Glutathione accelerates osteoclast differentiation and inflammatory bone destruction.

Chronic inflammation associated with bone tissues often destructs bones, which is essentially performed by osteoclasts in the presence of immunoregulatory molecules. Hence, regulating osteoclastogenesis is crucial to develop therapeutics for bone-destructive inflammatory diseases. It is believed that reactive oxygen species (ROS) are involved in receptor activator of NF-κB (RANK) ligand (RANKL)-induced osteoclast differentiation, and, therefore, glutathione (GSH), the most abundant endogenous antioxidant, suppresses osteoclast differentiation and bone resorption by RANKL. Interestingly, GSH also contributes to inflammatory responses, and the effects of GSH on osteoclast differentiation and bone destruction under inflammatory conditions have not yet been determined. Here, we investigated how GSH affects inflammatory cytokine-stimulated osteoclast differentiation in vitro and in a mouse model of inflammatory bone destruction. We found that GSH significantly promoted TNFα-stimulated osteoclast formation, while an inhibitor of GSH synthesis, buthionine sulfoximine, suppressed it. GSH facilitated the nuclear localisation of the nuclear factor of activated T cells c1 (NFATc1) protein, a master regulator of osteoclastogenesis, as well as the expression of osteoclast marker genes in a dose-dependent manner. N-acetylcysteine, a substrate of GSH synthesis, also stimulated osteoclast formation and NFATc1 nuclear localisation. GSH did not suppress cell death after osteoclast differentiation. In mouse calvaria injected with lipopolysaccharide, GSH treatment resulted in a fivefold increase in the osteolytic lesion area. These results indicate that GSH accelerates osteoclast differentiation and inflammatory bone destruction, suggesting GSH appears to be an important molecule in the mechanisms responsible for inflammatory bone destruction by osteoclasts.

Successful management of unresectable small bowel lymphoma with laparoscopy-assisted surgical exclusion of the affected intestine.

The incidence of small bowel lymphoma (SBL) is increasing worldwide. In contrast to resectable SBL, the treatment of unresectable SBL is still contentious. Here, we report a case of unresectable SBL that was treated by laparoscopic exclusion of the affected intestine before systemic chemotherapy was administered. An 84-year-old man was diagnosed with primary SBL involving extranodal dissemination. The patient received prophylactic surgery, namely exclusion of the affected intestine. This therapy diminishes well-known and life-threatening complications, such as perforation, bleeding, and obstruction, which may still occur after chemotherapy, and it makes the administration of chemotherapy safer. In addition, the surgery provides easy access for direct endoscopic observation and biopsy, which are otherwise difficult to perform. Follow-up after two courses of chemotherapy showed that the patient had achieved complete remission. In conclusion, the procedure described here may be an effective strategy for unresectable SBL.

Phytoestrogen Suppresses Efflux of the Diagnostic Marker Protoporphyrin IX in Lung Carcinoma.

One promising method to visualize cancer cells is based on the detection of the fluorescent photosensitizer protoporphyrin IX (PpIX) synthesized from 5-aminolevulinic acid (ALA), but this method cannot be used in cancers that exhibit poor PpIX accumulation. PpIX appears to be pumped out of cancer cells by the ABC transporter G2 (ABCG2), which is associated with multidrug resistance. Genistein is a phytoestrogen that appears to competitively inhibit ABCG2 activity. Therefore, we investigated whether genistein can promote PpIX accumulation in human lung carcinoma cells. Here we report that treatment of A549 lung carcinoma cells with genistein or a specific ABCG2 inhibitor promoted ALA-mediated accumulation of PpIX by approximately 2-fold. ABCG2 depletion and overexpression studies further revealed that genistein promoted PpIX accumulation via functional repression of ABCG2. After an extended period of genistein treatment, a significant increase in PpIX accumulation was observed in A549 cells (3.7-fold) and in other cell lines. Systemic preconditioning with genistein in a mouse xenograft model of lung carcinoma resulted in a 1.8-fold increase in accumulated PpIX. Long-term genistein treatment stimulated the expression of genes encoding enzymes involved in PpIX synthesis, such as porphobilinogen deaminase, uroporphyrinogen decarboxylase, and protoporphyrinogen oxidase. Accordingly, the rate of PpIX synthesis was also accelerated by genistein pretreatment. Thus, our results suggest that genistein treatment effectively enhances ALA-induced PpIX accumulation by preventing the ABCG2-mediated efflux of PpIX from lung cancer cells and may represent a promising strategy to improve ALA-based diagnostic approaches in a broader set of malignancies. Cancer Res; 76(7); 1837-46. ©2016 AACR.

Necrotic and apoptotic cells serve as nuclei for calcification on osteoblastic differentiation of human mesenchymal stem cells in vitro.

A close relationship between cell death and pathological calcification has recently been reported, such as vascular calcification in atherosclerosis. However, the roles of cell death in calcification by osteoblast lineage have not been elucidated in detail. In this study, we investigated whether cell death is involved in the calcification on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hMSC) under osteogenic culture in vitro. Apoptosis and necrosis occurred in an osteogenic culture of hMSC, and cell death preceded calcification. The generation of intracellular reactive oxygen species, chromatin condensation and fragmentation, and caspase-3 activation increased in this culture. A pan-caspase inhibitor (Z-VAD-FMK) and anti-oxidants (Tiron and n-acetylcysteine) inhibited osteogenic culture-induced cell death and calcification. Furthermore, calcification was significantly promoted by the addition of necrotic dead cells or its membrane fraction. Spontaneously dead cells by osteogenic culture and exogenously added necrotic cells were surrounded by calcium deposits. Induction of localized cell death by photodynamic treatment in the osteogenic culture resulted in co-localized calcification. These findings show that necrotic and apoptotic cell deaths were induced in an osteogenic culture of hMSC and indicated that both necrotic and apoptotic cells of osteoblast lineage served as nuclei for calcification on osteoblastic differentiation of hMSC in vitro.

Mitochondrial localization of ABC transporter ABCG2 and its function in 5-aminolevulinic acid-mediated protoporphyrin IX accumulation.

Accumulation of protoporphyrin IX (PpIX) in malignant cells is the basis of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy. We studied the expression of proteins that possibly affect ALA-mediated PpIX accumulation, namely oligopeptide transporter-1 and -2, ferrochelatase and ATP-binding cassette transporter G2 (ABCG2), in several tumor cell lines. Among these proteins, only ABCG2 correlated negatively with ALA-mediated PpIX accumulation. Both a subcellular fractionation study and confocal laser microscopic analysis revealed that ABCG2 was distributed not only in the plasma membrane but also intracellular organelles, including mitochondria. In addition, mitochondrial ABCG2 regulated the content of ALA-mediated PpIX in mitochondria, and Ko143, a specific inhibitor of ABCG2, enhanced mitochondrial PpIX accumulation. To clarify the possible roles of mitochondrial ABCG2, we characterized stably transfected-HEK (ST-HEK) cells overexpressing ABCG2. In these ST-HEK cells, functionally active ABCG2 was detected in mitochondria, and treatment with Ko143 increased ALA-mediated mitochondrial PpIX accumulation. Moreover, the mitochondria isolated from ST-HEK cells exported doxorubicin probably through ABCG2, because the export of doxorubicin was inhibited by Ko143. The susceptibility of ABCG2 distributed in mitochondria to proteinase K, endoglycosidase H and peptide-N-glycosidase F suggested that ABCG2 in mitochondrial fraction is modified by N-glycans and trafficked through the endoplasmic reticulum and Golgi apparatus and finally localizes within the mitochondria. Thus, it was found that ABCG2 distributed in mitochondria is a functional transporter and that the mitochondrial ABCG2 regulates ALA-mediated PpIX level through PpIX export from mitochondria to the cytosol.

Favorable response of heavily treated Wilms' tumor to paclitaxel and carboplatin.

BACKGROUND: Heavily treated Wilms' tumor responding to the combination of paclitaxel and carboplatin has not yet been reported. CASE REPORT: A 17-year-old man presented with hematuria. He received a diagnosis of Wilms' tumor with multiple lung metastases and was treated with preoperative chemotherapy including vincristine, dactinomycin, and doxorubicin, a right nephrectomy, and adjuvant chemotherapy, followed by pulmonary metastasectomy. During the next 8 years, he suffered from 4 relapses and has been treated with multiple anticancer agents including high-dose chemotherapy with autologous peripheral blood stem cell transplantation. Finally, the disease progressed due to peritoneal and pleural metastases. With opioid administration for left shoulder pain due to pleural metastasis, he received combination chemotherapy with carboplatin (area under the curve = 4) and paclitaxel (175 mg/m(2)) on day 1. After 2 cycles, he achieved a partial response with mild toxicity. He received 7 cycles of the chemotherapy and the time to progression was 200 days. CONCLUSION: In a refractory case after intensive treatments, we succeeded to control the disease for a while.

[A case of advanced gastric cancer showing a complete histological response after S-1/CDDP neoadjuvant chemotherapy].

We experienced a case of advanced gastric cancer treated by curative operation after neoadjuvant chemotherapy with S-1/ CDDP. Gastric endoscopy was carried out on a 76-year-old man with epigastric discomfort and revealed a type 1 lesion in his stomach. Papillary adenocarcinoma was pathologically shown by endoscopic biopsy. The patient was initially treated by two courses of neoadjuvant chemotherapy with S-1/CDDP due to the large lymph node metastases around the lesser curvature of the stomach and celiac axis. Completion of chemotherapy resulted in a marked shrinkage of the primary lesion and a reduction of lymph node metastases. Later, total gastrectomy, splenectomy and D2 lymph node dissection were performed. Histopathological examination revealed no cancer cells in either the primary lesion of the stomach or dissected lymph nodes, confirming a pathologically complete response.

Serum-dependent export of protoporphyrin IX by ATP-binding cassette transporter G2 in T24 cells.

Accumulation of protoporphyrin IX (PpIX) in cancer cells is a basis of 5-aminolevulinic acid (ALA)-induced photodymanic therapy. We studied factors that affect PpIX accumulation in human urothelial carcinoma cell line T24, with particular emphasis on ATP-binding cassette transporter G2 (ABCG2) and serum in the medium. When the medium had no fetal bovine serum (FBS), ALA induced PpIX accumulation in a time- and ALA concentration-dependent manner. Inhibition of heme-synthesizing enzyme, ferrochelatase, by nitric oxide donor (Noc18) or deferoxamine resulted in a substantial increase in the cellular PpIX accumulation, whereas ABCG2 inhibition by fumitremorgin C or verapamil induced a slight PpIX increase. When the medium was added with FBS, cellular accumulation of PpIX stopped at a lower level with an increase of PpIX in the medium, which suggested PpIX efflux. ABCG2 inhibitors restored the cellular PpIX level to that of FBS(-) samples, whereas ferrochelatase inhibitors had little effects. Bovine serum albumin showed similar effects to FBS. Fluorescence microscopic observation revealed that inhibitors of ABC transporter affected the intracellular distribution of PpIX. These results indicated that ABCG2-mediated PpIX efflux was a major factor that prevented PpIX accumulation in cancer cells in the presence of serum. Inhibition of ABCG2 transporter system could be a new target for the improvement of photodynamic therapy.

Effect of risedronate on osteoblast differentiation, expression of receptor activator of NF-κB ligand and apoptosis in mesenchymal stem cells.

Nitrogen-containing bisphosphonates (BPs) are antiresorptive drugs used for the treatment of metabolic bone diseases. Bone marrow stromal cells such as mesenchymal stem cells (MSCs) and MSC-derived osteoblasts that originate from MSCs are known to regulate osteoclast differentiation and activation via the expression of receptor activator of NF-κB ligand (RANKL). Although the effects of nitrogen-containing BPs on osteoclasts and osteoblasts have been well investigated, their effects in MSCs have not been clarified. In this study, we investigated the effects of risedronate (RIS), a nitrogen-containing BP, on osteoblast differentiation, RANKL expression and apoptosis in human and rat MSCs. RIS suppressed the formation of mineralized nodules and mRNA expression of differentiation marker genes such as bone sialoprotein and osteocalcin in MSC-derived osteoblasts. The RANKL expression induced by 1,25-(OH)(2) vitamin D(3) was not affected by RIS in human MSC-derived osteoblasts. In addition, treatment with high-concentration RIS induced chromatin condensation, an apoptosis feature, in MSCs. RIS-induced chromatin condensation was suppressed by a pan-caspase inhibitor zVAD-FMK and a cell-permeable isoprenoid analogue geranylgeraniol. These results indicate that RIS suppressed osteoblast differentiation and induced caspase- and isoprenoid depletion-dependent apoptosis and suggest that the antiresorptive effect of RIS is not mediated by a decrease in the RANKL expression in MSC-derived osteoblasts.

Oxidative stress enhances granulocytic differentiation in HL 60 cells, an acute promyelocytic leukemia cell line.

This paper studied the effects of physiologically available oxidants on HL 60 differentiation induced by all-trans retinoic acid (ATRA) or dimethyl sulfoxide (DMSO). Hydrogen peroxide (15 µM) and taurine chloramine (200 µM) induced HL 60 differentiation, which was detected by CD11b expression and superoxide production. Cd11b and p67phox mRNA expression was also augmented by these oxidants. In contrast, reducing chemicals, such as dithiothreitol, 2,3-dimercapto-1-propanol and N-acetylcysteine inhibited CD11b expression. Notably, DMSO inhibited methionine sulfoxide reductase activity, induced heme oxygenase-1 (ho-1) mRNA and enhanced oxidant-induced cell death, which indicated that DMSO intensified oxidative stress. After the addition of oxidants, ho-1 expression preceded the cd11b expression. Vicinal dithiol-reactive phenylarsine oxide (50 nM) also increased CD11b expression induced by DMSO or ATRA. These observations suggested that oxidative stress enhanced granulocytic differentiation of HL 60 cells and that leukaemic cell differentiation was affected by cellular redox status.

p66(Shc) has a pivotal function in impaired liver regeneration in aged mice by a redox-dependent mechanism.

Liver regeneration involves complicated processes and is affected by various patho-physiological conditions. This study was designed to examine the molecular mechanisms underlying the aging-associated impairment of liver regeneration. Male C57BL/6J mice were used as young and aged mice (<10 weeks and >20 months old, respectively). These mice were subjected to 70% partial hepatectomy (PH). Liver regeneration and liver injury/stresses were evaluated chronologically after PH. Post-hepatectomy liver regeneration was markedly impaired in aged mice. Though the extent of hepatocyte proliferation in the regenerating liver was similar in aged and young mice, cell growth was absent in aged mice. Oxidative stress (OS) was observed immediately after hepatectomy, followed by marked apoptosis in aged mice. Signaling molecules regarding cell proliferation (mitogen-activated protein kinase, STAT3, p46/52(Shc)) and anti-oxidation (catalase, superoxide dismutase, Ref-1, glutathione peroxidase) were expressed/activated after hepatectomy in livers of both aged and young mice. Akt was not activated in aged-mouse liver, but its expression was similar to that in young mice. p66(Shc), known as an age-/oxidant-associated protein, was strongly phosphorylated. By knocking down p66(Shc), the impairment of liver regeneration was normalized. OS immediately after hepatectomy induced subsequent liver injury (apoptosis), and deletion of p66(Shc) suppressed both OS and hepatocyte apoptosis in the regenerating liver of aged mice. Though we need additional data in other animal models to fully understand the mechanism, p66(Shc) may have a pivotal function in the impairment of liver regeneration in aged mice by triggering OS and subsequent apoptosis. This data may provide a clue to understanding the mechanism underlying the association between aging and the impairment of liver regeneration.

Alpha-tocopheryl succinate induces rapid and reversible phosphatidylserine externalization in histiocytic lymphoma through the caspase-independent pathway.

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 alpha-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 Ca(2+)-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.

Mechanism of cell death by 5-aminolevulinic acid-based photodynamic action and its enhancement by ferrochelatase inhibitors in human histiocytic lymphoma cell line U937.

Photodynamic therapy (PDT) for tumors is based on the tumor-selective accumulation of a photosensitizer, protoporphyrin IX (PpIX), followed by irradiation with visible light. However, the molecular mechanism of cell death caused by PDT has not been fully elucidated. The 5-aminolevulinic acid (ALA)-based photodynamic action (PDA) was dependent on the accumulation of PpIX, the level of which decreased rapidly by eliminating ALA from the incubation medium in human histiocytic lymphoma U937 cells. PDA induced apoptosis characterized by lipid peroxidation, increase in Bak and Bax/Bcl-xL, decrease in Bid, membrane depolarization, cytochrome c release, caspase-3 activation, phosphatidylserine (PS) externalization. PDT-induced cell death seemed to occur predominantly via apoptosis through distribution of PpIX in mitochondria. These cell death events were enhanced by ferrochelatase inhibitors. These results indicated that ALA-based-PDA induced apoptotic cell death through a mitochondrial pathway and that ferrochelatase inhibitors might enhanced the effect of PDT for tumors even at low concentrations of ALA.

Immunohistochemical staining of liver grafts with a monoclonal antibody against HCV-Envelope 2 for recurrent hepatitis C after living donor liver transplantation.

AIM: We evaluated the expression of hepatitis C virus (HCV) antigen on liver grafts by immunohistochemical staining (IHS) using IG222 monoclonal antibody (mAb) against HCV-envelope 2 (E2). METHODS: The study material was 84 liver biopsy specimens obtained from 28 patients who underwent living donor liver transplantation (LDLT) for HCV infection. The biopsy samples were examined histopathologically, and by IHS using IG222 mAb against HCV-E2. Serum HCV-RNA level was measured in all patients. The IHS grades were compared among the three groups classified according to the time elapsed from LDLT (at 1-30, 31-179 and > or =180 days post-LDLT) and among four post-transplant conditions, including acute cellular rejection (ACR). RESULTS: Immunoreactivity to IG222 was detected in 78.6% of the specimens obtained during the first month after LDLT, and there were no significant differences on the IHS grades between the three groups classified according to the time elapsed from LDLT. The IHS grades were significantly stronger in definite recurrent HCV (n = 12) and probable recurrent HCV (n = 7) than in definite ACR (n = 7) and other complications (n = 8). There were no significant differences in serum HCV-RNA levels among the four post-transplant conditions. There was no significant correlation between the IHS grades using IG222 mAb and serum HCV-RNA levels when data of 84 liver biopsy specimens were analyzed. CONCLUSIONS: Constant HCV-E2 expression was observed in liver biopsy specimens obtained 1 month or longer. The strong HCV-E2 expression on liver grafts were associated with recurrent hepatitis C after LDLT, but the serum HCV-RNA levels were not.

Activation of c-Jun N-terminal kinase is essential for oxidative stress-induced Jurkat cell apoptosis by monochloramine.

Leukemic cell apoptosis may be enhanced by appropriate oxidative stress. We report here the mechanism of Jurkat cell apoptosis by monochloramine (NH(2)Cl), a neutrophil-derived oxidant. NH(2)Cl induced caspase-dependent apoptosis, which was preceded by cytochrome c and Smac/Diablo release from mitochondria. Within 10min of NH(2)Cl treatment, c-Jun N-terminal kinase (JNK) activation and elevation of cytosolic Ca(2+) were observed. JNK inhibitors (SP600125 or JNK inhibitor VIII) significantly suppressed the apoptosis as well as caspase cleavage and cytochrome c release. In contrast, Ca(2+) chelation by EGTA+acetoxymethyl-EGTA had no effects on apoptosis. Our results indicated that JNK activation contributed most importantly to the NH(2)Cl-induced apoptosis.

Alpha-lipoic acid suppresses 6-hydroxydopamine-induced ROS generation and apoptosis through the stimulation of glutathione synthesis but not by the expression of heme oxygenase-1.

We previously reported that the generation of reactive oxygen species (ROS) is the initial event in cell death induced by 6-hydroxydopamine (6-OHDA), an experimental model of Parkinsonism. Since recent studies suggested the important role of antioxidant activity of alpha-lipoic acid (LA) in the suppression of apoptosis of various types, we studied the effect on 6-OHDA-induced apoptosis of PC12 cells. Biochemical analysis revealed that LA suppressed the 6-OHDA-induced ROS generation, increase of caspase-like activity and chromatin condensation. The suppression of 6-OHDA-induced apoptosis by LA required pre-incubation of PC12 cells with LA for 12-24 h. LA increased the intracellular levels of heme oxygenase-1 (HO-1) and glutathione (GSH) and stimulated the expression of GSH synthesis-related genes such as cystine/glutamate antiporter and gamma-glutamylcysteine synthetase (gamma-GCS). However, Sn-mesoporphyrin IX, an inhibitor of HO-1, did not attenuate the LA-induced suppression of apoptosis. In contrast, buthionine sulfoximine, an inhibitor of gamma-GCS, attenuated the LA-induced suppression of ROS generation and chromatin condensation. In addition, a transcription factor Nrf2, which regulates the expression of antioxidant enzymes such as gamma-GCS, translocated to the nucleus by LA. These results suggested that LA suppressed the 6-OHDA induced-apoptosis by the increase in cellular glutathione through stimulation of the GSH synthesis system but not by the expression of HO-1.

Regulation of 5-aminolevulinic acid-dependent protoporphyrin IX accumulations in human histiocytic lymphoma U937 cells.

The aim of the present work is to clarify the mechanism(s) that regulates the accumulation of protoporphyrin IX (PpIX) in human histiocytic lymphoma cell line U937 incubated with 5-aminolevulinic acid (ALA). Biosynthesis and accumulation of PpIX in the cells was determined after incubation with 0.1-5 mM ALA using a flow cytometric technique. The synthesized endogenous PpIX was found to localize predominantly in the mitochondrial region of the cells. The ALA-enhanced PpIX synthesis was suppressed by the presence of either beta-alanine, a competitive inhibitor of beta-transporters on cell membranes, or carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, an uncoupler of mitochondrial oxidative phosphorylation. In contrast, cellular accumulation of PpIX was enhanced by the presence of either deferoxamine (an iron chelater), MnCl2 (a ferrochelatase inhibitor), or Sn-mesoporphyrin (heme oxygenase inhibitor). These results suggest that ALA-enhanced accumulation of PpIX in U937 cells was regulated by cellular uptake and conversion of ALA to PpIX and by degradation of Heme.

Angiomyofibroblastoma of the vulva: a large pedunculated mass formation.

Angiomyofibroblastoma is a rare, usually small benign mesenchymal tumor that occurs in vulvar lesions of premenopausal women. A case of angiomyofibroblastoma that arose as a unique pedunculated and particularly large mass in the left vulva of a 48-year-old woman is presented herein. The patient had been aware of a gradually enlarged mass of 7 years duration without any other gynecological symptoms or signs. The maximum dimension of the tumor measured 11 cm. The resected tumor was well circumscribed with a bulging and glistening cut surface. Histological examination revealed an admixture of irregularly distributed hypercellular and hypocellular areas with spindled, plump spindled, or plasmacytoid stromal cells and abundant venular or capillary-sized vessels. Stromal cells characteristically cluster around delicate vessels within an edematous to collagenous matrix. In the present case, intralesional adipose tissue was present throughout the tumor. There was no significant nuclear atypia, and mitotic figures were very sparse. There was little stromal mucin throughout the tumor. Immunohistochemically, the stromal cells were characterized by strong reactivity for vimentin and CD34, with focal reactivity for desmin and alpha smooth muscle actin. Both estrogen and progesterone receptors were diffusely expressed in the stromal cells. These histological findings are consistent with angiomyofibroblastoma and support the hypothesis that angiomyofibroblastoma originates from perivascular stem cells with a capacity for myofibroblastic and fatty differentiation.

Cell-permeable cAMP analog suppresses 6-hydroxydopamine-induced apoptosis in PC12 cells through the activation of the Akt pathway.

Although cAMP protects neuronal cells from various apoptotic stimulations, its mechanism is not fully elucidated. We report here the molecular mechanism of the 6-hydroxydopamine (6-OHDA)-induced apoptosis of pheochromocytoma PC12 cells and its suppression by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (pCPT-cAMP), which is a membrane permeable cAMP analog. Treatment of PC12 cells with 6-OHDA resulted in the activation of caspases and apoptosis, as detected by chromatin condensation. 6-OHDA also induced superoxide generation, Bid cleavage and mitochondrial membrane depolarization. In addition, Akt phosphorylation that was favorable to cell survival was decreased and p38 MAPK phosphorylation was increased by 6-OHDA. PC12 cell apoptosis was inhibited by pCPT-cAMP, Z-VAD-fmk (a broad-range caspase inhibitor) and tiron (a superoxide scavenger), although PC12 cell apoptosis was not inhibited by cyclosporine A (an inhibitor of mitochondrial membrane permeability transition). Moreover, pCPT-cAMP promoted Akt phosphorylation, but it did not prevent superoxide generation and mitochondrial membrane depolarization. Conversely, LY294002, an inhibitor of Akt upstream molecule PI3-kinase, enhanced 6-OHDA-induced apoptosis. These results indicated that the 6-OHDA-induced apoptosis of PC12 cells was initiated by superoxide generation followed by caspase cascade activation, which was associated with the suppressed Akt phosphorylation and increased p38 phosphorylation. It is likely that pCPT-cAMP prevented the 6-OHDA-induced apoptosis via activation of the PI3-kinase/Akt pathway without any effect on superoxide generation or mitochondrial membrane depolarization.