Necrocide 1 Mediates a Non-Apoptotic Necrotic Cell Death and Immunogenic Response in Human Cancer Cells

doi:10.21203/rs.3.rs-2090541/v1

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Necrocide 1 Mediates a Non-Apoptotic Necrotic Cell Death and Immunogenic Response in Human Cancer Cells

https://doi.org/10.21203/rs.3.rs-2090541/v1

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Many anticancer agents induce apoptosis, mitotic catastrophe or senescence. Here, we report the functional characterization of an experimental inducer of TNF-independent necrosis, necrocide-1 (NC1). NC1 (but not its stereoisomer) killed a panel of human cancer cell lines (but not normal cells) at nanomolar concentrations and induced a non-apoptotic, necrotic morphotype, both in vitro and in vivo, in human cancer cells and xenograft models. NC1-induced killing was not inhibited by caspase inhibitors, Bcl-2 overexpression or TNF neutralization, suggesting that NC1 elicits a bona fide necrotic pathway. However, pharmacological or genetic inhibition of necroptosis, pyroptosis and ferroptosis faild to block NC1-mediated regulated necrosis. Instead, NC1 elicited mitochondria ROS production, either elimination of mitochondrial DNA or chemical inhibition of mitochondrial ROS production blocked NC1-induced necrotic cell death. NC1 induced all hallmarks of immunogenic cell death (calreticulin exposure, ATP release and HMGB1 release) in vitro, and NC1-killed mouse cancer cells were able to induce a protective anticancer immune response when injected into histo-compatible, immunocompetent mice. Altogether, we identify a previously uncharacterized signaling cascade leading to necrotic cell death and provided further support to the notion that the induction of programmed necrosis may constitute a future approach for anticancer therapy.

programmed cell death

TNF independent

necrosis

mitochondria

immunogenic cell death

Anticancer chemotherapeutics have been developed for long in a mostly empiric fashion, by identifying chemical compounds that reduce tumor growth or that kill human cancer cells in vitro ^{1, 2}. Only recently, so-called targeted agents have been identified in cell-based and molecular screening assays ^3–5. It has been commonly thought that efficient anticancer regimens would either trigger the apoptotic demise of tumor cells ^{6, 7}, to stimulate mitotic catastrophe⁸, and/or to induce a permanent arrest in the cell cycle that is generally referred to as ‘senescence’ ^{9, 10}. Unfortunately, tumor cells develop multiple strategies to evade apoptosis, mostly by upregulating anti-apoptotic proteins or by downregulating pro-apoptotic proteins so that they become increasingly therapy-resistant ^{7, 11}. Moreover, tumor cells can escape the cell cycle arrest imposed on them by mitotic inhibitors or senescence-inducing drugs ^12–14. This capacity to subvert therapeutic effects may explain why tumors often do not respond or only respond transiently to conventional or targeted anticancer agents.

The recent discovery that necrosis can occur in a regulated fashion and the ever more precise characterization of the underlying molecular mechanisms have spurred great interest^15–17, because non-apoptotic pathways might be instrumental to circumvent the resistance of cancer cells to conventional, pro-apoptotic therapeutic regimens ^18–20. In addition, necrotic cell death has been involved in prominent human diseases including neurodegenerative conditions such as Alzheimer’s and Parkinson’s disease as well as ischemic and infectious disorders ^21–23.

Although regulated necrosis has been originally identified as a tumor necrosis factor receptor 1 (TNFR1)-dependent cell death modality, emerging evidence suggests that necrosis can occur in a regulated fashion also in response to other stimuli including alkylating DNA damage, reactive oxygen species (ROS) overgeneration and calcium overload ^24–27. Recently, several distinct forms of programmed cell death (PCD) have been defined with unique cellular mechanisms. An important role downstream of the RIPK1-RIPK3-containining complex leading to necroptosis has been highlighted for the mixed lineage kinase like (MLKL) protein ^28–31. Gasdermins have been shown to form pores that cause pyroptosis after cleavage by inflammatory caspases including caspase 1, 4, 5 and 11^{32, 33}. The research field of ferroptosis, driven by iron-dependent phospholipid peroxidation, and regulated by multiple cellular metabolic pathways including redox homeostasis, iron handling, mitochondrial activity and metabolism of amino acids and lipids, has seen exponential growth over the past few years since the term was coined in 2012^{24, 34}. Intriguingly, therapy-resistant cancer cells, particularly those in the mesenchymal state and prone to metastasis, are exquisitely vulnerable to ferroptosis.

Here, we report the identification of a small molecule compound, 3-cycloheptyl-3-(4-hydroxyphenyl)6-methoxy-7-methyl-1,3-dihydroindole-2-one (which we named Necrocide 1, NC1) that operates as a potent inducer of regulated necrosis distinct from necroptosis, ferroptosis and pyroptosis, in vitro and in vivo, as well as the characterization of the signaling pathways underlying the pro-necrotic activity of NC1.

NC1 induces necrosis in vitro and in vivo.

3-cycloheptyl-3-(4-hydroxyphenyl)6-methoxy-7-methyl-1,3-dihydroindole-2-one (NC1; Fig. 1A), but not its inactive stereoisomer (NC1i), reduced the metabolic activity of human breast carcinoma MCF-7 cells in a dose- and time-dependent fashion (Fig. 1B, C), as it induced cell killing, as indicated by the release of lactate dehydrogenase into the culture medium (Fig. 1D). Tetrazolium conversion assays revealed the IC₅₀ of NC1 to be below 15 nM for nine of the ten human cancer cell lines tested. In contrast, NC1 failed to kill non-transformed IMR-90 fibroblasts and human umbilical vein endothelial cells (HUVECs), even at a concentration of 10 µM (Fig. 1E).

NC1 caused ballooning of MCF-7 cells (Fig. 1F), which became translucent in phase contrast microscopy, accompanied by an increase in nuclear diameter as well as by the formation of Hoechst 33342-positive chromatin clumps adjacent to the nuclear envelope (Fig. 1F, G). This contrasted with the appearance of MCF-7 cells succumbing to TNF, which induced apoptosis-associated morphological alterations such as chromatin condensation and cellular shrinkage (18) (Fig. 1F, G). Transmission electron microscopy confirmed the necrotic appearance of NC1-treated cells, manifesting a prominent swelling of cytoplasmic organelles, extranuclear vacuolization, peripheral chromatin condensation and plasma membrane rupture (Fig. 1H, I).

Cytochrome c and cathepsin L, which are usually sequestered within mitochondria or lysosomes, respectively, diffusely distributed throughout the cytoplasm upon NC1 treatment, as assessed by immunofluorescence microscopy upon staining with specific antibodies (Fig. 1J-L). Thus, we hypothesized that NC1 would induce both mitochondrial membrane permeabilization (MMP) and lysosomal membrane permeabilization (LMP). MMP was confirmed by staining NC1-treated MCF-7 cells with the mitochondrial transmembrane potential (Δψ_m)-sensitive fluorophore tetramethyl rhodamine methylester (TMRM), and LMP on MCF-7 cells succumbing to NC1 was corroborated by means of Lysotracker red staining. Thus, NC1 caused a drastic time-dependent reduction in Δψ_m and lysosomal membrane integrity (Fig. 1M). Altogether, these results indicate that NC1 stimulates the necrotic demise of human cancer cells.

We investigated the capacity of NC1 to induce necrotic cell death in vivo, in human cancers xenotransplanted in immunodeficient nu/nu mice. Intravenous injections or oral gavage of NC1 consistently reduced the growth of both PC3 and MCF-7 xenografts (Fig. 2A-C). Histochemical analyses of NC1-treated tumors revealed a massive necrotic response, featuring isolated swollen nuclei in tissues that were highly infiltrated by macrophages (Fig. 2D). Thus, NC1 can induce therapeutic necrosis of human cancer xenografts in vivo.

Molecular mechanisms of NC1-induced necrosis.

Next, we aimed at further characterizing the necrotic cell death subroutine triggered by NC1. In vivo live cell imaging analysis observed the ballooning of MCF7 cells upon NC1 treatment, and the rupture of plasma membrane is the hallmark of this NC1-induced cell death (Fig. 3A and Movie 1). These data suggest MCF7 cells upon NC1 treatment likely undergo necrosis rather than apoptosis. Contrarily to TNF induced caspase activation, NC1 failed to activate the cleavage of neither caspase-7 nor Poly (ADP-ribose) polymerase 1 (PARP1), two biochemical events displaying caspase activity associated with the execution of apoptosis in MCF-7 cells (Fig. 3B). Two necrotic markers, CyPA and HMGB1, were released from NC1-treated or tBH/zVAD cells into the extracellular medium (Fig. 3B). Both the broad-spectrum caspase inhibitor Z-VAD-fmk and the autophagy inhibitor Chloroquine (CQ) and 3-methyladenine (3-MA) failed to inhibit NC1-induced necrosis, suggesting that NC1-induced necrosis is different from apoptosis and autophagic cell death (Fig. 3C). Genetic depletion of the pro-apoptotic factors BAX and BAK by specific siRNAs was unable to rescue NC1-induced cell death (Fig. 3D). While the transfection-enforced overexpression of the anti-apoptotic protein BCL-2 successfully prevented TNF-induced apoptosis, it failed to protect cells from NC1-induced killing (Fig. 3E). Along similar lines, a TNFR1-blocking antibody failed to prevent NC1 induced necrosis in MCF7 cells (Fig. 3F), suggesting that NC1-induced cell death is independent of TNF signaling pathway. Together, all these data demonstrate that NC1-mediated cell death is different from apoptosis.

Several regulated necrosis pathways have been recently characterized, including necroptosis, ferroptosis and pyroptosis. Necroptosis requires the activation and translocation of receptor-interacting serine/threonine-protein kinases RIPK1 and RIPK3 from the plasma membrane to cytosol ^9–18, where Mixed lineage kinase like (MLKL) protein is phosphorylated by the RIPK1-RIPK3 complex and activated to execute necrosis in a caspase independent manner^14,16. Ferroptosis is characterized by iron-dependent accumulation of oxidized polyunsaturated fatty acid-containing phospholipids on the plasma membrane, which is negatively regulated by the phospholipid hydroperoxide-reducing enzyme glutathione peoxidase 4 (GPX4) and coenzyme Q10 catalyzing enzyme ferroptosis suppressor protein 1 (FSP1) in parallel systems. Pyroptosis is marked by activation of inflammation-activated caspase-1 and lipopolysaccharide (LPS)-activated caspase-11/4/5^22,23, and cleavage of pyroptosis executioner, the pore-forming protein gasdermin D (GSDMD) ^24,25.

We used chemical approaches to define NC1 induced necrosis. The necroptosis inhibitor NSA, ferroptosis inhibitor Ferrostain-1, iron chelator desferrioxamine (DFO) and Liproxstatin-1, pyroptosis inhibitor z-YVAD all failed to inhibit NC1-induced necrosis (Fig. 4A), indicating that NC1-induced necrosis is different from necroptosis, ferroptosis and pyroptosis. In addition, PARP inhibitor Olaparib failed to inhibit NC1-induced cell death, while it effectively blocked MNNG-mediated parthonatos (Fig. 4B). In line with this observation, suppression of MLKL by CRISPR/Cas9 was unable to antagonize NC1 induced cell death as demonstrated by cell viability, suggesting that NC1-induced cell death is different from necroptosis (Fig. 4C and 4D). Loss of GSDMD did not block NC1-induced cell death as well, suggesting that NC1-induced cell death is different from pyroptosis (Fig. 4E and 4F). Moreover, suppression of ACSL4 failed to block NC1-induced cell death, although ACSL4 defficiency dramatically rescued RSL3-induced ferroptosis as reported (Fig. 4G and 4H). Taken together, these data suggest that NC1-induced necrosis represents a previous undescribed cell death other than apoptosis, autophagy, necroptosis, ferroptosis, pyroptosis and parthonatos.

Mitochondria is important for NC1-induced necrosis.

To further decipher the mechanism of NC1-induced necrosis, RNA sequencing (RNA-seq) was executed. Functional analysis of RNA seq data identified a large proportion of the altered genes important for oxidative phosphorylation (Fig. 5A and 5B). The mitochondrial transmembrane potential (∆ᴪm) dissipates upon NC1 treatment, which occurs prior to loss of plasma membrane integrity. ∆ᴪm was examined during NC1-induced necrosis and we found that loss of ∆ᴪm occurred before the plasma membrane rupture (Fig. 5C), suggesting that mitochondrial permeability transition likely contributes to NC1-induced necrosis. Therefore, we next investigated if mitochondria participate in NC1-induced necrosis, we generated mitochondria-deficient cells as previously reported³² and observed that mitochondria-deficient cells were resistant to NC1-induced necrosis, suggesting that mitochondria are involved in NC1-induced necrosis (Fig. 5D). As we demonstrated, inhibition of mitochondria fission partially suppressed the cytotoxicity of NC1 (Fig. 5E). Importantly, genetic targeting of cyclophilin D blocked NC1-induced killing (Fig. 5F). Moreover, Cyclosporin A (CsA), which attenuates mitochondrial permeability transition, dramatically blocked NC1-induced cell death (Fig. 5G). Together, these data suggest the important role of mitochondria in NC1-induced cytotoxicity.

NC1 provoked a rapid increase in the production of ROS in sensitive MCF-7 cells, as detected by flow cytometry upon staining with the ROS-sensitive dye 2',7'-dichlorofluorescein-diacetate (DCFH-DA) (Fig. 6A). The ROS production is largely from the mitochondria, as we observed robust increase in MitoSOX-sensitive mitochondrial ROS but no increase in BODIPY-sensitive lipid ROS production (Fig. 6B and 6C). ROS production correlates with cellular sensitivity to NC1, since we observed no obvious general ROS or mitochondria ROS production in NC1-insensitive U₂OS and MEF cells (Fig. 6D-6F). To test this hypothesis, we treated 6 cancer cell lines as well as normal cell lines with NC1, and compared their ROS production and sensitivity to NC1 (Fig. 6G and 6H). Four cell lines including one prostate cancer cell line PC-3, and three human breast cancer cell lines MB468, MCF-7 and HCC1143 highly sensitive to NC1, exhibited high ROS production at the basal levels (Fig. 6G and 6H). In cancer cells that are insensitive to NC1, including lung cancer cell line A549 and osteosarcoma cell line U₂OS, their basal ROS levels were much lower (Fig. 6G and 6H). These data suggest that the basal ROS levels in the cancer cell lines likely correlate with their sensitivity to NC1.

Further, we investigate if mitochondrial reactive oxygen species (ROS) plays a role in NC1 induced necrosis. Two mitochondrial ROS driven necrosis inhibitors Necro-X2 and Necro-X5 both blunted NC1 cytotoxicity (Fig. 6I). All these data suggested that ROS production is likely involved in NC1-induced necrosis.

Immunogenic cell death induced by NC1

Immunogenic cell death (ICD) induced by anthracyclines is characterized by the exposure of calreticulin (CRT) on the cell surface, the release of ATP during the blebbing phase of apoptosis, as well as the release of HMGB1 from the nucleus via the permeabilized plasma membrane ³⁵. NC1 was capable of inducing these hallmarks on PC3 prostate cancer cells as efficiently as did the anthracyline, mitoxanthrone (MTX) (Fig. 7A-7C). This phenomenon was accompanied by an endoplasmic reticulum stress response (as illustrated by the phosphorylation of the eukaryotic translation initiation factor, eIF2α) (Fig. 7D). NC1 and MTX both were equivalent in inducing the ICD hallmarks in murine MCA205 fibrosarcoma cells, although through distinct signal transduction pathways (Fig. 7E-7G). NC1-induced CRT exposure, ATP and HMGB1 release were dramatically inhibited by cyclosporinA and necroX5 (Fig. 7E-7G). Conversely, MTX-induced CRT exposure and HMGB1 release were only partially suppressed by Z-VAD-fmk (Fig. 7E-7G). In accordance with the in vitro data, NC1-killed MCA205 cells were able to elicit a protective anticancer immune response when they were injected subcutaneously into histocompatible, immunocompetent C57Bl/6 mice (Fig. 7H). In conclusion, NC1 can induce a type of cell death that bears biochemical and functional hallmarks of ICD.

The dysregulation of programmed cell death is closely related to a variety of human diseases inlcuding cancers. Recent evidence shows that programmed cell death is not only limited to apoptosis, but also necrotic cell death are comprehensively regulated. Here, we identified a small molecular compound, 3-cycloheptyl-3-(4-hydroxyphenyl)6-methoxy-7-methyl-1,3-dihydroindole-2-one (NC1), which kills a panel of human cancer cells by inducing regulated necrosis. The necrotic demise induced by NC1 is distinct from necroptosis in thus far that it neither depends on TNFR1 signaling nor it requires caspase inhibition. Characterization of NC1-elicited signals point to the involvement of mitochondria through a non-conventional pathway leading to regulated necrosis. The robust antitumor effects of NC1 suggest that the induction of regulated necrosis holds promising potential for the development of future anticancer therapies.

Resisting cell death, especially apoptosis is a hallmark of cancer. One mechanism by which tumor cells develop resistance to cytotoxic agents and radiation is related to resistance to apoptosis. Our results presented here extend our understanding of cell death in that NC1 did not activate apoptotic signaling pathways, but directly induced necrotic cell death in a series of cancer cells. NC1 fails to induce the hallmarks of apoptosis (cellular and nuclear shrinkage with global chromatin condensation) and rather triggers the morphological characteristics of necrosis (oncosis, rather partial peripheral chromatin condensation without nuclear pyknosis, plasma membrane rupture without preceding blebbing)^36–38. At the functional levels, NC1 fails to activate caspases and kills cells in a fashion that is not perturbed by the addition of caspase inhibitors, nor modulated by changes in the expression level of prominent multidomain proteins from the Bcl-2 family including Bcl-2 itself, Bax and Bak. NC1 also kills in a p53-independent fashion because p53-mutant MDA-MB-468 cells³⁹ readily succumbed to NC1.

Several forms of necrotic cell death have been reported recently. The phosphorylated MLKL binds to phosphatidylinositol lipids, moves from the cytosol to the plasma and intracellular membranes and directly disrupts membrane integrity, resulting in necroptosis⁴⁰. Gasdermin D (GSDMD), a gasdermin-family member, is cleaved by both caspase-1 and caspase-11/4/5 to release its gasdermin-N domain that perforates the plasma membrane to induce cell swelling and pyroptosis. Herein, MCF-7 cells demonstrated the necrotic appearance upon NC1 treatment, manifesting a prominent swelling of cytoplasmic organelles. Neither suppression of MLKL nor GSDMD blocked NC1-induced cell death, suggesting it’s neither necroptosis nor pyroptosis. Ferroptosis is usually accompanied by a large amount of iron accumulation and lipid peroxidation during the cell death process. However, we were unable to detect the lipid peroxidation upon NC1 treatment. Moreover, the iron chelator DFO failed to block NC1-induced cell death, suggesting it’s different from ferroptosis. Alternatively, our data demonstrated that mitochondria play a critical role in NC1 induced cell death. Knockdown of cyclophilin D, the mitochondrion-specific cyclophilin that is required for the pro-necrotic permeabilization of mitochondrial membranes (so the called ‘permeability transition’) confers partial protection against NC1-mediated cell killing. Moreover, Cyclosporin A (CsA), which attenuates mitochondrial permeability transition, dramatically blocked NC1-induced cell death. Togethre, these data suggest the obligate contribution of mitochondria to the lethal signaling cascade.

In summary, NC1 induces necrotic cell death in a series of cancer cell lines. Moreover, NC1 induces therapeutic necrosis of human cancer xenografts in vivo. Interestingly, NC1 did not activate apoptotic signaling pathways, but directly induced necrotic cell death in cancer cells. Therefore, we identified a previously uncharacterized signaling cascade leading to necrotic cell death and provided further support to the notion that the induction of programmed necrosis may constitute a future approach for anticancer therapy.

Reagents

The Necrocide 1 (TP202377) compound was synthesized as described previously⁴¹. The following reagents are purchased from companies: Hematoxylin, eosin, Acridine Orange and Hoechst 33342 (Sigma). SYTOX Green (Invitrogen). z-VAD-fmk (ApexBio). Necrostatin-1, Necrostatin-5, Ferrostatin-1, Desferrioxamine (DFO), 3-MA, α-Tocopherol (Sigma)， Necrox-2 and Necrox-5 (Enzo Life Sciences). Human recombinant TNF-α (R&D Systems). ENLITEN ATP assay kit (Promega). OxiSelect ROS assay kit (Cellbiolabs). The following antibodies were used: PARP (Cell Signaling Technology, 9542), caspase-7 (Cell Signalling, 9746), CyPA (Santa Cruz), HMGB1 (Cell Signaling Technology, 3935), MLKL (Novus Biologicals, NBP3-03885), GSDMD (Thermo Fisher), beta tubulin (Sigma, T2200), TNF blocking antibody (R&D Systems), Cathepsin L (BD Transduction Laboratories, 611084). Smac mimetics was kindly provided by Dr. Xiaodong Wang at NIBS.

Cell Culture

All cell lines were obtained from ATCC (ATCC/LGC Promochem, Borås, Sweden). Cell culture reagents were obtained from Invitrogen except otherwise stated. All cell lines were maintained according to American Type Culture Collection guidelines in RPMI medium containing 10% Fetal Bovine Serum (FBS) 100 U/ml penicillin, and 100 mg/ml streptomycin. For experiments, cells were trypsinized and seeded in dishes (Santa Cruz) and allowed to grow to reach 70% confluence at 37˚C with 95%/5% air/CO₂ and 80% relative humidity. Cells were treated with compound for indicated concentrations and periods (necrocide 1, TNF (20 ng/ml), TNF antibody (2 ìg/ml), necrostatin-1 (20 μM), necrostatin-5 (20 μM), z- VAD-fmk (10 μM), 3-MA (10 mM), smac mimetics (100nM)). Cells were rinsed with cold PBS, harvested by scraping into PBS and isolated by centrifugation at 700 x g for 5 min, and cell pellets were stored at -80˚C until processing. Cell transfection was performed with Lipofectamine 2000 (Invitrogen) or HiPerfect (Qiagen) according to the protocol provided by the manufacturer.

Immunofluorescence Staining

Cells were grown to 70-80% confluence in 6-well dishes with coverslides, treated with necrocide 1 for 24 hours and were fixed with 4% paraformaldehyde solution in PBS at room temperature for 20 min. After permeabilization with PBS buffer containing 0.1% Triton X- 100 at room temperature for 20 min, cells were incubated with primary antibodies (anti-Myc or anti-Cathepsin L) at 37 °C for 2 h. After washing with PBS buffer containing 0.1% Triton X-100, cells were incubated with Rhodamine red- conjugated secondary antibodies at 37 °C for 2 hours. Slides were examined by using a laser scanning confocal microscope (Zeiss LSM 510 META UV/Vis).

Transmission Electron Microscope Analysis

For electron micros- copy, cells were fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 2 h followed by 1% osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7.2) for 2 h. Samples were enblocked with 0.5% aqueous uranyl acetate overnight and treated by low-temperature dehydration and infiltration with a graded series of Epon/Araldite, which was followed by the embedment in 100% Epon/Araldite. Thin sections (60 nM) were cut and stained with Reynalds lead citrate and analyzed with a FEI Tecnai 12 Transmission electron microscope.

Viability and cell death assays

Cell viability and dose-response curves were measured by WST-1 proliferation assay (Roche), lactate dehydrogenase assay (LDH cytotoxicity detection kit, Roche) or Cell Titer Glo (Cell Titer Glo Assay Kit, Promega) according to manufacturers’ instructions. Cells were treated at indicated concentrations for 72h. The IC₅₀ [nM] was estimated from the dose-response curves by nonlinear regression using GraphPad Prism. For necrocide 1 induced death in Bcl-2 ovexpressing cells, MCF-7 cells stable transfected with empty vector (MCF7-neo2) or Bcl-2 (MCF7-Bcl2-wt) were left untreated or treated with 50 and 500 nM necrocide 1 for 10h. Viability was determined by counting Trypan blue negative cells. For each condition, three randomly chosen fields of 100 cells were counted. Columns, mean from three independent triplicate experiments. Necrotic cell death was analysed by an Olympus IX microscopewith the UV channel. Cells were stainedwith cell-permeable Hoeschst 33342 2,5μg/ml (Sigma) for 10 min. Survival was determined by counting of trypan blue negative cells. Three randomly chosen fields containing a minimumof 100 cells were counted.

Establishment of mtDNA-depleted MCF-7 cells

As previously reported⁴², to develop partially mtDNA-depleted cell lines, we exposed MCF-7 cells to EtBr (0.2 µg/ml) in DMEM medium supplemented for 8 weeks with pyruvate and uridine, which are shown to be essential for the growth of mtDNA-depleted cells. The mtDNA content from MCF-7 cells cultured with or without EtBr was monitored routinely by amplifying total DNA using the following primers: tRNA-Leu (UUR)-F: CAC CCA AGA ACA GGG TTT GT; tRNA-Leu (UUR)-R: TGG CCA TGG GTA TGT TGT TA; microglobulin-F: TGC TGT CTC CAT GTT TGA TGT ATC T; microglobulin-R: TCT CTG CTC CCC ACC TCT AAG T.

Measurement of reactive oxygen species

Production of reactive oxygen species was determined using H2DCFDA (H2-DCF, DCF, Invitrogen), MitoSox (Invitrogen), BODIPY 581/591 C11(Invitrogen) and measured by FACS flowcytometry according to manufactures instructions. Briefly, MCF-7 cells were seeded 5*104 cells/well in a 12 wells plate. Cells were incubated with drug/medium for 2 hours at the final indicated concentrations.

In vivo efficacy studies

The anti-tumor effect in vivo was tested in a PC-3 prostate, MCF-7 breast, A2780 ovary and MiaPaca2 pancreas subcutaneous (s.c.) xenograft models. Female NMRI nude mice (6-8 weeks) were obtained from Taconic. After at least one week acclimatizing period, each mouse was injected with 1e7 in vitro grown cells washed once with PBS and suspended in 100 ml of PBS + 100 ml matrigel (BD). Necrocide 1 was formulated in 2% DMSO, 20% HP-b-CD and isotonic saline and given i.v. or oral at 10 ml/kg. Female 4-week-old NIHRNU-M nude rats were obtained from Taconic. One week after arrival each rat was injected at 4 sites, 2 in each flank, with 1e7 PC-3 human prostate cancer cells. The PC-3 cells were grown in RPMI + 10% FBS, washed once with PBS and suspended in 100 ml of PBS + 100 ml matrigel (BD). Tumor volume (tv) is determined from perpendicular tumor diameters D1 and D2. Tv=p×((D1+D2)/2)3 ×1/6. Treatment started at large tumor volumes around up to 1000 mm3. The experiments were conducted at TopoTarget Copenhagen, and approved by the Experimental Animal Inspectorate, Danish Ministry of Justice.

Author contributions

Christina Trojel-Hansen started the project and contributed with animal experiments; Jing Zhang performed the mechanism related experiments in cell lines with help from Jianghuang Wang, Zili Zhang, Xing Wang, Yuhui Qiao, and Huike Jiao. Mickaël Michaud, Oliver Kepp, Lorenzo Galluzzi, Maria Hoyer-Hansen and Marja Jäättelä contributed to the early characterization of NC1; Jing Zhang, Christina Trojel-Hansen, Guido Kroemer and Qing Zhong conceived the project, designed the experiments, analyzed the data and wrote the manuscript with the help of all authors. All authors discussed the results and commented on the manuscript.

Acknowledgements.

The work was supported in part by grants from NSFC (91754205, 91957204, 31771523 and M-1040) to Q. Z.; MOST (2019YFA0508602) to Q. Z. and Shanghai Municipal Science and Technology Project (20JC1411100, 19XD1402200) to Q. Z., the Shanghai Pujiang Program (20PJ1409500), grants from State Key Laboratory of Oncogenes and Related Genes (KF2101-93), National Natural Science Foundation of China (32070734), Natural Science Foundation of Shanghai (20ZR1430800) to J. Z., and NIH R01 (CA133228), and to G.K. from the Ligue Nationale contre le Cancer (Equipes labellisée), Agence Nationale pour la Recherche (ANR), European Commission (Active p53, Apo-Sys, ChemoRes, ApopTrain), Fondation pour la Recherche Médicale (FRM), Institut National du Cancer (INCa), Cancéropôle Ile-de-France, Fondation Bettencourt-Schueller and the LabEx Immuno-Oncology. This work is also supported by Shanghai Frontiers Science Center of Cellular Homeostasis and Human Diseases and the innovative research team of high-level local universities in Shanghai (SHSMU-ZDCX20212000).

Conflict of interest statement

The authors have declared that no conflict of interest exists.

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Necrocide 1 Mediates a Non-Apoptotic Necrotic Cell Death and Immunogenic Response in Human Cancer Cells

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Immunogenic cell death induced by NC1

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