CTX)3 induced apoptotic death of U937 cells. (A)Effect of CTX3 on the viabilityFigure 1. of Cobra U937 cells.cardiotoxin Cells were (CTX)3 incubated induced with apoptotic indicated death CTX3 of U937 concentrations cells. (A) Effect for 4 h.of (Inset)CTX3 on U937 the cellsviability were of treated U937 withcells. 150Cells nM were CTX3 incubated for indicated with indicated time periods. CTX3 Cell concentrations viability was for determined 4 h. (Inset) using U937 methlythiazolyldiphenyl-tetrazoliumcells were treated with 150 nM CTX3 bromide for indicated (MTT) time assay. periods. Results Cell are viability expressed was as determined the percentage using of cellmethlythiazolyldiphenyl-tetrazolium survival relative to the control. Each bromide value is the(MTT) mean assay.SD Results of three are independent expressed experimentsas the percentage with ± triplicateof cell survival measurements; relative to (B the) Flow control. cytometry Each va analyseslue is the of mean±SD annexin V-PI of three double independent staining CTX3-treated experiments cells.with U937triplicate cells measurements; were incubated (B with) Flow 150 cytometry nM CTX3 analyses for 4 h. Onof annexin the flow V-PI cytometric double scatterstaining graphs, CTX3- thetreated left lowercells. U937 quadrant cells representswere incubated remaining with 150 live nM cells. CTX3 The for right 4 h. lower On the quadrant flow cytometric represents scatter the populationgraphs, the ofleft early lower apoptotic quadrant cells. represents The right remaining upper quadrantlive cells. representsThe right lower the accumulation quadrant represents of late apoptoticthe population cells; (ofC) early Western apoptotic blot analyses cells. The of right procaspase-3 upper quadrant and poly(ADP-ribose) represents the accumulation polymerase (PARP) of late degradationapoptotic cells; in CTX3-treated (C) Western blot cells. analyses U937 cells of procaspase-3 were incubated and with poly(ADP-ribose) 150 nM CTX3 for polymerase 4 h; (D) Viability (PARP) ofdegradation CTX3-treated in CTX3-treated cells was restored cells. by U937 pretreatment cells were with incubated caspase with inhibitors. 150 nM U937CTX3 cells for 4 were h; (D pretreated) Viability withof CTX3-treated 10 µM Z-VAD-FMK cells was (pan-caspase restored by inhibitor)pretreatment or Z-DEVD-FMK with caspase inhibitors. (caspase-3 inhibitor)U937 cells for were 1 h, pretreated and then incubatedwith 10 μM with Z-VAD-FMK 150 nM CTX3 (pan-caspase for 4 h. Each inhibitor) value is or the Z-DE meanVD-FMKSD of (caspase-3 three independent inhibitor) experiments for 1 h, and ± with triplicate measurements (* p < 0.05). Toxins 2019, 11, x FOR PEER REVIEW 3 of 12 then incubated with 150 nM CTX3 for 4 h. Each value is the mean ± SD of three independent Toxins 2019experiments, 11, 527 with triplicate measurements (* p < 0.05). 3 of 12 To examine whether CTX3-induced apoptosis is related to mitochondrial dysfunction, the To examine whether CTX3-induced apoptosis is related to mitochondrial dysfunction, mitochondrial membrane potential (ΔΨm) of CTX3-treated cells was thus measured using TMRM the mitochondrial membrane potential (DYm) of CTX3-treated

Load more