Targeting immunogenic cell death in cancer

Immunogenic cell death (ICD) is a type of cancer cell death triggered by certain chemotherapeutic drugs, oncolytic viruses, physicochemical therapies, photodynamic therapy, and radiotherapy. It involves the activation of the immune system against cancer in immunocompetent hosts. ICD comprises the release of damage‐associated molecular patterns (DAMPs) from dying tumor cells that result in the activation of tumor‐specific immune responses, thus eliciting long‐term efficacy of anticancer drugs by combining direct cancer cell killing and antitumor immunity. Remarkably, subcutaneous injection of dying tumor cells undergoing ICD has been shown to provoke anticancer vaccine effects in vivo. DAMPs include the cell surface exposure of calreticulin (CRT) and heat‐shock proteins (HSP70 and HSP90), extracellular release of adenosine triphosphate (ATP), high‐mobility group box‐1 (HMGB1), type I IFNs and members of the IL‐1 cytokine family. In this review, we discuss the cell death modalities connected to ICD, the DAMPs exposed during ICD, and the mechanism by which they activate the immune system. Finally, we discuss the therapeutic potential and challenges of harnessing ICD in cancer immunotherapy.

Abbreviations

ATP

adenosine triphosphate

BAK

BCL‐2 homologous antagonist killer

BAX

BCL‐2‐associated X protein

BCL‐2

B‐cell lymphoma 2

BID

BH3‐interacting domain death agonist

c‐FLIP

cellular FLICE‐like inhibitory protein

cGAMP

cyclic guanosine monophosphate–adenosine monophosphate

cGAS

cyclic GMP‐AMP synthase

CRT

calreticulin

CXCL10

chemokine C‐X‐C motif ligand 10

DAMPs

damage‐associated molecular patterns

DCs

dendritic cells

DISC

death‐inducing signaling complex

ER

endoplasmic reticulum

FADD

FAS‐associated protein with death domain

FASL

FAS ligand

GSDMD

gasdermin D

GSDMD^NT

N‐terminal fragment of gasdermin D

GSDME

gasdermin E

HMGB1

high‐mobility group box‐1

HSP

heat‐shock proteins

Hyp‐PDT

hypericin‐based photodynamic therapy

ICD

immunogenic cell death

IFN

interferon

IFNAR

IFN‐α and IFN‐β receptors

IL

interleukin

IRF3

interferon regulatory factor 3

ISGs

IFN‐stimulated genes

LPS

lipopolysaccharide

MAPK

mitogen‐activated protein kinase

MHC

major histocompatibility complex

MLKL

mixed‐lineage kinase‐like

MOMP

mitochondrial outer membrane permeabilization

mtDNA

mitochondrial DNA

NF‐κB

nuclear factor kappa‐light‐chain‐enhancer of activated B cells

NK cells

natural killer cells

NLR

NOD‐like receptor

NLRP3

NOD‐like receptor family, pyrin domain‐containing 3 protein

P2RX7

purinergic receptor P2X 7

PD‐L1

programmed death ligand

PRRs

pattern recognition receptors

PS

phosphatidyl serine

RCD

regulated cell death

RIPK1

receptor‐interacting serine/threonine protein kinase 1

RIPK3

receptor‐interacting serine/threonine protein kinase 3

ROS

reactive oxygen species

STING

stimulator of interferon genes

tBID

truncated form of BID

TBK1

TANK‐binding kinase 1

TLR

Toll‐like receptor

TNF

tumor necrosis factor

TRAIL

TNF‐related apoptosis‐inducing ligand

ZBP

Z‐DNA‐binding protein

     

Paclitaxel triggers cell death primarily via caspase-independent routes in the non-small cell lung cancer cell line NCI-H460.

PURPOSE: Here we report on the role of mitochondria, death receptors (DRs), and caspases in exerting the cytotoxic effect of clinically relevant concentrations of paclitaxel in the non-small cell lung cancer cell line NCI-H460. EXPERIMENTAL DESIGN: We have characterized paclitaxel-induced cell death with annexin V, propidium iodide staining, and poly(ADP-ribose) polymerase cleavage assays. The involvement of the mitochondria pathway was studied by monitoring cytochrome c release and using H460 cells stable in overexpressing Bcl-2 or Bcl-x(L). DR dependency was analyzed in FADD dominant-negative or cytokine response modifier A-overexpressing cells, and a possible role for DR4 and DR5 was investigated by antagonistic antibodies. Caspase activity and cleavage assays and treatment with the synthetic inhibitor zVAD-fmk were used to determine the involvement of caspases. RESULTS: Paclitaxel-treated cells displayed several features of apoptosis, including annexin V staining and poly(ADP-ribose) polymerase cleavage. The sequence of events suggested the involvement of a DR, as indicated by an early role for Fas-associated death domain and caspase-8, followed by cleavage of Bid and the disruption of mitochondria; nonetheless, we failed to demonstrate the involvement of DR4 and DR5. Interestingly, inhibition of either one of these routes only resulted in a 30% reduction of cell death that was in line with the observed small effect of caspase inhibition by zVAD-fmk on H460 cell survival. CONCLUSION: Paclitaxel triggers cell death in H460 cells mainly via a currently unidentified caspase-independent mechanism in which the basic apoptotic machinery is merely coactivated. This finding is in sharp contrast with the largely caspase-dependent response elicited by DNA-damaging agents in these cells. We speculate on therapeutic implications.     

Arsenic trioxide triggers a regulated form of caspase-independent necrotic cell death via the mitochondrial death pathway

Cell death is generally believed to occur either by accidental, lytic necrosis or by programmed cell death, that is, apoptosis. The initiation and execution of cell death, however, is far more complex and includes pathways like caspase-independent apoptosis or actively triggered necrosis. In this study, we investigated the mechanisms of cell death induced by arsenic trioxide (arsenite, As2O3), a clinically efficient agent in anticancer therapy. As2O3-induced cell death coincides with cytochrome c release, facilitates mitochondrial permeability transition and is sensitive to inhibition by Bcl-x(L), indicating that cell demise is regulated through the mitochondrial apoptosis pathway. Nevertheless, only little caspase-3 activation was observed and As2O3-induced cell death was only weakly obstructed by the broad spectrum caspase inhibitor z-VAD-fmk. Moreover, disruption of caspase-9 or -2 failed to decrease the amount of As2O3-mediated cell death. Interestingly, As2O3-induced cell death had a predominantly necrosis-like phenotype as assessed by Annexin-V/propidium iodide staining and LDH release. Finally, blocking glutathione synthetase by buthionine sulfoximine enhanced the As2O3-mediated necrosis-like cell death without increasing caspase-3 cleavage. As2O3 does, however, not directly inhibit caspases, but appears to interfere with caspase activation. Altogether, our data clearly delineate a mode of As2O3-triggered cell death that differs considerably from that induced by conventional anticancer drugs. These findings may explain the capability of As2O3 to efficiently kill even chemoresistant tumor cells with disturbed apoptosis signaling and caspase activation, a frequent finding in malignancy.     

Isatis indigotica induces hepatocellular cancer cell death via caspase-independent apoptosis-inducing factor translocation apoptotic pathway in vitro and in vivo

Isatis indigotica is a biennial herbaceous cruciferous medical herb with antipyretic, antiviral, anti-inflammatory, and anti-endotoxin activity. This study explored the chemotherapeutic potential of I indigotica on human hepatoma cells and investigated the mechanism by which metabolites from I indigotica inhibit hepatoma cell growth. Antitumor activity was discovered in dried I indigotica leaf chloroform extracts (CEDLI). In nude mice xenotransplanted with human hepatoma cells, CEDLI supplementation inhibited tumor growth by ~40% compared with nonsupplemented animals without affecting body weight/food intake. CEDLI induced sub-G1 cell cycle arrest and apoptosis in hepatoma cells. Furthermore, CEDLI activates p53 and Bax, reduces Bcl-2 expression, and causes mitochondrial stress and the release of apoptosis-inducing factor into the cytosol followed by its translocation into the nucleus, resulting in hepatoma cell apoptosis. This study provides novel in vivo evidence of I indigotica's antitumor activity. The chemotherapeutic activity against human hepatoma tumorigenesis was because of a distinguished caspase-independent apoptotic pathway.     

Harnessing the cell death pathway for targeted cancer treatment

Genotoxic agents have long targeted apoptotic cell death as a primary means of treating cancer. However, the presence of cellular defects in many cancers has contributed to an acquired resistance to apoptotic cell death, lowering the effectiveness of chemo- and radiotherapies. The mechanisms by which cells achieve this resistance to treatment are still being investigated, but an alternative approach is the study of cell death pathways that are mechanistically distinct from apoptosis. These pathways, including autophagy and necrosis, have arisen as attractive targets for cancer therapy. This review will discuss apoptosis, autophagy, and necrosis in the context of tumorigenesis and drug resistance, as well as provide an up-to-date preclinical and clinical review of inhibitors targeting these cell death pathways for multiple cancer types. The goal of these studies is to identify molecular targets that will enhance the efficacy and specificity of current cancer therapies.     

7-Bromoindirubin-3'-oxime induces caspase-independent cell death

Indirubin, an isomer of indigo, is a reported inhibitor of cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3 (GSK-3) as well as an agonist of the aryl hydrocarbon receptor (AhR). Indirubin is the active ingredient of a traditional Chinese medicinal recipe used against chronic myelocytic leukemia. Numerous indirubin analogs have been synthesized to optimize this promising kinase inhibitor scaffold. We report here on the cellular effects of 7-bromoindirubin-3'-oxime (7BIO). In contrast to its 5-bromo- and 6-bromo- isomers, and to indirubin-3'-oxime, 7BIO has only a marginal inhibitory activity towards CDKs and GSK-3. Unexpectedly, 7BIO triggers a rapid cell death process distinct from apoptosis. 7-Bromoindirubin-3'-oxime induces the appearance of large pycnotic nuclei, without classical features of apoptosis such as chromatin condensation and nuclear fragmentation. 7-Bromoindirubin-3'-oxime-induced cell death is not accompanied by cytochrome c release neither by any measurable effector caspase activation. Furthermore, the death process is not altered either by the presence of Q-VD-OPh, a broad-spectrum caspase inhibitor, or the overexpression of Bcl-2 and Bcl-XL proteins. Neither AhR nor p53 is required during 7BIO-induced cell death. Thus, in contrast to previously described indirubins, 7BIO triggers the activation of non-apoptotic cell death, possibly through necroptosis or autophagy. Although their molecular targets remain to be identified, 7-substituted indirubins may constitute a new class of potential antitumor compounds that would retain their activity in cells refractory to apoptosis.     

化疗与肿瘤免疫原性细胞死亡

化疗耐药是临床难题,研究发现某些蒽环类化合物和奥沙利铂等化疗药物不仅诱导肿瘤细胞凋亡,而且可以引起免疫原性细胞死亡(ICD),通过诱导肿瘤细胞自噬,释放3类信号:钙网蛋白暴露在细胞表面,刺激树突状细胞(DC)吞噬;三磷酸腺苷释放,招募DC进入肿瘤灶;高迁徙率族蛋白B1促进DC与垂死肿瘤细胞形成稳定结合,诱导机体产生特异性的T细胞抗肿瘤免疫.深入理解化疗诱导的ICD,对于完善化疗方案具有重要意义.     

Restoration of ASC expression sensitizes colorectal cancer cells to genotoxic stress-induced caspase-independent cell death

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), an essential component of the inflammasome complex, is frequently silenced by epigenetic methylation in many tumor cells. Here, we demonstrate that restoration of ASC expression in human colorectal cancer DLD-1 cells, in which ASC is silenced by aberrant methylation, potentiated cell death mediated by DNA damaging agent. Contrarily, ASC knockdown in HT-29 cells rendered cells less susceptible to etoposide toxicity. The increased susceptibility of ASC-expressing DLD-1 cells to genotoxic stress was independent of inflammasome or caspase activation, but partially dependent on mitochondrial ROS production and JNK activation. Thus, our data suggest that ASC expression in cancer cells is an important factor in determining their susceptibility to chemotherapy.     

Onconase induces caspase-independent cell death in chemoresistant neuroblastoma cells

Hypoxia-inducible factor-1alpha (HIF-1alpha) is believed to promote tumor growth, and thus, is viewed as one of the most compelling cancer therapy targets. YC-1 is widely used as a potent inhibitor of HIF-1alpha both in vitro and in vivo, and is also being developed as a novel anticancer drug. However, little is known about the effects of YC-1 on tumor invasion or metastasis. In the present study, we found that the Hep3B cell migration-stimulatory effect of hypoxia was abolished by HIF-1alpha siRNA or YC-1. YC-1 also significantly inhibited the migrations of other cancer cells. Furthermore, YC-1 effectively inhibited cell invasion through Matrigel. In nude mice, GFP-expressing stable cell-lines of Hep3B or H1299 were inoculated into spleens to induce liver metastasis or into the pleural cavity to induce lung invasion. In untreated mice, many tumor lesions emitting strong fluorescence were found in livers or lungs, and fluorescence intensities and tumor lesion numbers were markedly reduced in YC-1-treated mice. These results suggest that YC-1 effectively inhibits tumor invasion and metastasis, and imply that YC-1 is worth while to further develop as a multipurpose anticancer drug.     

Role of AIF in caspase-dependent and caspase-independent cell death

The major challenge in treating cancer is that many tumor cells carry mutations in key apoptotic genes such as p53, Bcl family proteins or those affecting caspase signaling. Such defects render treatment with traditional chemotherapeutic agents ineffective. Many studies have demonstrated the importance of caspase-independent cell death pathways in injury, degenerative diseases and tumor tissue. It is now recognized that in addition to their critical role in the production of cellular energy, mitochondria are also the source of key proapoptotic molecules involved in caspase activation. More recently, it has been discovered that in response to apoptotic stimuli, mitochondria can also release caspase-independent cell death effectors such as AIF and Endonuclease G. In this review, we examine the role of Bcl family proteins and poly(ADP-ribose) polymerase-1 signaling in the regulation of these apoptotic pathways and address the ongoing controversies in this field. Continued study of the mechanisms of apoptosis including caspase-independent death processes are likely to reveal novel therapeutic targets for the treatment of diverse human pathologies including cancer, neurodegenerative diseases and acute injuries such as stroke or myocardial infarction.     

Inducing immunogenic cell death in immuno-oncological therapies

Immunotherapy has revolutionized cancer treatment and substantially improved patient outcomes with respect to multiple types of tumors. However, most patients cannot benefit from such therapies, mainly due to the intrinsic low immunogenicity of cancer cells(CCs) that allows them to escape recognition by immune cells of the body.Immunogenic cell death(ICD), which is a form of regulated cell death, engages in a complex dialogue between dying CCs and immune cells in the tumor microenvironment(TME),...     

Programmed cell death in an estrogen-independent human breast cancer cell line, MDA-MB-468.

Previous studies have demonstrated that estrogen-responsive human breast cancer cells can be induced to undergo an energy-dependent, genetically programmed series of biochemical changes that result in the active suicide of the cells following estrogen ablation. In contrast, estrogen-independent human breast cancer cells do not activate this programmed cell death pathway following estrogen ablation. This could be due either to the absence of the cellular machinery required for programmed cell death or simply to the inability of estrogen ablation to activate this machinery. To discriminate between these two possibilities, the MDA-MB-468 estrogen-independent human mammary adenocarcinoma cell line was used as a model system to study the mechanism of cell death following cytotoxic drug treatment. Exposure of these cells to the fluorinated pyrimidines, 5-fluoro-2'-deoxyuridine or trifluorothymidine, resulted in growth inhibition and loss of proliferative capacity within 24 h. These changes occurred while cell membrane integrity was intact as measured by either cellular morphology or trypan blue exclusion. After 48 h of drug treatment, loss of cell membrane integrity was followed by cell lysis and a rapid decline in cell number. The addition of 16 microM thymidine prior to drug treatment prevented cell death, but thymidine did not rescue these cells once drug treatment was initiated. Analysis of DNA revealed the characteristic fragmentation into nucleosomal oligomers that is a hallmark of programmed cell death. Associated with this death pathway was a 15-fold induction of transforming growth factor beta 1 gene expression that has been previously observed in a variety of cellular systems undergoing programmed cell death. These results indicate that MDA-MB-468 estrogen-independent human mammary carcinoma cells retain the ability to undergo programmed cell death after treatment with cytotoxic drugs that induce a "thymineless" state.     

Cathepsin B mediates caspase-independent cell death induced by microtubule stabilizing agents in non-small cell lung cancer cells

We have previously reported that the microtubule stabilizing agents (MSAs) paclitaxel, epothilone B and discodermolide induce caspase-independent cell death in non-small cell lung cancer (NSCLC) cells. Here we present two lines of evidence indicating a central role for the lysosomal protease cathepsin B in mediating cell death. First, inhibition of cathepsin B, and not of caspases or other proteases, such as cathepsin D or calpains, results in a strong protection against drug-induced cell death in several NSCLC cells. Second, MSAs trigger disruption of lysosomes and release and activation of cathepsin B. Interestingly, inhibition of cathepsin B prevents the appearance of multinucleated cells, an early characteristic of MSA-induced cell death, pointing to a central, proximal role for cathepsin B in this novel cell death pathway.     

Mitochondrial p53 phosphorylation induces Bak-mediated and caspase-independent cell death

Chemoresistance in cancer has previously been attributed to gene mutations or deficiency. Caspase mutations or Bax deficiency can lead to resistance to cancer drugs. We recently demonstrated that Bak initiates a caspase/Bax-independent cell death pathway. We show that Plumbagin (PL) (5-hydroxy-2-methyl-1,4-napthoquinone), a medicinal plant-derived naphthoquinone that is known to have anti-tumor activity in a variety of models, induces caspase-independent cell death in HCT116 Bax knockout (KO) or MCF-7 Bax knockdown (KD) cells that express wild-type (WT) Bak. The re-expression of Bax in HCT116 Bax KO cells fails to enhance the PL-induced cell death. Additionally, Bak knockdown by shRNA efficiently attenuates PL-induced cell death. These results suggest that PL-induced cell death depends primarily on Bak, not Bax, in these cells. Further experimentation demonstrated that p53 Ser15 phosphorylation and mitochondrial translocation mediated Bak activation and subsequent cell death. Knockdown of p53 or a p53 Ser15 mutant significantly inhibited p53 mitochondrial translocation and cell death. Furthermore, we found that Akt mediated p53 phosphorylation and the subsequent mitochondrial accumulation. Taken together, our data elaborate the role of Bak in caspase/Bax-independent cell death and suggest that PL may be an effective agent for overcoming chemoresistance in cancer cells with dysfunctional caspases.     

CD44 ligation induces caspase-independent cell death via a novel calpain/AIF pathway in human erythroleukemia cells

Ligation of the cell surface molecule CD44 by anti-CD44 monoclonal antibodies (mAbs) has been shown to induce cell differentiation, cell growth inhibition and in some cases, apoptosis in myeloid leukemic cells. We report, herein, that exposure of human erythroleukemic HEL cells to the anti-CD44 mAb A3D8 resulted in cell growth inhibition followed by caspase-independent apoptosis-like cell death. This process was associated with the disruption of mitochondrial membrane potential (Delta Psi m), the mitochondrial release of apoptosis-inducing factor (AIF), but not of cytochrome c, and the nuclear translocation of AIF. All these effects including cell death, loss of mitochondrial Delta Psi m and AIF release were blocked by pretreatment with the poly (ADP-ribose) polymerase inhibitor isoquinoline. A significant protection against cell death was also observed by using small interfering RNA for AIF. Moreover, we show that calpain protease was activated before the appearance of apoptosis, and that calpain inhibitors or transfection of calpain-siRNA decrease A3D8-induced cell death, and block AIF release. These data suggest that CD44 ligation triggers a novel caspase-independent cell death pathway via calpain-dependent AIF release in erythroleukemic HEL cells.     

Oncogenic Ras triggers cell suicide through the activation of a caspase-independent cell death program in human cancer cells.

To prevent neoplasia, cells of multicellular organisms activate cellular disposal programs such as apoptosis in response to deregulated oncogene expression, making the suppression of such programs an essential step for potentially neoplastic cells to become established as clinically relevant tumors. Since the mutation of ras proto-oncogenes, the most frequently mutated proto-oncogenes in human tumors, is very rare in some tumor types such as glioblastomas and gastric cancers, we hypothesized that mutated ras genes might activate a cell death program that cannot be overcome by these tumor types. Here we show that the expression of oncogenically mutated ras gene induces cellular degeneration accompanied by cytoplasmic vacuoles in human glioma and gastric cancer cell lines. Cells dying as a result of oncogenic Ras expression had relatively well-preserved nuclei that were negative for TUNEL staining. An immunocytochemical analysis demonstrated that the cytoplasmic vacuoles are derived mainly from lysosomes. This oncogenic Ras-induced cell death occurred in the absence of caspase activation, and was not inhibited by the overexpression of anti-apoptotic Bcl-2 protein. These observations suggested that oncogenic Ras-induced cell death is most consistent with a type of programmed cell death designated 'type 2 physiological cell death' or 'autophagic degeneration', and that this cell death is regulated by a molecular mechanism distinct from that of apoptosis. Our findings suggest a possible role for this non-apoptotic cell death in the prevention of neoplasia, and the activation of the non-apoptotic cell death program may become a potential cancer therapy complementing apoptosis-based therapies. In addition, the approach used in this study may be a valuable way to find genetically-regulated cell suicide programs that cannot be overcome by particular tumor types.     

The c-Myc-interacting adaptor protein Bin1 activates a caspase-independent cell death program

Cell death processes are progressively inactivated during malignant development, in part by loss of tumor suppressors that can promote cell death. The Bin1 gene encodes a nucleocytosolic adaptor protein with tumor suppressor properties, initially identified through its ability to interact with and inhibit malignant transformation by c-Myc and other oncogenes. Bin1 is frequently missing or functionally inactivated in breast and prostate cancers and in melanoma. In this study, we show that Bin1 engages a caspase-independent cell death process similar to type II apoptosis, characterized by cell shrinkage, substratum detachment, vacuolated cytoplasm, and DNA degradation. Cell death induction was relieved by mutation of the BAR domain, a putative effector domain, or by a missplicing event that occurs in melanoma and inactivates suppressor activity. Cells in all phases of the cell cycle were susceptible to death and p53 and Rb were dispensable. Notably, Bin1 did not activate caspases and the broad spectrum caspase inhibitor ZVAD.fmk did not block cell death. Consistent with the lack of caspase involvement, dying cells lacked nucleosomal DNA cleavage and nuclear lamina degradation. Moreover, neither Bcl-2 or dominant inhibition of the Fas pathway had any effect. In previous work, we showed that Bin1 could not suppress cell transformation by SV40 large T antigen. Consistent with this finding, we observed that T antigen suppressed the death program engaged by Bin1. This observation was interesting in light of emerging evidence that T antigen has roles in cell immortalization and human cell transformation beyond Rb and p53 inactivation. In support of a link to c-Myc-induced death processes, AEBSF, a serine protease inhibitor that inhibits apoptosis by c-Myc, potently suppressed DNA degradation by Bin1. Our findings suggest that the tumor suppressor activity of Bin1 reflects engagement of a unique cell death program. We propose that loss of Bin1 may promote malignancy by blunting death penalties associated with oncogene activation.     

Molecular determinants of immunogenic cell death elicited by anticancer chemotherapy

The success of some chemo- and radiotherapeutic regimens relies on the induction of immunogenic tumor cell death and on the induction of an anticancer immune response. Cells succumbing to immunogenic cell death undergo specific changes in their surface characteristics and release pro-immunogenic factors according to a defined spatiotemporal pattern. This stimulates antigen presenting cells such as dendritic cells to efficiently take up tumor antigens, process them, and cross-prime cytotoxic T lymphocytes, thus eliciting a tumor-specific cognate immune response. Such a response can also target therapy-resistant tumor (stem) cells, thereby leading, at least in some instances, to tumor eradication. In this review, we shed some light on the molecular identity of the factors that are required for cell death to be perceived as immunogenic. We discuss the intriguing observations that the most abundant endoplasmic reticulum protein, calreticulin, the most abundant intracellular metabolite, ATP, and the most abundant non-histone chromatin-binding protein, HMGB1, can determine whether cell death is immunogenic as they appear on the surface or in the microenvironment of dying cells.     

S-trityl-L-cysteine derivative induces caspase-independent cell death in K562 human chronic myeloid leukemia cell line

Effect of CF₃-STLC, a potent kinesin spindle protein (KSP) inhibitor, on K562 human CML cell line was investigated. Treatment with CF₃-STLC induced mitotic arrest of the cell cycle with the appearance of characteristic monoastral spindles, subsequent apoptotic cell death and cleavage of PARP-1, caspase-3, and 4E-BP1. The wide ranging caspase inhibitor z-VAD fmk prevented the cleavage of caspase-3 and 4E-BP1, but failed to attenuate PARP-1 cleavage or cell death triggered by CF₃-STLC. These results suggest that CF₃-STLC can induce apoptotic cell death in a caspase-independent manner, and may work effectively as an anti-cancer agent for hematological malignancies.