Capsaicin induces apoptosis by generating reactive oxygen species and disrupting mitochondrial transmembrane potential in human colon cancer cell lines

Although genetic factors are a well-known cause of colorectal cancer, environmental factors contribute more to its development. Despite advances in the fields of surgery, radiotherapy and chemotherapy, the cure rates for colon cancer have not substantially improved over the past few decades. Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), the principal pungent ingredient of hot chili pepper, has exhibited an anti-tumor effect in many cell types. However, the mechanisms responsible for the anti-tumor effect of capsaicin are not yet completely understood. In this study, we investigated whether capsaicin induces apoptosis in colon cancer cell lines. Capsaicin decreased cell viability in a dose-dependent manner in Colo320DM and LoVo cells. In addition, capsaicin produced cell morphology changes and DNA fragmentation, decreased the DNA contents, and induced phosphatidylserine translocation, which is a hallmark of apoptotic cell death. We showed that capsaicin-induced apoptosis is associated with an increase in ROS generation and a disruption of the mitochondrial transmenbrane potential. A possible mechanism of capsaicin-induced apoptosis is the activation of caspase 3, a major apoptosis-executing enzyme. Treatment with capsaicin induced a dramatic increase in caspase 3 activity, as assessed by the cleavage of Ac-DEVD-AMC, a fluorogenic substrate. In conclusion, our results clearly showed that capsaicin induced apoptosis in colon cancer cells. Although the actual mechanisms of capsaicin-induced apoptosis remain uncertain, it may be a beneficial agent for colon cancer treatment and chemoprevention.     

Ionizing radiation accelerates Drp1-dependent mitochondrial fission, which involves delayed mitochondrial reactive oxygen species production in normal human fibroblast-like cells

Ionizing radiation is known to increase intracellular level of reactive oxygen species (ROS) through mitochondrial dysfunction. Although it has been as a basis of radiation-induced genetic instability, the mechanism involving mitochondrial dysfunction remains unclear. Here we studied the dynamics of mitochondrial structure in normal human fibroblast like cells exposed to ionizing radiation. Delayed mitochondrial production was peaked 3 days after irradiation, which was coupled with accelerated mitochondrial fission. We found that radiation exposure accumulated dynamin-related protein 1 (Drp1) to mitochondria. Knocking down of Drp1 expression prevented radiation induced acceleration of mitochondrial fission. Furthermore, knockdown of Drp1 significantly suppressed delayed production of mitochondrial . Since the loss of mitochondrial membrane potential, which was induced by radiation was prevented in cells knocking down of Drp1 expression, indicating that the excessive mitochondrial fission was involved in delayed mitochondrial dysfunction after irradiation.     

N-t-Butyl hydroxylamine regulates ionizing radiation-induced apoptosis in U937 cells

Ionizing radiation induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Therefore, compounds that scavenge reactive oxygen species may confer regulatory effects on apoptosis. Recently, it has been shown that the decomposition product of the spin-trapping agent α-phenyl-N-t-butylnitrone, N-t-butyl hydroxylamine (NtBHA), mimics α-phenyl-N-t-butylnitrone and is much more potent in delaying reactive oxygen species-associated senescence. We investigated the effects of NtBHA on ionizing radiation-induced apoptosis. Upon exposure to 2 Gy of γ-irradiation, there was a distinct difference between the control cells and the cells pre-treated with 0.1 mM NtBHA for 2 h in regard to apoptotic parameters, cellular redox status, mitochondria function, and oxidative damage to cells. NtBHA effectively suppressed morphological evidence of apoptosis and DNA fragmentation in U937 cells exposed to ionizing radiation. The generation of intracellular reactive oxygen species was higher and the GSH level was lower in control cells compared to NtBHA-treated cells. The ionizing radiation-induced mitochondrial damage reflected by the altered mitochondrial permeability transition, the increase in the accumulation of reactive oxygen species, and the reduction of ATP production were significantly higher in control cells compared to NtBHA-treated cells. NtBHA pre-treated cells showed significant inhibition of apoptotic features such as activation of caspase-3, up-regulation of Bax and p53, and down-regulation of Bcl-2 compared to control cells upon exposure to ionizing radiation. This study indicates that NtBHA may play an important role in regulating the apoptosis induced by ionizing radiation presumably through scavenging of reactive oxygen species.     

Autophagy protein Ulk1 promotes mitochondrial apoptosis through reactive oxygen species

Regardless of rapid progression in the field of autophagy, it remains a challenging task to understand the cross talk with apoptosis. In this study, we overexpressed Ulk1 in HeLa cells and evaluated the apoptosis-inducing potential of the Ulk1 gene in the presence of cisplatin. The gain of function of Ulk1 gene showed a decline in cell viability and colony formation in HeLa cells. The Ulk1-overexpressing cells showed higher apoptotic attributes by an increase in the percentage of annexin V, escalated expression of Bax/Bcl2 ratio, and caspase-9, -3/7 activities. Further, reactive oxygen species (ROS) generation was found to be much higher in HeLa-Ulk1 than in the mock group. Scavenging the ROS by N-acetyl-L-cysteine increased cell viability and colony number as well as mitochondrial membrane potential (MMP). Our data showed that Ulk1 on entering into mitochondria inhibits the manganese dismutase activity and intensifies the mitochondrial superoxide level. The Ulk1-triggered autophagy (particularly mitophagy) resulted in a fall in ATP; thus the nonmitophagic mitochondria overwork the electron-transport cycle to replenish energy demand and are inadvertently involved in ROS overproduction that led to apoptosis. In this present investigation, our results decipher a previously unrecognized perspective of apoptosis induction by a key autophagy protein Ulk1 that may contribute to identification of its tumor-suppressor properties through dissecting the connection among cellular bioenergetics, ROS, and MMP.     

Cdk2 activity is associated with depolarization of mitochondrial membrane potential during apoptosis

In this study we show that panaxadiol, a ginseng saponin with a dammarane skeleton, induces apoptotic cell death by depolarization of mitochondrial membrane potential in human hepatoma SK-HEP-1 cells. Sequential activation of caspases-9, -3, and -7, but not of caspase-8, occurs after mitochondrial membrane depolarization and cytochrome c release from the mitochondria of panaxadiol-treated cells. Moreover, Cdk2 kinase activity, but not Cdc2 kinase activity, is markedly upregulated in the early stages of apoptosis. Olomoucine or roscovitine, specific Cdks inhibitors, effectively prevent mitochondrial membrane depolarization as well as apoptotic cell death in panaxadiol-treated cells. Thus, panaxadiol-treatment induces cell death-dependent activation of Cdk2 kinase activity, which is functionally associated with depolarization of mitochondrial membrane potential and subsequent cytochrome c release.     

Reactive oxygen species induced by proteasome inhibition in neuronal cells mediate mitochondrial dysfunction and a caspase-independent cell death

While increasing evidence shows that proteasome inhibition triggers oxidative damage, mitochondrial dysfunction and death in neuronal cells, the regulatory relationship among these events is unclear. Using mouse neuronal cells we show that the cytotoxicity induced by mild (0.25 μM) and potent (5.0 μM) doses of the proteasome inhibitor, N-Benzyloxycarbonyl-Ile-Glu (O-t-butyl)-Ala-leucinal, (PSI) involved a dose-dependent increase in caspase activation, overproduction of reactive oxygen species (ROS) and a mitochondrial dysfunction manifested by the translocation of the proapoptotic protein, Bax, from the cytoplasm to the mitochondria, membrane depolarization and the release of cytochrome c and the apoptosis inducing factor (AIF) from mitochondria to the cytoplasm and nucleus, respectively. Whereas caspase or Bax inhibition failed to prevent mitochondrial membrane depolarization and neuronal cell death, pretreatments with the antioxidant N-acetyl-l-cysteine (NAC) or overexpression of the antiapoptotic protein Bcl-xL abrogated these events in cells exposed to mild levels of PSI. These findings implicated ROS as a mediator of PSI-induced cytotoxicity. However, depletions in glutathione and Bcl-xL with potent proteasome inhibition exacerbated this response whereupon survival required the cooperative protection of NAC with Bcl-xL overexpression. Collectively, ROS induced by proteasome inhibition mediates a mitochondrial dysfunction in neuronal cells that culminates in death through caspase- and Bax-independent mechanisms. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fast neutrons-induced apoptosis is Fas-independent in lymphoblastoid cells

We have previously shown that ionizing radiation-induced apoptosis in human lymphoblastoid cells differs according to their p53 status, and that caspase 8-mediated cleavage of BID is involved in the p53-dependent pathway. In the present study, we investigated the role of Fas signaling in caspase 8 activation induced by fast neutrons irradiation in these cells. Fas and FasL expression was assessed by flow cytometry and by immunoblot. We also measured Fas aggregation after irradiation by fluorescence microscopy. We found a decrease of Fas expression after irradiation, but no change in Fas ligand expression. We also showed that, in contrast to the stimulation of Fas by an agonistic antibody, Fas aggregation did not occur after irradiation. Altogether, our data strongly suggest that fast neutrons induced-apoptosis is Fas-independent, even in p53-dependent apoptosis.     

Mechanisms of mitochondrial apoptosis induced by peripheral benzodiazepine receptor ligands in human colorectal cancer cells

Specific ligands of the peripheral benzodiazepine receptor (PBR) have been shown to induce apoptosis in gastrointestinal cancers. The aim of this study was to characterize the signaling pathways of PBR ligand-induced apoptosis. FGIN-1-27 but not PK 11195-induced apoptosis was associated with a decrease of mitochondrial membrane potential and an increase of mitochondrial volume in HT29 colorectal cancer cells. However, PK 11195-elicited apoptosis was associated with a downregulation of Bcl-2, translocation of Bax to the mitochondria including subsequent oligomerization, and activation of caspase-9, indicating the involvement of mitochondria in PK 11195-induced apoptosis. Moreover, PK 11195-induced apoptosis was associated with the generation of reactive oxygen species. This study demonstrates a novel mechanism of PK 11195-induced mitochondrial apoptosis without alteration of the mitochondrial membrane potential. The characterization of signaling pathways associated with PBR ligand-induced apoptosis will build the base for a future use of these ligands in anti-neoplastic therapeutic approaches.     

Regulation of ionizing radiation-induced apoptosis by a manganese porphyrin complex

Ionizing radiation induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Therefore, compounds that scavenge reactive oxygen species may confer regulatory effects on apoptosis. Superoxide dismutase (SOD) mimetics have been shown to be protective against cell injury caused by reactive oxygen species. We investigated the effects of the manganese (III) tetrakis(N-methyl-2-pyridyl)porphyrin (MnTMPyP), a cell-permeable SOD mimetic, on ionizing radiation-induced apoptosis. Upon exposure to 2 Gy of gamma-irradiation, there was a distinct difference between the control cells and the cells pre-treated with 5 microM MnTMPyP for 2 h with regard to apoptotic parameters, cellular redox status, mitochondria function, and oxidative damage to cells. MnTMPyP effectively suppressed morphological evidence of apoptosis and DNA fragmentation in U937 cells exposed to ionizing radiation. The [GSSG]/[GSH+GSSG] ratio and the generation of intracellular reactive oxygen species were higher and the [NADPH]/[NADP(+)+NADPH] ratio was lower in control cells compared to MnTMPyP-treated cells. The ionizing radiation-induced mitochondrial damage reflected by the altered mitochondrial permeability transition, the increase in the accumulation of reactive oxygen species, and the reduction of ATP production were significantly higher in control cells compared to MnTMPyP-treated cells. MnTMPyP pre-treated cells showed significant inhibition of apoptotic features such as activation of caspase-3, up-regulation of Bax and p53, and down-regulation of Bcl-2 compared to control cells upon exposure to ionizing radiation. This study indicates that MnTMPyP may play an important role in regulating the apoptosis induced by ionizing radiation presumably through scavenging of reactive oxygen species.     

Sulindac-derived reactive oxygen species induce apoptosis of human multiple myeloma cells via p38 mitogen activated protein kinase-induced mitochondrial dysfunction

Non-steroidal anti-inflammatory drugs are well known to induce apoptosis of cancer cells independent of their ability to inhibit cyclooxygenase-2, but the molecular mechanism for this effect has not yet been fully elucidated. The purpose of this study was to elucidate the potential signaling components underlying sulindac-induced apoptosis in human multiple myeloma (MM) cells. We found that sulindac induces apoptosis by promoting ROS generation, accompanied by opening of mitochondrial permeability transition pores, release of cytochrome c and apoptosis inducing factor from mitochondria, followed by caspase activation. Bcl-2 cleavage and down-regulation of the inhibitor of apoptosis proteins (IAPs) family including cIAP-1/2, XIAP, and survivin, occurred downstream of ROS production during sulindac-induced apoptosis. Forced expression of survivin and Bcl-2 blocked sulindac-induced apoptosis. Most importantly, sulindac-derived ROS activated p38 mitogen-activated protein kinase and p53. SB203580, a p38 mitogen-activated protein kinase inhibitor, and RNA inhibition of p53 inhibited the sulindac-induced apoptosis. Furthermore, p53, Bax, and Bak accumulated in mitochondria during sulindac-induced apoptosis. All of these events were significantly suppressed by SB203580. Our results demonstrate a novel mechanism of sulindac-induced apoptosis in human MM cells, namely, accumulation of p53, Bax, and Bak in mitochondria mediated by p38 MAPK activation downstream of ROS production.     

Reactive oxygen species-mediated endoplasmic reticulum stress and mitochondrial dysfunction contribute to polydatin-induced apoptosis in human nasopharyngeal carcinoma CNE cells

Previous studies revealed that polydatin, a natural small compound, possessed protective effect against ischemia/reperfusion injury and inflammation. However, the action and molecular mechanism of its potent anti-cancer activity remain poorly understood. In the present study, polydatin significantly killed several human tumor cell lines in a dose- and time-dependent manner. The compound also dose-dependently caused mitochondrial apoptosis in human nasopharyngeal carcinoma CNE cells. In addition, polydatin triggered endoplasmic reticulum (ER) stress and down-regulated the phosphorylation of Akt in CNE cells, while knock-down of CCAAT/enhancer-binding protein homologous protein (CHOP) dramatically abrogated the inactivation of Akt and reversed the pro-apoptotic effect of polydatin. Furthermore, polydatin provoked the generation of reactive oxygen species in CNE cells, while the antioxidant N-acetyl cysteine almost completely blocked the activation of ER stress and apoptosis, suggesting polydatin-induced reactive oxygen species is an early event that triggers ER stress mitochondrial apoptotic pathways in CNE cells. Taken together, these findings strongly suggest that polydatin might be a promising anti-tumor drug and our data provide the molecular theoretical basis for clinical application of polydatin.     

Nitric oxide-induced eosinophil apoptosis is dependent on mitochondrial permeability transition (mPT), JNK and oxidative stress: apoptosis is preceded but not mediated by early mPT-dependent JNK activation

Background

Eosinophils are critically involved in the pathogenesis of asthma. Nitric oxide (NO) is produced in high amounts in asthmatic lungs and has an important role as a regulator of lung inflammation. NO was previously shown to induce eosinophil apoptosis mediated via c-jun N-terminal kinase (JNK) and caspases. Our aim was to clarify the cascade of events leading to NO-induced apoptosis in granulocyte macrophage-colony stimulating factor (GM-CSF)-treated human eosinophils concentrating on the role of mitochondria, reactive oxygen species (ROS) and JNK.

Methods

Apoptosis was determined by flow cytometric analysis of relative DNA content, by Annexin-V labelling and/or morphological analysis. Immunoblotting was used to study phospho-JNK (pJNK) expression. Mitochondrial membrane potential was assessed by JC-1-staining and mitochondrial permeability transition (mPT) by loading cells with calcein acetoxymethyl ester (AM) and CoCl₂ after which flow cytometric analysis was conducted. Statistical significance was calculated by repeated measures analysis of variance (ANOVA) or paired t-test.

Results

NO-donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) induced late apoptosis in GM-CSF-treated eosinophils. SNAP-induced apoptosis was suppressed by inhibitor of mPT bongkrekic acid (BA), inhibitor of JNK SP600125 and superoxide dismutase-mimetic AEOL 10150. Treatment with SNAP led to late loss of mitochondrial membrane potential. Additionally, we found that SNAP induces early partial mPT (1 h) that was followed by a strong increase in pJNK levels (2 h). Both events were prevented by BA. However, these events were not related to apoptosis because SNAP-induced apoptosis was prevented as efficiently when BA was added 16 h after SNAP. In addition to the early and strong rise, pJNK levels were less prominently increased at 20–30 h.

Conclusions

Here we demonstrated that NO-induced eosinophil apoptosis is mediated via ROS, JNK and late mPT. Additionally, our results suggest that NO induces early transient mPT (flickerings) that leads to JNK activation but is not significant for apoptosis. Thereby, we showed some interesting early events in NO-stimulated eosinophils that may take place even if the threshold for irreversible mPT and apoptosis is not crossed. This study also revealed a previously unknown physiological function for transient mPT by showing that it may function as initiator of non-apoptotic JNK signalling.     

Cholesterol secoaldehyde induces apoptosis in J774 macrophages via mitochondrial pathway but not involving reactive oxygen species as mediators

Cholesterol secoaldehyde (3β-hydroxy-5-oxo-5,6-secocholestan-6-al or ChSeco) is an oxysterol known to be formed in reactions of ozone with cholesterol and also when cholesterol-5α-hydroperoxide undergoes Hock cleavage. In view of its widespread occurrence and atherogenic potential, we examined the effects of ChSeco on mouse J774 macrophage viability and events associated with apoptosis. A dose-dependent decrease in cell viability, disruptions in mitochondrial transmembrane potential (64 ± 5.5%; mean ± SD, n = 3), increased levels of cytosolic cytochrome c (8.8 ± 0.84 ng/ml; mean ± SD, n = 3), activation of caspase-3 (ca. 3.6-fold) and caspase-9 (ca.1.8-fold), and increased DNA fragmentation (ca. 5-fold), all indicative of apoptosis, were observed in response to exposure to ChSeco. The apoptotic nature of cell death in macrophages was confirmed by dual staining with acridine orange and ethidium bromide. However, unlike the case with cardiomyoblasts and neuronal cells, the apoptotic process in these immune cells was not mediated by increased levels of reactive oxygen species as indicated by a minimal or no increase in 2′,7′-dichlorofluorescein fluorescence. It is suggested that the apoptotic process is mediated via the mitochondrial pathway and that ChSeco formed in biological environments contributes to the initiation, progression, and culmination of atherosclerotic plaque formation, as these processes are critically dependent on macrophage apoptosis.     

Pyridoxamine protects intestinal epithelium from ionizing radiation-induced apoptosis

Reactive oxygen species (ROS) and reactive carbonyl species (RCS) are the major causes of biological tissue damage during exposure to ionizing radiation (IR). The existing strategies to protect normal tissues from the detrimental effects of IR suffer from several shortcomings including highly toxic side effects, unfavorable administration routes, and low efficacy. These shortcomings emphasize a need for radioprotective treatments that combine effectiveness with safety and ease of use. In this paper, we demonstrate that pyridoxamine, a ROS and RCS scavenger with a very favorable safety profile, can inhibit IR-induced gastrointestinal epithelial apoptosis in cell culture and in an animal model. Pyridoxamine was more effective at protecting from radiation-induced apoptosis than amifostine, a synthetic thiol compound and the only FDA-approved radioprotector. We suggest that pyridoxamine has potential as an effective and safe radioprotective agent.     

Involvement of Mst1 in tumor necrosis factor-alpha-induced apoptosis of endothelial cells

Mammalian sterile 20-kinase 1 (Mst1), a member of the sterile-20 family protein kinase, plays an important role in the induction of apoptosis. However, little is know about the physiological activator of Mst1 and the role of Mst1 in endothelial cells (ECs). We examined whether Mst1 is involved in the tumor necrosis factor (TNF)-α-induced apoptosis of ECs. Western blot analysis revealed that TNF-α induced activation of caspase 3 and Mst1 in a time- and dose-dependent manner. TNF-α-induced Mst1 activation is almost completely prevented by pretreatment with Z-DEVD-FMK, a caspase 3 inhibitor. Nuclear staining with Hoechst 33258 and fluorescence-activated cell sorting of propidium iodide-stained cells showed that TNF-α induced apoptosis of EC. Diphenyleneiodonium, an inhibitor of NADPH oxidase, and N-acetylcysteine, a potent antioxidant, also inhibited TNF-α-induced activation of Mst1 and caspase 3, as well as apoptosis. Knockdown of Mst1 expression by short interfering RNA attenuated TNF-α-induced apoptosis but not cleavage of caspase 3. These results suggest that Mst1 plays an important role in the induction of TNF-α-induced apoptosis of EC. However, positive feedback mechanism between Mst1 and caspase 3, which was shown in the previous studies, was not observed. Inhibition of Mst1 function may be beneficial for maintaining the endothelial integrity and inhibition of atherogenesis.     

Withaferin A enhances radiation-induced apoptosis in Caki cells through induction of reactive oxygen species, Bcl-2 downregulation and Akt inhibition

Withaferin A (Wit A), a natural compound derived from the medicinal plant Withania somnifera, has been reported for the anti-tumor effects, including the inhibition of tumor cell growth, metastasis and angiogenesis. In this study, we investigated the effect of Wit A on radiation-induced apoptosis in human renal cancer cells (Caki cells). Our results showed that, compared with Wit A or radiation alone, the combination of both resulted in a significant enhancement of apoptosis, showing the increase in the cleavage of caspase-3 and PARP as well as sub-G1 cell population. In addition, reactive oxygen species (ROS) generation was correlated with the enhancement of radiation-induced apoptosis by Wit A. Wit A downregulated Bcl-2 protein levels and ectopic expression of Bcl-2 in Caki cells attenuated the apoptosis induced by Wit A plus radiation. Taken together, these results indicate that Wit A enhanced radiation-induced apoptosis in Caki cells through ROS generation, down-regulation of Bcl-2 and Akt dephosphorylation. Thus, our study shows that Wit A may be used as an effective radiosensitizer in cancer therapy.     

Enhancement of radiation-induced apoptosis of human lymphoma U937 cells by sanazole

Sanazole has been tested clinically as a hypoxic cell radiosensitizer. In this study, we determined whether sanazole enhances the radiation-induced apoptosis of human lymphoma U937 cells. Our results revealed that, compared with 10 mM sanazole or radiation alone, the combination of both resulted in a significant enhancement of apoptosis after 6 h, which was evaluated on the basis of DNA fragmentation, morphological changes, and phosphatidylserine externalization. Sanazole alone enhanced intracellular superoxide and hydrogen peroxide formation, which further increased when the cells were irradiated. Significant enhancement of Fas externalization, loss of mitochondrial membrane potential (MMP), and activation of caspase-3 and caspase-8 were observed after the combined treatment. Moreover, this combination could also enhance Bid activation, reduction of Hsp70 expression level and release of cytochrome c from the mitochondria to the cytosol. An immediate increase in the intracellular Ca²⁺ concentration ([Ca²⁺] _i ) was observed after the combined treatment. These results suggest that the intracellular superoxide and peroxide generated by sanazole might be involved in the enhancement of radiation-induced apoptosis, and that these effects are associated with modulation of the Fas-mitochondria-caspase-dependent pathway, an increase in [Ca²⁺] _i , and a decrease in the Hsp70 expression levels.     

Radiation-induced apoptosis of stem/progenitor cells in human umbilical cord blood is associated with alterations in reactive oxygen and intracellular pH

To investigate the sensitivity of human hematopoietic stem cell populations to radiation and its relevance to intracellular events, specifically alteration in cellular energy production systems, we examined the frequency of apoptotic cells, generation of superoxide anions (O*2-), and changes in cytosol pH in umbilical cord blood (UCB) CD34+/CD38-, CD34+/CD38+ and CD34-/CD38+ cells before and after 5Gy of X-irradiation. Human UCB mononucleated cells were used in this study. After X-irradiation and staining subgroups of the cells with fluorescence (FITC, PE, or CY)-labeled anti-CD34 and anti-CD38 antibodies, analyses were performed by FACScan using as stains 7-amino-actinomycin D (7-AAD) for the detection of apoptosis, and hydroethidine (HE) for the measurement of O*2- generation in the cells. For intracellular pH, image analysis was conducted using confocal laser microscopy after irradiation and staining with carboxy-SNAFR-1. The frequency of apoptotic cells, as determined by cell staining with 7-AAD, was highest in the irradiated CD34+/CD38- cell population, where the level of O*2- detected by the oxidation of HE was also most highly elevated. Intracellular pH measured with carboxy-SNARF-1-AM by image cytometer appeared to be lowest in the same irradiated CD34+/CD38- cell population, and this intracellular pH decreased as early as 4 h post-irradiation, virtually simultaneous with the significant elevation of O*2- generation. These results suggest that the CD34+/CD38- stem cell population is sensitive to radiation-induced apoptosis as well as production of intracellular O*2-, compare to more differentiated CD34+/CD38+ and CD34-/CD38+ cells and that its intracellular pH declines at an early phase in the apoptosis process.