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Bengalin initiates autophagic cell death through ERK–MAPK pathway following suppression of apoptosis in human leukemic U937 cells
Life Sciences 93 (2013) 271–276
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Bengalin initiates autophagic cell death through ERK–MAPK pathway following suppression of apoptosis in human leukemic U937 cells Shubho Das Gupta a, Babli Halder a, Antony Gomes b, Aparna Gomes a,⁎ a Drug Development/Diagnostics and Biotechnology Division, Council of Scientific and Industrial Research (CSIR)—Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata-700032, India b Laboratory of Toxinology and Experimental Pharmacodynamics, Department of Physiology, University of Calcutta, 92, APC Roy Road, Kolkata-700009, India
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Article history: Received 26 December 2012 Accepted 24 June 2013 Keywords: Scorpion venom (Bengalin) Autophagy Caspase-3 Apoptosis ERK–MAPK LC3
a b s t r a c t Aims: The aim of this study was to assess the autophagy inducing ability of the scorpion venom toxin Bengalin in human leukemic U937 cells. The same toxin was previously shown to induce apoptosis in human leukemic cells. Main methods: Bengalin was purified from Indian black scorpion (Heterometrus bengalensis) venom by ion exchange chromatography and HPLC. In human leukemic U937 cells, Bengalin associated MAPK (mitogen activated protein kinase) pathway was determined by western blotting. Downstream to MAPK, the Bengalin induced apoptosis-mediator caspase-3 was blocked by chemical inhibitor and reconfirmed by siRNA mediated gene knockdown. Subsequent to caspase-3 blocking, the autophagic response was evaluated by quantification of acidic vesicle organelles formation and modulations of Atg's, Beclin-1, LC3-1 and LC3-II expression by western blotting. Key findings: In U937 cells, Bengalin increased ERK1/2 expression to bring about cell death. However in subsequent caspase-3 blocked conditions, Bengalin downregulated p-Akt, p-mTOR and decreased apoptosis. It had also increased the percentage of acidic vesicle organelles positive cells. Bengalin could induce autophagic response by augmenting Beclin-1, Atg12, Atg7, Atg5 and Atg3 in U937 cells. Moreover a time dependant reciprocal relation was observed between LC3-I and LC3-II expression upon Bengalin treatment. The decrease in LC3-II was inhibited in the presence of lysozomal enzyme blockers thereby suggesting lysosome involvement in the autophagic response. Significance: We have for the first time demonstrated that scorpion venom-component could induce an alternate cell death pathway other than apoptosis in the form of autophagy in human leukemic U937 cells. © 2013 Elsevier Inc. All rights reserved.
Introduction Among the different modes of cell death, apoptosis and autophagy have been highly investigated (Kourtis and Tavernarakis, 2009). Apoptosis is the type I PCD (programmed cell death) where cells are killed in a highly orchestrated manner upon receiving death stimulus. Apoptosis is mediated by several cellular pathways of which extrinsic, intrinsic and modulations of MAPK including Erk's are few important ones (Santarpia et al., 2012; Kohno et al., 2011). In most of the pathways, an imperative effector molecule is caspase-3, which is a cysteine aspartate protease. Apart from playing a crucial role in apoptosis, the ERK pathway is also implicated in a different mode of cell death via modulation of Akt and mTOR (Wang et al., 2009; Aoki et al., 2007a,b).
⁎ Corresponding author at: Drug Development/Diagnostics and Biotechnology Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata-700032, India. Tel.: +91 33 24995708; fax: +91 33 24730284. E-mail address: [email protected] (A. Gomes). 0024-3205/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2013.06.022
This cell death is type II PCD or autophagy, whereby activation of a cascade of events leads to the formation of autophagosomes, autophagolysosomes and ultimately culminates in cell death. Few of the critical stimulators of autophagy are serum starvation (Komatsu et al., 2005), pathogens and different types of toxins (Kirkegaard et al., 2004). During autophagy, the autophagy related gene (Atg) products get activated and play a crucial role in the initiation and its subsequent progression. Activated Atg's further trigger the downstream molecule LC3 (microtubule-associated protein 1 light chain 3) and help in the formation of autophagosomes. In our previous study we had showed that Bengalin induced apoptosis by increasing caspase-3 activity (Das Gupta et al., 2010). Hence we wanted to observe whether caspase-3 blocking could lead to cell death through any alternate pathway. Although some studies are being conducted on the possible role of scorpion venom and its component(s) in inhibiting cancer cell growth (D'Suze et al., 2010; Zargan et al., 2011; Zhao et al., 2011), none of the studies have reported any alternate mode of cell death other than apoptosis. In fact venom from the scorpion Tityus discrepans has shown to cause
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autophagic changes in adrenal cortex cells upon envenomation in normal murine systems (Pulido-Méndez et al., 2002). However there is no report of scorpion venom component mediated autophagy induction in cancer cells whatsoever. The present study is unique in the sense this is the first report of a pure protein Bengalin from scorpion venom, that primarily causes human leukemic U937 cell death by apoptosis, however upon suppression of which still eradicates the cells through autophagy.
were cultured at an initial concentration of 106 cells/ml. This was followed by Lipofectamin (Invitrogen) mediated transfection of the cells with scrambled siRNA, which would serve as nonspecific negative control while caspase-3 siRNA to block its activity. After 24 h of transfection, U937 cells were treated with Bengalin at 3.7 μg/ml concentration. The treated cells were then processed for detecting cell viability, apoptosis and western blot analysis. Quantification of acidic vesicle organelles (AVO) formation
Materials and methods Reagents Primary antibodies ERK-1/2, JNK-1/2, p-38, p-ERK-1/2, p-JNK1/2, p-p38, p-Akt (phospho-Akt) (Ser473), Akt (Ser473), p-mTOR, mTOR, caspase-3, β-actin, goat anti-mouse secondary antibodies, scrambled siRNA and caspase-3 specific siRNA were procured from Santa Cruz Biotechnology Inc. (CA, USA). Autophagy sampler kit was procured from Cell Signaling Technology (Danvers, MA, USA). MTT (3[4-dimethylthiazol-2-71]-2-5-diphenyl tetrazolium bromide), caspase-3 inhibitor (DEVD-CHO) and MEK blocker PD98059 from Calbiochem (La Jolla, CA, USA), E64d and pepstatin A were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Cytoplasmic proteins are collected in AVO for their lysosomal degradation during autophagy. Flow cytometric estimation of AVO was carried out (Aoki et al., 2007a,b). In brief, initially 106 cells were treated with caspase-3 blocker for desired period followed by Bengalin treatment for 24 h. After treatment, the cells were incubated for 15 min with acridine orange (1 μg/ml) and analyzed in a Becton Dickinson flow cytometer (San Jose, CA, USA). Western immunoblot analysis Western immunoblot was performed to assay the expression level of various proteins associated with the autophagic process (Das Gupta et al., 2010). Briefly, the cells after treatment with Bengalin
Purification of Bengalin Bengalin was purified from the whole venom of Heterometrus bengalensis by the method as described before (Das Gupta et al., 2010). Cell culture Human histiocytic lymphoma U937 cell was purchased from National Facility for Animal Tissue and Cell Culture, Pune, India. Cells were cultured in RPM-1640 (Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) with penicillin, streptomycin and gentamycin used as antibiotics (Sigma-Aldrich, St. Louis, MO, USA). Incubation of cells was done at 5% CO2 and 95% humidified atmosphere in a CO2 incubator. MTT assay The cytotoxicity effect of Bengalin on U937 cells both pre and post caspase-3 knockdown was analyzed by MTT assay (Mosmann, 1983). Briefly about 106 cells/ml of either pre or post caspase-3 knockdown were treated with Bengalin (0–3.7 μg/ml) for 48 h followed by MTT incubation. Absorbance was recorded in a Bio-Rad microplate reader: Model 680XR (Philadelphia, PA, USA) with references serving as blank. Each treatment condition was carried out in quadruplicate, while every experiment was independently repeated thrice. Apoptosis assay Analyses for apoptosis were done to determine the mode of cell death after ERK-1/2 and caspase-3 activation in U937 cells following 24 h treatment with Bengalin. Assay for apoptosis was done by AnnexinV-PI Apoptosis Detection Kit (Sigma-Aldrich) as per the manufacturer's instruction. The untreated control and treated cells were acquired in a flow cytometer (Beckton Dickinson, San Jose, USA). Obtained results were analyzed by the Cell Quest software platform (Becton Dickinson, USA). siRNA mediated gene knockdown siRNA transfection was performed to specifically knockdown the activity of the targeted gene products. Cells from both the cell lines
Fig. 1. Bengalin mediated augmentation of ERK1/2 expression, downregulation of both p-Akt and p-mTOR level. (A) U937 cells were treated with 3.7 μg/ml concentration of Bengalin for 0, 12 and 24 h. Expression level of p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38 and p-38 was seen after treatment by western blot. Here β-actin serves as loading control. (B) Western blot analysis was done to observe the effect of Bengalin on p-Akt, Akt, p-mTOR, mTOR in U937 cells after 24 h of treatment. β-Actin served as a loading control. Here the blot is a single representative of Bengalin treatment from three independent experiments with similar results.
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(3.7 μg/ml) for 24 h were washed and lysed with chilled RIPA buffer (Sigma-Aldrich) containing protease inhibitor cocktail (Fermentas, USA). Equal amount of proteins were loaded, ran in a 10% gel and transferred electrophoretically to a nitrocellulose membrane. The nonspecific sites were blocked with BSA and the membrane was incubated with primary antibodies (ERK-1/2, JNK-1/2, p-38, p-ERK-1/2, p-JNK1/2, p-p38, p-Akt (Ser473), Akt (Ser473), p-mTOR, mTOR, caspase-3, LC3-I, LC3-II, Beclin-1, Atg3, Atg5, Atg7, Atg12 and β-actin) and then with alkaline phosphatase conjugated secondary antibodies. The bands were developed and visualized by NBT-BCIP ready mix (Sigma-Aldrich).
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Results Bengalin increased ERK1/2 expression, downregulated p-Akt, p-mTOR and decreased apoptosis in caspase-3 blocked U937 cells MAPK pathway has been implicated in initiation and progression of apoptosis in human leukemic cells. Hence our intention was to determine whether any of the specific MAPK pathways was responsible for initiating Bengalin mediated cell death. Time dependant changes in the expression levels of ERK1/2 and p-ERK were assayed by western immunoblot. Upon incubation with Bengalin (3.7 μg/ml for
Fig. 2. Apoptosis and cell mortality were not significantly attenuated upon Bengalin treatment in ERK and/or caspase-3 blocked cells, while higher cell viability was observed in U937 cells which were treated with ERK and/or caspase-3 inhibitors along with Bengalin. (A) Cells were treated with ERK blocker and the effect on apoptosis was observed upon 24 h of Bengalin treatment. (B) U937 cells were treated with caspase-3 specific siRNA and then effect of Bengalin on apoptosis was observed after 24 h of treatment. (C) Effect of Bengalin on cell viability of caspase-3 blocked cells either by caspase-3 specific siRNA or (D) caspase-3 inhibitor. (E) Effect of Bengalin on cell viability which was blocked by both caspase-3 as well as ERK inhibitors. Data are expressed as mean ± SD from at least three independent experiments. p-Values were calculated using Dunnett's multiple comparison test. *p b 0.01. (F) Effect of Bengalin on caspase-3 specific siRNA-treated U937 cells. Cell lysate was taken for western blot analysis to observe the expression level of caspase-3. The cells were preincubated with either scrambled siRNA or caspase-3 specific siRNA followed by Bengalin treatment at a concentration of 3.7 μg/ml for 24 h. β-Actin served as the loading control.
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U937 cells), it was observed that p-ERK1/2 levels increased significantly after 12 h of treatment and subsided later on (Fig. 1A). However there were no alterations in the levels of p-p38 and p-JNK. Also the levels of p38, JNK and ERK remained unchanged after Bengalin treatment (Fig. 1A). Further Bengalin also inhibited the expression of p-Akt (Ser473) and p-mTOR (Fig. 1B). It was observed that the percentage of apoptosis declined when U937 cells were treated with the ERK blocker and Bengalin (Fig. 2A). Moreover when in individual experiments, caspase-3 was blocked using caspase-3 specific siRNA, there was a decrease in apoptosis of the Bengalin treated U937 cells (Fig. 2B). However MTT assay of caspase-3 inhibited (caspase-3 specific siRNA or DEVD-CHO) U937 cells still showed a significant cell death following Bengalin treatment (Figs. 2C and D). However there was a cessation of cell death when U937 cells were treated with combination of PD98059 and DEVD-CHO (Fig. 2E). Expression of caspase-3 was reduced when the U937 cells were treated with caspase-3 specific siRNA prior to treatment with Bengalin in comparison to cells treated with Bengalin only (Fig. 2F). Bengalin increased percentage of AVO positive cells in caspase-3 blocked U937 cells In order to confirm and quantify autophagy in Bengalin treated U937 cells we had evaluated the formation of AVO's by flow cytometric method. There was a significant increase of percentage of AVO positive cells after caspase-3 blocking and Bengalin treatment as compared to the caspase-3 blocked and Bengalin untreated counterparts (Fig. 3). However when both ERK1/2 and caspase-3 were blocked, there was a marked decrease in the percentage of AVO positive cells (Fig. 3). Bengalin mediated alteration of autophagy associated proteins in U937 cells The expression of several autophagy related proteins was analyzed using western blot after blocking caspase-3 activity using DEVD-CHO. It was evidenced from the results that in Bengalin treated U937 cells, Beclin-1 level was augmented. Similarly increase in the expression of Atg12, Atg7, Atg5 and Atg3 was also observed in the caspase-3 inhibited Bengalin treated U937 cells (Fig. 4A). However Bengalin treatment could not up-regulate Beclin-1, Atg12, Atg7, Atg5, Atg3 expressions in U937 cells which were treated with PD98059 prior to DEVD-CHO blocking (Fig. 4B). Again upon incubation with Bengalin, the LC3-I level reflected a reciprocal relation
with LC3-II expression over time. LC3-I expression was decreased while LC3-II expression level peaked after 24 h treatment and the level inversed after 36 h (Fig. 4B). Upon blocking of lysozomal enzyme activity with pepstatin, this decrease in LC3-II expression could not be observed (Fig. 4B). PD98059 pre-treated U937 cells could not show any expression change of LC3I and LC3II due to Bengalin treatment (Fig. 4C).
Discussion The high molecular weight protein Bengalin, purified from the venom of the Indian black scorpion, H. bengalensis had effectively brought about human leukemic U937 and K562 cell death (Das Gupta et al., 2007, 2010). In our previous study we had established that the mode of cell death was primarily mitochondria mediated apoptotic in nature. This fact also raised our inquisitiveness to investigate whether any other pathway was responsible for inducing apoptotic cell death in U937 cells. We had started our preliminary investigations with the MAPK pathway. Here we observed that Bengalin treatment could effectively modulate the ERK1/2 pathway, which in turn caused cell death. Our earlier observations had suggested that Bengalin induced mitochondrial apoptosis was in fact caspase-3 mediated (Das Gupta et al., 2010). Hence we further wanted to observe whether caspase-3 was indispensible in causing Bengalin mediated cell death of human leukemic cells. To investigate this, we inhibited the activity of caspase-3 using chemical inhibitor or siRNAs and even under these circumstances it was seen that Bengalin caused leukemic cell death. Flow cytometric analyses of the Bengalin treated caspase-3 blocked U937 cells failed to show an accumulation in either necrotic or apoptotic quadrants. Thus the probability of necrosis and apoptosis could be ruled out. Under this condition of Bengalin treatment and caspase3- blockade, cells were still dying. Thus it could be presumed that Bengalin mediated cell death was occurring by some alternate pathway other than apoptosis. So we investigated the role of ERK1/2 in this cell death process and upon blocking the activity of ERK1/2 with the inhibitor, PD98059, we found that the cell death process ceased considerably. Thus the probable role of ERK1/2 in the cell death cascade could be confirmed. Earlier reports suggested an involvement of ERK1/2 in inducing autophagic cell death (Cagnol and Chambard, 2010; Choi et al., 2010). Hence we decided to investigate the role of autophagy as an alternate pathway for apoptosis in causing Bengalin mediated cell death of the caspase-3 blocked cells. All our investigations were subsequently carried out with the caspase-3 blocked condition.
Fig. 3. Bengalin treatment increased percentage of AVO in caspase-3 and caspase-3 plus ERK1/2 inhibited U937 cells. Acridine orange staining was done for flow cytometric studies after 24 h of treatment of Bengalin to observe the percentage of AVO in caspase-3 blocked as well as cells which were blocked by caspase-3 as well as ERK inhibitors. Data are expressed as mean ± SD from at least three independent experiments. p-values were calculated using Dunnett's multiple comparison test. *p b 0.01.
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Fig. 4. Bengalin treatment increased the level of Beclin-1, Atg12, Atg7, Atg5, Atg3 and LC3-II and declined LC3-I expression. (A) Whole cell lysate was taken for western blot analysis to observe the expression level of Beclin-1, Atg12, Atg7, Atg5 and Atg3 protein in caspase-3 blocked U937 cells after 24 h of Bengalin treatment at a concentration 3.7 μg/ml. β-Actin served as a loading control. (B) Cells were initially treated with PD98059 and DEVD-CHO and then with Bengalin. Whole cell lysate was taken for western blot analysis to observe the expression level of Beclin-1, Atg12, Atg7, Atg5 and Atg3 protein. β-actin was used as loading control. (C) Caspase-3 blocked cells were treated with Bengalin for 0, 6, 12, 24, 36 h. Level of LC3-I and LC3-II was observed by western blot. Cells were also treated with ERK blocker as well as caspase-3 blocker followed by Bengalin treatment for different time intervals. Expression of LC3I and LC3II from the treated cells was observed by western blotting. β-Actin served as a loading control. Here the blot is a single representative of Bengalin treatment from three independent experiments with similar results.
Among the critical mediator of autophagic response is the PI3K/ Akt/mTOR pathway. Activation of PI3K/Akt/mTOR is known to negatively regulate autophagy (Saiki et al., 2011; Huang and Manning, 2009; Shinojima et al., 2007). In our studies there was a definite decrease of p-Akt (Ser473) and p-mTOR upon Bengalin treatment in U937 cells thereby suggesting a tentative role of their involvement in the autophagic response. Hence upon discovering a Bengalin initiated probable trend towards autophagic cell death in the caspase-3 blocked cells, we further proceeded towards analyzing the involvement of other downstream molecules engaged in the autophagic cascade. Autophagic process involves critical steps viz. initiation, elongation, maturation, autophagolysosome–lysosome fusion and ultimately degradation of the autophagolysosome (Tanida, 2011; Radoshevich and Debnath, 2011). This entire cascade is controlled by several
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proteins of which Beclin-1, Atg's and LC3 are critical effectors and help in proper progression of the process. Inactivated mTOR facilitates the formation of the active PI3KC-III and Beclin-1 complex required for nucleation (Kim et al., 2006). Beclin-1 again is negatively regulated by Bcl-2 (Pattingre et al., 2005). In our previous study we had observed that Bengalin could reduce the action of Bcl-2 (Das Gupta et al., 2010) in human leukemic cells while causing apoptosis. In our present study we observed a distinct augmentation of Beclin-1 which could be related to the Bengalin induced suppression of Bcl-2. For autophagy induction, the endogenous conjugate of Atg12 and Atg5 complex is required (Tanida, 2011). This complex in association with LC3-II is responsible for the formation of intracellular autophagosomes. The LC3-I gets activated by Atg-7 and then undergoes lipidation with phosphatidylethanolamine (PE) by Atg-3. This is followed by the completion stage where the cytoplasmic targets for autophagy are encapsulated in the autophagosome (Yu et al., 2004; Apel et al., 2009; Janku et al., 2011). Increase of Atg-12 and Atg-5 expressions due to Bengalin treatment could be suggestive of an induction of autophagy. However upon blocking ERK with PD98059 no alteration of Atg's and Beclin-1 could be seen. Our one of the major target molecule in determining the mode of cell death was the microtubule associated protein light chain 3 (LC3) molecule. LC3 exists in 2 forms viz. LC3-I and LC3-II. LC3-I is found ubiquitously expressed in the cytosol. On receiving autophagic stimulus, the LC3-I gets lipidated with phosphatidylethanolamine and thus converts to LC3-II. The produced LC3-II associates with both the inner and outer membranes of autophagosomes. Bengalin caused reduction in LC3-I expression followed by an upregulation of LC3-II, both of which were time dependant. The flux of LC3-II is transient as the autophagosomes with which it was present, fuse with the lysosomes and produce autophagolysosome where the inner membrane LC3-II gets degraded. However the outer membrane LC3-II detaches from PE by Atg-4 and converts back to LC3-I in the cytosol (Mizushima and Yoshimori, 2007). Our data with LC3-II are in conformation to this statement as Bengalin caused a time dependant decrease of LC3-I and a subsequent increase in LC3-II, the relation between the two molecules being reciprocal in Bengalin treated U937 cells. Since an increase of LC3-II expression is directly proportional to the formation of autophagosomes, suggesting a trend towards autophagic cell death hence it might be presumed that Bengalin induced an autophagic change in the caspase-3 blocked leukemic cells. Again upon blocking the activity of lysosomal proteases in the Bengalin treated cells, the LC3-II level did not decrease significantly underlying the involvement of lysosomes in the Bengalin induced cell death. Moreover the increase in the expression level of Atg-7 also highlights the fact of LC3-I activation and the subsequent increase in the Atg-3 corroborates the hypothesis of LC3-I lipidation to LC3-II. Further to this finding, blocking of ERK with PD98059 failed to show any time dependent alteration of LC3I or LC3II. This is suggestive of an absence of autophagy, which could be due to the inhibition of ERK upstream of caspase-3 irrespective of Bengalin treatment in U937 cells. The major efficacy of Bengalin is reflected in its capability to cause leukemic cell death. In our previous study, Bengalin caused apoptosis in leukemic cells through the intrinsic pathway. Moreover upon blocking the pivotal effector molecule caspase-3, Bengalin could still eradicate the human leukemic cells through an alternate pathway, which could be autophagic in nature. Hence the study is unique in the sense that it is the first report of any scorpion venom-component mediated autophagic cell death of human cancer cells. However further studies are warranted for an even better understanding of the detailed molecular mechanism of the Bengalin mediated autophagic response. Conclusion In conclusion, our studies demonstrated that Bengalin, a pure protein from scorpion venom could initiate a probable ERK1/2 mediated
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autophagic cell death pathway in human leukemic U937 cells, upon blocking its caspase-3 induced apoptogenic effect. When studies related to the whole scorpion venom or venom component induced apoptotic cancer cell death is still in its early stage, the present study with Bengalin is a crucial leap in unfolding an alternate mechanism of scorpion venom component mediated cancer cell eradication. This study might provide preliminary knowledge for further understanding of the highly sought after area of chemotherapeutic drug development from natural resources like venoms and toxins. Conflict of interest All authors declare no conflict of interest whatsoever.
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Bengalin initiates autophagic cell death through ERK–MAPK pathway following suppression of apoptosis in human leukemic U937 cells Shubho Das Gupta a, Babli Halder a, Antony Gomes b, Aparna Gomes a,⁎ a Drug Development/Diagnostics and Biotechnology Division, Council of Scientific and Industrial Research (CSIR)—Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata-700032, India b Laboratory of Toxinology and Experimental Pharmacodynamics, Department of Physiology, University of Calcutta, 92, APC Roy Road, Kolkata-700009, India
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Article history: Received 26 December 2012 Accepted 24 June 2013 Keywords: Scorpion venom (Bengalin) Autophagy Caspase-3 Apoptosis ERK–MAPK LC3
a b s t r a c t Aims: The aim of this study was to assess the autophagy inducing ability of the scorpion venom toxin Bengalin in human leukemic U937 cells. The same toxin was previously shown to induce apoptosis in human leukemic cells. Main methods: Bengalin was purified from Indian black scorpion (Heterometrus bengalensis) venom by ion exchange chromatography and HPLC. In human leukemic U937 cells, Bengalin associated MAPK (mitogen activated protein kinase) pathway was determined by western blotting. Downstream to MAPK, the Bengalin induced apoptosis-mediator caspase-3 was blocked by chemical inhibitor and reconfirmed by siRNA mediated gene knockdown. Subsequent to caspase-3 blocking, the autophagic response was evaluated by quantification of acidic vesicle organelles formation and modulations of Atg's, Beclin-1, LC3-1 and LC3-II expression by western blotting. Key findings: In U937 cells, Bengalin increased ERK1/2 expression to bring about cell death. However in subsequent caspase-3 blocked conditions, Bengalin downregulated p-Akt, p-mTOR and decreased apoptosis. It had also increased the percentage of acidic vesicle organelles positive cells. Bengalin could induce autophagic response by augmenting Beclin-1, Atg12, Atg7, Atg5 and Atg3 in U937 cells. Moreover a time dependant reciprocal relation was observed between LC3-I and LC3-II expression upon Bengalin treatment. The decrease in LC3-II was inhibited in the presence of lysozomal enzyme blockers thereby suggesting lysosome involvement in the autophagic response. Significance: We have for the first time demonstrated that scorpion venom-component could induce an alternate cell death pathway other than apoptosis in the form of autophagy in human leukemic U937 cells. © 2013 Elsevier Inc. All rights reserved.
Introduction Among the different modes of cell death, apoptosis and autophagy have been highly investigated (Kourtis and Tavernarakis, 2009). Apoptosis is the type I PCD (programmed cell death) where cells are killed in a highly orchestrated manner upon receiving death stimulus. Apoptosis is mediated by several cellular pathways of which extrinsic, intrinsic and modulations of MAPK including Erk's are few important ones (Santarpia et al., 2012; Kohno et al., 2011). In most of the pathways, an imperative effector molecule is caspase-3, which is a cysteine aspartate protease. Apart from playing a crucial role in apoptosis, the ERK pathway is also implicated in a different mode of cell death via modulation of Akt and mTOR (Wang et al., 2009; Aoki et al., 2007a,b).
⁎ Corresponding author at: Drug Development/Diagnostics and Biotechnology Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata-700032, India. Tel.: +91 33 24995708; fax: +91 33 24730284. E-mail address: [email protected] (A. Gomes). 0024-3205/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2013.06.022
This cell death is type II PCD or autophagy, whereby activation of a cascade of events leads to the formation of autophagosomes, autophagolysosomes and ultimately culminates in cell death. Few of the critical stimulators of autophagy are serum starvation (Komatsu et al., 2005), pathogens and different types of toxins (Kirkegaard et al., 2004). During autophagy, the autophagy related gene (Atg) products get activated and play a crucial role in the initiation and its subsequent progression. Activated Atg's further trigger the downstream molecule LC3 (microtubule-associated protein 1 light chain 3) and help in the formation of autophagosomes. In our previous study we had showed that Bengalin induced apoptosis by increasing caspase-3 activity (Das Gupta et al., 2010). Hence we wanted to observe whether caspase-3 blocking could lead to cell death through any alternate pathway. Although some studies are being conducted on the possible role of scorpion venom and its component(s) in inhibiting cancer cell growth (D'Suze et al., 2010; Zargan et al., 2011; Zhao et al., 2011), none of the studies have reported any alternate mode of cell death other than apoptosis. In fact venom from the scorpion Tityus discrepans has shown to cause
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autophagic changes in adrenal cortex cells upon envenomation in normal murine systems (Pulido-Méndez et al., 2002). However there is no report of scorpion venom component mediated autophagy induction in cancer cells whatsoever. The present study is unique in the sense this is the first report of a pure protein Bengalin from scorpion venom, that primarily causes human leukemic U937 cell death by apoptosis, however upon suppression of which still eradicates the cells through autophagy.
were cultured at an initial concentration of 106 cells/ml. This was followed by Lipofectamin (Invitrogen) mediated transfection of the cells with scrambled siRNA, which would serve as nonspecific negative control while caspase-3 siRNA to block its activity. After 24 h of transfection, U937 cells were treated with Bengalin at 3.7 μg/ml concentration. The treated cells were then processed for detecting cell viability, apoptosis and western blot analysis. Quantification of acidic vesicle organelles (AVO) formation
Materials and methods Reagents Primary antibodies ERK-1/2, JNK-1/2, p-38, p-ERK-1/2, p-JNK1/2, p-p38, p-Akt (phospho-Akt) (Ser473), Akt (Ser473), p-mTOR, mTOR, caspase-3, β-actin, goat anti-mouse secondary antibodies, scrambled siRNA and caspase-3 specific siRNA were procured from Santa Cruz Biotechnology Inc. (CA, USA). Autophagy sampler kit was procured from Cell Signaling Technology (Danvers, MA, USA). MTT (3[4-dimethylthiazol-2-71]-2-5-diphenyl tetrazolium bromide), caspase-3 inhibitor (DEVD-CHO) and MEK blocker PD98059 from Calbiochem (La Jolla, CA, USA), E64d and pepstatin A were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Cytoplasmic proteins are collected in AVO for their lysosomal degradation during autophagy. Flow cytometric estimation of AVO was carried out (Aoki et al., 2007a,b). In brief, initially 106 cells were treated with caspase-3 blocker for desired period followed by Bengalin treatment for 24 h. After treatment, the cells were incubated for 15 min with acridine orange (1 μg/ml) and analyzed in a Becton Dickinson flow cytometer (San Jose, CA, USA). Western immunoblot analysis Western immunoblot was performed to assay the expression level of various proteins associated with the autophagic process (Das Gupta et al., 2010). Briefly, the cells after treatment with Bengalin
Purification of Bengalin Bengalin was purified from the whole venom of Heterometrus bengalensis by the method as described before (Das Gupta et al., 2010). Cell culture Human histiocytic lymphoma U937 cell was purchased from National Facility for Animal Tissue and Cell Culture, Pune, India. Cells were cultured in RPM-1640 (Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) with penicillin, streptomycin and gentamycin used as antibiotics (Sigma-Aldrich, St. Louis, MO, USA). Incubation of cells was done at 5% CO2 and 95% humidified atmosphere in a CO2 incubator. MTT assay The cytotoxicity effect of Bengalin on U937 cells both pre and post caspase-3 knockdown was analyzed by MTT assay (Mosmann, 1983). Briefly about 106 cells/ml of either pre or post caspase-3 knockdown were treated with Bengalin (0–3.7 μg/ml) for 48 h followed by MTT incubation. Absorbance was recorded in a Bio-Rad microplate reader: Model 680XR (Philadelphia, PA, USA) with references serving as blank. Each treatment condition was carried out in quadruplicate, while every experiment was independently repeated thrice. Apoptosis assay Analyses for apoptosis were done to determine the mode of cell death after ERK-1/2 and caspase-3 activation in U937 cells following 24 h treatment with Bengalin. Assay for apoptosis was done by AnnexinV-PI Apoptosis Detection Kit (Sigma-Aldrich) as per the manufacturer's instruction. The untreated control and treated cells were acquired in a flow cytometer (Beckton Dickinson, San Jose, USA). Obtained results were analyzed by the Cell Quest software platform (Becton Dickinson, USA). siRNA mediated gene knockdown siRNA transfection was performed to specifically knockdown the activity of the targeted gene products. Cells from both the cell lines
Fig. 1. Bengalin mediated augmentation of ERK1/2 expression, downregulation of both p-Akt and p-mTOR level. (A) U937 cells were treated with 3.7 μg/ml concentration of Bengalin for 0, 12 and 24 h. Expression level of p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38 and p-38 was seen after treatment by western blot. Here β-actin serves as loading control. (B) Western blot analysis was done to observe the effect of Bengalin on p-Akt, Akt, p-mTOR, mTOR in U937 cells after 24 h of treatment. β-Actin served as a loading control. Here the blot is a single representative of Bengalin treatment from three independent experiments with similar results.
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(3.7 μg/ml) for 24 h were washed and lysed with chilled RIPA buffer (Sigma-Aldrich) containing protease inhibitor cocktail (Fermentas, USA). Equal amount of proteins were loaded, ran in a 10% gel and transferred electrophoretically to a nitrocellulose membrane. The nonspecific sites were blocked with BSA and the membrane was incubated with primary antibodies (ERK-1/2, JNK-1/2, p-38, p-ERK-1/2, p-JNK1/2, p-p38, p-Akt (Ser473), Akt (Ser473), p-mTOR, mTOR, caspase-3, LC3-I, LC3-II, Beclin-1, Atg3, Atg5, Atg7, Atg12 and β-actin) and then with alkaline phosphatase conjugated secondary antibodies. The bands were developed and visualized by NBT-BCIP ready mix (Sigma-Aldrich).
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Results Bengalin increased ERK1/2 expression, downregulated p-Akt, p-mTOR and decreased apoptosis in caspase-3 blocked U937 cells MAPK pathway has been implicated in initiation and progression of apoptosis in human leukemic cells. Hence our intention was to determine whether any of the specific MAPK pathways was responsible for initiating Bengalin mediated cell death. Time dependant changes in the expression levels of ERK1/2 and p-ERK were assayed by western immunoblot. Upon incubation with Bengalin (3.7 μg/ml for
Fig. 2. Apoptosis and cell mortality were not significantly attenuated upon Bengalin treatment in ERK and/or caspase-3 blocked cells, while higher cell viability was observed in U937 cells which were treated with ERK and/or caspase-3 inhibitors along with Bengalin. (A) Cells were treated with ERK blocker and the effect on apoptosis was observed upon 24 h of Bengalin treatment. (B) U937 cells were treated with caspase-3 specific siRNA and then effect of Bengalin on apoptosis was observed after 24 h of treatment. (C) Effect of Bengalin on cell viability of caspase-3 blocked cells either by caspase-3 specific siRNA or (D) caspase-3 inhibitor. (E) Effect of Bengalin on cell viability which was blocked by both caspase-3 as well as ERK inhibitors. Data are expressed as mean ± SD from at least three independent experiments. p-Values were calculated using Dunnett's multiple comparison test. *p b 0.01. (F) Effect of Bengalin on caspase-3 specific siRNA-treated U937 cells. Cell lysate was taken for western blot analysis to observe the expression level of caspase-3. The cells were preincubated with either scrambled siRNA or caspase-3 specific siRNA followed by Bengalin treatment at a concentration of 3.7 μg/ml for 24 h. β-Actin served as the loading control.
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U937 cells), it was observed that p-ERK1/2 levels increased significantly after 12 h of treatment and subsided later on (Fig. 1A). However there were no alterations in the levels of p-p38 and p-JNK. Also the levels of p38, JNK and ERK remained unchanged after Bengalin treatment (Fig. 1A). Further Bengalin also inhibited the expression of p-Akt (Ser473) and p-mTOR (Fig. 1B). It was observed that the percentage of apoptosis declined when U937 cells were treated with the ERK blocker and Bengalin (Fig. 2A). Moreover when in individual experiments, caspase-3 was blocked using caspase-3 specific siRNA, there was a decrease in apoptosis of the Bengalin treated U937 cells (Fig. 2B). However MTT assay of caspase-3 inhibited (caspase-3 specific siRNA or DEVD-CHO) U937 cells still showed a significant cell death following Bengalin treatment (Figs. 2C and D). However there was a cessation of cell death when U937 cells were treated with combination of PD98059 and DEVD-CHO (Fig. 2E). Expression of caspase-3 was reduced when the U937 cells were treated with caspase-3 specific siRNA prior to treatment with Bengalin in comparison to cells treated with Bengalin only (Fig. 2F). Bengalin increased percentage of AVO positive cells in caspase-3 blocked U937 cells In order to confirm and quantify autophagy in Bengalin treated U937 cells we had evaluated the formation of AVO's by flow cytometric method. There was a significant increase of percentage of AVO positive cells after caspase-3 blocking and Bengalin treatment as compared to the caspase-3 blocked and Bengalin untreated counterparts (Fig. 3). However when both ERK1/2 and caspase-3 were blocked, there was a marked decrease in the percentage of AVO positive cells (Fig. 3). Bengalin mediated alteration of autophagy associated proteins in U937 cells The expression of several autophagy related proteins was analyzed using western blot after blocking caspase-3 activity using DEVD-CHO. It was evidenced from the results that in Bengalin treated U937 cells, Beclin-1 level was augmented. Similarly increase in the expression of Atg12, Atg7, Atg5 and Atg3 was also observed in the caspase-3 inhibited Bengalin treated U937 cells (Fig. 4A). However Bengalin treatment could not up-regulate Beclin-1, Atg12, Atg7, Atg5, Atg3 expressions in U937 cells which were treated with PD98059 prior to DEVD-CHO blocking (Fig. 4B). Again upon incubation with Bengalin, the LC3-I level reflected a reciprocal relation
with LC3-II expression over time. LC3-I expression was decreased while LC3-II expression level peaked after 24 h treatment and the level inversed after 36 h (Fig. 4B). Upon blocking of lysozomal enzyme activity with pepstatin, this decrease in LC3-II expression could not be observed (Fig. 4B). PD98059 pre-treated U937 cells could not show any expression change of LC3I and LC3II due to Bengalin treatment (Fig. 4C).
Discussion The high molecular weight protein Bengalin, purified from the venom of the Indian black scorpion, H. bengalensis had effectively brought about human leukemic U937 and K562 cell death (Das Gupta et al., 2007, 2010). In our previous study we had established that the mode of cell death was primarily mitochondria mediated apoptotic in nature. This fact also raised our inquisitiveness to investigate whether any other pathway was responsible for inducing apoptotic cell death in U937 cells. We had started our preliminary investigations with the MAPK pathway. Here we observed that Bengalin treatment could effectively modulate the ERK1/2 pathway, which in turn caused cell death. Our earlier observations had suggested that Bengalin induced mitochondrial apoptosis was in fact caspase-3 mediated (Das Gupta et al., 2010). Hence we further wanted to observe whether caspase-3 was indispensible in causing Bengalin mediated cell death of human leukemic cells. To investigate this, we inhibited the activity of caspase-3 using chemical inhibitor or siRNAs and even under these circumstances it was seen that Bengalin caused leukemic cell death. Flow cytometric analyses of the Bengalin treated caspase-3 blocked U937 cells failed to show an accumulation in either necrotic or apoptotic quadrants. Thus the probability of necrosis and apoptosis could be ruled out. Under this condition of Bengalin treatment and caspase3- blockade, cells were still dying. Thus it could be presumed that Bengalin mediated cell death was occurring by some alternate pathway other than apoptosis. So we investigated the role of ERK1/2 in this cell death process and upon blocking the activity of ERK1/2 with the inhibitor, PD98059, we found that the cell death process ceased considerably. Thus the probable role of ERK1/2 in the cell death cascade could be confirmed. Earlier reports suggested an involvement of ERK1/2 in inducing autophagic cell death (Cagnol and Chambard, 2010; Choi et al., 2010). Hence we decided to investigate the role of autophagy as an alternate pathway for apoptosis in causing Bengalin mediated cell death of the caspase-3 blocked cells. All our investigations were subsequently carried out with the caspase-3 blocked condition.
Fig. 3. Bengalin treatment increased percentage of AVO in caspase-3 and caspase-3 plus ERK1/2 inhibited U937 cells. Acridine orange staining was done for flow cytometric studies after 24 h of treatment of Bengalin to observe the percentage of AVO in caspase-3 blocked as well as cells which were blocked by caspase-3 as well as ERK inhibitors. Data are expressed as mean ± SD from at least three independent experiments. p-values were calculated using Dunnett's multiple comparison test. *p b 0.01.
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Fig. 4. Bengalin treatment increased the level of Beclin-1, Atg12, Atg7, Atg5, Atg3 and LC3-II and declined LC3-I expression. (A) Whole cell lysate was taken for western blot analysis to observe the expression level of Beclin-1, Atg12, Atg7, Atg5 and Atg3 protein in caspase-3 blocked U937 cells after 24 h of Bengalin treatment at a concentration 3.7 μg/ml. β-Actin served as a loading control. (B) Cells were initially treated with PD98059 and DEVD-CHO and then with Bengalin. Whole cell lysate was taken for western blot analysis to observe the expression level of Beclin-1, Atg12, Atg7, Atg5 and Atg3 protein. β-actin was used as loading control. (C) Caspase-3 blocked cells were treated with Bengalin for 0, 6, 12, 24, 36 h. Level of LC3-I and LC3-II was observed by western blot. Cells were also treated with ERK blocker as well as caspase-3 blocker followed by Bengalin treatment for different time intervals. Expression of LC3I and LC3II from the treated cells was observed by western blotting. β-Actin served as a loading control. Here the blot is a single representative of Bengalin treatment from three independent experiments with similar results.
Among the critical mediator of autophagic response is the PI3K/ Akt/mTOR pathway. Activation of PI3K/Akt/mTOR is known to negatively regulate autophagy (Saiki et al., 2011; Huang and Manning, 2009; Shinojima et al., 2007). In our studies there was a definite decrease of p-Akt (Ser473) and p-mTOR upon Bengalin treatment in U937 cells thereby suggesting a tentative role of their involvement in the autophagic response. Hence upon discovering a Bengalin initiated probable trend towards autophagic cell death in the caspase-3 blocked cells, we further proceeded towards analyzing the involvement of other downstream molecules engaged in the autophagic cascade. Autophagic process involves critical steps viz. initiation, elongation, maturation, autophagolysosome–lysosome fusion and ultimately degradation of the autophagolysosome (Tanida, 2011; Radoshevich and Debnath, 2011). This entire cascade is controlled by several
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proteins of which Beclin-1, Atg's and LC3 are critical effectors and help in proper progression of the process. Inactivated mTOR facilitates the formation of the active PI3KC-III and Beclin-1 complex required for nucleation (Kim et al., 2006). Beclin-1 again is negatively regulated by Bcl-2 (Pattingre et al., 2005). In our previous study we had observed that Bengalin could reduce the action of Bcl-2 (Das Gupta et al., 2010) in human leukemic cells while causing apoptosis. In our present study we observed a distinct augmentation of Beclin-1 which could be related to the Bengalin induced suppression of Bcl-2. For autophagy induction, the endogenous conjugate of Atg12 and Atg5 complex is required (Tanida, 2011). This complex in association with LC3-II is responsible for the formation of intracellular autophagosomes. The LC3-I gets activated by Atg-7 and then undergoes lipidation with phosphatidylethanolamine (PE) by Atg-3. This is followed by the completion stage where the cytoplasmic targets for autophagy are encapsulated in the autophagosome (Yu et al., 2004; Apel et al., 2009; Janku et al., 2011). Increase of Atg-12 and Atg-5 expressions due to Bengalin treatment could be suggestive of an induction of autophagy. However upon blocking ERK with PD98059 no alteration of Atg's and Beclin-1 could be seen. Our one of the major target molecule in determining the mode of cell death was the microtubule associated protein light chain 3 (LC3) molecule. LC3 exists in 2 forms viz. LC3-I and LC3-II. LC3-I is found ubiquitously expressed in the cytosol. On receiving autophagic stimulus, the LC3-I gets lipidated with phosphatidylethanolamine and thus converts to LC3-II. The produced LC3-II associates with both the inner and outer membranes of autophagosomes. Bengalin caused reduction in LC3-I expression followed by an upregulation of LC3-II, both of which were time dependant. The flux of LC3-II is transient as the autophagosomes with which it was present, fuse with the lysosomes and produce autophagolysosome where the inner membrane LC3-II gets degraded. However the outer membrane LC3-II detaches from PE by Atg-4 and converts back to LC3-I in the cytosol (Mizushima and Yoshimori, 2007). Our data with LC3-II are in conformation to this statement as Bengalin caused a time dependant decrease of LC3-I and a subsequent increase in LC3-II, the relation between the two molecules being reciprocal in Bengalin treated U937 cells. Since an increase of LC3-II expression is directly proportional to the formation of autophagosomes, suggesting a trend towards autophagic cell death hence it might be presumed that Bengalin induced an autophagic change in the caspase-3 blocked leukemic cells. Again upon blocking the activity of lysosomal proteases in the Bengalin treated cells, the LC3-II level did not decrease significantly underlying the involvement of lysosomes in the Bengalin induced cell death. Moreover the increase in the expression level of Atg-7 also highlights the fact of LC3-I activation and the subsequent increase in the Atg-3 corroborates the hypothesis of LC3-I lipidation to LC3-II. Further to this finding, blocking of ERK with PD98059 failed to show any time dependent alteration of LC3I or LC3II. This is suggestive of an absence of autophagy, which could be due to the inhibition of ERK upstream of caspase-3 irrespective of Bengalin treatment in U937 cells. The major efficacy of Bengalin is reflected in its capability to cause leukemic cell death. In our previous study, Bengalin caused apoptosis in leukemic cells through the intrinsic pathway. Moreover upon blocking the pivotal effector molecule caspase-3, Bengalin could still eradicate the human leukemic cells through an alternate pathway, which could be autophagic in nature. Hence the study is unique in the sense that it is the first report of any scorpion venom-component mediated autophagic cell death of human cancer cells. However further studies are warranted for an even better understanding of the detailed molecular mechanism of the Bengalin mediated autophagic response. Conclusion In conclusion, our studies demonstrated that Bengalin, a pure protein from scorpion venom could initiate a probable ERK1/2 mediated
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autophagic cell death pathway in human leukemic U937 cells, upon blocking its caspase-3 induced apoptogenic effect. When studies related to the whole scorpion venom or venom component induced apoptotic cancer cell death is still in its early stage, the present study with Bengalin is a crucial leap in unfolding an alternate mechanism of scorpion venom component mediated cancer cell eradication. This study might provide preliminary knowledge for further understanding of the highly sought after area of chemotherapeutic drug development from natural resources like venoms and toxins. Conflict of interest All authors declare no conflict of interest whatsoever.
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