Biomedical subjects

O Inanami

Publications and source records attributed to O Inanami.

At least 19 recordsLinked to original sources

PubMed2001

Induction of neurite outgrowth in PC12 cells by alpha -phenyl-N-tert-butylnitron through activation of protein kinase C and the Ras-extracellular signal-regulated kinase pathway.

The spin trap alpha-phenyl-N-tert-butylnitron (PBN) is widely used for studies of the biological effects of free radicals. We previously reported the protective effects of PBN against ischemia-reperfusion injury in gerbil hippocampus by its activation of extracellular signal-regulated kinase (ERK) and suppression of both stress-activated protein kinase and p38 mitogen-activated protein kinase. In the present study, we found that PBN induced neurite outgrowth accompanied by ERK activation in PC12 cells in a dose-dependent manner. The induction of neurite outgrowth was inhibited significantly not only by transient transfection of PC12 cells with dominant negative Ras, but also by treatment with mitogen-activated protein kinase/ERK kinase inhibitor PD98059. The activation of receptor tyrosine kinase TrkA was not involved in PBN-induced neurite outgrowth. A protein kinase C (PKC) inhibitor, GF109203X, was found to inhibit neurite outgrowth. The activation of PKCepsilon was observed after PBN stimulation. PBN-induced neurite outgrowth and ERK activation were counteracted by the thiol-based antioxidant N-acetylcysteine. From these results, it was concluded that PBN induced neurite outgrowth in PC12 cells through activation of the Ras-ERK pathway and PKC.

Acetylcysteine↗

PubMed2001

Isolation, characterization, and cDNA cloning of chicken turpentine-induced protein, a new member of the scavenger receptor cysteine-rich (SRCR) family of proteins.

Acute-phase serum proteins were induced by administrating a chicken with turpentine oil. One of these proteins was a new protein that appeared in front of albumin in polyacrylamide disc gel electrophoresis using a 4.5-16% gel. To purify this protein, turpentine-administrated chicken serum was fractionated by ammonium sulfate precipitation at 50% saturation, and the supernatant fraction was chromatographed on a DEAE-Toyopearl 650S column. The purified protein is a mannose-glycoprotein, and its N-terminal sequence, determined by the Edoman method, is not homologous from that of other reported acute-phase proteins. An analysis of physiological function with two different test systems, chemiluminescence measurement and electron spin resonance spectroscopy, showed that the purified protein has antioxidant activity and inhibits superoxide (O(2)) mediated by activation of the receptor. In support of these results, the complete amino acid sequence of 18-B is homologous to the scavenger receptor cysteine-rich (SRCR) family of proteins that participate in the regulation of leukocyte function. 18-B is composed of four SRCR domains, which is different from the previously characterized SRCR family of proteins such as Spalpha, CD6, and CD163. These findings indicate that turpentine-induced 18-B, a new member of scavenger receptor cysteine-rich family, may be implicated in regulation of cell function in a manner of inhibition of the overproduction of the reactive oxygen species.

Amino Acid Sequence↗

PubMed2001

ESR detection of intraphagosomal superoxide in polymorphonuclear leukocytes using 5-(diethoxyphosphoryl)-5-methyl-l-pyrroline-N-oxide.

We applied a spin trap, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), to detect O2*- generation during phagocytosis in human polymorphonuclear leukocytes (PMNs). PMNs were activated with serum-opsonized zymosan (sOZ) in the presence of DEPMPO. The ESR spectra mainly consisted of Cu,Zn-SOD-sensitive DEPMPO-OOH spin adducts. To clarify where these spin-adducts were present, cells after stimulation were separated from extracellular fluid by brief centrifugation and resuspended in Hanks' balanced salt solution. ESR examination showed that DEPMPO-OOH adducts were present in both fractions. When cells were stimulated by phorbol myristate acetate (PMA), the DEPMPO-OOH was detected in extracellular fluid but not in the cell fraction. Furthermore, DEPMPO-OOH adducts were quickly converted into ESR-silent compounds by addition of cell lysate of PMNs. These results indicate that DEPMPO is useful to detect O2*- of extracellular space including the intraphagosome but not that of intracellular space in sOZ-stimulated phagocytes.

Cyclic N-Oxides↗

PubMed2001

Superoxide generation from human polymorphonuclear leukocytes by liposome-encapsulated hemoglobin.

We investigated the interactions between liposome-encapsulated hemoglobin (Neo Red Cells: NRC) and human polymorphonuclear leukocytes as assessed by superoxide generation. NRC triggered superoxide generation from neutrophils in a dose-dependent manner. Empty liposomes also induced superoxide production of neutrophils. Superoxide generation of neutrophils induced by phorbol myristate acetate (PMA) was delayed but intensified both by NRC and empty liposomes. The intensity of superoxide generation induced by NRC was smaller than that by the empty liposomes. As NRC contained superoxide dismutase (SOD) that was copurified with hemoglobin from red blood cells and its activity remained, SOD contained in NRC may partially eliminate superoxide.

Blood Substitutes↗

PubMed2000

Neuroprotective effect of alpha-phenyl-N-tert-butylnitrone in gerbil hippocampus is mediated by the mitogen-activated protein kinase pathway and heat shock proteins.

alpha-Phenyl-N-tert-butylnitrone (PBN), a spin trap, is known as a protective agent against delayed-neuronal death after ischemia-reperfusion. To investigate this neuroprotective effect of PBN, we examined the effect of PBN on the mitogen-activated protein kinase (MAPK) signaling pathway and the expression of heat shock proteins (HSPs) in the gerbil hippocampus following transient (5 min) ischemia. Immunoblot analysis revealed that intraperitoneal (i. p.) injection of PBN (200 mg/kg) enhanced the activation of extracellular-response kinase (ERK) and suppressed the activation of stress-activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK) and p38 mitogen-activated protein kinase (p38) at 6 h after ischemia. Elevated levels of HSP27 and HSP70 were seen at the same period. These data suggest that PBN protects against delayed-neuronal death not only by its inherent radical-trapping activity but also by regulating the MAPK pathway and up-regulating HSPs.

Animals↗

PubMed2000

Roles of p38 MAPK, PKC and PI3-K in the signaling pathways of NADPH oxidase activation and phagocytosis in bovine polymorphonuclear leukocytes.

Stimulation of bovine polymorphonuclear leukocytes (PMN) with serum-opsonized zymosan (sOZ) induced the activation of p38 mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3-K) and sOZ-induced O(2)(-) production was significantly attenuated by their inhibitors (SB203580 for p38 MAPK, GF109203X for PKC and wortmannin for PI3-K). They caused significant attenuation of sOZ-induced phosphorylation of p47phox as well. Flow cytometric analysis, however, revealed that SB203580 and wortmannin attenuated phagocytosis, but GF109203X facilitated it. The results suggest that p38 MAPK and PI3-K participated in both signaling pathways of NADPH oxidase activation (O(2)(-) production) and phagocytosis, and PKC participated in the signaling pathway of NADPH oxidase activation alone.

Animals↗

PubMed2000

FMLP-induced formation of F-actin in HL60 cells is dependent on PI3-K but not on intracellular Ca2+, PKC, ERK or p38 MAPK.

OBJECTIVE AND DESIGN: To further understand the mechanisms of signal transduction pathways for the formation of F-actin (polymerization of actin) and the activation of NADPH oxidase in phagocytic cells, the effects of various inhibitors on them were studied. MATERIALS AND METHODS: Differentiated HL60 cells were studied to examine their N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated formation of F-actin and activation of NADPH oxidase following treatment with various inhibitors. These included a protein kinase C (PKC) inhibitor (GF 109203X), a phosphatidylinositide 3 kinase (PI3-K) inhibitor (wortmannin), an extracellular response kinase (ERK) inhibitor (PD 98059), a p38 mitogen-activated protein kinase (MAPK) inhibitor (SB 203580) and an intracellular Ca2+ -chelator (BAPTA-AM). RESULTS: The treatment with wortmannin suppressed the formation of F-actin, with less suppression of the activation of NADPH oxidase. BAPTA-AM and GF 109203X did not attenuate the formation of F-actin but completely inhibited the activation of NADPH oxidase. PD 98059 and SB 203580 partially inhibited the activation of NADPH oxidase without influence on the formation of F-actin. Furthermore, wortmannin but not BAPTA-AM and GF 109203X inhibited the fMLP-induced activation of Akt, which is known to regulate NADPH oxidase. CONCLUSIONS: These results suggest that the formation of F-actin is dependent on PI3-K and independent of PKC, ERK and p38 MAPK as well as the increase in intracellular Ca2+, whereas the activation of NADPH oxidase is partly dependent on ERK, p38 MAPK, Akt regulated by PI3-K, and strongly dependent on the activation of PKC and the increase in intracellular Ca2+.

Actins↗

PubMed2000

2-Chloro-2'-deoxyadenosine induces apoptosis through the Fas/Fas ligand pathway in human leukemia cell line MOLT-4.

The mechanism of apoptosis induced by 2-chloro-2'-deoxyadenosine (2CdA) in human leukemia cell line MOLT-4 was investigated. 2CdA induced increases of 3'-OH ends of genomic DNA, ladder-like DNA fragmentation and phosphatidylserine translocation to the outer membrane, which are apoptotic characteristics. These apoptotic phenomena induced by 2CdA were inhibited by cycloheximide (CHX; a protein synthesis inhibitor), deoxycytidine (dC; a substrate of deoxycytidine kinase), acetyl Ile-Glu-Thr-Asp aldehyde (Ac-IETD-CHO; a caspase-8 inhibitor) and acetyl Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO; a caspase-3 inhibitor). The protein synthesis-dependent expression of Fas and Fas ligand (Fas-L) was detected by treatment with 2CdA. The proteolytic processing of procaspases-8 and -3 to produce active fragments, caspases-8 (p18) and -3 (p17), respectively, was observed after treatment with 2CdA, and suppressed by cycloheximide. Increases in the activities of caspases-8 and -3 were observed after 2CdA treatment. Their activation was also dependent on protein synthesis. These results indicated that 2CdA-induced apoptosis was triggered by phosphorylation of 2CdA followed by the protein synthesis-dependent expression of Fas and Fas-L and activation of caspases-8 and -3.

Antineoplastic Agents↗

PubMed2000

Radiation sensitivity of megakaryocyte colony-forming cells in human placental and umbilical cord blood.

The in vitro radiation sensitivity of CFU-Meg isolated from human placental and umbilical cord blood was evaluated in plasma clot cultures stimulated by recombinant human cytokines, including thrombopoietin, the FLT3 ligand (FLT3LG), interleukin-3, interleukin-11 and stem cell factor. The CD34(+) cells were irradiated with X rays at a dose rate of 73 cGy/ min. The megakaryocyte colonies were identified by using an FITC-conjugated antibody to glycoprotein IIbIIIa and were classified into two groups based on colony size: large colonies (immature CFU-Meg) and small colonies (mature CFU-Meg). Treatment with thrombopoietin alone or in combination with FLT3LG and/or interleukin-11 gave exponential radiation survival curves (D(0) for immature CFU-Meg = 56-77 cGy, D(0) for mature CFU-Meg = 86 cGy-1.12 Gy), while marked shoulders were observed on the survival curves for colonies supported by the combination of thrombopoietin, interleukin-3 and stem cell factor (D(0) for immature CFU-Meg = 89- 98 cGy; D(0) for mature CFU-Meg = 1. 25-1.31 Gy). Our results showed that the immature CFU-Meg were more radiosensitive than the mature CFU-Meg and that the combination of cytokines, including thrombopoietin, interleukin-3 and stem cell factor, affected the radiation sensitivity of CFU-Meg to the same extent as with thrombopoietin alone or in combination with FLT3LG and/or interleukin-11.

Antigens, CD34↗

PubMed2000

Spin-trapping detection of superoxides in polymorphonuclear leukocytes stimulated with serum-opsonized zymosan.

To clarify where oxygen radicals are generated in polymorphonuclear leukocytes (PMNs) during phagocytosis, superoxides (O2-) from opsonized symosan (OZ)--stimulated human PMNs were detected by the ESR and spin-trapping methods. PMNs were preactivated with OZ for the indicated periods of time at 37 degrees C. Then a spin-trapping agent, 5, 5-dimethyl-1-pyrroline N-oxide (DMPO), was added to them, and they were further incubated for 30 sec for ESR observations. The ESR spectra consisted of two components due to the DMPO-OOH and DMPO-OH spin adducts. To clarify where these spin-adducts were present, cells were separated from extracellular fluid by brief centrifugation and resuspended in Hanks' balanced salt solution. ESR examination of two fractions showed that the DMPO-OOH adducts was present in the cell fraction, whereas the DMPO-OH adducts were present in the extracellular fluid. When DMSO was used as a scavenger of hydroxyl radicals (.OH), DMPO-CH3 adducts were observed in the fluid fraction but not in the cell fraction. Both spin adducts were completely abolished by Cu, Zn-SOD but not catalase. These results indicated that O2- were produced inside phagosomes of OZ-stimulated PMNs and .OH were produced outside them by spontaneous decomposition of the DMPO-OOH adducts.

Electron Spin Resonance Spectroscopy↗

PubMed1999

Inflammatory cell-mediated tumour progression and minisatellite mutation correlate with the decrease of antioxidative enzymes in murine fibrosarcoma cells.

We isolated six clones of weakly tumorigenic fibrosarcoma (QR) from the tumorigenic clone BMT-11 cl-9. The QR clones were unable to grow in normal C57BL/6 mice when injected s.c. (1x10(5) cells). However, they formed aggressive tumours upon co-implantation with a 'foreign body', i.e. a gelatin sponge, and the rate of tumour take ranged from 8% to 58% among QR clones. The enhanced tumorigenicity was due to host cell-mediated reaction to the gelatin sponge (inflammation). Immunoblot analysis and enzyme activity assay revealed a significant inverse correlation between the frequencies of tumour formation by QR clones and the levels of manganese superoxide dismutase (Mn-SOD, P<0.005) and glutathione peroxidase (GPchi, P<0.01) in the respective tumour clones. Electron spin resonance (ESR) revealed that superoxide-scavenging ability of cell lysates of the QR clone with high level of Mn-SOD was significantly higher than that with low level of the antioxidative enzyme in the presence of potassium cyanide, an inhibitor for copper-zinc superoxide dismutase (CuZn-SOD) (P<0.001). Minisatellite mutation (MSM) induced by the inflammatory cells in tumour cells were investigated by DNA fingerprint analysis after QR clones had been co-cultured with gelatin-sponge-reactive cells. The MSM rate was significantly higher in the subclones with low levels of Mn-SOD and GPchi (P<0.05) than in the subclones with high levels of both enzymes. The MSM of the subclones with low levels of both enzymes was inhibited in the presence of mannitol, a hydroxyl radical scavenger. The content of 8-hydroxydeoxyguanosine (8-OHdG) by which the cellular DNA damage caused by active oxygen species can be assessed was significantly low in the tumours arising from the QR clone with high levels of Mn-SOD and GPchi even if the clone had been co-implanted with gelatin sponge, compared with the arising tumour from the QR clone with low levels of those antioxidative enzymes (P<0.001). In contrast, CuZn-SOD and catalase levels in the six QR clones did not have any correlation with tumour progression parameters. These results suggest that tumour progression is accelerated by inflammation-induced active oxygen species particularly accompanied with declined levels of intracellular antioxidative enzymes in tumour cells.

Animals↗

PubMed1999

Effects of intracellular calcium chelator BAPTA-AM on radiation-induced apoptosis regulated by activation of SAPK/JNK and caspase-3 in MOLT-4 cells.

PURPOSE: To examine the roles ofintracellular calcium in radiation-induced apoptosis of MOLT-4 cells, the effects of intracellular calcium chelator BAPTA-AM on the induction of apoptosis and the activation of apoptosis-relating enzymes SAPK/JNK and caspase-3 were studied. MATERIALS AND METHODS: MOLT-4 cells pretreated with 5 microM BAPTA-AM were exposed to X-rays. DNA fragmentation, the expression of phosphorylated SAPK/JNK and the activation of caspase-3 and calcium concentration were measured by agarose gel electrophoresis, Western blotting and spectrofluorometry. RESULTS: Time-dependent ladder-like DNA fragmentation was observed at 4h, 5 h and 6 h after exposure to 15 Gy of X-rays. This fragmentation was significantly attenuated by pretreatment with BAPTA-AM up to 5 h after irradiation, but the attenuation due to BAPTA-AM was no longer detectable at 6 h. Activation of SAPK/JNK and caspase-3 was observed at 1 and 4 h after X-irradiation, respectively, and BAPTA-AM retarded the activation for 2 h. The pretreatment with BAPTA-AM was found to suppress the increase of calcium concentration for 6h after irradiation. CONCLUSION: These results revealed that chelation of calcium merely delayed the onset of the radiation-induced apoptosis regulated by the activation of SAPK/JNK and caspase-3, and calcium was not essential for the induction of apoptosis in X-irradiated MOLT-4 cells.

Apoptosis↗

PubMed1999

Attenuation of caspase-3-dependent apoptosis by Trolox post-treatment of X-irradiated MOLT-4 cells.

PURPOSE: The relationship between post-irradiation treatment with Trolox, an antioxidant that inhibits lipid peroxidation, and X-ray-induced apoptosis, with regard to signal transduction pathways, was examined in MOLT-4, a human leukaemia cell line. MATERIALS AND METHODS: In MOLT-4 cells treated with Trolox after X-irradiation, viability, DNA fragmentation, expression of p53, BCL-2, BAX, active SAPK/JNK, active caspase-3 and the cleavage of PARP were measured by the trypan blue exclusion test, agarose gel electrophoresis and Western blotting. RESULTS: Stained cells and ladder-like DNA cleavage were observed after X-irradiation. Cell death and DNA fragmentation were significantly inhibited by the post-irradiation treatment with Trolox. The expression of p53 and active SAPK/JNK was increased after X-irradiation, and fragments of PARP and the activated fragment of caspase-3 were produced. Post-irradiation treatment with Trolox attenuated the X-irradiation-induced expression, fragmentation or activation of these apoptosis-related biomolecules. The expression of BCL-2 and BAX, which would occur downstream from p53, was not changed by irradiation and Trolox treatment. Furthermore, cell death was associated with caspase-3 because the ladder-like DNA cleavage was completely inhibited by Ac-DEVD-CHO but not Ac-YVAD-CHO, TLCK and PMSF. CONCLUSION: Post-irradiation events such as membrane damage induce caspase-3-dependent apoptosis, which might be mediated by the activation of SAPK/JNK and be independent of p53.

Antioxidants↗

PubMed1999

Hydroxyl radical generation and lipid peroxidation in C2C12 myotube treated with iodoacetate and cyanide.

To mimic exercise-induced events such as energetic impairment, free radical generation, and lipid peroxidation in vitro, mouse-derived C2C12 myotubes were submitted to the inhibition of glycolytic and/or oxidative metabolism with 1 mM iodoacetate (IAA) and/or 2 mM sodium cyanide (CN), respectively, under 5% CO2/95% air up to 180 min. Electron spin resonance (ESR) analysis with a spin-trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) revealed time-course increases in spin adducts from hydroxyl radical (DMPO-OH) and carbon-centered radical (DMPO-R) in the supernatant of C2C12 myotubes treated with the combination of IAA + CN. In this condition, malondialdehyde (MDA) and lactate dehydrogenase (LDH) were released into the supernatant. By the addition of iron-chelating 1 mM deferoxamine to the C2C12 preparation with IAA + CN, both ESR signals of DMPO-OH and DMPO-R were completely abolished, and the release of MDA and LDH were significantly reduced, while cyanide-resistant manganese superoxide dismutase had negligible effects on these parameters. Hence, a part of the injury of C2C12 myotube under IAA + CN was considered to result from the lipid peroxidation, which was induced by hydroxyl radical generated from iron-catalyzed systems such as the Fenton-type reaction. This in vitro model would be a helpful tool for investigating the free radical-related muscle injury.

Animals↗

PubMed1999

Hydrogen peroxide-induced activation of SAPK/JNK regulated by phosphatidylinositol 3-kinase in Chinese hamster V79 cells.

To clarify activation mechanisms of stress-activated protein kinase/C-Jun N-terminal kinase (SAPK/JNK) during oxidative stress, the roles of phosphatidylinositol 3-kinase (PI 3-kinase), concentration of intracellular calcium ([Ca2+]i), and cyclic AMP-dependent kinase (PKA) in hydrogen peroxide (H2O2)-induced SAPK/JNK activation were examined in Chinese hamster V79 cells. SAPK/JNK was dose-dependently activated after H2O2 treatment (from 10 microM to 1 mM), and a PI 3-kinase inhibitor (wortmaninn), intracellular calcium chelator (BAPTA-AM), and PKA activator (dibutyl cyclic AMP and forskolin) inhibited this activation. An increase in [Ca2+], was observed after treatment with H2O2. Immunoprecipitation revealed that a PI 3-kinase regulatory subunit, p85alpha, was associated with insulin receptor substance 1 (IRS-1) phosphorylated by H2O2 treatment. Furthermore, the formation of this complex of p85alpha and phospho-IRS-1 was abolished by the presence of BAPTA-AM but not forskolin. These results indicated that the PI 3-kinase activated through phosphorylation of IRS-1 upstream of SAPK/JNK after H2O2 treatment of V79 cells and that [Ca2+]i was a regulation factor for phosphorylation of IRS-1.

Androstadienes↗

PubMed1999

Elevation of intracellular calcium ions is essential for the H2O2-induced activation of SAPK/JNK but not for that of p38 and ERK in Chinese hamster V79 cells.

The mitogen-activated protein kinases (MAPK), including stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38, and extracellular signal-related kinase (ERK), are believed to be important biomolecules in cell proliferation, survival, and apoptosis induced by extracellular stimuli. In Chinese hamster V79 cells exposed to hydrogen peroxide (H2O2), we recently demonstrated that SAPK/JNK was activated by tyrosine kinase and intracellular Ca2+ ([Ca2+]i). In this study, we report that [Ca2+]i release from intracellular stores is important in the activation of SAPK/JNK but not p38 and ERK. H2O2-induced elevation of [Ca2+]i was observed in Ca2+-free medium. Pretreatment with thapsigargin, a Ca2+-ATPase inhibition of endoplasmic reticulum (ER), did not influence H2O2-induced elevation of [Ca2+]i in the absence of external Ca2+. An intracellular Ca2+ chelator (BAPTA-AM) inhibited H2O2-induced phosphorylation of SAPK/JNK, but an extracellular Ca2+ chelator (EDTA) or a Ca2+ entry blocker (NiCl2) did not. Activation of p38 and ERK in V79 cells exposed to H2O2 was observed in the presence of these inhibitors. These results suggest that [Ca2+]i release from intracellular stores such as mitochondria or nuclei but not ER, occurred after H2O2 treatment and Ca2+-dependent tyrosine kinase-induced activation of SAPK/JNK, although [Ca2+]i was unnecessary for the H2O2-induced activation of p38 and ERK.

Animals↗

PubMed1999

Lipid peroxides and antioxidants in serum of neonatal calves.

OBJECTIVE: To examine the association between oxidative stress and antioxidants in neonatal calves after birth. SAMPLE POPULATION: Sera from 6 healthy Holstein-Friesian cows and 7 of their newborn calves were obtained at various intervals after birth. PROCEDURE: Lipid peroxides in serum of cows and their newborn calves were estimated by measuring concentrations of thiobarbituric acid-reactive substances (TBARS). The antioxidative activities of neonatal sera were evaluated by measuring their superoxide-scavenging activities, ferroxidase activities, and the concentration of bilirubin-associated albumin. RESULTS: Concentration of TBARS in neonatal sera within 1 day after birth was significantly higher than concentrations > or = 2 days after birth and concentrations in serum of the dams. In contrast, antioxidative activities of neonatal sera, evaluated on the basis of superoxide-scavenging activities, ferroxidase activities, and concentration of bilirubin-associated albumin 3 hours after birth, were significantly lower than antioxidative activities in sera of the dams. CONCLUSIONS: Susceptibility of calves to oxidative stress during the neonatal period may be explained by the immature defense system against superoxide radicals.

Animals↗

PubMed1999

Effects of BAPTA-AM and forskolin on apoptosis and cytochrome c release in photosensitized Chinese hamster V79 cells.

The mechanism of caspase-3-dependent apoptosis induced by photodynamic therapy (PDT) of cultured Chinese hamster V79 cells with pheophorbide a (PPa) was investigated. The PPa-PDT induced rapid apoptosis within 30 min after irradiation of cells. This apoptosis was inhibited by the 1O2 quencher N3- and caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde, suggesting that 1O2 activated caspase-3 and then caused apoptosis. The intracellular calcium [Ca2+]i chelator (acetoxymethyl)-1,2-bis(o-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA-AM) and the cyclic adenosine monophosphate (cAMP)-increasing agent forskolin also inhibited not only the PPa-PDT-induced activation of caspase-3 but also apoptosis in V79 cells. Furthermore, PPa-PDT-induced cytochrome c release from mitochondria was found to be inhibited by the treatment with BAPTA-AM but not forskolin. These results indicated that [Ca2+]i and cAMP independently serve as regulators for PPa-PDT-induced apoptosis in the upstream of caspase-3.

Animals↗