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.     

Novel inosine monophosphate dehydrogenase inhibitor VX-944 induces apoptosis in multiple myeloma cells primarily via caspase-independent AIF/Endo G pathway

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme required for the de novo synthesis of guanine nucleotides from IMP. VX-944 (Vertex Pharmaceuticals, Cambridge, MA, USA) is a small-molecule, selective, noncompetitive inhibitor directed against human IMPDH. In this report, we show that VX-944 inhibits in vitro growth of human multiple myeloma (MM) cell lines via induction of apoptosis. Interleukin-6, insulin-like growth factor-1, or co-culture with bone marrow stromal cells (BMSCs) do not protect against VX-944-induced MM cell growth inhibition. VX-944 induced apoptosis in MM cell lines with only modest activation of caspases 3, 8, and 9. Furthermore, the pan-caspase inhibitor z-VAD-fmk did not inhibit VX-944-induced apoptosis and cell death. During VX-944-induced apoptosis, expressions of Bax and Bak were enhanced, and both apoptosis-inducing factor (AIF) and endonuclease G (Endo G) were released from the mitochondria to cytosol, suggesting that VX-944 triggers apoptosis in MM cells primarily via a caspase-independent, Bax/AIF/Endo G pathway. Importantly, VX-944 augments the cytotoxicity of doxorubicin and melphalan even in the presence of BMSCs. Taken together, our data demonstrate a primarily non-caspase-dependent apoptotic pathway triggered by VX-944, thereby providing a rationale to enhance MM cell cytotoxicity by combining this agent with conventional agents which trigger caspase activation.     

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.     

IFN-gamma induces cell death in human hepatoma cells through a TRAIL/death receptor-mediated apoptotic pathway.

We demonstrated the induction of cell death in a hepatoma cell line by IFN-gamma and its possible mechanism. Among the 2 hepatitis B virus (HBV)-associated hepatoma cell lines, SNU-354 and SNU-368, IFN-gamma induced cell death and increased caspase-3 activity in SNU-368 but not in SNU-354. IFN-gamma induced several changes in the mRNA expression level of apoptosis-regulating genes, e.g., increased expression of Fas, caspase-1 and TNF-related apoptosis-inducing ligand (TRAIL). In particular, IFN-gamma potently increased the mRNA expression of TRAIL in both cell lines. However, it did not change the mRNA expression level of death-mediating TRAIL receptors, e.g., DR4 and DR5, which were constitutively expressed in both cell lines. In contrast, the decoy receptor DcR1 was expressed in SNU-354 but not in SNU-368, and its expression level in SNU-354 was increased by IFN-gamma. Another decoy receptor, DcR2, was constitutively expressed in both cell lines; however, its expression level in SNU-368 was decreased by IFN-gamma. In addition, exogenous recombinant TRAIL reduced viability in SNU-368, but not in SNU-354, cells. From these findings, we speculated that TRAIL up-regulation and the subsequent TRAIL-mediated apoptosis serve as a mechanism of IFN-gamma-induced cell death in SNU-368. To confirm this hypothesis, we demonstrated that soluble DR4-Fc fusion protein, a TRAIL pathway inhibitor, inhibited IFN-gamma-induced cell death in SNU-368. Our results demonstrated that IFN-gamma acts as an inducer of cell death through TRAIL-mediated apoptosis.     

Silibinin induces apoptosis via calpain-dependent AIF nuclear translocation in U87MG human glioma cell death

Background

Silibinin, a natural polyphenolic flavonoid, has been reported to induce cell death in various cancer cell types. However, the molecular mechanism is not clearly defined. Our previous study showed that silibinin induces glioma cell death and its effect was effectively prevented by calpain inhibitor. The present study was therefore undertaken to examine the role of calpain in the silibinin-induced glioma cell death.

Methods

U87MG cells were grown on well tissue culture plates and cell viability was measured by MTT assay. ROS generation and △ψ_m were estimated using the fluorescence dyes. PKC activation and Bax expression were measured by Western blot analysis. AIF nuclear translocation was determined by Western blot and immunocytochemistry.

Results

Silibinin induced activation of calpain, which was blocked by EGTA and the calpain inhibitor Z-Leu-Leu-CHO. Silibinin caused ROS generation and its effect was inhibited by calpain inhibitor, the general PKC inhibitor GF 109203X, the specific PKC_δ inhibitor rottlerin, and catalase. Silibinin-induce cell death was blocked by calpain inhibitor and PKC inhibitors. Silibinin-induced PKC_δ activation and disruption of △ψ_m were prevented by the calpain inhibitor. Silibinin induced AIF nuclear translocation and its effect was prevented by calpain inhibitor. Transfection of vector expressing microRNA of AIF prevented the silibinin-induced cell death.

Conclusions

Silibinin induces apoptotic cell death through a calpain-dependent mechanism involving PKC, ROS, and AIF nuclear translocation in U87MG human glioma cells.     

Alemtuzumab induces caspase-independent cell death in human chronic lymphocytic leukemia cells through a lipid raft-dependent mechanism.

Alemtuzumab is a humanized IgG1 kappa antibody directed against CD52, a glycosyl-phosphatidylinositol linked cell-membrane protein of unknown function. Herein, we demonstrate that alemtuzumab promotes rapid death of chronic lymphocytic leukemia (CLL) cells in vitro, in a complement and accessory cell free system. Using minimal detergent solubilization of CLL membranes, we found that CD52 colocalizes with ganglioside GM-1, a marker of membrane rafts. Fluorescence microscopy revealed that upon crosslinking CD52 with alemtuzumab+anti-Fc IgG, large patches, and in many cases caps, enriched in CD52 and GM-1 formed upon the CLL cell plasma membrane. Depletion of membrane cholesterol or inhibition of actin polymerization significantly diminished the formation of alemtuzumab-induced caps and reduced alemtuzumab-mediated CLL cell death. We compared alemtuzumab-induced direct cytotoxicity, effector cell-mediated toxicity and complement-mediated cytotoxicity of CLL cells to normal T cells. The direct cytotoxicity and observed capping was significantly greater for CLL cells as compared to normal T cells. Cell-mediated and complement-mediated cytotoxicity did not significantly differ between the two cell types. In summary, our data support the hypothesis that alemtuzumab can initiate CLL cell death by crosslinking CD52-enriched lipid rafts. Furthermore, the differential direct cytotoxic effect suggests that CD52 directed antibodies could possibly be engineered to more specifically target CLL cells.     

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.     

CD44 ligation induces apoptosis via caspase- and serine protease-dependent pathways in acute promyelocytic leukemia cells.

We have recently reported that ligation of the CD44 cell surface antigen with A3D8 monoclonal antibody (mAb) triggers incomplete differentiation and apoptosis of the acute promyelocytic leukemia (APL)-derived NB4 cells. The present study characterizes the mechanisms underlying the apoptotic effect of A3D8 in NB4 cells. We show that A3D8 induces activation of both initiator caspase-8 and -9 and effector caspase-3 and -7 but only inhibition of caspase-3/7 and caspase-8 reduces A3D8-induced apoptosis. Moreover, A3D8 induces mitochondrial alterations (decrease in mitochondrial membrane potential DeltaPsi m and cytochrome c release), which are reduced by caspase-8 inhibitor, suggesting that caspase-8 is primarily involved in A3D8-induced apoptosis of NB4 cells. However, the apoptotic process is independent of TNF-family death receptor signalling. Interestingly, the general serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) decreases A3D8-induced apoptosis and when combined with general caspase inhibitor displays an additive effect resulting in complete prevention of apoptosis. These results suggest that both caspase-dependent and serine protease-dependent pathways contribute to A3D8-induced apoptosis. Finally, A3D8 induces apoptosis in all-trans-retinoic acid-resistant NB4-derived cells and in APL primary blasts, characterizing the A3D8 anti-CD44 mAb as a novel class of apoptosis-inducing agent in APL.     

Berberine induces cell death in human hepatoma cells in vitro by downregulating CD147

The isoquinoline plant alkaloid berberine has anti-tumor effects on a variety of carcinoma cells, mainly through inhibition of cell proliferation, apoptosis induction and cell cycle arrest. However, the mechanisms underlying its role in tumor progression are unknown. In the present study, we investigated the molecular mechanisms involved in berberine-induced cell death in human hepatoma carcinoma cell (HCC) lines HepG2 and SMMC7721. Our results showed that berberine inhibited tumor cell viability in a dose- and time-dependent manner, and induced cell death via apoptosis and autophagy. Moreover, berberine treatment significantly inhibited CD147 expression by HCC cells in a dose-dependent manner. Overexpression of CD147 protein markedly reduced berberine-induced cell death. Our data provide the first experimental evidence that berberine induces cell death in HCC cells via downregulation of CD147 and suggest a new mechanism to explain its anti-tumor effects.     

交联CD44通过Fas途径促进人肥大细胞白血病HMC-1细胞的凋亡

目的:探讨交联CD44分子对人肥大细胞白血病HMC-1细胞株Fas表达及其介导的细胞凋亡的影响.方法:应用流式细胞术检测HMC-1细胞表面CD44及Fas的表达,并观察透明质酸(native hyaluronan,HA)、低分子质量透明质酸(fragmented hyaluronan,F-HA)处理或抗体交联CD44分子后细胞表面Fas表达的变化,进而检测PI3K、PKC及MAPK信号通路抑制剂、蛋白质合成抑制剂和肌动蛋白聚合抑制剂对CD44分子交联后细胞表面Fas表达的影响,用Northern杂交技术检测交联CD44对Fas mRNA表达的影响,用Annexin V/PI双染法检测交联CD44、HA或F-HA对Fas介导的细胞凋亡的影响.结果:HMC-1细胞高表达CD44而低表达Fas,交联CD44分子显著上调细胞表面Fas的表达(P＜0.05),而Fas mRNA转录水平没有明显变化;肌动蛋白聚合抑制剂细胞松弛素B能抑制CD44上调的Fas表达(P＜0.05),所检测细胞信号通路抑制剂和蛋白质合成抑制剂均未能抑制Fas表达的上调(P ＞0.05);F-HA和交联CD44均能够促进Fas介导的细胞凋亡(P＜0.05).结论:CD44分子可上调HMC-1细胞的Fas表达并放大Fas介导的细胞凋亡,CD44分子可能成为肥大细胞白血病治疗的新靶点.     

A novel synthetic drug, LB-18, closely related to lembehyne-A derived from a marine sponge, induces caspase-independent cell death to human neuroblastoma cells

Neuroblastoma is a common solid tumor of children that arises from the sympathetic nervous system. Much work has consequently focused on the possibility of inducing marked cell death in neuroblastoma, and the new effective drugs are required. We have newly synthesized LB-18, closely related to lembehyne A (LB-A), a polyacetylene derived from a kind of marine sponge. LB-A has been shown to induce p21/WAF1 and causes G1 phase arrest in mouse neuroblastoma Neuro2A cells; however, we show here that LB-18 causes cell death in human neuroblastoma KP-N-TK cells in a dose-dependent manner. TUNEL assay and flow cytometric analysis showed that the cell death caused by LB-18 was associated with the DNA damage but the pan-caspase inhibitor, zVAD-fmk, could not prevent the cell death. Western blot analysis and cleavage of the caspase-3 or -7 substrate assay showed that LB-18 could not activate caspases 3, 7, 8 and 9. These results suggest that LB-18 causes caspase-independent cell death in human neuroblastoma cells. In the future, LB-18 may be useful for cancer therapeutics, especially for neuroblastoma.     

Doxazosin induces apoptosis of benign and malignant prostate cells via a death receptor-mediated pathway

Quinazoline-based alpha1-adrenoceptor antagonists such as doxazosin and terazosin have been previously shown to induce apoptosis in prostate cancer cells via an alpha1-adrenoceptor-independent pathway, involving activation of transforming growth factor-beta1 (TGF-beta1) signaling. In this study, the molecular events initiating this apoptotic effect were further investigated in vitro using the human androgen-independent prostate cancer cells PC-3 and the human benign prostate epithelial cells BPH-1. Quantitative microarray assays were done in PC-3 and BPH-1 cells after treatment with doxazosin (25 micromol/L, 6 and 24 hours) to identify the early gene changes. Transient changes in the expression of several apoptosis regulators were identified, including up-regulation of Bax and Fas/CD95 and down-regulation of Bcl-xL and TRAMP/Apo3. Moreover, there were significant changes in the expression pattern of signaling components of the extracellular matrix such as integrins alpha2, alphaV, beta1, and beta8. Western blot analysis revealed activation of caspase-8 and caspase-3 within the first 6 to 12 hours of treatment with doxazosin in both PC-3 and BPH-1 cells. Doxazosin-induced apoptosis was blocked by specific caspase-8 inhibitors, supporting the functional involvement of caspase-8 in doxazosin-induced apoptosis. The effect of doxazosin on recruitment of Fas-associated death domain (FADD) and procaspase-8 to the Fas receptor was examined via analysis of death-inducing signaling complex formation. Doxazosin increased FADD recruitment and subsequent caspase-8 activation, implicating Fas-mediated apoptosis as the underlying mechanism of the effect of doxazosin in prostate cells. These results show that doxazosin exerts its apoptotic effects against benign and malignant prostate cells via a death receptor-mediated mechanism with a potential integrin contribution towards cell survival outcomes.     

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.     

Proteasome inhibition by MG132 induces growth inhibition and death of human pulmonary fibroblast cells in a caspase-independent manner

MG132 as a proteasome inhibitor that can induce apoptotic cell death in various cell types including lung cancer cells. We investigated the cellular effects of MG132 on human pulmonary fibroblast (HPF) cells in relation to cell growth inhibition and death, and described the molecular mechanisms of MG132 in HPF cell death. This agent dose-dependently inhibited the growth of HPF cells with an IC50 of approximately 20 μM at 24 h and induced cell death accompanied by the loss of mitochondrial membrane potential (MMP; ?Ψm) and an increase in caspase-3 and -8 activities. MG132 increased intracellular ROS levels and GSH-depleted cell numbers. However, all the tested caspase inhibitors intensified HPF growth inhibition by MG132 and caspase-9 inhibitor also enhanced cell death and MMP (?Ψm) loss. Moreover, the administration of Bcl-2 siRNA augmented HPF cell death by MG132 whereas p53, Bax, caspase-3 and -8 siRNAs did not strongly affect cell death. In addition, each caspase inhibitor and siRNA differently affects ROS levels including O2?- regardless of cell growth inhibition and cell death levels. Caspase-8 and -9 inhibitors increased the number of GSH-depleted cells in MG132-treated HPF cells. In conclusion, MG132 induced growth inhibition and death in HPF cells in a caspase-independent manner. The growth inhibition and death of HPF cells by MG132 and/or each caspase inhibitor or apoptosis-related siRNA were not tightly related to the changes in ROS levels.     

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.     

BH3-only protein BIK induces caspase-independent cell death with autophagic features in Bcl-2 null cells

The BH3-only protein BIK normally induces apoptotic cell death. Here, we have investigated the role of BCL-2 in BIK-induced cell death using Bcl-2+/+ and Bcl-2-/- mouse embryo fibroblasts. Ectopic expression of BIK in Bcl-2-/- cells resulted in enhanced cell death compared to Bcl-2+/+ cells. In these cells, while caspase-8 was activated, there was no significant activation of caspase-9 and 3. There was no detectable mitochondrial to cytosolic release of cytochrome-c. However, there was significant redistribution of AIF from mitochondria to the nucleus. The extent of BIK-induced cell death was augmented by treatment with the pancaspase inhibitor, zVAD-fmk. The Bcl-2 null cells expressing BIK exhibited autophagic features such as cytosolic vacuoles, punctate distribution of LC3 and enhanced expression of Beclin-1. The survival of BIK-expressing Bcl-2-/- cells was enhanced in the presence of PI3 kinase inhibitors 3-methyladenine and Wortmannin and also by depletion of Atg5 and Beclin-1. Death of BIK-expressing Bcl-2-/- cells treated with zVAD-fmk was increased under caspase-8 depletion. Our results suggest enhanced expression of BIK in the Bcl-2 deficient cells leads to cell death with autophagic features and the extent of such cell death could be increased by inhibition of caspases.     

Hyaluronic acid/CD44H interaction induces cell detachment and stimulates migration and invasion of human glioma cells in vitro

The mechanisms underlying the invasive properties of glio-mas, the major form of intrinsic brain tumours in humans, are poorly understood. We have reported that CD44 plays an important role in this behaviour in vitro. In the present work, we investigated the role of its ligand, hyaluronic acid (HA), in invasion in 8 human glioma cell lines. We found that HA mediates cell detachment via its interaction with its high affinity receptor, CD44H. Using 8 μm porosity polycarbonate filter transwells, we demonstrate that HA strongly stimulates migration in all 8 cell lines. This effect was found to be partially counteracted by a CD44H monoclonal antibody (MAb), suggesting the involvement of CD44H, as well as other HA receptors, in this process. Furthermore, incorporation of increasing concentrations of HA in Matrigel in an in vitro invasion assay resulted in a substantial increase in the invasive propensity of the glioma cell lines. Moreover, blocking experiments with the CD44H MAb suggest that CD44H and other receptors interact with HA to promote cell invasion in vitro. Our results show that HA induces cell detachment, stimulates migration and promotes invasion via its interaction with CD44H and other HA receptors in vrtro. These effects could be prevented by use of specific HA receptor antibodies.     

CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

CD44 is a cell surface antigen expressed on acute myeloid leukemia cells and is used as a marker to isolate leukemia stem cells. CD44 ligation with the antibody A3D8 has been found to induce apoptosis in human acute promyelocytic leukemia (APL) cells via activation of caspase-8. The mechanism of A3D8-induced caspase-8 activation was studied in APL NB4 cells. A3D8 induces lipid raft clustering which causes Fas aggregation as determined with a confocal microscope. A3D8-induced apoptosis is abrogated by the lipid raft disrupting agent methyl-β-cyclodextrin and the caspase-8 inhibitor Z-IETD-fmk. Western blot analysis reveals that A3D8 binds to the standard form of CD44 (CD44s). HL-60 cells without detectable CD44s protein are not responsive to A3D8-induced apoptosis. SKNO-1 cells containing higher level of CD44s protein are more sensitive to A3D8-induced apoptosis than NB4 cells. These results indicate that A3D8 induces apoptosis in leukemia cells through caspase-8 activation by binding to CD44s protein and inducing lipid raft clustering.