RESUMO
Introns represent almost half of the human genome, although they are eliminated from transcripts through RNA splicing. Yet, different classes of non-canonical miRNAs have been proposed to originate directly from intron splicing. Here, we considered the alternative splicing of introns as an interesting source of miRNAs, compatible with a developmental switch. We report computational prediction of new Short Intron-Derived ncRNAs (SID), defined as precursors of smaller ncRNAs like miRNAs and snoRNAs produced directly by splicing, and tested their dependence on each key factor in canonical or alternative miRNAs biogenesis (Drosha, DGCR8, DBR1, snRNP70, U2AF65, PRP8, Dicer, Ago2). We found that about half of predicted SID rely on debranching of the excised intron-lariat by the enzyme DBR1, as proposed for mirtrons. However, we identified new classes of SID for which miRNAs biogenesis may rely on intermingling between canonical and alternative pathways. We validated selected SID as putative miRNAs precursors and identified new endogenous miRNAs produced by non-canonical pathways, including one hosted in the first intron of SRA (Steroid Receptor RNA activator). Consistent with increased SRA intron retention during myogenic differentiation, release of SRA intron and its associated mature miRNA decreased in cells from healthy subjects but not from myotonic dystrophy patients with splicing defects.
Assuntos
Íntrons/genética , MicroRNAs/genética , RNA não Traduzido/genética , Processamento Alternativo/genética , Biologia Computacional , Genoma Humano , Humanos , MicroRNAs/biossíntese , Precursores de RNA/genéticaRESUMO
Autophagy inducers can prevent cardiovascular aging and age-associated diseases including atherosclerosis. Therefore, we hypothesized that autophagy-inducing compounds that act on atherosclerosis-relevant cells might have a protective role in the development of atherosclerosis. Here we identified 3,4-dimethoxychalcone (3,4-DC) as an inducer of autophagy in several cell lines from endothelial, myocardial and myeloid/macrophagic origin, as demonstrated by the aggregation of the autophagosome marker GFP-LC3 in the cytoplasm of cells, as well as the downregulation of its nuclear pool indicative of autophagic flux. In this respect, 3,4-DC showed a broader autophagy-inducing activity than another chalcone (4,4- dimethoxychalcone), spermidine and triethylene tetramine. Thus, we characterized the potential antiatherogenic activity of 3,4-DC in two different mouse models, namely, (i) neointima formation with smooth muscle expansion of vein segments grafted to the carotid artery and (ii) genetically predisposed ApoE-/- mice fed an atherogenic diet. In the vein graft model, local application of 3,4-DC was able to maintain the lumen of vessels and to reduce neointima lesions. In the diet-induced model, intraperitoneal injections of 3,4-DC significantly reduced the number of atherosclerotic lesions in the aorta. In conclusion, 3,4-DC stands out as an autophagy inducer with potent antiatherogenic activity.
Assuntos
Aterosclerose , Neointima , Camundongos , Animais , Neointima/tratamento farmacológico , Neointima/patologia , Hiperplasia/patologia , Aterosclerose/patologia , Aorta/patologia , Modelos Animais de Doenças , Autofagia , Camundongos Endogâmicos C57BLRESUMO
The radiochemotherapy- or chemotherapy-induced stimulation of immunogenic cell death (ICD) affecting malignant cells ignites antitumor immune responses that are clinically relevant as they allow to achieve durable responses beyond treatment discontinuation. The mechanistic exploration of ICD and the discovery of agents and interventions that are endowed with the capacity to elicit ICD is of the utmost importance. Here, we describe an assay for the assessment of type I interferon (IFN) production, which is one of the salient features of ICD. Biosensor cells that express GFP under the control of the IFN-inducible MX dynamin like GTPase 1 (MX1) gene promoter are employed, and the fluorescent signal is assessed by automated microscopy. The described workflow is automation-friendly, rendering it compatible with high-throughput screening (HTS) for drug discovery.
Assuntos
Interferon Tipo I , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Morte Celular ImunogênicaRESUMO
Antibody-drug conjugates (ADCs) are used to target cancer cells by means of antibodies directed to tumor-associated antigens, causing the incorporation of a cytotoxic payload into target cells. Here, we characterized the mode of action of ADC costing of a TWEAKR-specific monoclonal antibody conjugated to a small molecule kinesin spindle protein (KSP) inhibitor (KSPi). These TWEAKR-KSPi-ADCs showed strong efficacy in a TWEAKR expressing CT26 colon cancer model in mice. TWEAKR-KSPi-ADCs controlled the growth of CT26 colon cancers in immunodeficient as well as in immunocompetent mice. However, when treated with suboptimal doses, TWEAKR-KSPi-ADCs were still active in immunocompetent but not in immunodeficient mice, indicating that TWEAKR-KSPi-ADCs act - in addition to the cytotoxic mode of action - through an immunological mechanism. Indeed, in vitro experiments performed with a cell-permeable small molecule KSPi closely related to the active payload released from the TWEAKR-KSPi-ADCs revealed that KSPi was capable of stimulating several hallmarks of immunogenic cell death (ICD) on three different human cancer cell lines: cellular release of adenosine triphosphate (ATP) and high mobility group B1 protein (HMGB1), exposure of calreticulin on the cell surface as well as a transcriptional type-I interferon response. Further, in vivo experiments confirmed that treatment with TWEAKR-KSPi-ADCs activated immune responses via enhancing the infiltration of CD4+ and CD8+ T lymphocytes in tumors and the local production of interferon-γ, interleukin-2, and tumor necrosis factor-α. In conclusion, the antineoplastic effects of TWEAKR-KSPi-ADCs can partly be attributed to its ICD-stimulatory properties.
Assuntos
Antineoplásicos , Imunoconjugados , Neoplasias , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/farmacologia , Imunoconjugados/metabolismo , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Cinesinas , Camundongos , Neoplasias/tratamento farmacológico , Receptor de TWEAKRESUMO
Robotized high throughput screening allows for the assessment of autophagy in a large number of samples. Here, we describe a drug discovery platform for the phenotypic identification of novel autophagy inducers by means of automated cell biology workflows employing robotized cell culture, sample preparation and data acquisition. In this setting, fluorescent biosensor cells that express microtubule-associated proteins 1A/1B light chain 3B (best known as LC3) conjugated to green fluorescent protein (GFP), are utilized together with automated high content microscopy for the image-based assessment of autophagy. In sum, we detail a drug discovery screening workflow from high throughput sample preparation and processing to data acquisition and analysis.
Assuntos
Autofagia , Ensaios de Triagem em Larga Escala , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Proteínas de Fluorescência Verde/genética , Proteínas Associadas aos MicrotúbulosRESUMO
BACKGROUND: Pharmacological autophagy enhancement constitutes a preclinically validated strategy for preventing or treating most major age-associated diseases. Driven by this consideration, we performed a high-content/high-throughput screen on 65 000 distinct compounds on a robotized fluorescence microscopy platform to identify novel autophagy inducers. RESULTS: Here, we report the discovery of picropodophyllin (PPP) as a potent inducer of autophagic flux that acts on-target, as an inhibitor of the tyrosine kinase activity of the insulin-like growth factor-1 receptor (IGF1R). Thus, PPP lost its autophagy-stimulatory activity in cells engineered to lack IGF1R or to express a constitutively active AKT serine/threonine kinase 1 (AKT1) mutant. When administered to cancer-bearing mice, PPP improved the therapeutic efficacy of chemoimmunotherapy with a combination of immunogenic cytotoxicants and programmed cell death 1 (PDCD1, better known as PD-1) blockade. These PPP effects were lost when tumors were rendered PPP-insensitive or autophagy-incompetent. In combination with chemotherapy, PPP enhanced the infiltration of tumors by cytotoxic T lymphocytes, while reducing regulatory T cells. In human triple-negative breast cancer patients, the activating phosphorylation of IGF1R correlated with inhibited autophagy, an unfavorable local immune profile, and poor prognosis. CONCLUSION: Altogether, these results suggest that IGF1R may constitute a novel and druggable therapeutic target for the treatment of cancer in conjunction with chemoimmunotherapies.
Assuntos
Antineoplásicos/uso terapêutico , Autofagia/genética , Receptor IGF Tipo 1/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Feminino , Humanos , CamundongosRESUMO
B-cell maturation antigen (BCMA) is an attractive therapeutic target highly expressed on differentiated plasma cells in multiple myeloma and other B-cell malignancies. GSK2857916 (belantamab mafodotin, BLENREP) is a BCMA-targeting antibody-drug conjugate approved for the treatment of relapsed/refractory multiple myeloma. We report that GSK2857916 induces immunogenic cell death in BCMA-expressing cancer cells and promotes dendritic cell activation in vitro and in vivo GSK2857916 treatment enhances intratumor immune cell infiltration and activation, delays tumor growth, and promotes durable complete regressions in immune-competent mice bearing EL4 lymphoma tumors expressing human BCMA (EL4-hBCMA). Responding mice are immune to rechallenge with EL4 parental and EL4-hBCMA cells, suggesting engagement of an adaptive immune response, immunologic memory, and tumor antigen spreading, which are abrogated upon depletion of endogenous CD8+ T cells. Combinations with OX40/OX86, an immune agonist antibody, significantly enhance antitumor activity and increase durable complete responses, providing a strong rationale for clinical evaluation of GSK2857916 combinations with immunotherapies targeting adaptive immune responses, including T-cell-directed checkpoint modulators.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antígeno de Maturação de Linfócitos B/antagonistas & inibidores , Linfócitos T CD8-Positivos/imunologia , Imunoconjugados/farmacologia , Morte Celular Imunogênica , Linfoma/tratamento farmacológico , Mieloma Múltiplo/tratamento farmacológico , Animais , Anticorpos Monoclonais/química , Apoptose , Antígeno de Maturação de Linfócitos B/imunologia , Proliferação de Células , Feminino , Humanos , Linfoma/imunologia , Linfoma/metabolismo , Linfoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Programmed cell death is regulated by the balance between activating and inhibitory signals. Here, we have identified RECS1 (responsive to centrifugal force and shear stress 1) [also known as TMBIM1 (transmembrane BAX inhibitor motif containing 1)] as a proapoptotic member of the TMBIM family. In contrast to other proteins of the TMBIM family, RECS1 expression induces cell death through the canonical mitochondrial apoptosis pathway. Unbiased screening indicated that RECS1 sensitizes cells to lysosomal perturbations. RECS1 localizes to lysosomes, where it regulates their acidification and calcium content, triggering lysosomal membrane permeabilization. Structural modeling and electrophysiological studies indicated that RECS1 is a pH-regulated calcium channel, an activity that is essential to trigger cell death. RECS1 also sensitizes whole animals to stress in vivo in Drosophila melanogaster and zebrafish models. Our results unveil an unanticipated function for RECS1 as a proapoptotic component of the TMBIM family that ignites cell death programs at lysosomes.
RESUMO
Several antineoplastic agents are endowed with the ability to induce immunogenic cell death (ICD), a modality of cellular demise that is accompanied by the release of danger associated molecular patterns such as adenosine triphosphate (ATP) into the tumor microenvironment. ATP-mediated ligation of purinergic P2R receptors then facilitates the chemotactic recruitment and activation of innate immune effectors, thus favoring the induction of anticancer immunity. Here, we provide a protocol for the fluorescence microscopy-based quantification of ICD-associated ATP secretion that is amenable to high-throughput screening. As compared to the traditional luciferase-based detection of ATP in cell culture supernatants, the analysis presented here is cost-efficient and can be combined with the parallel assessment of cellular morphology.
Assuntos
Trifosfato de Adenosina/análise , Antineoplásicos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Morte Celular Imunogênica/efeitos dos fármacos , Quinacrina/farmacologia , Trifosfato de Adenosina/imunologia , Trifosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Morte Celular Imunogênica/imunologia , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/patologiaRESUMO
Systemic anticancer immunity can be reinstated via the induction of immunogenic cell death (ICD) in malignant cells. Thus, certain classes of cytotoxic compounds, for example, anthracyclines, oxaliplatin and taxanes are endowed with the capacity to act on cancer cells to ignite premortem stress pathways that lead to the surface exposure of calreticulin (CALR) and the cellular release of adenosine triphosphate, annexin A1, high mobility group B1 and type-1 interferons. Altogether, these alterations constitute the hallmarks of ICD. Here we report the design of a discovery pipeline for the identification of novel ICD inducers by means of a phenotypic screening platform. The use of fluorescent biosensors as proxies for the manifestation of ICD hallmarks has enabled the exploration of large collections of chemical compounds by automatized screening routines. Imaging-based assessment and phenotypic selection led to the identification of potential ICD inducers that could be validated further invitro and invivo, confirming that bona fide ICD inducers possess the capacity to induce immunological long-term memory and to confer resistance against rechallenge with syngeneic tumors. Machine learning algorithms analyzing the physicochemical properties of ICD inducers can assist in the preselection of compounds with potential ICD-stimulatory properties, further accelerating the screening efforts designed to develop new immunotherapeutic agents.
RESUMO
Systemic treatment with the active transcription inhibitor lurbinectedin aims at inducing tumor cell death in hyperproliferative neoplasms. Here we show that cell death induced by lurbinectedin reinstates and enhances systemic anticancer immune responses. Lurbinectedin treatment showed traits of immunogenic cell death, including the exposure of calreticulin, the release of ATP, the exodus of high mobility group box 1 (HMGB1) and type 1 interferon responses in vitro. Lurbinectedin treated cells induced antitumor immunity when injected into immunocompetent animals and treatment of transplanted fibrosarcomas reduced tumor growth in immunocompetent yet not in immunodeficient hosts. Anticancer effects resulting from lurbinectedin treatment were boosted in combination with PD-1 and CTLA-4 double immune checkpoint blockade (ICB), and lurbinectedin combined with double ICB exhibited strong antineoplastic effects. Cured animals exhibited long term immune memory effects that rendered them resistant to rechallenge with syngeneic tumors underlining the potency of combination therapy with lurbinectedin.
RESUMO
The phosphorylation of eIF2α is essential for the endoplasmic reticulum (ER) stress response, the formation of stress granules, as well as macroautophagy. Several successful anticancer chemotherapeutics have the property to induce immunogenic cell death (ICD), thereby causing anticancer immune responses. ICD is accompanied by the translocation of calreticulin (CALR) from the ER lumen to the plasma membrane, which facilitates the transfer of tumor-associated antigens to dendritic cells. Here we systematically investigated the capacity of anticancer chemotherapeutics to induce signs of ER stress. ICD inducers including anthracyclines and agents that provoke tetraploidization were highly efficient in enhancing the phosphorylation of eIF2α, yet failed to stimulate other signs of ER stress including the transcriptional activation of activating transcription factor 4 (ATF4), the alternative splicing of X-box binding protein 1 (XBP1s) mRNA and the proteolytic cleavage of activating transcription factor 6 (ATF6) both in vitro and in cancers established in mice. Systematic analyses of clinically used anticancer chemotherapeutics revealed that only eIF2α phosphorylation, but none of the other signs of ER stress, correlated with CALR exposure. eIF2α phosphorylation induced by mitoxantrone, a prototype ICD-inducing anthracyline, was mediated by eIF2α kinase-3 (EIF2AK3). Machine-learning approaches were used to determine the physicochemical properties of drugs that induce ICD, revealing that the sole ER stress response relevant to the algorithm is eIF2α phosphorylation with its downstream consequences CALR exposure, stress granule formation and autophagy induction. Importantly, this approach could reduce the complexity of compound libraries to identify ICD inducers based on their physicochemical and structural characteristics. In summary, it appears that eIF2α phosphorylation constitutes a pathognomonic characteristic of ICD.
Assuntos
Apoptose , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Fator 6 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/metabolismo , Algoritmos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Calreticulina/farmacologia , Linhagem Celular , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/genética , Feminino , Humanos , Camundongos , Camundongos Nus , Mitoxantrona/farmacologia , Mitoxantrona/uso terapêutico , Neoplasias/tratamento farmacológico , Fosforilação/efeitos dos fármacos , Transplante Heterólogo , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
This corrects the article DOI: 10.1038/cdd.2016.86.
RESUMO
Oncolytic peptides and peptidomimetics are being optimized for the treatment of cancer by selecting agents with high cytotoxic potential to kill a maximum of tumor cells as well as the capacity to trigger anticancer immune responses and hence to achieve long-term effects beyond therapeutic discontinuation. Here, we report on the characterization of two novel oncolytic peptides, DTT-205 and DTT-304 that both selectively enrich in the lysosomal compartment of cancer cells yet differ to some extent in their cytotoxic mode of action. While DTT-304 can trigger the aggregation of RIP3 in ripoptosomes, coupled to the phosphorylation of MLKL by RIP3, DTT-205 fails to activate RIP3. Accordingly, knockout of either RIP3 or MLKL caused partial resistance against cell killing by DTT-304 but not DTT-205. In contrast, both agents shared common features in other aspects of pro-death signaling in the sense that their cytotoxic effects were strongly inhibited by both serum and antioxidants, partially reduced by lysosomal inhibition with bafilomycin A1 or double knockout of Bax and Bak, yet totally refractory to caspase inhibition. Both DTT-304 and DTT-205 caused the exposure of calreticulin at the cell surface, as well as the release of HMGB1 from the cells. Mice bearing established subcutaneous cancers could be cured by local injection of DTT-205 or DTT-304, and this effect depended on T lymphocytes, as it led to the establishment of a long-term memory response against tumor-associated antigens. Thus, mice that had been cured from cancer by the administration of DTT compounds were refractory against rechallenge with the same cancer type several months after the disappearance of the primary lesion. In summary, DTT-205 and DTT-304 both have the capacity to induce immunotherapeutic oncolysis.
Assuntos
Descoberta de Drogas/métodos , Memória Imunológica/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Animais , Antígenos de Neoplasias/metabolismo , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Feminino , Proteína HMGB1/metabolismo , Células HT29 , Humanos , Gotículas Lipídicas/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Necrose , Peptídeos/síntese química , Fosforilação , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais/efeitos dos fármacos , Resultado do TratamentoRESUMO
LTX-401 is an oncolytic amino acid derivative with potential immunogenic properties. Here, we demonstrate that LTX-401 selectively destroys the structure of the Golgi apparatus, as determined by means of ultrastructural analyses and fluorescence microscopic observation of cells expressing Golgi-targeted GFP reporters. Subcellular fractionation followed by mass spectrometric detection revealed that LTX-401 selectively enriched in the Golgi rather than in mitochondria or in the cytosol. The Golgi-dissociating agent Brefeldin A (BFA) reduced cell killing by LTX-401 as it partially inhibited LTX-401-induced mitochondrial release of cytochrome c and the activation of BAX. The cytotoxic effect of LTX-401 was attenuated by the double knockout of BAX and BAK, as well as the mitophagy-enforced depletion of mitochondria, yet was refractory to caspase inhibition. LTX-401 induced all major hallmarks of immunogenic cell death detectable with biosensor cell lines including calreticulin exposure, ATP release, HMGB1 exodus and a type-1 interferon response. Moreover, LTX-401-treated tumors manifested a strong lymphoid infiltration. Altogether these results support the contention that LTX-401 can stimulate immunogenic cell death through a pathway in which Golgi-localized LTX-401 operates upstream of mitochondrial membrane permeabilization.
Assuntos
Antineoplásicos/farmacologia , Complexo de Golgi/metabolismo , beta-Alanina/análogos & derivados , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Brefeldina A/farmacologia , Linhagem Celular Tumoral , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Humanos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Permeabilidade , Frações Subcelulares/metabolismo , beta-Alanina/química , beta-Alanina/farmacologiaRESUMO
The oncolytic peptide LTX-315 has been designed for killing human cancer cells and turned out to stimulate anti-cancer immune responses when locally injected into tumors established in immunocompetent mice. Here, we investigated the question whether LTX-315 induces apoptosis or necrosis. Transmission electron microscopy or morphometric analysis of chromatin-stained tumor cells revealed that LTX-315 failed to induce apoptotic nuclear condensation and rather induced a necrotic phenotype. Accordingly, LTX-315 failed to stimulate the activation of caspase-3, and inhibition of caspases by means of Z-VAD-fmk was unable to reduce cell killing by LTX-315. In addition, 2 prominent inhibitors of regulated necrosis (necroptosis), namely, necrostatin-1 and cycosporin A, failed to reduce LTX-315-induced cell death. In conclusion, it appears that LTX-315 triggers unregulated necrosis, which may contribute to its pro-inflammatory and pro-immune effects.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Oligopeptídeos/farmacologia , Osteossarcoma/tratamento farmacológico , Clorometilcetonas de Aminoácidos/farmacologia , Apoptose , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/ultraestrutura , Inibidores de Caspase/farmacologia , Linhagem Celular Tumoral , Ciclosporina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Microscopia Eletrônica de Transmissão , Necrose , Osteossarcoma/metabolismo , Osteossarcoma/ultraestrutura , Fatores de TempoRESUMO
LTX-315 has been developed as an amphipathic cationic peptide that kills cancer cells. Here, we investigated the putative involvement of mitochondria in the cytotoxic action of LTX-315. Subcellular fractionation of LTX-315-treated cells, followed by mass spectrometric quantification, revealed that the agent was enriched in mitochondria. LTX-315 caused an immediate arrest of mitochondrial respiration without any major uncoupling effect. Accordingly, LTX-315 disrupted the mitochondrial network, dissipated the mitochondrial inner transmembrane potential, and caused the release of mitochondrial intermembrane proteins into the cytosol. LTX-315 was relatively inefficient in stimulating mitophagy. Cells lacking the two pro-apoptotic multidomain proteins from the BCL-2 family, BAX and BAK, were less susceptible to LTX-315-mediated killing. Moreover, cells engineered to lose their mitochondria (by transfection with Parkin combined with treatment with a protonophore causing mitophagy) were relatively resistant against LTX-315, underscoring the importance of this organelle for LTX-315-mediated cytotoxicity. Altogether, these results support the notion that LTX-315 kills cancer cells by virtue of its capacity to permeabilize mitochondrial membranes.