CoCl2-induced Apoptosis Research Articles

Protective effects and mechanism of hyperin on CoCl2-induced PC12 cells

To investigate the neuroprotective effects and mechanism of hyperin on CoCl2-induced hypoxic/ischemic PC12 cells. CoCl2 was used to treat rat pheochromocytoma cell line (PC12) to investigate the protective effects of different concentrations of hyperin. Hyperin could significantly inhibit CoCl2-induced cytotoxicity and apoptosis on PC12 cells in a dose-dependent manner, by inhibiting reactive oxygen species (ROS) production, increasing mitochondrial membrane potential (MMP), and inactivating caspase-3 and poly ADP-ribose polymerase (PARP). Hyperin could inhibit CoCl2-induced cytotoxicity and apoptosis on PC12 cells, show neuroprotective effects on hypoxic/ischemic neural injuries.

Ginsenoside Rb1 protects cardiomyocytes against CoCl2-induced apoptosis in neonatal rats by inhibiting mitochondria permeability transition pore opening

To investigate whether mitochondria permeability transition pore (mPTP) opening was involved in ginsenoside Rb1 (Gs-Rb1) induced anti-hypoxia effects in neonatal rat cardiomyocytes ex vivo. Cardiomyocytes were randomly divided into 7 groups: control group, hypoxia group (500 micromol/L CoCl(2)), Gs-Rb1 200 micromol/L group (CoCl(2) intervention+Gs-Rb1), wortmannin (PI3K inhibitor) 0.5 micromol/L group, wortmannin+Gs-Rb1 group, adenine 9-beta-D-arabinofuranoside (Ara A, AMPK inhibitor) 500 micromol/L group, and Ara A and Gs-Rb1 group. Apoptosis rate was determined by using flow cytometry. The opening of the transient mPTP was assessed by using co-loading with calcein AM and CoCl(2) in high conductance mode. Expression of GSK-3beta, cytochrome c, caspase-3 and poly (ADP-ribose) polymerase (PARP) was measured by using Western blotting. DeltaGSK-3beta was defined as the ratio of p-Ser9-GSK-3beta to total GSK-3beta. CoCl(2) significantly stimulated mPTP opening and up-regulated the level of DeltaGSK-3beta. There was a statistically significant positive correlation between apoptosis rate and mPTP opening, between apoptosis rate and DeltaGSK-3beta, and between mPTP opening and DeltaGSK-3beta. Gs-Rb1 significantly inhibited mPTP opening induced by hypoxia (41.3%+/-2.0%, P<0.001) . Gs-Rb1 caused a 77.3%+/-3.2% reduction in the expression of GSK-3beta protein (P<0.001) and a significant increase of 1.182+/-0.007-fold (P=0.0001) in p-Ser9-GSK-3beta compared with control group. Wortmannin and Ara A significantly inhibited the effect of Gs-Rb1 on mPTP opening and DeltaGSK-3beta. Gs-Rb1 significantly decreased expression of cytochrome c (66.1%+/-1.7%, P=0.001), caspase-3 (56.5%+/-2.7%, P=0.001) and cleaved poly ADP-ribose polymerase (PARP) (57.9%+/-1.4%, P=0.001). Gs-Rb1 exerted anti-hypoxia effect on neonatal rat cardiomyocytes by inhibiting GSK-3beta-mediated mPTP opening.

Bone marrow derived stromal cells modified by adenovirus-mediated HIF-1α double mutant protect cardiac myocytes against CoCl 2-induced apoptosis

Bone marrow derived stromal cells (MSCs) can prevent the apoptosis of ischemic cardiomyocytes (CMCs). This anti-apoptosis activity may be related to an activation of the HIF-1α signal pathway in MSCs. Therefore, we investigated protective effects of an adenovirus (Ad)-mediated active form of HIF-1α (HIF-1α-Ala564-Ala803) modified MSCs on CMCs against CoCl 2-induced apoptosis. At normoxia, pAd-HIF1α-Ala564-Ala803 exhibited a stable HIF-1α protein expression in MSCs. Compared with the single CMC culture, the TGF-β1 level and the Bcl-2 expression were significantly increased, concomitant with a reduced expression of caspase-3, the LDH release and TUNEL-positive CMCs in CMC and MSC, β-galactosidase (LacZ)-MSC or HIF-1α-Ala564-Ala803-MSC coculture exposed to CoCl 2. Furthermore, these effects were more prominent in CMC and HIF-1α-Ala564-Ala803-MSC coculture than in CMC and MSC or LacZ-MSC coculture exposed to CoCl 2. Pre-transfection of TGF-β1-small interfering RNA (siRNA) effectively inhibited the TGF-β1 level, resulting in a dramatic reduction in the Bcl-2 expression as well as an increased level of apoptosis in CMC and HIF-1α-Ala564-Ala803-MSC coculture exposure to CoCl 2, whereas pre-transfection of green fluorescent protein (GFP)-siRNA had no such effects. These data suggest that HIF1α-Ala564-Ala803 modified MSCs have better protective effects of CMCs against the CoCl 2-induced apoptosis and these protective effects are at least partly TGF-β1-mediated.

Anti-hypoxic effect of ginsenoside Rbl on neonatal rat cardiomyocytes is mediated through the specific activation of glucose transporter-4 ex vivo

The aim of this study was to investigate whether Gs-Rbl relieves the CoCl(2)-induced apoptosis of hypoxic neonatal rat cardiomyocytes and in which the role of glucose transporter-4 (GLUT-4). Gs-Rbl (0, 10, 50, 100, 200, 400, and 500 micromol/L), adenine 9-beta-D-arabinofuranoside (ara A, 500 micromol/L; AMPK inhibitor) and wortmannin (0.5 micromol/L; PI3K inhibitor) only in combination with 200 micromol/L Gs-Rbl were administered in hypoxic cardiomyocytes, which were induced by 500 micromol/L CoCl(2) for 12 h. Then, the apoptotic rate (AR), 2-[(3)H]-deoxy-D-glucose (2-[(3)H]-DG) uptake, and the expression of GLUT-4 (including in plasma membrane, PM), phospho-AMPKalpha (Thr172), AMPKalpha and Akt in cells were assayed. Compared with simple hypoxia (0 micromol/L Gs-Rbl), Gs-Rb1 greater than 10 micromol/L significantly decreased the apoptotic rate (P<0.01) and significantly increased 2-[(3)H]-DG uptake (P<0.01), GLUT-4 content in cells and PM (P<0.01), AMPK activity (P<0.01) and Akt (P<0.01) levels in a dose-dependent manner. AMPK activity was completely suppressed by ara-A, just as Akt was suppressed by wortmannin. The AR, glucose uptake and GLUT-4 levels in cells and PM were partly down-regulated by ara-A or wortmannin. Gs-Rb1 may protect neonatal rat cardiomyocytes from apoptosis induced by CoCl(2). The anti-apoptotic effect of Gs-Rb1 may occur by improving glucose uptake, in which GLUT-4 translocation and expression played a key role. Both the AMPK and the PI3K/Akt pathways may take part in the anti-hypoxic efficacy of Gs-Rb1.

Downregulation of Telomerase in CoCl2-Induced Apoptosis of Myeloid Leukemic Cells

Many reports showed that hypoxia-inducible factor-1 (HIF-1 ), the main factor activated by hypoxia, plays a role in the transcriptional regulation of the human telomerase reverse transcriptase (hTERT), one of the critical elements of the oncogenic process. As a hypoxia-mimetic agent, cobalt chloride (CoCl2) can induce the accumulation of HIF-1 . Herein, we demonstrated that hTERT expression was greatly decreased during CoCl2-induced apoptosis in leukemic cell lines while overexpression of hTERT inhibited CoCl2-induced apoptosis. Knockdown of HIF-1 by shRNA in U937 cells did not abrogate CoCl2-induced hTERT downregulation nor CoCl2-induced apoptosis while inducible expression of HIF- 1 decreased the expression of hTERT. Furthermore, CoCl2 could decrease the c-myc protein in all the cells tested, no matter the HIF-1 was silenced or not. Taken together, we demonstrated that downregulation of hTERT contributes to CoCl2-induced apoptosis in leukemic cell lines and HIF-1 is not indispensable for CoCl2 induced downregulation of hTERT.

Estradiol protects PC12 cells against CoCl 2-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 (CoCl 2)-mediated hypoxic death in PC12 cells. Estradiol inhibits CoCl 2-induced cell death with genomic DNA fragmentation and morphologic changes such as cell shrinkage and condensed nuclei. Pre-incubation of estradiol prior to CoCl 2 treatment attenuated CoCl 2-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 CoCl 2-treated cells, indicating that estradiol affect on mitochondrial pathway. Estradiol attenuated also CoCl 2-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 CoCl 2-treated cells, demonstrating that estradiol has no affect on upstream signaling through the PI3K/Akt in inhibition of CoCl 2-induced apoptosis in PC12 cells. Taken together, estradiol was found to have a neuroprotective effect against CoCl 2-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.

Inhibition by epigallocatechin gallate of CoCl 2-induced apoptosis in rat PC12 cells

Epigallocatechin-3-gallate (EGCG) is a major constituent of green tea polyphenols. This study was aimed to investigate the possible mechanisms of EGCG-mediated inhibition against apoptosis in rat pheochromocytoma PC12 cells by exposure to CoCl 2. Exposure to CoCl 2 caused the generation of ROS and induced cell death with appearance of apoptotic morphology and DNA fragmentation. However, EGCG rescued the loss of viability in the cells exposed to CoCl 2 and led the reduction of DNA fragmentation and sub-G 1 fraction of cell cycle. Also, EGCG attenuated the CoCl 2-induced disruption of mitochondrial membrane potential (ΔΨm), release of cytochrome c from the mitochondria to cytosol and abolished the CoCl 2-stimulated activities of the caspase cascades, caspase-9 and caspase-3. In addition, EGCG ameliorated the increase in the Bax to Bcl-2 ratio, a marker of apoptosis proceeding, induced by CoCl 2 treatment. Taken together, the present results suggest that EGCG inhibit the CoCl 2-induced apoptosis of PC12 cells through the mitochondria-mediated apoptosis pathway involved in modulating the Bcl-2 family.

Bcl-2 Family and Caspases are Involved in CoCl2-Induced Apoptosis of PC12 Cells

Hypoxic/ischemic condition induces the neuronal apoptotic events, consequently resulting in neuronal damages. Cobalt chloride (CoCl₂) could mimic the hypoxic condition including the production of reactive oxygen species (ROS). This study aimed to investigate the roles of Bcl-2 family and caspases as central regulators of apoptosis, in CoCl₂-induced apoptosis of PC12 cells. Cell viability was determined by MTT assay and DNA fragmentation was detected by DNA laddering. The expression levels of Bcl-2, Bax, Bid, cytochrome c and Fas/APO-1 were determined by RT-PCR or Western blotting analysis in CoCl₂-treated PC12 cells. Caspase-9 and caspase-3 activities were assessed using spectrophotometry and caspase-8 activity was measured with fluorospectrocytometry. Administration of CoCl₂ decreased viability of cells in a dose- and time-dependent manner. Furthermore, fragmentation of the genomic DNA and apoptotic bodies were induced in CoCl₂-treated PC12 cells. Bcl-2, an anti-apoptotic Bcl-2 family, was downregulated, whereas Bax, pro-apoptotic molecule, was upregulated in CoCl₂-treated cells. Treatment of CoCl₂ augumented the release of cytochrome c into the cytoplasm and increase of caspase-8, -9, and -3 activities. In addition, CoCl₂ upregulated Fas and downregulated pro-Bid, which are known to be correlated with death receptor-mediated apoptotic signaling pathway. Therefore, these results suggest that Bcl-2 family and caspase play crucial roles in CoCl₂-induced apoptosis through mitochondria- and death receptor-dependent pathways in PC12 cells.

Involvement of mitochondrial- and Fas-mediated dual mechanism in CoCl 2-induced apoptosis of rat PC12 cells

Hypoxic/ischemic condition induces neuronal apoptotic events, which consequently lead to neuronal cell death. However, its specific mechanistic pathways remain obscure. Cobalt chloride (CoCl 2) could mimic the hypoxic condition including the production of reactive oxygen species (ROS). In this report, we investigated the signal pathway of CoCl 2-induced apoptosis in PC12 cells. The main mechanism for these apoptosis appeared to be mitochondria-mediated pathway accompanied with loss of the mitochondrial transmembrane potential (ΔΨm) followed by cytochrome c release from the mitochondria into the cytosol, resulting in the activation of caspase-9 and caspase-3. Also, upregulation of pro-apoptotic protein Bax, and downregulation of anti-apoptotic protein Bcl-2 by presence of CoCl 2 appeared significantly and it might result in activating mitochondria-mediated apoptosis. We showed that expression of Fas and Fas ligand was upregulated and caspase-8 was significantly activated in CoCl 2-induced apoptotic cells. In addition, ZB4, an antagonistic Fas-antibody, inhibited the activation of caspase-8 by CoCl 2, indicating that Fas receptor was involved in this pathway. These results demonstrate that CoCl 2 induce apoptosis in PC12 cells via different dual apoptosis pathway through death receptor as well as mitochondria.

Involvement of caspase-3 and p38 mitogen-activated protein kinase in cobalt chloride-induced apoptosis in PC12 cells.

Our previous study showed that cobalt chloride (CoCl2) could induce PC12 cell apoptosis and that the CoCl2-treated PC12 cells may serve as a simple in vitro model for the study of the mechanism of hypoxia-linked neuronal disorders. The aim of this study is to elucidate the mechanism of CoCl2-induced apoptosis in PC12 cells. Caspases are known to be involved in the apoptosis induced by various stimuli in many cell types. To investigate the involvement of caspases in CoCl2-induced apoptosis in PC12 cells, we generated PC12 cells that stably express the viral caspases inhibitor gene p35 and analyzed the effect of p35 on the process of apoptosis induced by CoCl2. We also examined the effect of cell-permeable peptide inhibitors of caspases. The results showed that the baculovirus p35 gene and the general caspases inhibitor Z-VAD-FMK significantly block apoptosis induced by CoCl2, confirming that caspase is involved in CoCl2-induced apoptosis. Further investigation showed that in this process the caspase-3-like activity is increased, as indicated by the cells' ability to cleave the fluorogenic peptide substrate Ac-Asp-Glu-Val-Asp-7-AMC and to degrade the DNA-repairing enzyme poly-(ADP-ribose) polymerase (PARP), an endogenous caspase-3 substrate. At the same time, caspase-3-specific inhibitors, namely, the peptide Ac-DEVD-CHO, Ac-DEVD-FMK, partially inhibit CoCl2-induced apoptosis. These findings suggested that caspase-3 or caspase-3-like proteases are involved in the apoptosis induced by CoCl2 in PC12 cells. Additionally, we have observed that another apoptotic marker, p38 mitogen-activated protein kinase (MAPK), is significantly activated in this process in a time-dependent manner and that a selective p38 MAPK inhibitor, SB203580, partially inhibits this cell death. The addition of SB203580 also partially suppresses caspase-3-like activity. All these results confirm that the CoCl2-treated PC12 cell is a useful in vitro model with which to study hypoxia-linked neuronal disorders. Furthermore, the results showing that the baculovirus p35 gene and caspase inhibitors possess a remarkable ability to rescue PC12 cells from CoCl2-induced cell death may have implications for future neuroprotective therapeutic approaches for the hypoxia-associated disorders.