Ac-cel, a Novel Antioxidant, Protects Against Hydrogen Peroxide-induced Injury in PC12 Cells via Attenuation of Mitochondrial Dysfunction

Oxidative stress has been implicated in pathophysiology of many neurodegenerative diseases (ND) and increased oxidative stress is closely associated with mitochondrial dysfunction. As a result, looking for potent antioxidants, especially those targeting mitochondria, has become an attractive strategy in ND therapy. In this study, we explored protective effects and potential mechanism of Ac-cel, a novel compound, against hydrogen peroxide (H₂O₂)-induced injury in PC12 cells. Pretreatment of PC12 cells with Ac-cel prior to 24 h of H₂O₂ exposure markedly attenuated cytotoxicity induced by H₂O₂ as evidenced by morphological changes and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Ac-cel also exhibited potent antiapoptotic effect demonstrated by results of annexin V and PI staining. The above beneficial effects of Ac-cel were accompanied by improved mitochondrial function, reduced caspase-3 cleavage as well as upregulated ratio of Bcl-2/Bax protein expression. Moreover, Ac-cel pretreatment markedly reversed intracellular reactive oxygen species (ROS) accumulation following 30 min of H₂O₂ exposure in PC12 cells. Further, subcellular investigation indicated that Ac-cel significantly reduced production of mitochondrial ROS in isolated rat cortical mitochondria. Taken together, the present study, for the first time, reports that Ac-cel pretreatment inhibits H₂O₂-stimulated early accumulation of intracellular ROS possibly via reducing mitochondrial ROS production directly and leads to subsequent preservation of mitochondrial function. These results indicate that Ac-cel is a potential drug candidate for treatment of oxidative stress-associated ND.     

17β-Estradiol Impedes Bax-Involved Mitochondrial Apoptosis of Retinal Nerve Cells Induced by Oxidative Damage via the Phosphatidylinositol 3-Kinase/Akt Signal Pathway

Oxidative stress leading to retinal nerve cells (RNCs) apoptosis is a major cause of neurodegenerative disorders of the retina. 17β-Estradiol (E2) has been suggested to be a neuroprotective agent in the central nervous system; however, at present, the underlying mechanisms are not well understood, and the related research on the RNCs is less reported. Here, in order to investigate the protective role and mechanism of E2 against oxidative stress-induced damage on RNCs, the transmission electron microscopy and annexin V-FITC/propidium iodide assay were applied to detect the RNCs apoptosis. Western blot and real-time PCR were used to determine the expression of the critical molecules in Bcl-2 and caspase family associated with apoptosis. The transmission electron microscopy results showed that H₂O₂ could induce typical features of apoptosis in RNCs, including formation of the apoptosome. E2 could, however, suppress the H₂O₂-induced morphological changes of apoptosis. Intriguingly, we observed E2-mediated phagocytic scavenging of apoptosome. In response to H₂O₂-induced apoptosis, Bax, acting as one of the pivotal pro-apoptotic members of Bcl-2 family, increased significantly, which directly resulted in an increased ratio of Bax to anti-apoptotic protein Bcl-2 (Bax/Bcl-2). Additionally, caspases 9 and 3, which are the critical molecules of the mitochondrial apoptosis pathway, were activated by H₂O₂. In contrast, E2 exerted anti-apoptotic effects by reducing the expression of Bax to decrease the ratio of Bax/Bcl-2 and impeded the caspases 9/3 activation. Moreover, LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, could sharply block the effect of E2 in reducing the percentage of apoptotic cells resistance to H₂O₂. And the attenuation of Bax, the reduced activities of caspases 9/3 and the impeded release of mitochondrial cytochrome c mediated by E2 resistance to H₂O₂ damage were significantly retrieved by LY294002 administration. Taken together, E2 protects the RNCs against H₂O₂-induced apoptosis by significantly inhibiting the Bax-involved mitochondrial apoptosis via the activation of PI3K/Akt signal pathway.     

Protective effects of edaravone against cobalt chloride-induced apoptosis in PC12 cells

Objective

To investigate the neuroprotective effects of edaravone (Eda) on cobalt chloride (CoCl₂)-induced oxidative stress and apoptosis in cultured PC12 cells as well as the underlying mechanisms.

Methods

PC12 cells impaired by CoCl₂ were used as the cell model of hypoxia. MTT (methyl thiazolyl tetrazolium) was used to assay the viability of the PC12 cells exposed to Eda with gradient concentrations; Hochest 33258 stain assay was used to analyze the apoptosis ratio of the PC12 cells; Bcl-2 and Bax protein levels in PC12 cells were examined by western blotting. ROS level, the mitochondrial transmembrane potential and caspase-3 activity in each group were detected by spectrofluorometer.

Results

CoCl₂ treatment caused the loss of cell viability in PC12 cells, which was associated with the elevation of apoptotic rate, the formation of ROS and the disruption of mitochondrial transmembrane potential. CoCl₂ also significantly induced the upregulation of Bax/Bcl-2 ratio and the activation of caspase-3. In contrast, Eda significantly reversed these phenotypes, with its maximum protective effect at 0.1 μmol/L.

Conclusion

These results indicated that Eda could protect PC12 cells from CoCl₂-induced cytotoxicity, and this protection might be ascribed to its anti-oxidative and anti-apoptotic activities.     

Dual Contradictory Effect of H-89 on Neuronal Retraction, Death and Inflammation in Differentiated PC12 Cells Subjected to Oxidative Stress

Interrelation between oxidative stress and neuro-inflammation has been discussed extensively to contribute to neuronal dysfunction in neurodegenerative disorders. In this manner, it seems that there is an intriguing link between protein kinase A (PKA), neuronal apoptosis and inflammation. Rat PC12 pheochromocytoma cell can be induced to differentiate into neuron-like cells possessing elongated neurites by nerve growth factor. In this study, we investigated the effect of H-89, a selective inhibitor of PKA, on the neurite retraction along with evaluation of cell death and inflammatory markers in the differentiated PC12 cells, exposed to H₂O₂. We found that dose-dependent inhibition of PKA by low and medium concentrations of H-89 (5, 7 and 10 μM) enhanced the parameters of neurite outgrowth and complexity in the cells co-treated with H₂O₂ as an oxidative stress. Similar concentrations of H-89 significantly inhibited cell death and neurite retraction induced by oxidative stress. Components of TNF-α–NFκB–COX-2 axis, a discussed pathway in neuroinflammation, downregulated dose-dependently by administration of H-89 in H₂O₂-induced PC12 cells. In this condition, PKA inhibition by the high concentrations of H-89 (15 and 20 μM) led to enhanced cell death and inflammation with decreased neurite outgrowth. These findings indicate that H-89 has a dual contradictory effect on oxidative stress and inflammation that affect neurite outgrowth and complexity in differentiated PC12 cells.     

促红细胞生成素对1-甲基-4-苯基吡啶损伤的PC12细胞的保护作用(英文)

目的探讨促红细胞生成素(erythropoietin,EPO)对1-甲基-4-苯基吡啶离子(MPP+)诱导的PC12细胞变性损伤的保护作用及机制。方法用MPP+处理PC12细胞制作帕金森病细胞模型,采用四甲基偶氮唑蓝法检测暴露于不同浓度EPO后细胞的活性;流式细胞术与DNA断端原位标记法(terminal deoxynucleotidyl transferase dUTPnick end labeling, TUNEL)检测各组的细胞凋亡率;免疫印迹法检测不同处理组PC12细胞Bcl-2和Bax的表达,并采用荧光法观察不同处理组PC12细胞活性氧(reactive oxygen species,ROS)与线粒体膜电位水平以及caspase-3活性的变化。结果 MPP+可以使PC12细胞存活率下降,凋亡率增高;同时PC12细胞内ROS增多,线粒体膜电位下降。MPP+还可以明显地提高Bax/Bcl-2比值并激活caspase-3。而EPO可以抑制这些由MPP+引发的改变,并在1 U/mL时发挥最大保护作用。结论 EPO可抑制MPP+诱导的PC12细胞死亡,其作用机制可能与其自身抗氧化和抗凋亡的特性有关。     

促红细胞生成素对1-甲基-4-苯基吡啶损伤的PC12细胞的保护作用

目的探讨促红细胞生成素（erythropoietin,EPO）对1-甲基-4-苯基吡啶离子（MPP^＋）诱导的PC12细胞,变性损伤的保护作用及机制。方法用MPP^＋处理PC12细胞制作帕金森病细胞模型,采用四甲基偶氮哗监泫检测暴露于不同浓度EPO后细胞的活性;流式细胞术与DNA断端原位标记法（terminal deoxynucleotidyl transferase dUTP nick end labeling,TUNEL）检测各组的细胞凋亡率;免疫印迹法检测不同处理组PC12细胞Bcl-2和Bax的表达,并采用荧光法观察不同处理组PC12细胞活性氧（reactive oxygen species,ROS）与线粒体膜电位水平以及caspase-3活性的变化。结果MPP^＋可以使PC12细胞存活率下降,凋亡率增高;同时PC12细胞内ROS增多,线粒体膜电位下降。MPP^＋还可以明显地提高Bax／Bcl-2比值并激活caspase-3。而EPO可以抑制这些由MPP^＋引发的改变,并在1U／mL时发挥最大保护作用。结论EPO可抑制MPP^＋诱导的PC12绌胞死亡,其作用机制可能其自身抗氧化和抗凋亡的特性有关。     

Protective effect of trifluoperazine on hydrogen peroxide-induced apoptosis in PC12 cells

This study investigated effects of trifluoperazine (TFP) against the cytotoxicity induced by H₂O₂ in PC12 cells and the mechanisms thereof. Different concentrations of H₂O₂ (100-500 μM) induced a significant decrease in cell viability accompanied by increased oxidative stress and cell apoptosis. Pretreatment with TFP inhibited H₂O₂-induced cell viability loss. The flow cytometric assay showed that TFP can inhibit intracellular reactive oxygen species (ROS) generation and reduce the cell apoptosis. The electrophysiological recordings indicated that when treated with H₂O₂, the calcium current was significantly increased. Pretreatment with TFP increased mitochondrial membrane potential (MMP) in cells of oxidative injury. These results suggested that TFP can reduce apoptosis by inhibiting ROS generation and preventing loss of MMP in cells. Meanwhile, the protective effect of TFP on the cell apoptosis may be related to the calcium overload. TFP may inhibit the calcium overload process to achieve the protection against apoptosis.     

EGb761 Protects Hydrogen Peroxide-induced Death of Spinal Cord Neurons through Inhibition of Intracellular ROS Production and Modulation of Apoptotic Regulating Genes

The present study was conducted to investigate whether Ginkgo biloba extract (EGb) 761 could protect spinal cord neurons from H₂O₂-induced toxicity. In primary spinal cord neurons isolated from embryonic day 14 rats, H₂O₂ administration resulted in a significant decrease in the survival of spinal cord neurons. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and Hoechst 33342 nuclear staining showed that these cells die by apoptosis. Such neuronal death, however, was significantly reversed by EGb761 in a dose-dependent manner. Moreover, a marked increase in intracellular free radical generation was found after the H₂O₂ administration which could be reversed almost completely by EGb761, indicating that inhibition of free radical generation is an important mechanism of the anti-apoptosis action of EGb761. Finally, treatment of cells with H₂O₂ for 12 h reduced the expression of Bcl-2, an anti-apoptotic gene, by 70% but showed no effect on the level of Bax, a pro-apoptotic gene. EGb76 treatment, however, significantly reversed H₂O₂-induced reduction of Bcl-2 expression and inhibited Bax expression by 2.3-fold. Thus, our study provided evidence showing that the protective effect of EGb761 on spinal cord neuronal apoptosis after oxidative stress is mediated, at least in part, by its anti-oxidative action and regulation of apoptosis-related genes Bcl-2 and Bax.     

Hydrogen peroxide–induced apoptosis mediated by p53 protein in glial cells

It is now generally accepted that massive neuronal death due to oxidative stress is a regular feature of brains in neurodegenerative diseases. However, much less attention has been given to the death of glial cells. In this study, we examined p53-sensitive apoptosis of cells by using human glioblastoma A172 cells and p53-deficient mouse astrocytes. In human A172 cells, hydrogen peroxide (H₂O₂) caused cell death in a time- and concentration-dependent manner, accompanied by nucleosomal DNA fragmentation and chromatin condensation. After treatment with H₂O₂, p53 protein was highly expressed and protein levels of Bak, p21^WAF1/CIP1 and GADD45 were also enhanced. However, the protein levels of Bcl-2 and Bax did not change. On the other hand, primary cultured astrocytes from p53-deficient mouse brain grew faster than wild-type and heterozygous astrocytes. In addition, p53-deficient astrocytes were more resistant to H₂O₂-induced apoptosis than wild-type and heterozygous astrocytes. These results suggest that glial proliferation and the repair of damaged DNA may be regulated by p53-induced p21^WAF1/CIP1 and GADD45, and that glial apoptosis caused by oxidative stress may be mediated by p53-induced Bak. GLIA 25:154–164, 1999. © 1999 Wiley-Liss, Inc.     

IMM-H004, a Novel Coumarin Derivative Compound,Inhibits H2O2-Induced Neurotoxicity via Antioxidant and Antiapoptosis in PC12 Cells

IMM-H004 [7-hydroxy-5-methoxy-4-methyl-3-(4-methylpiperazin-1-yl)-coumarin] is a novel derivative of coumarin, which played neuroprotective roles in brain ischemia in rats in previous studies. Although antiapoptosis and improving synapsis structure were proved, the effects and mechanisms of IMM-H004 in brain ischemia need further study. In this paper, the effect of IMM-H004 on H₂O₂-induced neurotoxicity in pheochromocytoma (PC12) cells was researched. Morphological observation, MTT method and PI/Hoechst staining were used to indicate cell viability and apoptosis. JC-1 and DCFH-DA were used to test mitochondrial membrane potential (MMP) and reactive oxygen species (ROS), respectively. The antioxidative activity was detected by Glutathione (GSH) and Total Antioxidant Capacity (TAC) Assay kits. Western blot was used to test apoptosis related proteins. Our results showed that treatment with 1-10 μM IMM-H004 markedly increased cell viability and decreased cell apoptosis induced by H₂O₂. Moreover, 1-10 μM IMM-H004 could enhance MMP and protect mitochondrial function. 1-10 μM IMM-H004 also could lower the ROS and raise the GSH and TAC level. Furthermore, 1-10 μM IMM-H004 could decrease the ratio of Bax/Bcl-2 and increase the ratio of p-AKT/AKT, which were related to apoptosis and survival. All these indicated that IMM-H004 protects PC12 cells against H₂O₂-induced neurotoxicity. Antioxidative and antiapoptosis may be the mechanisms of IMM-H004 in brain ischemia. These studies indicate that IMM-H004 might be a potential drug for treatment brain ischemia.     

Pretreatment of MQA,a caffeoylquinic acid derivative compound,protects against H2O2-induced oxidative stress in SH-SY5Y cells

Objectives: Compound MQA (1,5-O-dicaffeoyl-3-O-[4-malic acid methyl ester]-quinic acid) is a natural caffeoylquinic acid derivative isolated from Arctium lappa L. roots. This study aims to explore the neuroprotective effects of MQA against hydrogen peroxide (H₂O₂)-induced oxidative stress in SH-SY5Y neuroblastoma cells.

Methods: The SH-SY5Y cells were divided into four groups, including control, 20 μM MQA, 200 μM H2O2, 200 μM H2O2 + 20 μM MQA groups. The effects of MQA on H₂O₂-induced cell death were measured by MTT and LDH assays. Hoechst 33342 and Annexin V-PI double staining were used to observed H2O2-induced apoptosis. Also, the effects of MQA on antioxidant system and mitochondrial pathway were explored. Further, steady-state phosphorylation levels of ERK1/2, Akt and GSK-3β were examined by Western blot analysis.

Results: Pretreatment with MQA prevented cell death in SH-SY5Y cells exposed to 200 μM H2O2 for 3 h. Meanwhile, Hoechst 33342 and Annexin V-PI double staining showed that MQA attenuated H₂O₂-induced apoptosis. These changes are related to elevation in SOD activity, reduction in MDA production and ROS formation, and increases in mitochondrial membrane potential (MMP). In addition, the potential mechanisms of MQA against H₂O₂-induced apoptosis are associated with increases in the Bcl-2/Bax ratio, decreases in cytochrome c release, caspase-3 and caspase-9 expressions, phosphorylation of ERK1/2, and dephosphorylation of AKT and GSK-3β.

Conclusion: These findings suggest that protective effects of MQA against H₂O₂-induced apoptosis might be associated with mitochondrial apoptosis, ERK1/2 and AKT/GSK-3β pathway.     

Cyanidin suppresses amyloid beta-induced neurotoxicity by inhibiting reactive oxygen species-mediated DNA damage and apoptosis in PC12 cells

Amyloid beta(Aβ)-induced oxidative stress is a major pathologic hallmark of Alzheimer's disease. Cyanidin, a natural flavonoid compound, is neuroprotective against oxidative damage-mediated degeneration. However, its molecular mechanism remains unclear. Here, we investigated the effects of cyanidin pretreatment against Aβ-induced neurotoxicity in PC12 cells, and explored the underlying mechanisms. Cyanidin pretreatment significantly attenuated Aβ-induced cell mortality and morphological changes in PC12 cells. Mechanistically, cyanidin effectively blocked apoptosis induced by Aβ, by restoring the mitochondrial membrane potential via upregulation of Bcl-2 protein expression. Moreover, cyanidin markedly protected PC12 cells from Aβ-induced DNA damage by blocking reactive oxide species and superoxide accumulation. These results provide evidence that cyanidin suppresses Aβ-induced cytotoxicity, by preventing oxidative damage mediated by reactive oxide species, which in turn inhibits mitochondrial apoptosis. Our study demonstrates the therapeutic potential of cyanidin in the prevention of oxidative stress-mediated Aβ neurotoxicity.     

Estradiol protects PC12 cells against CoCl2-induced apoptosis

In hypoxic/ischemic conditions, neuronal apoptotic events are occurred, resulting in neuronal diseases. Estradiol is a female sex hormone with steroid structure known to provide neuroprotection through multiple mechanisms in the central nervous system. This study was aimed to investigate the signal transduction pathway leading to the inhibitory effects of estradiol against cobalt chloride (CoCl₂)-mediated hypoxic death in PC12 cells. Estradiol inhibits CoCl₂-induced cell death with genomic DNA fragmentation and morphologic changes such as cell shrinkage and condensed nuclei. Pre-incubation of estradiol prior to CoCl₂ treatment attenuated CoCl₂-mediated the reactive oxygen species (ROS) production and limited the activities of the caspase cascades, such as caspase-8, -9 and -3. Furthermore, estradiol downregulated the Bax:Bcl-2 ratio and decreased the release of cytochrome c from the mitochondria into the cytosol in CoCl₂-treated cells, indicating that estradiol affect on mitochondrial pathway. Estradiol attenuated also CoCl₂-induced upregulation of Fas-ligand (Fas-L) and truncated of Bid in sequence of death receptor-mediated pathway. In addition, estradiol increased the phosphorylation of Akt in CoCl₂-treated cells, demonstrating that estradiol has no affect on upstream signaling through the PI3K/Akt in inhibition of CoCl₂-induced apoptosis in PC12 cells.Taken together, estradiol was found to have a neuroprotective effect against CoCl₂-induced apoptosis of PC12 cells by the attenuating ROS production and the modulating apoptotic signal pathway through Bcl-2 family, cytochrome c, Fas/Fas-L as well as PI3K/Akt pathway.     

Conditioned Media of Choroid Plexus Epithelial Cells Induces Nrf2-Activated Phase II Antioxidant Response Proteins and Suppresses Oxidative Stress-Induced Apoptosis in PC12 Cells

Based on the critical role of the choroid plexus (CP) in detoxification processes in the central nervous system (CNS), herein we investigated the effect of choroid plexus epithelial cells conditioned media (CPECs-CM) under oxidative conditions. CPECs were isolated from rat brains, cultured, and the conditioned media were collected. Then pheochromocytoma neuron-like cells (PC12) were treated simultaneously with CPECs-CM and H₂O₂ as the oxidative stressor. Next, the effect of CPECs-CM on neurite outgrowth and cell differentiation in the presence of H₂O₂ was determined. Our results showed that CPECs-CM improved the expansion of neurites and differentiation in PC12 cells under oxidative stress conditions. Changes in apoptotic factors, nuclear factor erythroid 2-related factor 2 (Nrf2) and γ-glutamylcysteine synthetase as the highlighted pathway in the antioxidant defense system were determined by western blot. Also, the activity of antioxidant enzymes and lipid peroxidation level were determined. CPECs-CM-treated PC12 cells could survive after exposure to H₂O₂ by reduction of caspase-3 cleavage and Bax level and elevation of anti-apoptotic factor Bcl2. Our data also revealed that Nrf2 activation, and consequently its downstream protein levels, increased in the presence of CPECs-CM. Based on our data, we can conclude that CPECs-CM protects PC12 cells against oxidative stress and apoptosis. It seems that CPECs secrete antioxidative agents and neurotrophic factors that have a role in the health of the CNS.     

Polysaccharides from Angelica sinensis alleviate neuronal cell injury caused by oxidative stress

Angelica sinensis has antioxidative and neuroprotective effects. In the present study, we aimed to determine the neuroprotective effect of polysaccharides isolated from Angelica sinensis. In a pre-liminary experiment, Angelica sinensis polysaccharides not only protected PC12 neuronal cells from H202-induced cytotoxicity, but also reduced apoptosis and intracellular reactive oxygen species levels, and increased the mitochondrial membrane potential induced by H202 treatment. In a rat model of local cerebral ischemia, we further demonstrated that Angelica sinensis poly-saccharides enhanced the antioxidant activity in cerebral cortical neurons, increased the number of microvessels, and improved blood flow after ischemia. Our findings highlight the protective role of polysaccharides isolated from Angelica sinensis against nerve cell injury and impairment caused by oxidative stress.     

Protective effect of BAG5 on MPP+-induced apoptosis in PC12 cells

Abstract

Objectives: Parkinson’s disease (PD) is the most common neurodegenerative disease in humans, and an abundance of evidence has implicated apoptosis signaling pathways in the neurodegeneration of PD. The purpose of this study was to assess the role of B-cell lymphoma 2 (Bcl-2)-associated athanogene 5 (BAG5) protein, which was previously confirmed to play an important role in the pathogenesis of PD, in the regulation of apoptosis induced by 1-methyl-4-phenyl-pyridinium (MPP⁺) in PC12 cells.

Methods: PC12 cells were treated with MPP⁺ for 48 hours to induce apoptosis, and activation of Bcl-2, Bcl-xl, and caspase 3 was measured by western blot.

Results: The upregulation of BAG5 in PC12 cells inhibited apoptosis and increased the expression of anti-apoptotic proteins, including Bcl-2 and Bcl-xl, after MPP⁺ treatment. In addition, downregulation of BAG5 in PC12 cells enhanced apoptosis and decreased the expression of these proteins after MPP⁺ treatment.

Discussion: The data suggest that BAG5 inhibits MPP⁺-induced apoptosis through both endogenous and mitochondria-mediated pathways of apoptosis. Through this mechanism, the upregulation of BAG5 levels may occur through its anti-apoptotic activity in PD.     

Erythropoietin attenuates 6-hydroxydopamine-induced apoptosis via glycogen synthase kinase 3β-mediated mitochondrial translocation of Bax in PC12 cells

The aim of this study was to determine the mechanism by which erythropoietin (EPO) suppressed 6-hydroxydopamine (6-OHDA)-induced apoptosis. Our results showed that 6-OHDA remarkably decreased phosphorylation of glycogen synthase kinase 3β (GSK3β) as well as enhanced the level of Bax in the mitochondria. Besides, 6-OHDA decreased the mitochondrial expression of Bcl-2 without altering the cytoplasmic expression of Bcl-2. In line with these results, 6-OHDA treatment enhanced the apoptosis and caspase 3 activity in PC12 cells. These findings indicated that mitochondrial dysfunction was involved in the neurotoxicity of 6-OHDA and GSK3β might act upstream of Bax/Bcl-2 and the caspase 3 pathways in 6-OHDA-treated PC12 cells. Furthermore, EPO reduced 6-OHDA-induced growth inhibition. Western blot exhibited that GSK3β inhibitor 4-benzyl-2-methyl-1, 2,4-thiadiazolidine-3, 5-dione (TDZD8) and EPO not only increased the phosphorylation of GSK3β but also inhibited the mitochondrial translocation of Bax. In agreement with these results, EPO and TDZD8 obviously increased the mitochondrial expression of Bcl-2. Finally, TDZD-8 and EPO significantly suppressed the enhanced apoptosis and activity of caspase 3 induced by 6-OHDA. Taken together, GSK3β-mediated mitochondrial cell death pathway is involved in the neuroprotective effect of EPO against 6-OHDA-induced apoptosis.     

Roles of Ras-Erk in apoptosis of PC12 cells induced by trophic factor withdrawal or oxidative stress

To understand the role of Ras-MAPK (mitogen-activated protein kinase) in trophic factor withdrawal- and oxidative stress-induced apoptotic cell death processes, undifferentiated rat pheochromocytoma PC12 cells and a PC12 variant cell line stably expressing the Ras dominant-negative mutant (M-M17-26) were subjected to serum withdrawal in the absence or presence of H₂O₂ treatment. The extent of cell death was analyzed by lactate dehydrogenase release, internucleosomal DNA fragmentation, and caspase-3 assays. Both serum with-drawal and H₂O₂ treatment induced apoptotic cell death in PC12 cells, and the extent of cell death was greatly enhanced in M-M17-26 cells. DNA fragmentation induced by serum withdrawal or H₂O₂ treatment was blocked completely by a general caspase inhibitor, Z-VAD-FMK. A selective MAPK kinase inhibitor, U0126, blocked the H₂O₂-induced phosphorylation of Erk1/2 (extracellular signal-regulated kinase) in PC12 cells and increased the levels of active caspase-3 in M-M17-26 under serum withdrawal or H₂O₂ treatment. In addition, the short-term H₂O₂ treatment (5–30 min) was sufficient to cause DNA fragmentation in M-M17-26 cells even though H₂O₂ was removed and cells were incubated in regular growth medium with complete serum for 24 h. However, similar, short-term H₂O₂ treatment of PC12 cells did not induce DNA fragmentation 24 h later. These results suggest that the Ras-Erk pathway is critical in mediating protection against apoptotic cell death induced by either trophic factor withdrawal or increased oxidative stress.