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Axon guidance molecules play an important role in regulating proper neuronal networking during neuronal development. They also have non-neuronal properties, which include angiogenesis, inflammation, and tumor development. Semaphorin 3F (SEMA3F), a member of the class 3 semaphorins, was initially identified as an axon guidance factor, that repels axons and collapses growth cones. However, SEMA3F has similar effects on endothelial cells (ECs) and tumor cells. In this review, we discuss the novel molecular mechanisms underlying SEMA3F activity in vascular and tumor biology. Recent evidence suggests that SEMA3F functions as a PI3K-Akt-mTOR inhibitor in mammalian cells, including T cells, ECs, and tumor cells. Therefore, SEMA3F may have broad therapeutic implications. We also discuss the key role of axon guidance molecules as regulators of the tumor microenvironment. Netrin-1, a chemoattractant factor in the neuronal system, promotes tumor progression by enhancing angiogenesis and metastasis. Moreover, our recent studies demonstrate that netrin-1/neogenin interactions augment CD4+ T cell chemokinesis and elicit pro-inflammatory responses, suggesting that netrin-1 plays a key role in modulating the function of a tumor and its surrounding cells in the tumor microenvironment. Overall, this review focuses on SEMA3F and netrin-1 signaling mechanisms to understand the diverse biological functions of axon guidance molecules. [ABSTRACT FROM AUTHOR]

Abstract: In many developed countries including Japan, how to care the bedridden elderly people with chronic wounds such as decubitus becomes one of the most concerned issues. Although antioxidant micronutrients including vitamin E, especially α‐tocopherol (α‐Toc), are reported to shorten a period of wound closure, the promoting effect of α‐Toc on wound healing independent of its antioxidant activity remains to be fully elucidated. The aim of this study was to examine whether α‐Toc affects wound‐mediated HaCaT keratinocyte polarization process including the recruitment of polarity regulating proteins, leading to wound repair independently of its antioxidant activity. We investigated the effects of α‐Toc and other antioxidants such as Trolox, a cell‐permeable α‐Toc analog on the migration, proliferation, and cell polarization of HaCaT keratinocytes after wounding. We analyzed the localization and complex formation of polarity proteins, partitioning defective 3 (Par3), and atypical protein kinase C (aPKC), and aPKC activity by immunohistochemistry, immunoprecipitation analyses, and in vitro kinase assays, respectively. α‐Toc but not other antioxidants enhanced the wound closure and cell polarization in HaCaT keratinocytes after wounding. α‐Toc regulated the localization and complex formation of Par3 and aPKC during wound healing. Knockdown of aPKC or Par3 abrogated α‐Toc‐mediated promotion of the wound closure and cell polarization in HaCaT keratinocytes. Furthermore, aPKC kinase activity was significantly increased in α‐Toc‐treated cells through activation of phosphatidylinositol 3‐kinase/Akt signaling pathway. These results suggest that α‐Toc promotes HaCaT keratinocyte wound repair by regulating the aPKC kinase activity and the formation of aPKC–Par3 complex. © 2017 BioFactors, 44(2):180–191, 2018 [ABSTRACT FROM AUTHOR]

Ferulic Acid (FA) is a highly abundant phenolic phytochemical which is present in plant tissues. FA has biological effects on physiological and pathological processes due to its anti-apoptotic and anti-oxidative properties, however, the detailed mechanism(s) of function is poorly understood. We have identified FA as a molecule that inhibits apoptosis induced by hydrogen peroxide (H2O2) or actinomycin D (ActD) in rat pheochromocytoma, PC12 cell. We also found that FA reduces H2O2-induced reactive oxygen species (ROS) production in PC12 cell, thereby acting as an anti-oxidant. Then, we analyzed FA-mediated signaling responses in rat pheochromocytoma, PC12 cells using antibody arrays for phosphokinase and apoptosis related proteins. This FA signaling pathway in PC12 cells includes inactivation of pro-apoptotic proteins, SMAC/Diablo and Bad. In addition, FA attenuates the cell injury by H2O2 through the inhibition of phosphorylation of the extracellular signal-regulated kinase (ERK). Importantly, we find that FA restores expression levels of brain-derived neurotrophic factor (BDNF), a key neuroprotective effector, in H2O2-treated PC12 cells. As a possible mechanism, FA increases BDNF by regulating microRNA-10b expression following H2O2 stimulation. Taken together, FA has broad biological effects as a neuroprotective modulator to regulate the expression of phosphokinases, apoptosis-related proteins and microRNAs against oxidative stress in PC12 cells. [ABSTRACT FROM AUTHOR]

Netrin-1, the protein product of the NTN1 gene, is an axon guidance molecule implicated in regulation of cell survival and tumorigenesis. Expression of the netrin-1 receptors deleted in colorectal cancer (DCC) and uncoordinated 5 homolog (UNC5H) is frequently silenced in colorectal cancer (CRC) by either loss of heterozygosity or epigenetic mechanisms. However, netrin-1 expression and regulation in CRC are mostly unknown. Here, we report that NTN1 expression is significantly reduced in most CRC tissues compared to the adjacent normal intestinal mucosa, and that NTN1 DNA methylation is significantly higher in CRCs (24.6%) than in the adjacent normal intestinal mucosa (4.0%). In 6 CRC cell lines, NTN1 expression is low. Treatment with 5-Aza-2′-deoxycytidine increased expression of NTN1 in CRC cell lines, indicating that DNA methylation represses NTN1 transcription in CRCs. NTN1 DNA hypermethylation was significantly associated with advanced CRC disease. Median netrin-1 serum levels were significantly decreased in CRC patients (330.1 pg/mL) compared with normal individuals (438.6 pg/mL). Our results suggest that netrin-1 is a candidate biomarker for CRC. • Netrin-1 mRNA and protein levels are down-regulated in most CRCs. • Netrin-1 DNA methylation is higher in CRCs than in corresponding normal mucosa. • Hypermethylation of netrin-1 is associated with CRC patients with advanced TNM stage. • Serum levels of netrin-1 are significantly decreased in patients with CRC. [ABSTRACT FROM AUTHOR]

Background: An oxyntic gland neoplasm confined to the mucosal layer (T1a) is classified as an oxyntic gland adenoma, whereas that with submucosal invasion (T1b) is defined as gastric adenocarcinoma of the fundic gland type (GA-FG)., Methods: To reveal the differences in clinical features between them, we retrospectively investigated 136 patients with 150 oxyntic gland adenoma and GA-FG lesions., Results: The univariate analysis revealed that the mean size (GA-FG vs. oxyntic gland adenoma, 7.7±5.4 vs. 5.5±3.1 mm), the prevalence of elevated morphology (79.1% vs. 51.8%), black pigmentation within the lesion (23.9% vs. 9.6%), and non or closed-type atrophy (81.2% vs. 65.1%) were different between the two groups. A multivariate logistic regression analysis revealed that ≥5 mm lesion size (odds ratio, 2.96; 95% confidence interval: 1.21-7.23), elevated morphology (odds ratio, 2.40; 95% confidence interval: 1.06-5.45), and no or closed-type atrophy (odds ratio, 2.49; 95% confidence interval: 1.07-5.80) were factors in distinguishing GA-FG from oxyntic gland adenoma. When oxyntic gland neoplasms with no or one feature were judged as oxyntic gland adenomas and those with two or three features were judged as GA-FG, the sensitivity and specificity were 85.1% and 43.4% for GA-FG, respectively., Conclusions: We identified three possible distinctive features of GA-FG compared to oxyntic gland adenoma: lesion size ≥5 mm, elevated morphology, and no or closed-type atrophy., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-22-870/coif). The authors have no conflicts of interest to declare., (2023 Journal of Gastrointestinal Oncology. All rights reserved.)

Ferulic Acid (FA) is a highly abundant phenolic phytochemical which is present in plant tissues. FA has biological effects on physiological and pathological processes due to its anti-apoptotic and anti-oxidative properties, however, the detailed mechanism(s) of function is poorly understood. We have identified FA as a molecule that inhibits apoptosis induced by hydrogen peroxide (H 2 O 2 ) or actinomycin D (ActD) in rat pheochromocytoma, PC12 cell. We also found that FA reduces H 2 O 2 -induced reactive oxygen species (ROS) production in PC12 cell, thereby acting as an anti-oxidant. Then, we analyzed FA-mediated signaling responses in rat pheochromocytoma, PC12 cells using antibody arrays for phosphokinase and apoptosis related proteins. This FA signaling pathway in PC12 cells includes inactivation of pro-apoptotic proteins, SMAC/Diablo and Bad. In addition, FA attenuates the cell injury by H 2 O 2 through the inhibition of phosphorylation of the extracellular signal-regulated kinase (ERK). Importantly, we find that FA restores expression levels of brain-derived neurotrophic factor (BDNF), a key neuroprotective effector, in H 2 O 2 -treated PC12 cells. As a possible mechanism, FA increases BDNF by regulating microRNA-10b expression following H 2 O 2 stimulation. Taken together, FA has broad biological effects as a neuroprotective modulator to regulate the expression of phosphokinases, apoptosis-related proteins and microRNAs against oxidative stress in PC12 cells.

Most gastric bezoars can be treated with endoscopic fragmentation combined with or without cola dissolution, whereas laparotomy or laparoscopic surgery is generally inevitable for small intestinal bezoars because they cause small bowel obstruction. Therefore, early diagnosis and management of gastric bezoars are necessary to prevent bezoar-induced ileus. To investigate the incidence of overlooked gastric bezoars during the initial esophagogastroduodenoscopy, we retrospectively reviewed the cases of 27 patients diagnosed with gastrointestinal bezoars. The bezoars were diagnosed using esophagogastroduodenoscopy (n=25), abdominal ultrasonography (n=1), and barium follow-through examination (n=1). Bezoars were overlooked in 9/25 patients (36.0%) during the initial endoscopy examination because the bezoars were covered with debris in the stomach. Of the 9 patients, 8 had concomitant gastric ulcers, and the other patient had gastric lymphoma. Although a computed tomography (CT) scan was performed before the second-look endoscopy in 8 of the 9 patients, the bezoars were mistaken as food debris on CT findings and were overlooked in these patients. In conclusion, gastric bezoars may not be discovered during the initial esophagogastroduodenoscopy and CT scan. In cases with debris in the stomach, second-look endoscopy is essential to detect bezoars., Competing Interests: No potential conflict of interest relevant to this article was reported.

In this study, we examined whether compound 48/80 (C48/80), a mast cell degranulator, causes hepatic oxidative damage in rats. Serum and liver biochemical parameters were determined 0.5, 3 or 6 h after a single treatment with C48/80 (0.75 mg/kg). Serum histamine and serotonin levels increased 0.5 h after C48/80 treatment but diminished thereafter. Increases in serum vitamin C (VC) and transaminases and hepatic hydrogen peroxide, lipid peroxide, and myeloperoxidase levels and a decrease in hepatic reduced glutathione level occurred 0.5 h after C48/80 treatment and further proceeded at 3 h, but these changes diminished at 6 h. Serum lipid peroxide and hepatic VC levels increased 3 h after C48/80 treatment. Hepatic glycogen level decreased 0.5 h after C48/80 treatment and further decreased at 3 h. Pre-administered ketotifen diminished all these changes found at 3 h after treatment, while pre-administered NPC 14686 diminished these changes except changes in serum histamine and serotonin levels. Hepatocellular apoptosis observed at 3 h after C48/80 treatment was attenuated by pre-administered ketotifen and NPC 14686. These results indicate that C48/80 causes oxidative damage by enhancing VC synthesis via reduced glutathione depletion-dependent glycogenolysis and lipid peroxidation through neutrophil infiltration following mast cell degranulation in rat livers., Competing Interests: No potential conflicts of interest were disclosed.

Here we report the case of a 73-year-old male who had undergone esophagogastroduodenoscopy (EGD) at a nearby hospital or at our hospital every year since 2006. In 2013, EGD results revealed a discolored lesion, measuring 6mm in diameter, on the anterior side of the upper body in the stomach. Helicobacter pylori (HP) was eradicated in 2010, and the background mucosa around the lesion was endoscopically diagnosed as non-atrophic. We performed endoscopic biopsy of the lesion. Histological examination of the specimen confirmed gastric adenocarcinoma of the fundic gland type. Based on the findings of EGD, ultrasonic endoscopy, and upper gastrointestinal series, we diagnosed that the infiltration of the adenocarcinoma was limited to the mucosa. Hence, we performed endoscopic submucosal dissection (ESD). After ESD, the resected cancer was located in the mucosa and no invasive lesion was detected at any vessels. Therefore, complete resection was performed through ESD. Retrospectively, the lesion could be detected in the endoscopic images taken in 2006. The shape and diameter of the lesion did not seem to have significantly changed from 2006 to 2013. In this case, slow tumor progression was observed. In 2015, no new lesions in the stomach or metastatic area were detected. Here we report a rare case of gastric adenocarcinoma of the fundic gland type that showed very slow progression.

The formation of a bezoar is a relatively infrequent disorder that affects the gastrointestinal system. Bezoars are mainly classified into four types depending on the material constituting the indigestible mass of the bezoar: phytobezoars, trichobezoars, pharmacobezoars, and lactobezoars. Gastric bezoars often cause ulcerative lesions in the stomach and subsequent bleeding, whereas small intestinal bezoars present with small bowel obstruction and ileus. A number of articles have emphasized the usefulness of Coca-Cola(®) administration for the dissolution of phytobezoars. However, persimmon phytobezoars may be resistant to such dissolution treatment because of their harder consistency compared to other types of phytobezoars. Better understanding of the etiology and epidemiology of each type of bezoar will facilitate prompt diagnosis and management. Here we provide an overview of the prevalence, classification, predisposing factors, and manifestations of bezoars. Diagnosis and management strategies are also discussed, reviewing mainly our own case series. Recent progress in basic research regarding persimmon phytobezoars is also briefly reviewed.

We examined how dietary supplementation of vitamin E protects against liver oxidative damage in rats with water-immersion restraint stress (WIRS). Before WIRS exposure, rats received a normal diet (ND) or vitamin E-supplemented diet (VESD) (500 IU α-tocopherol/kg diet) at a mean dose of 15 g/animal/d for 4 wk. The two diet groups had serum transaminases and lactate dehydrogenase activities and adrenocorticotropic hormone, corticosterone, and glucose levels to a similar extent. VESD-fed rats had higher liver α-tocopherol concentrations and lower liver ascorbic acid, total coenzyme Q9 (CoQ9), reduced CoQ9, reduced CoQ10, and lipid peroxide (LPO) concentrations than ND-fed rats. When the two diet groups were exposed to 6 h of WIRS, the serum liver cell damage index enzyme activities increased more greatly in ND-fed rats than in VESD-fed rats but the serum stress marker levels increased to a similar extent. The WIRS exposure caused no change in liver LPO concentration with the further increase in liver α-tocopherol concentration in VESD-fed rats but increased liver LPO concentration without changing liver α-tocopherol concentration in ND-fed rats. Upon the WIRS exposure, liver reduced glutathione concentration decreased with the further decrease in liver ascorbic acid concentration in VESD-fed rats and those concentrations decreased in ND-fed rats. The WIRS exposure recovered the decreased liver total CoQ9 and reduced CoQ9 concentrations in VESD-fed rats but decreased liver total CoQ9, reduced CoQ9, and reduced CoQ10 concentrations in ND-fed rats. These results indicate that dietary vitamin E supplementation protects against liver oxidative damage without affecting the stress response in rats with WIRS.

We examined the effect of vitamin E depletion on liver oxidative damage in rats with water-immersion restraint stress (WIRS). Male Wistar rats were fed a normal diet (N) or vitamin E-depleted diet (VE-D) for 4 wk. N- and VE-D-fed rats were exposed to WIRS for 6 h. The activities of serum transaminases and lactate dehydrogenase and serum ascorbic acid concentration were similar in both diet groups. WIRS exposure increased these serum enzyme activities and the serum ascorbic acid concentration in both diet groups but the ratios of these increases were higher in VE-D-fed rats than in N-fed rats. Serum and liver α-tocopherol concentrations in VE-D-rats were approximately 50% and 30% of those in N-fed rats, respectively. WIRS exposure reduced liver α-tocopherol concentration in VE-D-fed rats, but not in N-fed rats. Liver ascorbic acid and reduced glutathione concentrations were higher in the VE-D-fed group than in the N-fed group. WIRS exposure reduced liver ascorbic acid and reduced glutathione concentrations in both diet groups. There were no differences in liver concentrations of coenzyme Q9 or coenzyme Q10 in the reduced form between the N- and VE-D-fed groups. WIRS exposure reduced liver concentrations of coenzyme Q9 and coenzyme Q10 in the reduced form in both diet groups. Liver lipid peroxide concentration was higher in the VE-D-fed group than in the N-fed group. WIRS exposure raised liver lipid peroxide concentration more in the VE-D-fed group than in the N-fed group. These results indicate that vitamin E depletion enhances liver oxidative damage in rats with WIRS.

Parkinson's disease is a major neurodegenerative disease involving the selective degeneration of dopaminergic neurons and α-synuclein containing Lewy bodies formation in the substantia nigra. Although α-synuclein is a key molecule for both dopaminergic neuron death and the formation of inclusion bodies, the mechanism of α-synuclein induction of Parkinson's disease-related pathogenesis is not understood. In the present study, we found that the interaction between dopamine and α-synuclein requires the oxidation of dopamine. Furthermore, we examined the protective effect of chlorogenic acid, a major polyphenol contained in coffee, against α-syn and dopamine-related toxicity. Chlorogenic acid inhibits several DA/α-synuclein-related phenomenon, including the oxidation of dopamine, the interaction of oxidized dopamine with α-synuclein, and the oligomerization of α-synuclein under dopamine existing conditions in vitro. Finally, we showed that the cytoprotective effect against α-synuclein-related toxicity in PC12 cells that can be controlled by the Tet-Off system. Although the induction of α-synuclein in catecholaminergic PC12 cells causes a decrease in cell viability, chlorogenic acid rescued this cytotoxicity significantly in a dose dependent manner. These results suggest that the interaction of oxidized DA with α-synuclein may be a novel therapeutic target for Parkinson's disease, and polyphenols, including chlorogenic acid, are candidates as protective and preventive agents for Parkinson's disease onset.

The present study was performed by using selective inhibitors of caspase-8 and caspase-3 functioning upstream and downstream from mitochondria, respectively to determine whether mitochondria are involved in the mechanisms underlying production and externalization of oxidized phosphatidylserine (PSox) during Fas-mediated apoptosis. Treatment with anti-Fas antibody induced caspase-3 activation, chromatin condensation, release of cytochrome c (cyt c) from mitochondria into the cytosol as well as production of PSox and its exposure to the cell surface in Jurkat cells. Inhibition of caspase-8 by pretreatment with Z-IETD-FMK, a membrane permeable selective caspase-8 inhibitor reduced mitochondrial cyt c release, the amount of PSox not only within but also on the surface of Jurkat cells, caspase-3 activation, and apoptotic cell number after treatment with anti-Fas antibody. In contrast, Z-DEVD-FMK, a membrane permeable selective caspase-3 inhibitor was unable to inhibit cyt c release, and the amount of PSox both within and on the surface of the cells after anti-Fas antibody, although it suppressed caspase-3 activation and apoptosis. Thus, these results strongly suggest that mitochondria play an important role in production of PSox and subsequent its externalization during apoptosis.

Polaprezinc (PZ), a chelate compound consisting of zinc and l-carnosine (Car), is an anti-ulcer drug developed in Japan. In the present study, we investigated whether PZ suppresses mortality, pulmonary inflammation, and plasma nitric oxide (NO) and tumor necrosis factor (TNF)-alpha levels in endotoxin shock mice after peritoneal injection of lipopolysaccharide (LPS), and how PZ protects against LPS-induced endotoxin shock. PZ pretreatment inhibited the decrease in the survival rate of mice after LPS injection. PZ inhibited the increases in plasma NO as well as TNF-alpha after LPS. Compatibly, PZ suppressed LPS-induced inducible NO synthase mRNA transcription in the mouse lungs. PZ also improved LPS-induced lung injury. However, PZ did not enhance the induction of heat shock protein (HSP) 70 in the mouse lungs after LPS. Pretreatment of RAW264 cells with PZ suppressed the production of NO and TNF-alpha after LPS addition. This inhibition likely resulted from the inhibitory effect of PZ on LPS-mediated nuclear factor-kappaB (NF-kappaB) activation. Zinc sulfate, but not Car, suppressed NO production after LPS. These results indicate that PZ, in particular its zinc subcomponent, inhibits LPS-induced endotoxin shock via the inhibition of NF-kappaB activation and subsequent induction of proinflammatory products such as NO and TNF-alpha, but not HSP induction.

Reduced coenzyme Q(10) (CoQ(10)H(2)) is known as a potent antioxidant in biological systems. However, it is not yet known whether CoQ(9)H(2) could act as an antioxidant in human cells. The aim of this study is to assess whether exogenously added CoQ(9) can protect human liver cells against injuries induced by a water-soluble radical initiator, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and a lipid-soluble radical initiator, 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN). CoQ(9)-enriched cells were obtained by treatment of HepG2 cells with 10 microM CoQ(9) liposomes for 24 h. CoQ(9)-enriched cells were exposed to 10 mM AAPH and 500 microM AMVN over 4 h and 24 h, respectively. The loss of viability after treatment with AAPH or AMVN was much less in CoQ(9)-enriched cells than in naive HepG2 cells. The decrease in glutathione and the increase in thiobarbituric acid-reactive substance after treatment with AAPH or AMVN were also suppressed in CoQ(9)-enriched cells. The incubation of CoQ(9)-enriched cells with AAPH or AMVN led to a decrease in cellular CoQ(9)H(2) and reciprocal increase in cellular CoQ(9) resulting from its antioxidant function. Taken together, it was demonstrated for the first time that exogenously added CoQ(9) could prevent oxidative stress-mediated damage to human cells by virtue of its antioxidant activity.

Polaprezinc, a chelate compound consisting of zinc and l-carnosine, is clinically used as a medicine for gastric ulcers. It has been shown that induction of heat shock protein (HSP) is involved in protective effects of polaprezinc against gastric mucosal injury. In the present study, we investigated whether polaprezinc and its components could induce HSP70 and prevent acetaminophen (APAP) toxicity in mouse primary cultured hepatocytes. Hepatocytes were treated with polaprezinc, zinc sulfate or l-carnosine at the concentration of 100 microM for 9 h, and then exposed to 10 mM APAP. Polaprezinc or zinc sulfate increased cellular HSP70 expression. However, l-carnosine had no influence on it. Pretreatment of the cells with polaprezinc or zinc sulfate significantly suppressed cell death as well as cellular lipid peroxidation after APAP treatment. In contrast, pretreatment with polaprezinc did not affect decrease in intracellular glutathione after APAP. Furthermore, treatment with KNK437, an HSP inhibitor, attenuated increase in HSP70 expression induced by polaprezinc, and abolished protective effect of polaprezinc on cell death after APAP. These results suggested that polaprezinc, in particular its zinc component, induces HSP70 expression in mouse primary cultured hepatocytes, and inhibits lipid peroxidation after APAP treatment, resulting in protection against APAP toxicity.

We investigated whether pretreatment with geranylgeranylacetone (GGA), a potent heat shock protein (HSP) inducer, could inhibit proinflammatory cytokine liberation and nitric oxide (NO) production in lipopolysaccharide (LPS)-treated murine macrophages. The levels of NO and tumor necrosis factor-alpha (TNF-alpha) released from murine macrophage RAW 264 cells were increased dose- and time-dependently following treatment with LPS (1 microg/ml). GGA (80 microM) treatment 2 h before LPS addition significantly suppressed TNF-alpha and NO productions at 12 h and 24 h after LPS, respectively, indicating that GGA inhibits activation of macrophages. However, replacement by fresh culture medium before LPS treatment abolished the inhibitory effect of GGA on NO production in LPS-treated cells. Furthermore, GGA inhibited both HSP70 and inducible NO synthase expressions induced by LPS treatment despite an HSP inducer. When it was examined whether GGA interacts with LPS and/or affects expression of Toll-like receptor 4 (TLR4) and CD14 on the cell surface, GGA inhibited the binding of LPS to the cell surface, while GGA did not affect TLR4 and CD14 expressions. These results indicate that GGA suppresses the binding of LPS to the cell surface of macrophages, resulting in inhibiting signal transduction downstream of TLR4.

Hydrogen peroxide (H(2)O(2)) is generated endogenously during execution of both intrinsic as well as extrinsic apoptotic programs suggesting that it may function as a secondary messenger in apoptotic pathways. In the present study, we investigated the role of endogenously generated H(2)O(2) by using two cell lines-HL-60 cells and its subclone, H(2)O(2) resistant HP100 cells overexpressing catalase (CAT). With the exception of CAT, we found no differences in the expression of other primary antioxidant enzymes (Cu/Zn-superoxide dismutase, Mn-superoxide dismutase, and glutathione peroxidase) or apoptosis-related proteins (Bcl-2 and Bax) in HP100 cells as compared with the parental HL-60 cells. Production of H(2)O(2) was readily detectable as early as 1 h after melphalan (Mel) exposure of HL-60 cells but not HP-100 cells. Biomarkers of apoptosis, such as release of cytochrome c, disruption of mitochondrial transmembrane potential, caspase-3 activation, and chromatin condensation, became apparent much later, 3 h and onward after Mel treatment of HL-60 cells. The emergence of essentially all biomarkers of apoptosis was dramatically delayed in HP100 cells as compared with HL-60 cells. A relatively minor phospholipid species, phosphatidylserine (PS), was markedly oxidized 3 h after Mel treatment in HL-60 cells (but not in HP100 cells) where it was significantly inhibited by exogenously added CAT. The two most abundant classes of membrane phospholipids, phosphatidylcholine and phosphatidyletanolamine, did not undergo any significant oxidation. PS oxidation took place 3 h after exposure of HL-60 cells to Mel and paralleled the appearance of cytochrome c in the cytosol. Neither cytochrome c release nor PS oxidation occurred in Mel-treated HP100 cells, indicating that both endogenous H(2)O(2) and cytochrome c were essential for selective PS oxidation detected in HL-60 cells. Mel-induced PS oxidation was also associated with externalization of PS on the surface of HL-60 cells. Given that 3-amino-1,2,4-triazole, a CAT inhibitor, suppressed the resistance of HP100 cells to apoptosis, production of reactive oxygen species, PS oxidation, and PS externalization induced by Mel, the results from the present study suggest that H(2)O(2) is critical for triggering the Mel-induced apoptotic program as well as PS oxidation and externalization.