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• W2088055025 abstract "Artesunate (ART) is an anti-malaria drug that has been shown to exhibit anti-tumor activity, and functional lysosomes are reported to be required for ART-induced cancer cell death, whereas the underlying molecular mechanisms remain largely elusive. In this study, we aimed to elucidate the molecular mechanisms underlying ART-induced cell death. We first confirmed that ART induces apoptotic cell death in cancer cells. Interestingly, we found that ART preferably accumulates in the lysosomes and is able to activate lysosomal function via promotion of lysosomal V-ATPase assembly. Furthermore, we found that lysosomes function upstream of mitochondria in reactive oxygen species production. Importantly, we provided evidence showing that lysosomal iron is required for the lysosomal activation and mitochondrial reactive oxygen species production induced by ART. Finally, we showed that ART-induced cell death is mediated by the release of iron in the lysosomes, which results from the lysosomal degradation of ferritin, an iron storage protein. Meanwhile, overexpression of ferritin heavy chain significantly protected cells from ART-induced cell death. In addition, knockdown of nuclear receptor coactivator 4, the adaptor protein for ferritin degradation, was able to block ART-mediated ferritin degradation and rescue the ART-induced cell death. In summary, our study demonstrates that ART treatment activates lysosomal function and then promotes ferritin degradation, subsequently leading to the increase of lysosomal iron that is utilized by ART for its cytotoxic effect on cancer cells. Thus, our data reveal a new mechanistic action underlying ART-induced cell death in cancer cells. Artesunate (ART) is an anti-malaria drug that has been shown to exhibit anti-tumor activity, and functional lysosomes are reported to be required for ART-induced cancer cell death, whereas the underlying molecular mechanisms remain largely elusive. In this study, we aimed to elucidate the molecular mechanisms underlying ART-induced cell death. We first confirmed that ART induces apoptotic cell death in cancer cells. Interestingly, we found that ART preferably accumulates in the lysosomes and is able to activate lysosomal function via promotion of lysosomal V-ATPase assembly. Furthermore, we found that lysosomes function upstream of mitochondria in reactive oxygen species production. Importantly, we provided evidence showing that lysosomal iron is required for the lysosomal activation and mitochondrial reactive oxygen species production induced by ART. Finally, we showed that ART-induced cell death is mediated by the release of iron in the lysosomes, which results from the lysosomal degradation of ferritin, an iron storage protein. Meanwhile, overexpression of ferritin heavy chain significantly protected cells from ART-induced cell death. In addition, knockdown of nuclear receptor coactivator 4, the adaptor protein for ferritin degradation, was able to block ART-mediated ferritin degradation and rescue the ART-induced cell death. In summary, our study demonstrates that ART treatment activates lysosomal function and then promotes ferritin degradation, subsequently leading to the increase of lysosomal iron that is utilized by ART for its cytotoxic effect on cancer cells. Thus, our data reveal a new mechanistic action underlying ART-induced cell death in cancer cells. Artesunate (ART), 3The abbreviations used are: ARTartesunateNCOA4nuclear receptor coactivator 4ROSreactive oxygen speciesBAFbafilomycin A1NACN-acetylcysteineDFOdeferoxamine mesylateFTHferritin heavy chainFTLferritin light chainV-ATPasevacuolar H+-ATPaseDHAdihydroartemisininMEFmouse embryonic fibroblastZN-benzyloxycarbonylfmkfluoromethylketoneLTRLysoTracker RedLTGLysoTracker GreenMTRMitoTracker RedMSRMitoSox RedTFEBtranscription factor EBCtrlcontrolPIpropidium iodideLC3light chain 3mTORC1mammalian or mechanistic target of rapamycin complex 1ATGautophagy-related geneIRPiron-responsive protein. a water-soluble derivative of artemisinin, has been widely used for treatment of malaria (1China Cooperative Research Group on Qinghaosu and Its Derivatives as AntimalarialsChemical studies on qinghaosu (artemisinine).J. Tradit. Chin. Med. 1982; 2: 3-8PubMed Google Scholar). ART, together with other derivatives of artemisinin, was first shown to exhibit toxicity to Ehrlich ascites tumor cells (2Woerdenbag H.J. Moskal T.A. Pras N. Malingré T.M. el-Feraly F.S. Kampinga H.H. Konings A.W. Cytotoxicity of artemisinin-related endoperoxides to Ehrlich ascites tumor cells.J. Nat. Prod. 1993; 56: 849-856Crossref PubMed Scopus (268) Google Scholar). At present, there is an increasing amount of evidence suggesting that ART has an anti-cancer function (3Ho W.E. Peh H.Y. Chan T.K. Wong W.S. Artemisinins: pharmacological actions beyond anti-malarial.Pharmacol. Ther. 2014; 142: 126-139Crossref PubMed Scopus (340) Google Scholar). For instance, ART has been shown to induce apoptosis and necrosis in multiple human cancer cells (4Du J.H. Zhang H.D. Ma Z.J. Ji K.M. Artesunate induces oncosis-like cell death in vitro and has antitumor activity against pancreatic cancer xenografts in vivo.Cancer Chemother. Pharmacol. 2010; 65: 895-902Crossref PubMed Scopus (126) Google Scholar, 5Zhou C. Pan W. Wang X.P. Chen T.S. Artesunate induces apoptosis via a Bak-mediated caspase-independent intrinsic pathway in human lung adenocarcinoma cells.J. Cell Physiol. 2012; 227: 3778-3786Crossref PubMed Scopus (66) Google Scholar). The anti-cancer effects of ART also include the following: cell cycle arrest (6Zhao Y. Jiang W. Li B. Yao Q. Dong J. Cen Y. Pan X. Li J. Zheng J. Pang X. Zhou H. Artesunate enhances radiosensitivity of human non-small cell lung cancer A549 cells via increasing NO production to induce cell cycle arrest at G2/M phase.Int. Immunopharmacol. 2011; 11: 2039-2046Crossref PubMed Scopus (64) Google Scholar), inhibition of angiogenesis (7Dell'Eva R. Pfeffer U. Vené R. Anfosso L. Forlani A. Albini A. Efferth T. Inhibition of angiogenesis in vivo and growth of Kaposi's sarcoma xenograft tumors by the anti-malarial artesunate.Biochem. Pharmacol. 2004; 68: 2359-2366Crossref PubMed Scopus (218) Google Scholar), and reduction of cell invasion and metastasis (8Rasheed S.A. Efferth T. Asangani I.A. Allgayer H. First evidence that the antimalarial drug artesunate inhibits invasion and in vivo metastasis in lung cancer by targeting essential extracellular proteases.Int. J. Cancer. 2010; 127: 1475-1485Crossref PubMed Scopus (98) Google Scholar). Moreover, the anti-cancer potential of ART has also been tested in a number of animal cancer models (4Du J.H. Zhang H.D. Ma Z.J. Ji K.M. Artesunate induces oncosis-like cell death in vitro and has antitumor activity against pancreatic cancer xenografts in vivo.Cancer Chemother. Pharmacol. 2010; 65: 895-902Crossref PubMed Scopus (126) Google Scholar, 6Zhao Y. Jiang W. Li B. Yao Q. Dong J. Cen Y. Pan X. Li J. Zheng J. Pang X. Zhou H. Artesunate enhances radiosensitivity of human non-small cell lung cancer A549 cells via increasing NO production to induce cell cycle arrest at G2/M phase.Int. Immunopharmacol. 2011; 11: 2039-2046Crossref PubMed Scopus (64) Google Scholar, 9Hou J. Wang D. Zhang R. Wang H. Experimental therapy of hepatoma with artemisinin and its derivatives: in vitro and in vivo activity, chemosensitization, and mechanisms of action.Clin. Cancer Res. 2008; 14: 5519-5530Crossref PubMed Scopus (272) Google Scholar). However, the exact mechanisms underlying ART-mediated cell death have not been fully elucidated. artesunate nuclear receptor coactivator 4 reactive oxygen species bafilomycin A1 N-acetylcysteine deferoxamine mesylate ferritin heavy chain ferritin light chain vacuolar H+-ATPase dihydroartemisinin mouse embryonic fibroblast N-benzyloxycarbonyl fluoromethylketone LysoTracker Red LysoTracker Green MitoTracker Red MitoSox Red transcription factor EB control propidium iodide light chain 3 mammalian or mechanistic target of rapamycin complex 1 autophagy-related gene iron-responsive protein. Lysosomes are the key intracellular organelles that perform the digestive function via the endocytic or autophagic pathway (10Eskelinen E.L. Tanaka Y. Saftig P. At the acidic edge: emerging functions for lysosomal membrane proteins.Trends Cell Biol. 2003; 13: 137-145Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). Lysosomal hydrolases and integral lysosomal membrane proteins are essential for lysosomal function (11Saftig P. Klumperman J. Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function.Nat. Rev. Mol. Cell Biol. 2009; 10: 623-635Crossref PubMed Scopus (1095) Google Scholar). The internal pH of lysosomes is acidic (below pH 5), which is mainly controlled by the vacuolar H+-ATPase (V-ATPase) complex (12Mindell J.A. Lysosomal acidification mechanisms.Annu. Rev. Physiol. 2012; 74: 69-86Crossref PubMed Scopus (708) Google Scholar). On the other hand, lysosomes are known to play a critical role in autophagy; at the maturation/degradation stage, autophagosomes fuse with lysosomes to form autolysosomes for degradation (13Mizushima N. Komatsu M. Autophagy: renovation of cells and tissues.Cell. 2011; 147: 728-741Abstract Full Text Full Text PDF PubMed Scopus (3920) Google Scholar, 14Settembre C. Fraldi A. Medina D.L. Ballabio A. Signals from the lysosome: a control centre for cellular clearance and energy metabolism.Nat. Rev. Mol. Cell Biol. 2013; 14: 283-296Crossref PubMed Scopus (1081) Google Scholar). At present, the effects of ART on autophagy and lysosome remain controversial. It has been reported that ART inhibits autophagy via inhibition of lysosomal turnover without affecting lysosomal functions in breast cancer cells (15Hamacher-Brady A. Stein H.A. Turschner S. Toegel I. Mora R. Jennewein N. Efferth T. Eils R. Brady N.R. Artesunate activates mitochondrial apoptosis in breast cancer cells via iron-catalyzed lysosomal reactive oxygen species production.J. Biol. Chem. 2011; 286: 6587-6601Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). In contrast, one recent report showed that ART promotes autophagy via up-regulating the expression of Beclin 1, which was also studied in breast cancer cells (16Chen K. Shou L.M. Lin F. Duan W.M. Wu M.Y. Xie X. Xie Y.F. Li W. Tao M. Artesunate induces G2/M cell cycle arrest through autophagy induction in breast cancer cells.Anticancer Drugs. 2014; 25: 652-662Crossref PubMed Scopus (75) Google Scholar). Moreover, ART-induced cancer cell death was shown to be dependent on lysosomes, although the mechanism is unclear (15Hamacher-Brady A. Stein H.A. Turschner S. Toegel I. Mora R. Jennewein N. Efferth T. Eils R. Brady N.R. Artesunate activates mitochondrial apoptosis in breast cancer cells via iron-catalyzed lysosomal reactive oxygen species production.J. Biol. Chem. 2011; 286: 6587-6601Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Iron is an essential nutrient, and its intracellular availability is tightly regulated. One of the key regulatory mechanisms involves ferritin (17Rouault T.A. The role of iron regulatory proteins in mammalian iron homeostasis and disease.Nat. Chem. Biol. 2006; 2: 406-414Crossref PubMed Scopus (788) Google Scholar). Ferritin functions as the major iron storage protein in mammals and consists of 24 protein subunits that can store up to 4,500 atoms of iron per ferritin (18Ford G.C. Harrison P.M. Rice D.W. Smith J.M. Treffry A. White J.L. Yariv J. Ferritin: design and formation of an iron-storage molecule.Philos. Trans. R. Soc. Lond. B Biol. Sci. 1984; 304: 551-565Crossref PubMed Scopus (482) Google Scholar). There are two subunits of ferritin, ferritin heavy chain (FTH) and ferritin light chain (FTL). FTH has a ferroxidase function that can catalyze extracellular iron into nontoxic ferric iron form and store this form of iron in the ferritin complex (19Harrison P.M. Arosio P. The ferritins: molecular properties, iron storage function and cellular regulation.Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2246) Google Scholar). Ferritin is stable in iron-rich conditions, whereas it is rapidly degraded under conditions of iron starvation (20Asano T. Komatsu M. Yamaguchi-Iwai Y. Ishikawa F. Mizushima N. Iwai K. Distinct mechanisms of ferritin delivery to lysosomes in iron-depleted and iron-replete cells.Mol. Cell Biol. 2011; 31: 2040-2052Crossref PubMed Scopus (164) Google Scholar). Ferritin has been reported to be degraded either in the lysosomes or by the proteasomes depending on the cellular stimulants (21De Domenico I. Ward D.M. Kaplan J. Specific iron chelators determine the route of ferritin degradation.Blood. 2009; 114: 4546-4551Crossref PubMed Scopus (134) Google Scholar). Recently, the nuclear receptor coactivator 4 (NCOA4) was identified as the cargo for the ferritin degradation via autophagic pathway, and the autophagic turnover of ferritin is termed ferritinophagy (22Mancias J.D. Wang X. Gygi S.P. Harper J.W. Kimmelman A.C. Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy.Nature. 2014; 509: 105-109Crossref PubMed Scopus (810) Google Scholar). To date, the effect of ART on ferritin and the link between free iron ions and lysosomal function in ART-induced cell death have not been well studied. In this study, we aimed to clarify the effect of ART on autophagy and lysosomal function as well as to elucidate the underlying molecular mechanisms of how lysosome is involved in ART-induced cell death. Here, we utilized a blue fluorescence-tagged ART to investigate its localization, and our results clearly showed that ART accumulates in lysosome. Interestingly, we found that ART promotes lysosomal function via engaging lysosomal V-ATPase. More importantly, we provided clear evidence demonstrating that ferritin degradation via lysosomes is essential for ART-induced cell death. The results from our study shed new light on the molecular mechanisms underlying ART-induced cell death and support the development of this important anti-malaria drug as a cancer therapeutic agent. HeLa and HepG2 cells were obtained from American Type Culture Collection. The GFP-LC3-expressing stable HeLa cells were provided by Dr. N. Mizushima (Tokyo Medical and Dental University, Japan), TSC2-WT and TSC2-KO MEFs were obtained from Dr. D. J. Kwiatkowski (23Zhang H. Cicchetti G. Onda H. Koon H.B. Asrican K. Bajraszewski N. Vazquez F. Carpenter C.L. Kwiatkowski D.J. Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR.J. Clin. Invest. 2003; 112: 1223-1233Crossref PubMed Scopus (443) Google Scholar). All cell lines were maintained in DMEM (Sigma-Aldrich, D1152) containing 10% fetal bovine serum (HyClone, SV30160.03) in a 5% CO2 atmosphere at 37 °C. After the indicated times of designated treatments, cells were collected and rinsed with PBS. The whole cell lysates were prepared in the Laemmli buffer (62.5 mm Tris-HCl, pH 6.8, 20% glycerol, 2% SDS, 2 mm DTT, phosphatase inhibitor, and proteinase inhibitor mixture). Protein concentrations were determined by the DCTM protein assay (Bio-Rad, 162-0177). An equal amount of protein was resolved by SDS-PAGE and transferred onto PVDF membrane (Bio-Rad). After blocking with StartingBlock blocking buffers (Thermo Scientific, 37538), the membrane was probed with designated first and second antibodies, developed with the enhanced chemiluminescence method (Thermo Scientific, 34076), and visualized using Kodak Image Station 4000R (Eastman Kodak Co.). Antibodies were obtained as follows: anti-microtubule-associated protein 1 light chain 3 (LC3) antibody (Sigma-Aldrich, L7543), anti-ATG7 antibody (ProScience, 3617), anti-tubulin (Sigma-Aldrich, T6199), anti-FLAG (Sigma-Aldrich, F3165), anti-β-actin (Sigma-Aldrich, A5441), anti-V-ATPase D1 (Santa Cruz Biotechnology, Inc., SC-69105), anti-V-ATPase B2 (Santa Cruz Biotechnology, SC-166122), anti-iron-responsive protein 2 (IRP2) (Santa Cruz Biotechnology, SC-33682), anti-FTH (Abcam, ab65080), anti-FTL (Abcam, ab69090), anti-TFEB (Bethyl Laboratories, A303-673A), and anti-NCOA4 (Sigma, SAB1404569). All of the other antibodies were purchased from Cell Signaling Technology: anti-lysosome-associated membrane protein 1 (LAMP1) antibody (catalog nos. 3243S and 9091S), anti-phospho-S6 (catalog no. 2211), anti-S6 (catalog no. 2217), caspase 3 (catalog no. 9662), and anti-PARP-1 (catalog no. 9542). Briefly, cells were first cultured on 8-well Lab-TekTM chambered coverglass (Thermo Scientific, 155411) overnight, followed by designated treatment. All of the confocal images were obtained with 60× oil objective (numerical aperture 1.4) lenses of Olympus Fluoview FV1000. The images were processed with FV10-ASW 3.0 Viewer software. Cell death was estimated by morphological changes under phase-contrast microscopy and quantified by a propidium iodide (PI; 5 μg/ml) exclusion assay coupled with flow cytometry (BD Biosciences). Western blotting was also used to indicate the cell death via PARP-1 and caspase-3 cleavages. The cells were treated with blue fluorescent ART (20 μm, LynxTag-ARTTMASBlue, from BioLynx Technologies) with or without bafilomycin A1 (BAF) (50 nm) in full DMEM for 30 min. Subsequently, 50 nm LysoTracker Red DND-99 (LTR; Invitrogen, L7528) or MitoTracker Red CMXRos (MTR; Invitrogen, M7512) was added for 30 min. The cells were washed with PBS twice, and DMEM full medium was added into the well, followed by observation under the confocal microscope. After the designated treatments, cells were incubated with 50 nm LTR or LysoTracker Green DND-26 (LTG; Invitrogen, L7526) reagents in full DMEM for 30 min for labeling and tracking acidic organelles in live cells. For labeling mitochondria, the cells were incubated with 50 nm MTR in PBS for 15 min and then washed twice with PBS followed by incubation of full DMEM for the imaging or collection for flow cytometry. The cells in the chambered coverglass were observed under a confocal microscope. The cells from the 24-well plate were collected, and the fluorescence intensities of 10,000 cells/sample were measured by flow cytometry using the BD FACS cytometer (BD Biosciences). We recorded the fluorescence of LTR and MTR using the FL-2 channel of FACS (BD Biosciences). Lysosomal function was also estimated by the cathepsin B and L enzymatic activity. After designated treatment, cells were further loaded with Magic RedTM cathepsin B (Immunochemistry Technologies, 938) or cathepsin L (Immunochemistry Technologies, 942) reagents for 30 min. The cells in the chambered coverglass were observed under a confocal microscope. The cells from the 24-well plate were collected, and the fluorescence intensities of 10,000 cells per sample were measured by flow cytometry. We recorded the fluorescence of Magic Red using the FL-2 channel of FACS. Lysosomal protein proteolysis was estimated by DQ Red BSA (Invitrogen, D12051) staining. Cells were first incubated with DQ Red BSA for 1 h and then washed with PBS two times, followed by the designated treatment. The cells in the chambered coverglass were observed under a confocal microscopy. The cells from the 24-well plate were collected, and the fluorescence intensities of 10,000 cells/sample were measured by flow cytometry. We recorded the fluorescence of DQ Red BSA using the FL-3 channel of FACS. Cells were first cultured in an 8-well chambered coverglass overnight. After the designated treatment, cells were first fixed with 4% paraformaldehyde in PBS for 15 min at 37 °C and then permeabilized with 0.01% saponin in PBS for 10 min. After blocking with 1% BSA in PBS for 30 min, cells were incubated with V-ATPase B2 primary antibody (Santa Cruz Biotechnology, SC-166122) in a 1:100 dilution overnight at 4 °C. On the second day, the cells were then incubated with LAMP1 in a 1:100 dilution (Cell Signaling Technology, 9091S) for 3 h at room temperature, followed by Alexa Fluor 488 goat anti-mouse secondary antibody (Invitrogen, A-11029) and Alexa Fluor 555 donkey anti-rabbit secondary antibody (Invitrogen, A-31572). The cells were examined using a confocal microscope, and representative cells were selected and photographed. The use of two different proximity ligation assay probes with amplifiable DNA reporter enabled us to visualize and quantify the protein-protein interactions in situ (24Jarvius M. Paulsson J. Weibrecht I. Leuchowius K.J. Andersson A.C. Wählby C. Gullberg M. Botling J. Sjöblom T. Markova B. Ostman A. Landegren U. Söderberg O. In situ detection of phosphorylated platelet-derived growth factor receptor β using a generalized proximity ligation method.Mol. Cell Proteomics. 2007; 6: 1500-1509Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). The HeLa cells were first seeded in a 16-well chamber. Treated cells were first fixed with 4% paraformaldehyde for 15 min at 37 °C and then permeabilized with 0.01% saponin in PBS for 10 min, followed by blocking with 1% BSA in PBS for 30 min. Cells were then incubated with anti V-ATPase V1 domain subunit B2 (V1B2) and anti-V-ATPase V0 domain subunit D1 (V0D1) in a 1:100 dilution, incubated overnight at 4 °C. The chamber was then performed with the procedure based on the manufacturer's instructions (Olink Bioscience). The scrambled RNAi oligonucleotides (Dharmacon, ON-TARGETplus Non-targeting Pool, D-001810–10-05) and siRNAs targeting ATG7 (Dharmacon, SMARTpool, ON-TARGETplus human ATG7, L-020112-00-0005; target sequences: CCAACACACUCGAGUCUUU, GAUCUAAAUCUCAAACUGA, GCCCACAGAUGGAGUAGCA, and GCCAGAGGAUUCAACAUGA), TFEB (Dharmacon, SMARTpool, ON-TARGETplus human TFEB, L-009798-00-0005; target sequences: CAACAGUGCUCCCAAUAGC, GCAGCCACCUGAAUGUGUA, UGAAAGGAGACGAAGGUUC, and GCAGAUGCCCAACACGCUA), and NCOA4 (Dharmacon, SMARTpool, ON-TARGETplus human NCOA4, L-010321-00-0005; target sequences: CAGAUUCACAGUUGCAUAA, ACAAAGAUCUAGCCAAUCA, ACAAGUGGCUGCUUCGAAA, and GAGAAGUGGUUAUAUCGAA) were transfected into HeLa cells using the DharmaFECT 4 Transfection Reagent (Dharmacon, T-2001-02) according to the manufacturer's protocol. After 48 h, the cells were subjected to the designated treatment. For plasmid transfection, HeLa cells were transiently transfected with pcDNA or FTH-FLAG plasmid using LipofectamineTM 2000 according to the manufacturer's protocol. After 24 h, the cells were treated as indicated. CM-H2DCFDA (Invitrogen, C6827) and MitoSOXTM Red (MSR; Invitrogen, M36008) were chosen for the detection of intracellular ROS and mitochondrial superoxide production, respectively. When CM-H2DCFDA passively diffuses into cells, its acetate groups are cleaved by intracellular esterases and subsequently oxidized by ROS and yield a fluorescent adduct, CM-DCF (25Aon M.A. Cortassa S. Marbán E. O'Rourke B. Synchronized whole cell oscillations in mitochondrial metabolism triggered by a local release of reactive oxygen species in cardiac myocytes.J. Biol. Chem. 2003; 278: 44735-44744Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar). MSR is a fluoroprobe for detection of superoxide in the mitochondria of live cells (26Mukhopadhyay P. Rajesh M. Yoshihiro K. Haskó G. Pacher P. Simple quantitative detection of mitochondrial superoxide production in live cells.Biochem. Biophys. Res. Commun. 2007; 358: 203-208Crossref PubMed Scopus (258) Google Scholar). Briefly, cells were first cultured in a Lab-TekTM chambered coverglass or 24-well plate overnight. After the designated treatments, cells were incubated with 5 μm MSR or 1 μm CM-H2DCFDA in PBS for 10 min. Then the MSR or CM-H2DCFDA was removed, and the cells were washed with PBS twice. The cells in the coverglass were incubated in full medium and observed under a confocal microscope. The cells in the 24-well plate were collected, and fluorescence intensity was measured. We recorded the fluorescence of CM-DCF using the FL-1 channel and MSR with the FL-2 channel of FACS (BD Biosciences). TFEB luciferase vector was provided by Dr. A. Ballabio (27Sardiello M. Palmieri M. di Ronza A. Medina D.L. Valenza M. Gennarino V.A. Di Malta C. Donaudy F. Embrione V. Polishchuk R.S. Banfi S. Parenti G. Cattaneo E. Ballabio A. A gene network regulating lysosomal biogenesis and function.Science. 2009; 325: 473-477Crossref PubMed Scopus (1568) Google Scholar). The transient transfection of the TFEB luciferase vector was done in HeLa cells using LipofectamineTM 2000 transfection reagent according to the manufacturer's protocols. Renilla luciferase vector was used as a transfection control. The luciferase activity was measured at 48 h after transfection using the Dual-Luciferase reporter assay system (Promega, E1960) based on the protocol provided by the manufacturer. Briefly, following the treatments, the cell lysate was collected from each well after the addition of cell lysis reagent. After the addition of luciferase assay substrate, the firefly luciferase activity was determined using a luminometer (Promega), and the Renilla luciferase activity was then measured by adding the Stop & Glo substrate. RNA was extracted with the RNeasy kit (Qiagen, 217004). A reverse transcription reaction was performed using 1 μg of total RNA with iScriptTM Reverse Transcription Supermix for RT-qPCR (Bio-Rad, 170-8841). The mRNA expression levels were determined by real-time PCR using SsoFast EvaGreen Supermix (Bio-Rad, 172-5201AP) and the CFX96 Touch Real-time PCR Detection System (Bio-Rad). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control of RNA integrity. Real-time PCR was performed in triplicate. The primers used for FTH and FTL were purchased from Qiagen: Hs_FTH1_1_SG QuantiTect primer assay (QT00072681) and Hs_FTL_1_SG QuantiTect Primer Assay (QT00055860). All Western blot data and image data presented are representative of three independent experiments. The numeric data except for quantitative RT-PCR data are presented as mean ± S.D. from three independent experiments and analyzed using Student's t test. Quantitative RT-PCR data are presented as mean ± S.D. from two independent experiments (each in triplicate). ART has been shown to inhibit cell growth, induce radiosensitivity, and enhance TRAIL-induced apoptosis in human cervical cancer HeLa cells (28Luo J. Zhu W. Tang Y. Cao H. Zhou Y. Ji R. Zhou X. Lu Z. Yang H. Zhang S. Cao J. Artemisinin derivative artesunate induces radiosensitivity in cervical cancer cells in vitro and in vivo.Radiat. Oncol. 2014; 9: 84Crossref PubMed Scopus (34) Google Scholar, 29Thanaketpaisarn O. Waiwut P. Sakurai H. Saiki I. Artesunate enhances TRAIL-induced apoptosis in human cervical carcinoma cells through inhibition of the NF-κB and PI3K/Akt signaling pathways.Int. J. Oncol. 2011; 39: 279-285PubMed Google Scholar, 30Chen H.H. Zhou H.J. Fang X. Inhibition of human cancer cell line growth and human umbilical vein endothelial cell angiogenesis by artemisinin derivatives in vitro.Pharmacol. Res. 2003; 48: 231-236Crossref PubMed Scopus (207) Google Scholar). In this study, we first examined the cytotoxicity of ART on HeLa cells. We found that ART (50 μm for 48 h) caused evident cell death in HeLa cells, as assessed by morphological changes (Fig. 1A, top), Hoechst staining for chromatin condensation (Fig. 1A, bottom), and quantitatively through PI-live cell exclusion test (Fig. 1, B and C). ART-induced cell death was also found to be dose-dependent in HeLa cells (Fig. 1D). In addition to its toxicity, treatment with ART alone or combined with N-benzyloxycarbonyl-Phe-Ala-fluoromethylketone (Z-VAD-fmk) was also shown to reduce the number of cells (Fig. 1A), which is consistent with the earlier findings that ART inhibits cancer cell proliferation (6Zhao Y. Jiang W. Li B. Yao Q. Dong J. Cen Y. Pan X. Li J. Zheng J. Pang X. Zhou H. Artesunate enhances radiosensitivity of human non-small cell lung cancer A549 cells via increasing NO production to induce cell cycle arrest at G2/M phase.Int. Immunopharmacol. 2011; 11: 2039-2046Crossref PubMed Scopus (64) Google Scholar, 16Chen K. Shou L.M. Lin F. Duan W.M. Wu M.Y. Xie X. Xie Y.F. Li W. Tao M. Artesunate induces G2/M cell cycle arrest through autophagy induction in breast cancer cells.Anticancer Drugs. 2014; 25: 652-662Crossref PubMed Scopus (75) Google Scholar). To further examine the type of cell death induced by ART, we tested the protective effect of the general caspase inhibitor Z-VAD-fmk on ART-mediated cell death. As shown in Fig. 1, A–C, the cell death induced by ART was dramatically protected by Z-VAD-fmk. Consistently, we also observed cleavage of both caspase-3 (17 kDa) and PARP-1 (89 kDa), both classical markers of apoptotic cell death in HeLa cells treated with ART (Fig. 1E). The caspase-3 and PARP cleavage can also be blocked by Z-VAD-fmk (Fig. 1F). Similar results were also observed in HepG2 cells (data not shown). In order to understand the molecular mechanisms underlying ART-mediated cell death, we utilized LynxTag-ARTTM ASBlue, a blue fluorescence-tagged ART, to investigate its cellular localization. As shown in Fig. 2, ART was found to be predominantly localized in lysosomes, as evidenced by its co-staining with LTR (Fig. 2A) and LAMP1 (Fig. 2B). There was little colocalization of ART with MTR (Fig. 2C). Altogether, the data showed that ART accumulates in the lysosomes. Notably, treatment with BAF, an inhibitor of lysosomal acidification via inhibition of the lysosomal V-ATPase activity (31Yoshimori T. Yamamoto A. Moriyama Y. Futai M. Tashiro Y. Bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells.J. Biol. Chem. 1991; 266: 17707-17712Abstract Full Text PDF PubMed Google Scholar), abolished LTR staining of the lysosomes (Fig. 2A), whereas accumulation of ART was not affected by BAF treatment (Fig. 2B), suggesting that ART accumulation in lysosomes is independent of lysosomal pH. An e" @default.
• W2088055025 created "2016-06-24" @default.
• W2088055025 creator A5019344890 @default.
• W2088055025 creator A5027631499 @default.
• W2088055025 creator A5028525767 @default.
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• W2088055025 date "2014-11-01" @default.
• W2088055025 modified "2023-10-15" @default.
• W2088055025 title "Artesunate Induces Cell Death in Human Cancer Cells via Enhancing Lysosomal Function and Lysosomal Degradation of Ferritin" @default.
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