Disruption of mitochondria is an early event during dolichyl monophosphate-induced apoptosis in U937 cells

Dolichyl monophosphate (Dol-P) is involved in the attachment of carbohydrate chains to proteins in the formation of N-linked glycoprotein. We found that this compound induces apoptosis in human leukemia U937 cells. During this apoptotic execution, the increase of plasma membrane fluidity (5-20 min), reduction in mitochondrial transmembrane potential (delta psi m) and translocation of apoptosis-inducing factor (1-3 hr), caspase-3-like protease activation (2-4 hr), chromatin condensation and DNA ladder formation (3-4 hr) were observed successively. In this study, we examined mitochondrial morphological changes by electron microscopy and delta psi m by JC-1 from immediately after treatment of Dol-P. After 5 min of treatment, we observed clearly that mitochondrial cristae began to be disrupted ultrastructurally and almost all the cristae were disintegrated after 1 hr of treatment. The delta psi m of Dol-P treated cells was reduced to 34% as compared with that of control cells immediately after treatment and was quartered within 1 hr. The reduction in delta psi m was not inhibited by cyclosporin A, N-acetyl-L-cysteine and vitamin E. These results indicate that mitochondrial disruption is one of the first triggering events of Dol-P-induced apoptosis.     

Mechanism of UV-Induced Apoptosis in Human Leukemia Cells: Roles of Ca/Mg-Dependent Endonuclease, Caspase-3, and Stress-Activated Protein Kinases

Ultraviolet light (UV) induced rapid apoptosis of U937 leukemia cells, concurrent with DNA fragmentation and cleavage of poly(ADP-ribose)polymerase (PARP) by activated caspase-3. Thein vitroreconstitution of intact HeLa S3 nuclei and apoptotic U937 cytosolic extract (CE) revealed that (i) Ca²⁺/Mg²⁺-dependent, Zn²⁺-sensitive endonuclease activated in the apoptotic CE induced DNA ladder in HeLa nuclei at pH 6.8–7.4, (ii) activated caspase-3 cleaved PARP in HeLa nuclei, and (iii) when the apoptotic CE was treated with the caspase-3 inhibitor (1 μM Ac-DEVD-CHO) or the caspase-1 inhibitor (10 μM Ac-YVAD-CHO), the former, but not the latter, caused a 50% inhibition of DNA fragmentation and the complete inhibition of PARP cleavage in HeLa nuclei. Similarly, Ac-DEVD-CHO (100 μM) inhibited apoptosis and DNA ladder by 50% and PARP cleavage completely in UV-irradiated U937 cells, but Ac-YVAD-CHO (100 μM) did not. Thus, UV-induced apoptosis of U937 cells involves the Ca²⁺/Mg²⁺-dependent endonuclease pathway and the caspase-3–PARP cleavage–Ca²⁺/Mg²⁺-dependent endonuclease pathway. The former pathway produced directly 50% of apoptotic DNA ladder, and the latter involved activated caspase-3 and PARP cleavage, followed by formation of the remaining 50% DNA ladder by the activated endonuclease. In UV-irradiated B-cell lines, further, p53-dependent increase of Bax resulted in a greater caspase-3 activation compared to its absence. However, UV-induced activation of JNK1 and p38 was not affected by the caspase-1 and -3 inhibitors in U937 cells, so that caspases-1 and -3 do not function upstream of JNK1 and p38.     

Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis.

Apoptosis inducing factor (AIF) is a novel apoptotic effector protein that induces chromatin condensation and large-scale ( approximately 50 kbp) DNA fragmentation when added to purified nuclei in vitro. Confocal and electron microscopy reveal that, in normal cells, AIF is strictly confined to mitochondria and thus colocalizes with heat shock protein 60 (hsp60). On induction of apoptosis by staurosporin, c-Myc, etoposide, or ceramide, AIF (but not hsp60) translocates to the nucleus. This suggests that only the outer mitochondrial membrane (which retains AIF in the intermembrane space) but not the inner membrane (which retains hsp60 in the matrix) becomes protein permeable. The mitochondrio-nuclear redistribution of AIF is prevented by a Bcl-2 protein specifically targeted to mitochondrial membranes. The pan-caspase inhibitor Z-VAD. fmk does not prevent the staurosporin-induced translocation of AIF, although it does inhibit oligonucleosomal DNA fragmentation and arrests chromatin condensation at an early stage. ATP depletion is sufficient to cause AIF translocation to the nucleus, and this phenomenon is accelerated by the apoptosis inducer staurosporin. However, in conditions in which both glycolytic and respiratory ATP generation is inhibited, cells fail to manifest any sign of chromatin condensation and advanced DNA fragmentation, thus manifesting a 'necrotic' phenotype. Both in the presence of Z-VAD. fmk and in conditions of ATP depletion, AIF translocation correlates with the appearance of large-scale DNA fragmentation. Altogether, these data are compatible with the hypothesis that AIF is a caspase-independent mitochondrial death effector responsible for partial chromatinolysis.     

Apoptosis-inducing factor (AIF): a novel caspase-independent death effector released from mitochondria

Apoptosis-inducing factor (AIF) is a phylogenetically ancient mitochondrial intermembrane flavoprotein endowed with the unique capacity to induce caspase-independent peripheral chromatin condensation and large-scale DNA fragmentation when added to purified nuclei. In addition to its apoptogenic activity on nuclei, AIF can also participate in the regulation of apoptotic mitochondrial membrane permeabilization and exhibits an NADH oxidase activity. Under normal circumstances, AIF is secluded behind the outer mitochondrial membrane. However, upon apoptosis induction AIF translocates to the cytosol and the nucleus. Injection of anti-AIF antibodies or knockout of the AIF gene have demonstrated that AIF may be required for cell death occurring in response to some stimuli. In particular, inactivation of AIF renders embryonic stem cells resistant to cell death following growth factor withdrawal. Moreover, AIF is essential for programmed cell death during cavitation of embryoid bodies, the very first wave of (caspase-independent) cell death indispensable for mouse morphogenesis. We have recently found that AIF is neutralized by heat-shock protein (HSP) 70, in a reaction that appears to be independent of ATP or the ATP-binding domain (ABD) of HSP70 and thus differs from the previously described Apaf-1/HSP70 interaction (which requires ATP and the HSP70 ABD). Intriguingly, HSP70 lacking ABD (HSP70 Delta ABD) inhibits apoptosis induced by serum withdrawal, staurosporin, and menadione, three models of apoptosis which are also affected by micro-injection of anti-AIF antibody or genetic ablation of AIF. Altogether, these data suggest that AIF plays a role in the regulation of caspase-independent cell death.     

凋亡诱导因子与细胞凋亡

凋亡诱导因子 (apoptosisinducefactor,AIF)是定位于线粒体膜间隙中的一种氧化还原酶 ,含有线粒体定位信号和核定位信号序列 ,具有很强的促凋亡活性 ,在类胚体成腔和胚胎早期分化过程中具有重要作用。在死亡信号或细胞胁迫的刺激下 ,线粒体通透性转变孔开放 ,释放AIF及细胞色素c至细胞质溶质中 ,具有核定位信号序列的AIF便进入细胞核内 ,引起染色质的初步凝集和DNA大规模断片化 (约 5 0kb) ,进而引发不依赖于胱冬肽酶的细胞凋亡途径 ;线粒体膜间隙释放出来的细胞色素c则可引起染色质的进一步凝集和DNA的寡核小体断片化 ,从而引发依赖于胱冬肽酶的细胞凋亡途径 ;与此同时 ,从线粒体膜间隙释放出来的AIF又可反馈放大线粒体通透性转变孔的渗透性 ,引起AIF与细胞色素c的进一步释放从而加快细胞死亡的进程。此外 ,细胞胁迫还可激活由多聚 (ADP 核糖 )聚合酶 1(PARP 1)所引发的细胞凋亡途径 ,通过AIF和细胞色素c引发细胞凋亡。最新研究结果表明 ,AIF同源线粒体关联死亡诱导者 (AIF homologousmitochondria associatedinducerofdeath ,AMID)与p5 3应答基因的编码产物 (p5 3 responsivegene 3,PRG3)均为AIF的同源蛋白质 ,可直接诱导人类细胞的凋亡。线虫的凋亡诱导因子WAH 1所诱导的细胞凋亡途径依赖于胱冬肽酶     

Apoptotic pathways involved in U937 cells exposed to LDL oxidized by hypochlorous acid

Oxidized low-density lipoproteins (oxLDL) play a critical role in atherogenesis. One oxidative pathway of LDL involves myeloperoxidase, which catalyzes the production of hypochlorous acid (HOCl) in monocytes. We investigated the apoptotic mechanism induced by oxLDL, generated by HOCl treatment of native LDL, in human monocytic U937 cell line. The involvement of the mitochondrial apoptotic pathway was analyzed in Bcl-2-overexpressing clones, generated from U937 cells. HOCl-oxLDL induced in U937 cells (i) a marked caspase-dependent increase of apoptosis, (ii) a loss of mitochondrial membrane potential, (iii) a specific activation of caspase-2, -3, -8, and -9, and (iv) a similar degree of apoptosis in presence or absence of anti-Fas and anti-TNF-R1 antibodies. Moreover, the degree of HOCl-oxLDL-induced caspase-3 and -8 activation, and apoptosis was significantly reduced in U937/Bcl-2 cells, with no activation of caspase-9. By contrast, Cu-oxLDL-mediated apoptosis in U937 cells involved exclusively the mitochondrial pathway. In conclusion, the mechanism of HOCl-oxLDL-induced apoptosis in monocytic U937 cells involves the two pathways of apical caspase activation: (i) death receptor-mediated caspase-8 and (ii) mitochondria-mediated caspase-9. This converges in the activation of executing caspases, including caspase-3, and apoptosis. The interference of Bcl-2 overexpression with HOCl-oxLDL-induced apoptosis suggests the importance of mitochondrial involvement in this apoptotic mechanism.     

Generation of reactive oxygen species is an early event in dolichyl phosphate-induced apoptosis

The mechanism of induction of apoptosis by dolichyl phosphate (Dol-P) was investigated in U937 cells. Studies using isolated mitochondria revealed that the respiratory complex II activity was almost completely inhibited by 20 microg/ml of Dol-P but not by the same concentration of dolichol. Activities of complex I and III were also inhibited by Dol-P, but nearly 50% of activity still remained at 20 microg/ml. Dol-P induced release of cytochrome-c from the isolated mitochondria. Fluorometric microtiter plate assay revealed that generation of reactive oxygen species (ROS) increased in a time-dependent manner. Flow cytometric analysis also indicated that Dol-P caused loss of mitochondrial membrane potential (Deltapsi(m)) and increased ROS generation. The addition of the antioxidant pyrrolidine dithiocarbamate (PDTC) significantly inhibited Dol-P-induced ROS generation and activation of caspase-3. A specific inhibitor of respiratory complex II, thenoyltrifluoroacetone (TTFA), increased ROS generation, potentially mimicking the consequence of inhibition of electron flow at complex II by Dol-P in U937 cells. Electron microscopy revealed that mitochondria became swollen and spherical in shape by the treatment with Dol-P. Neither the tyrosine kinase inhibitor k252a nor mitogen activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitors PD98059 and U0126 inhibited the Dol-P-induced apoptosis. Together, these results suggest that the direct disruption of mitochondrial respiratory complexes and the consequent ROS generation play a critical role in the initiation of Dol-P-induced apoptosis.     

Inhibition of K+ efflux prevents mitochondrial dysfunction, and suppresses caspase-3-, apoptosis-inducing factor-, and endonuclease G-mediated constitutive apoptosis in human neutrophils

Neutrophils die rapidly via apoptosis and their survival is contingent upon rescue from constitutive programmed cell death by signals from the microenvironment. In these experiments, we investigated whether prevention of K⁺ efflux could affect the apoptotic machinery in human neutrophils. Disruption of the natural K⁺ electrochemical gradient suppressed neutrophil apoptosis (assessed by annexin V binding, nuclear DNA content and nucleosomal DNA fragmentation) and prolonged cell survival within 24–48 h of culture. High extracellular K⁺ (10–100 mM) did not activate extracellular signal-regulated kinase (ERK) and Akt, nor affected phosphorylation of p38 MAPK associated with constitutive apoptosis. Consistently, pharmacological blockade of ERK kinase or phosphatidylinositol 3-kinase (PI 3-kinase) did not affect the anti-apoptotic action of KCl. Inhibition of K⁺ efflux effectively reduced, though never completely inhibited, decreases in mitochondrial transmembrane potential (ΔΨ_m) that preceded development of apoptotic morphology. Changes in ΔΨ_m resulted in attenuation of cytochrome c release from mitochondria into the cytosol and decreases in caspase-3 activity. Culture of neutrophils in medium containing 80 mM KCl with the pan-caspase inhibitor Z-VAD-FMK resulted in slightly greater suppression of apoptosis than KCl alone. High extracellular KCl also attenuated translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) from mitochondria to nuclei. The DNase inhibitor, aurintricarboxylic acid (ATA) partially inhibited nucleosomal DNA fragmentation, and the effects of ATA and 80 mM KCl were not additive. These results show that prevention of K⁺ efflux promotes neutrophil survival by suppressing apoptosis through preventing mitochondrial dysfunction and release of the pro-apoptotic proteins cytochrome c, AIF and EndoG independent of ERK, PI 3-kinase and p38 MAPK. Thus, K⁺ released locally from damaged cells may function as a survival signal for neutrophils.     

CD437, a synthetic retinoid, induces apoptosis in human respiratory epithelial cells via caspase-independent mitochondrial and caspase-8-dependent pathways both up-regulated by JNK signaling pathway

The synthetic retinoid-related molecule CD437-induced apoptosis in human epithelial airway respiratory cells: the 16HBE bronchial cell line and normal nasal epithelial cells. CD437 caused apoptosis in S-phase cells and cell cycle arrest in S phase. Apoptosis was abolished by caspase-8 inhibitor z-IETD-fmk which preserved S-phase cells but was weakly inhibited by others selective caspase-inhibitors, indicating that caspase-8 activation was involved. z-VAD and z-IETD prevented the nuclear envelope fragmentation but did not block the chromatin condensation. The disruption of mitochondrial transmembrane potential was also induced by CD437 treatment. The translocation of Bax to mitochondria was demonstrated, as well as the release of cytochrome c into the cytosol and of apoptosis-inducing factor (AIF) translocated into the nucleus. z-VAD and z-IETD did not inhibit mitochondrial depolarization, Bax translocation or release of cytochrome c and AIF from mitochondria. These results suggest that CD437-induced apoptosis is executed by two converging pathways. AIF release is responsible for chromatin condensation, the first stage of apoptotic cell, via a mitochondrial pathway independent of caspase. But final stage of apoptosis requires the caspase-8-dependent nuclear envelope fragmentation. In addition, using SP600125, JNK inhibitor, we demonstrated that CD437 activates the JNK-MAP kinase signaling pathway upstream to mitochondrial and caspase-8 pathways. Conversely, JNK pathway inhibition, which suppresses S-phase apoptosis, did not prevent cell cycle arrest within S phase, confirming that these processes are triggered by distinct mechanisms.     

Thiosulfinates from Allium tuberosum L. induce apoptosis via caspase-dependent and -independent pathways in PC-3 human prostate cancer cells

This study was aimed to evaluate the apoptotic effects of thiosulfinates purified from Allium tuberosum L. on PC-3 human prostate cancer cells, and to elucidate detailed apoptosis mechanisms. Thiosulfinates significantly decrease viable cell numbers in dose- and time-dependent manners by apoptotic cell death via DNA fragmentation, chromatin condensation, and an increased sub-G1 phase. Apoptosis induced by thiosulfinates is associated with the activation of initiator caspase-8 and -9, and the effector caspase-3. In this study, thiosulfinates stimulated Bid cleavage, indicating that the apoptotic action of caspase-8-mediated Bid cleavage leads to the activation of caspase-9. Thiosulfinates decreased the expression of the anti-apoptotic protein Bcl-2 and increased the expression of the pro-apoptotic protein Bax. Thiosulfinates also increased the expression of AIF, a caspase-independent mitochondrial apoptosis factor, in PC-3 cells. These results indicate that thiosulfinates from A. tuberosum L. inhibit cell proliferation and induce apoptosis in PC-3 cells, which may be mediated via both caspase-dependent and -independent pathways.     

Involvement of hydroperoxide in mitochondria in the induction of apoptosis by the eicosapentaenoic acid

Eicosapentaenoic acid (EPA) induced apoptosis of rat basophilic leukemia cells (RBL2H3 cells), whereas 100 μM linoleic acid (LA) had no significant effect. Cytochrome c was released at 4 h. Apoptosis was detected at 6 h after exposure to EPA and docosahexaenoic acid (DHA), and preceded the activation of caspase-3. Liberation of apoptosis-inducing factor (AIF) from mitochondria and its translocation into the nucleus were observed at 4 h. A broad-specificity caspase inhibitor, z-VAD-fmk, failed to suppress the apoptosis, suggesting that EPA induced caspase-independent apoptosis. On other hand, a poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor that blocks AIF translocation to the nucleus suppressed EPA-induced apoptosis. The level of hydroperoxide in the cells and mitochondria increased at the early phase of apoptosis within 2 h. On the contrary, elevation of hydroperoxide in mitochondria was not observed after treatment with LA. The EPA-induced apoptosis was abolished by prevention of the hydroperoxide elevation in mitochondria via overexpression of mitochondrial phospholipid hydroperoxide glutathione peroxidase (PHGPx). Neither cytochrome c nor AIF were released from mitochondria in the mitochondrial PHGPx-overexpressing cells. EPA also induced apoptosis in HeLa cells, but not in L929 or RAW264.7 cells. Enhancement of the hydroperoxide level in mitochondria was found in the EPA-sensitive HeLa cells after treatment with EPA, whereas no such enhancement was observed in the apoptosis-resistant L929 and RAW264.7 cells. These results suggest that the generation of hydroperoxide in mitochondria induced by EPA is associated with AIF release from mitochondria and the induction of apoptosis.     

Differential apoptotic pathways activated in response to Cu-induced or HOCl-induced LDL oxidation in U937 monocytic cell line

We compared the apoptotic mechanism involved in U937 human monocytic cell line in presence of oxidized low-density lipoproteins (oxLDL) obtained after treatment with hypochlorous acid (HOCl) or copper (Cu).Both types of oxLDL induced U937 apoptotic cell death via the mitochondrial pathway. In contrast to HOCl-oxLDL, Cu-oxLDL induced apoptosis via a caspase-independent mechanism, with no activation of pro-caspase-3, but via the release of apoptosis inducing factor (AIF) from mitochondria.The apoptotic program of the monocyte differs depending on the mode of LDL oxidation, based on differences in the oxidatively modified components of the two oxLDL types.     

Mitochondria-mediated caspase-independent apoptosis induced by cadmium in normal human lung cells

Cadmium, a well-known environmental hazard, has caused serious health problems in humans and animals. Accumulating evidence suggests the cadmium toxicity is mediated by oxidative stress-induced cell death. However, the molecular signaling underlying cadmium-induced apoptosis remains unclear. In this study, we demonstrate here that cadmium induced mixed types of cell death including primary apoptosis (early apoptosis), secondary necrosis (late apoptosis), and necrosis in normal human lung cells, MRC-5, as revealed by chromatin condensation, phosphatidylserine (PS) externalization, and hypodiploid DNA content. The total apoptotic cells reached a plateau of around 40.0% after 24 h exposure of 100 microM cadmium. Pretreatment with Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), a broad spectrum of caspase inhibitor, could not rescue apoptotic cells from cadmium toxicity. Coincidently, we failed to detect the activation of pro-caspase-3 and cleavage of PARP by immunoblot, which implies the apoptogenic activity of cadmium in MRC-5 cells is caspase-independent. JC-1 staining also indicated that mitochondrial depolarization is a prelude to cadmium-induced apoptosis, which was accompanied by a translocation of caspase-independent pro-apoptotic factor apoptosis-inducing factor (AIF) into the nucleus as revealed by the immunofluorescence assay. In summary, this study demonstrated for the first time that cadmium induced a caspase-independent apoptotic pathway through mitochondria-mediated AIF translocation into the nucleus.     

Telomerase activity and hepatic functions of rat embryonic liver progenitor cell in nanoscaffold-coated model bioreactor

Phosphatidylserine (PS) externalization is a key feature of apoptotic cell death and plays an important role in clearance of apoptotic cells by phagocytes. PS externalization during apoptosis is generally an irreversible event mediated by caspase activation and is accompanied by other apoptotic events. We report here that an apoptosis inducer α-tocopheryl succinate (TOS) can induce PS externalization that is independent of apoptosis and reversible in the absence of fetal bovine serum (FBS) in histiocytic lymphoma U937 cells. In the presence of FBS, TOS induced PS externalization via a caspase-dependent mechanism accompanied by mitochondrial depolarization, cell shrinkage, increase of caspase-3 activity, and chromatin condensation. In contrast, in the absence of FBS, TOS induced the rapid PS externalization which was not accompanied by other apoptotic events. The PS externalization was reversible by removing TOS and was not involved in Ca²⁺-dependent scramblase activation and thiol oxidation of aminophospholipid translocase. A similar PS externalization was also induced by cholesteryl hemisuccinate (CS), the other succinate ester. These results suggested that the mechanism of TOS- and CS-induced PS externalization in the absence of FBS was different from it occurring during typical apoptosis.     

Evidence in favour of a role for peripheral-type benzodiazepine receptor ligands in amplification of neuronal apoptosis

The mitochondrial peripheral benzodiazepine receptor (PBR) is involved in a functional structure designated as the mitochondrial permeability transition (MPT) pore, which controls apoptosis. PBR expression in nervous system has been reported in glial and immune cells. We now show expression of both PBR mRNA and protein, and the appearance of binding of a synthetic ligand fluo-FGIN-1-27 in mitochondria of rat cerebellar granule cells (CGCs). Additionally, the effect of PBR ligands on colchicine-induced apoptosis was investigated. Colchicine-induced neurotoxicity in CGCs was measured at 24 h. We show that, in vitro, PBR ligands 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195), 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4- benzodiazepin-2-one (Ro5-4864) and diazepam (25– 50 M) enhanced apoptosis induced by colchicine, as demonstrated by viability experiments, flow cytometry and nuclear chromatin condensation. Enhancement of colchicine-induced apoptosis was characterized by an increase in mitochondrial release of cytochrome c and AIF proteins and an enhanced activation of caspase-3, suggesting mitochondrion dependent mechanism that is involved in apoptotic process. Our results indicate that exposure of neural cells to PBR ligands generates an amplification of apoptotic process induced by colchicine and that the MPT pore may be involved in this process.     

Heat-shock protein 70 antagonizes apoptosis-inducing factor

Heat-shock protein 70 (Hsp70) has been reported to block apoptosis by binding apoptosis protease activating factor-1 (Apaf-1), thereby preventing constitution of the apoptosome, the Apaf-1/cytochrome c/caspase-9 activation complex [1,2]. Here we show that overexpression of Hsp70 protects Apaf-1-/- cells against death induced by serum withdrawal, indicating that Apaf-1 is not the only target of the anti-apoptotic action of Hsp70. We investigated the effect of Hsp70 on apoptosis mediated by the caspase-independent death effector apoptosis inducing factor (AIF), which is a mitochondrial intermembrane flavoprotein [3,4]. In a cell-free system, Hsp70 prevented the AIF-induced chromatin condensation of purified nuclei. Hsp70 specifically interacted with AIF, as shown by ligand blots and co-immunoprecipitation. Cells overexpressing Hsp70 were protected against the apoptogenic effects of AIF targeted to the extramitochondrial compartment. In contrast, an anti-sense Hsp70 complementary DNA, which reduced the expression of endogenous Hsp70, increased sensitivity to the lethal effect of AIF. The ATP-binding domain of Hsp70 seemed to be dispensable for inhibiting cell death induced by serum withdrawal, AIF binding and AIF inhibition, although it was required for Apaf-1 binding. Together, our data indicate that Hsp70 can inhibit apoptosis by interfering with target proteins other than Apaf-1, one of which is AIF.     

A highly polar xanthophyll of 9′-cis-neoxanthin induces apoptosis in HCT116 human colon cancer cells through mitochondrial dysfunction

Highly polar xanthophylls of 9′-cis-neoxanthin (neoxanthin) and fucoxanthin, which have the characteristic structure of an epoxy group and an allenic bond, were previously found to induce apoptosis in human prostate cancer cells. In the present study, we found apoptosis induction by neoxanthin in HCT116 human colon cancer cells and examined the induction mechanism. The cells exposed to 20 μM neoxanthin clearly showed chromatin condensation, DNA fragmentation, and an increase in hypodiploid cells. Neoxanthin treatment increased the activities of caspase-3, -8 and -9, and the protein levels of their active subunits, except in the case of caspase-8. The treatment also caused the loss of mitochondrial transmembrane potential at an early stage and subsequently the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria to cytosol. The exposure of neoxanthin directly to mitochondria isolated from the cells enhanced the release of cytochrome c and AIF in a dose-dependent manner. Approximately 50% of the neoxanthin taken up into the HCT116 cells accumulated in the mitochondrial fraction. These results suggest that the accumulation of neoxanthin in mitochondria causes the loss of mitochondrial transmembrane potential and thereafter releases cytochrome c and AIF, leading to the execution of apoptosis.