C. elegans-based screen identifies lysosome-damaging alkaloids that induce STAT3-dependent lysosomal cell death
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Lysosomes are degradation and signaling centers within the cell, and their dysfunction impairs a wide variety of cellular processes. To understand the cellular effect of lysosome damage, we screened natural small-molecule compounds that induce lysosomal abnormality using Caenorhabditis elegans (C. elegans) as a model system. A group of vobasinyl-ibogan type bisindole alkaloids (ervachinines A–D) were identified that caused lysosome enlargement in C. elegans macrophage-like cells. Intriguingly, these compounds triggered cell death in the germ line independently of the canonical apoptosis pathway. In mammalian cells, ervachinines A–D induced lysosomal enlargement and damage, leading to leakage of cathepsin proteases, inhibition of autophagosome degradation and necrotic cell death. Further analysis revealed that this ervachinine-induced lysosome damage and lysosomal cell death depended on STAT3 signaling, but not RIP1 or RIP3 signaling. These findings suggest that lysosome-damaging compounds are promising reagents for dissecting signaling mechanisms underlying lysosome homeostasis and lysosome-related human disorders.
Keywordslysosome alkaloids lysosomal cell death STAT3 Caenorhabditis elegans
Lysosomes are acidic single-membrane organelles that function as the major sites for digesting cargoes received from several pathways, including endocytosis, phagocytosis and autophagy. The lysosome contains >60 different hydrolytic enzymes, many of which are activated at low pH. The acidity of the lysosomal lumen is generated and maintained by v-ATPase, an ATP-dependent proton pump. The lysosome contains over 150 membrane proteins that are required for the integrity and homeostasis of the organelle (Saftig and Klumperman, 2009). Impairment of lysosomal function is responsible for more than 70 lysosomal storage diseases (LSDs) (Macauley, 2016) and contributes to many other human diseases, such as neurodegenerative disorders and cancers (Nixon, 2013; Perera et al., 2015). Lysosomes also participate in several types of cell death including apoptosis and necroptosis (Taniguchi et al., 2015; Kreuzaler et al., 2011). In particular, lysosomal damage leads to lysosomal cell death (LCD) under specific physiological or pathological conditions, for instance, mammary gland involution after lactation, neutrophil aging, and bacterial infection (Sargeant et al., 2014; Loison et al., 2014; Prince et al., 2008).
LCD is characterized by lysosomal membrane permeabilization (LMP) and release of cathepsin proteases into the cytoplasm (Boya and Kroemer, 2008). In the cytoplasm, cathepsins act as executioners of cell death by mechanisms that are not well understood. Interestingly, while LCD is known to occur independently of caspases, cytoplasmic cathepsins can cleave Bid, promoting mitochondrial translocation of the proapoptotic proteins Bax and Bak, which in turn induce mitochondrial membrane permeabilization and caspase-dependent apoptosis (Oberle et al., 2010). Cathepsins can also promote the degradation of antiapoptotic proteins such as XIAP to facilitate apoptosis (Oberle et al., 2010). In addition, cathepsins promote LMP and thus amplify LCD signals (Oberle et al., 2010). The caspase-independent feature of LCD offers an important alternative for designing therapeutic strategies for cancer treatment. Because cancer cells generally carry mutations in proapoptotic factors or overexpression of antiapoptotic factors, they are usually resistant to apoptosis. However, it has been found that LCD can be induced in such apoptosis-resistant cells (Gonzalez et al., 2012). Induction of LCD was also found to restrict propagation of invading bacterial pathogens (Almaguel et al., 2010; Zhu et al., 2015). Thus, identifying potent LCD-inducing compounds may provide valuable reagents both for dissecting mechanisms underlying LCD and for treating lysosome-related human diseases.
In this study, we took advantage of the unique endo-lysosome system in macrophage-like cells in Caenorhabditis elegans (C. elegans) to screen for natural compounds that induced lysosomal abnormality. Our screen identified a group of vobasinyl-ibogan type bisindole alkaloids (ervachinines A–D) that caused abnormal lysosome enlargement in both C. elegans and mammalian cells. We further found that these lysosome-targeting natural compounds induced LMP and LCD in a STAT3-dependent manner. These findings suggest that ervachinines A–D are promising candidates for dissecting the signals underlying lysosome homeostasis and for developing therapeutic reagents for human disorders resulting from defective apoptosis.
Using C. elegans as a model to screen for natural compounds that induce lysosomal abnormality
To determine the identities of the enlarged vacuoles induced by HEC-23, we treated worms expressing endosome- or lysosome-specific proteins tagged with fluorescent proteins. HEC-23-enlarged vacuoles were positive for mCherry::CUP-5 (lysosomal calcium channel), LMP-1::GFP (lysosomal membrane protein) and ASP-1::dsRed (lysosomal hydrolase) (Fig. 1G–J). However, HEC-23 did not change the sizes of early endosomes labeled by 2xFYVE::GFP, an indicator of early endosome-specific phosphatidylinositol 3-phosphate (PI3P) (Fig. 1G). These results indicate that HEC-23 specifically enlarged lysosomes in coelomocytes.
HEC-23 impairs lysosomal degradation and increases the number of cell corpses in the germline
Because dysfunction of lysosomes contributes to cell death and affects cell corpse clearance, we tested whether HEC-23 affects apoptosis in germlines. In wild-type animals, HEC-23 treatment caused a significant increase in button-like structures that were encircled by GFP-tagged engulfment receptor CED-1 (CED-1::GFP) or the F-actin-binding protein moesin (GFP::moesin) (Xu et al., 2014) (Fig. 2E–I, and Table S1). This suggests that the button-like structures were dying cells. The ced-4(n1162) and ced-3(n717) mutants are respectively deficient in CED-4/Apaf1 and CED-3/Caspase, which are required for apoptosis, HEC-23 also induced button-like cell corpses in these mutants (Fig. 2J, K). Thus, HEC-23 likely induced cell death independently of the canonical apoptosis pathway (Horvitz et al., 1994). These cell corpses persisted much longer than the cell corpses resulting from physiological cell death (Fig. 2L), suggesting that HEC-23-induced lysosome damage also compromised the clearance of cell corpses.
HEC-23 induces lysosome enlargement in mammalian cells
HEC-23 inhibits autophagosome degradation
HEC-23-induced lysosomal enlargement depends on STAT3 activation
HEC-23 induces cathepsin-dependent necrosis through STAT3 signaling
In this study, we present C. elegans as a model for screening natural compounds that target lysosomes. We found that a group of alkaloids, named HECs, enlarge the size and impair the integrity, acidification and digestion capacity of lysosomes, and these effects are conserved in different species. HEC-induced lysosomal impairment results in the accumulation of cell corpses and non-apoptotic cell death in C. elegans and lysosomal cell death in mammalian cells.
The use of C. elegans to identify lysosome-targeting small-molecule compounds has many advantages. Firstly, the macrophage-like coelomocytes are active in fluid-phase endocytosis and pinocytosis, which facilitate the entry of compounds into the cell. Secondly, lysosomes are easily identified in a living animal using DIC optics or fluorescently labeled makers. Thirdly, the powerful genetic approaches and the availability of numerous mutant alleles make it possible to dissect the mechanisms underlying the function of the compounds.
Apoptosis and necrosis have been extensively studied during the development of organisms and the pathogenesis of diseases. In the present study, we find that mutation of neither ced-3 nor ced-4 could totally block the HEC-23-induced elevation of cell corpses. This demonstrates that HEC-23 induces non-apoptotic cell death in C. elegans. We also performed knock-down of RIP1 and RIP3, which did not reverse HEC-23-induced necrosis in mammalian cells. These data demonstrate that HEC-23-induced necrosis is independent of the RIP1/3 pathway.
Intriguingly, LMP also plays a crucial physiological role in regression of the mammary gland. Conditional deletion of STAT3 causes an obviously delay in involution of the mammary gland and reduces the level of cell death (Kreuzaler et al., 2011; Sargeant et al., 2014). In the present study, we found that HEC-23 induces the activation of STAT3, up-regulation of cathepsins B and L and down-regulation of Spi2A. Importantly, knock-down of STAT3 abolished lysosomal enlargement and necrosis induced by HEC-23 treatment. Furthermore, inhibition of cathepsins reverses the HEC-23-induced reduction in cell viability. These findings indicate that HEC23-induced necrosis is dependent on the STAT3-cathepsin pathway.
We found that HEC-23 treatment significantly promotes the phosphorylation of STAT3 at Y705. Unfortunately, we failed to obtain functional biotin-labeled compounds related to HEC-23, since they all lost the ability to enlarge lysosomes in cells. However, we can still propose how HEC-23 may interact with potential targets in the JAK-STAT3 signaling pathways. Firstly, HEC-23 may function as an agonist to directly bind with and activate JAK (Janus kinases), which in turn phosphorylates STAT3 at the Y705 site (Vainchenker and Constantinescu, 2013). Secondly, HEC-23 may function as an antagonist to bind with Src homology region 2 domain-containing phosphatase-1 (SHP-1) or suppressor of cytokine signaling (SOCS), which inhibits JAK activity in the cytoplasm (Vainchenker and Constantinescu, 2013). Thirdly, HEC-23 may function as an antagonist to bind with E3 SUMO-protein ligases (PIASs) or protein tyrosine phosphatases (PTPs), which inhibit STAT3 transcription activity in the nucleus (Vainchenker and Constantinescu, 2013).
Usually, LCD remains functional in apoptosis-resistant cancer cells. The fact that HEC-23 induced non-apoptotic cell death in several different cancer cell lines suggests that HEC-23 family compounds can potentially be used to develop therapeutic reagents for cancers that are resistant to apoptosis-based therapy. Notably, the antimalarial agent mefloquine was found to induce LMP and release of cathepsins into the cytoplasm of human acute myeloid leukemia (AML) cells, which provides a novel and promising therapeutic strategy for AML (Sukhai et al., 2013). Further studies are needed to investigate the application of HEC-23 and STAT3 activation to the therapeutic treatment of AML and other cancers.
Materials and Methods
C. elegans strains and genetics
The Bristol strain N2 is used as wild type. The mutant alleles used in this study are vps-18(tm1125), arl-8(tm2388), ced-3(n717) and ced-4(n1162). The integrated arrays are: smIs34(P ced-1 ced-1::gfp), yqIs121(P ced-1 gfp::moesin), cdIs85(Punc-1222xfyve::gfp), cdIs97(P unc-122 mCherry::cup-5), cdIs131(P unc-122 gfp::rab-5), pwIs50(P lmp-1 lmp-1::gfp), and tmIs225(P asp-1 asp-1::dsRed). C. elegans cultures and genetic crosses were performed according to standard procedures.
Screen for natural compounds that induce enlargement of lysosomes
All compounds used in this study were isolated from plants and compound structures were determined by means of 1D NMR, 2D NMR and MS as previously reported (Li et al., 2016). In total, 257 natural compounds (35 alkaloids, 23 triterpenes, 46 diterpenes, 22 sesquiterpenes, 17 monoterpenes, 18 tetranortriterpenoids, 12 flavonoids, 11 coumarins, 25 sterols, 27 lignanoids, 11 saponins, and 10 cyclic peptides) were tested individually for their ability to induce lysosome enlargement. Worms were cultured in M9 solution (1 L contains 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl and 1 mmol/L MgSO4) with X1666. 20–30 worms at L4 stage were transferred into liquid culture with or without different natural compounds in 24-well plates in a shaker (120 rpm, 20°C). 48 h or 72 h later, worms were examined for lysosomal changes in coelomocytes under a fluorescence microscope with DIC optics. The investigators were blinded to compound identities during the screen.
Microscopy and trafficking experiments in coelomocytes
Adult worms were immobilized with 2.5 mmol/L levamisole in M9 solution and mounted on 2% agarose pads for imaging. DIC pictures were captured by using an AxioImager M1 (Carl Zeiss). Fluorescence images were obtained by using an inverted FV1000 confocal microscope system (IX81; Olympus). TR-BSA trafficking assays in C. elegans coelomocytes were performed as previously described29. In brief, TR-BSA (Sigma-Aldrich; 1 mg/mL in water) was injected into the body cavity of adult worms. Then worms were cultured on NGM plates seeded with Escherichia coli OP50. Worms were imaged by confocal microscopy at different time points after injection. For each time point, similar results were obtained in more than 30 coelomocytes from 15–20 different worms.
Germ cell corpse analysis
Animals synchronized to different adult stages were scored for germ cell corpses under Nomarski optics. Animals were grown in liquid culture at 20°C unless otherwise indicated. For cell corpse analysis in HEC-23-treated worms, animals were scored for germ cell corpses after 12 h, 24 h, 36 h, 48 h and 72 h. At each time point, germ cell corpses in the meiotic region of one gonad arm were counted for every animal, and ≥30 animals were analyzed.
Cell culture, transfection and reagents
All cell lines were cultured at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (HyClone), 100 U/mL penicillin and 100 mg/mL streptomycin. No cell lines used in this study were found in the database of commonly misidentified cell lines that is maintained by ICLAC and NCBI Biosample. All cell lines were from American type culture collection (ATCC). Transient transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturers’ instructions. TR-BSA and CA-074-Me were from Tocris Bioscience (Bristol, UK). bafilomycin A1, 3-MA, z-VAD, necrosis inhibitor IM-54 and cisplatin were from Calbiochem (Darmstadt, Germany). The mature lysosome dye BODIPY-pepstatin A, LysoSensor, Annexin V and PI were purchased from Invitrogen Life Technologies (Carlsbad, CA). The antibody against LC3 was from MBL. Antibodies against p-STAT3 and STAT3 were from Cell Signaling Technology. Mouse monoclonal antibodies for α-tubulin were purchased from Sigma-Aldrich (St. Louis, MO). HRP-, Cy3- and FITC-conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).
Cells were lysed in ice-cold RIPA buffer (20 mmol/L Tris-HCl pH 7.5, 100 mmol/L NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1 mmol/L PMSF) containing Complete Protease Inhibitor Cocktail (1 tablet in 50 mL RIPA buffer) and Phosphatase Inhibitor Cocktail Tablets (1 tablet in 10 mL RIPA buffer) (Roche, Basel, Switzerland). Cell lysates were spun down at 12,000 rpm for 10 min at 4°C. 20 μg of each supernatant were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and probed with the indicated antibodies. α-Tubulin was used as the internal control.
Small interfering RNA (siRNA)
Cells were transfected with 100 pmol RNA oligos twice (at 0 h and 24 h) using Lipofectamine 2000 in 6-well plates or confocal culture dishes. The efficiency of siRNAs was evaluated by Western blot or qRT-PCR.
Quantitative real-time reverse-transcription PCR (qPCR)
RNA was isolated from cells by using TRIzol reagent (Invitrogen) as recommended by the manufacturer. A reverse transcription kit (Promega) was used to reverse transcribe RNA (1 μg) in a 20 μL-reaction mixture. Quantification of gene expression was performed using a real-time PCR system (7900HT Fast; Applied Biosystems) in triplicate. Amplification of the sequence of interest was normalized with the reference endogenous GAPDH gene.
The mammalian expression vector pEGFP-N2-cathepsin L was constructed by inserting the cDNA of cathepsin L between the HindIII and KpnI sites of the pEGFP-N2 vector using standard protocols confirmed by sequencing.
The following vectors were kindly provided by other scientists: mCherry-LAMP1 (Dr. Li Yu, Tsinghua University, China), EGFP-Galectin3 (Dr. Tamotsu Yoshimori, Osaka University, Japan), RFP-GFP-LC3 (Dr. Hong Zhang, Institute of Biophysics, CAS). All expression constructs were confirmed by DNA sequencing.
Transmission electron microscopy
HeLa cells were cultured on plastic cover slices with DMEM containing 10% FBS. After HEC-23 treatment for 3 h, the cells were fixed in fixation buffer (2.5% glutaraldehyde, 1% paraformaldehyde in PBS) on ice for 1 h. The samples were then post-fixed by 1% OsO4 for 2 h, followed by dehydration in a graded ethanol series (30%, 50%, 70%, 90% and 100%). After rinsing with propylene oxide (100%) for 3 times, the samples were infiltrated stepwise in increasing concentrations of embed 812 resin (propylene oxide:resin 2:1 for 3 h and 1:1 for 5 h). Then, the samples were incubated in 100% fresh resin twice for 8 h, and transferred into fresh resin in an embedding mold and polymerized in a 60°C oven for 3 days. Ultrathin sections (70 nm) were generated with a diamond knife (Diatome) on an ultramicrotome (Ultracut UCT; Leica Microsystems), and collected on copper grids (EMS). The slices on copper grids were stained with 2% UAc and 1% citric acid for 10 min. Then, the samples were visualized with a JEM-1400 TEM at 80 kV. Pictures were recorded with a Gatan832 (4k × 2.7k) CCD camera.
MTT assay for cell viability
Cells were cultured in 96-well plates with DMEM containing 10% FBS. After HEC-23 treatment for 24 h, 15 μL of dye solution was added into each well. Then the plates were incubated at 37°C for 2 h in a humidified CO2 incubator. 100 μL of stop solution was added to each well, and the absorbance was recorded at 570 nm using a 96-well plate reader. A reference wavelength at 630 nm was used. The MTT kit was purchased from Promega (Cat# G4002).
Cell death quantification by flow cytometry
After treatment with HEC-23 for 24 h, the cells were harvested and washed with PBS. Cells were stained in binding buffer containing Annexin V (5 μL) and PI (10 μL) for 15–30 min in the dark. After extensive washing, cells were suspended in PBS and transferred into tubes for quantification of cell death by flow cytometry using a FACS AriaII machine (BD Biosciences). Data were analyzed by using FlowJo software (FLOWJO, LLC).
Statistics and reproducibility
Data were analyzed with Prism (GraphPad Software) to generate curves and bar charts. Statistical analyses were performed using t-tests or ANOVA. P < 0.05, indicated with *, was considered statistically significant. P < 0.01, indicated with **, was considered significant. P < 0.001, indicated with ***, was considered extremely significant. P > 0.05 was considered not significant (NS).
This research was supported by grants 31230043 (to C.Yang), 21432010 (to X. Hao and C. Yang), and 81473122 (to Y. Zhang) from the National Natural Science Foundation of China, 2013CB910102 from the National Basic Research Program of China, and the CAS Interdisciplinary Innovation Team (to C. Yang), the Youth Innovation Promotion Association of CAS (2015323), CAS “Light of West China” Program (to Y. Zhang), the Young Academic and Technical Leader Raising Foundation of Yunnan Province (to Y. Zhang), the Technological Leading Talent Project of Yunnan Province (to X. Hao), the Startup Funding of Fudan University (to Y. Li), and Funding for Construction of Outstanding Universities in Shanghai (to Y. Li). We also appreciate Dr. Isabel Hanson to proofread this manuscript.
AML, acute myeloid leukemia; C. elegans, Caenorhabditis elegans; DIC, differential interference contrast; FBS, fetal bovine serum; LCD, lysosomal cell death; LMP, lysosomal membrane permeabilization; LSDs, lysosomal storage diseases; PI, propidium iodide; PTPs, protein tyrosine phosphatases; SHP-1, Src homology region 2 domain-containing phosphatase-1; SOCS, suppressor of cytokine signaling; TEM, transmission electron microscopy
Y Li, C. Yang, Y. Zhang and X. Hao designed the experiments and wrote the paper. Q. Gan and M. Xu contributed to the experiments shown on Fig. 2E–H and J–L. X. Ding and G. Tang contributed to compound screening. K. Liu, X. Liu, L. Guo and Z. Gao contributed materials. J. Liang and X. Wang performed transmission electron microscopy. Y. Li and Y. Zhang analyzed results. All authors reviewed and approved the final version of the manuscript.
COMPLIANCE WITH ETHICS GUIDELINES
Yang Li, Yu Zhang, Qiwen Gan, Meng Xu, Xiao Ding, Guihua Tang, Jingjing Liang, Kai Liu, Xuezhao Liu, Xin Wang, Lingli Guo, Zhiyang Gao, Xiaojiang Hao, and Chonglin Yang declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed.
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