CoCl2 simulated hypoxia induce cell proliferation and alter the expression pattern of hypoxia associated genes involved in angiogenesis and apoptosis

BACKGROUND/AIMS: Hypoxia microenvironment plays a crucial role during tumor progression and it tends to exhibit poor prognosis and make resistant to various conventional therapies. HIF-1α acts as an important transcriptional regulator directly or indirectly associated with genes involved in cell proliferation, angiogenesis, apoptosis and energy metabolism during tumor progression in hypoxic microenvironment. This study was aimed to investigate the expression pattern of the hypoxia associated genes and their association during breast cancer progression under hypoxic microenvironment in breast cancer cells. METHODS: Cell proliferation in MCF-7 and MDA-MB-231 cell lines treated with different concentration of CoCl2 was analyzed by MTT assay. Flow cytometry was performed to check cell cycle distribution, whereas cell morphology was examined by phase contrast microscopy in both the cells during hypoxia induction. Expression of hypoxia associated genes HIF-1α, VEGF, p53 and BAX were determined by semiquantitative RT-PCR and real-time PCR. Western blotting was performed to detect the expression at protein level. RESULTS: Our study revealed that cell proliferation in CoCl2 treated breast cancer cells were concentration dependent and varies with different cell types, further increase in CoCl2 concentration leads to apoptotic cell death. Further, accumulation of p53 protein in response to hypoxia as compare to normoxia showed that induction of p53 in breast cancer cells is HIF-1α dependent. HIF-1α dependent BAX expression during hypoxia revealed that after certain extent of hypoxia induction, over expression of BAX conquers the effect of anti-apoptotic proteins and ultimately leads to apoptosis in breast cancer cells. CONCLUSION: In conclusion our results clearly indicate that CoCl2 simulated hypoxia induce the accumulation of HIF-1α protein and alter the expression of hypoxia associated genes involved in angiogenesis and apoptosis.

The Mechanisms of Cobaltous Chloride-Induced Hypoxia on Muscle Atrophy / 中国运动医学杂志

Objective To study the regulating mechanisms of cobaltous chloride (CoCl2)induced hypoxia on muscle atrophy.Methods The mouse skeletal C2C12 myotube line was used as the cell model and divided into a control group,a CoCl2 group,a 3-Methyladenine(3MA)group and a CoCl2+3MA group.The control group was not given any treatment,the CoCl2 group was treated with 200 μM CoCl2 to induce hypoxia,and the 3MA group or CoCl2+3MA group was treated with 5 mM 3MA in the absence or presence of 200 μM CoCl2.The Giemsa stain was conducted to observe the morphology of myotubes.The multifunctional microplate reader was utilized to quantify the reactive oxygen species (ROS)expression.The autophagosome was observed by using the transmission electron microscopy.The mRNA and protein expression of hypoxia-inducible factor-1α(HIF-1α),Bcl2/adenovirus E1B19kDa interacting protein 3(BNIP3),microtubule associated protein1 light chain 3(LC3)were detected using the quantitative real time polymerase chain reaction (QRT-PCR)and Western blotting (WB).Moreover,3-Methyladenine(3MA)was used to suppress autophagy and the expression of muscle atrophy F-box(MAF-bx)was detected.Results More spindly ring-shaped myotubes were formed in the control group,while CoCl2 induced myotubes atrophy and rupture.The expression of ROS increased significantly in the CoCl2 group compared with the control group(t=-4.965,P=0.008).Transmission electron microscopy results showed autophagosome formation induced by CoCl2,and significant improvement in the HIF-1α,BNIP3 and LC3 in the CoCl2 group compared with the control group(P＜0.05).The protein expression of MAFbx decreased when co-cultured with CoCl2 and 3MA(F=18.246,P=0.001).Conclusions The skeletal muscle atrophy caused by CoCl2 induced hypoxia may be associated with autophagy promoted by the HIF-1α/BNIP3 signal pathway,and inhibition of hypoxia induced autophagy can partly reduce the atrophy in skeletal C2C12 myotubes.

Effect of hypoxia and PDGF-BB siRNA transfected on the production of PDGF-BB and proliferation in cultured human RPE cells / 中华实验眼科杂志

Background Proliferative vitreoretinopathy (PVR):no blood vessels,fibrous membrane cell proliferation occurs retinal surface.The development of specific mechanisms has not been completely clarified,but the retinal pigment epithelium(RPE) and platelet-derived growth factor(PDGF) for the past few years are the research hotspot.Objective To explore the effects of hypoxia on the production of PDGF-BB and proliferation in cultured human RPE (hRPE) cells.Methods In the experimental group,10,15,20,30,40 μmol/L CoCl2 were used to mimic hypoxia environment of hRPE cells,the control group did not add CoCl2 on cultured hRPE cells.By using reverse transcriptive PCR (RT-PCR) and enzyme linked immunosorbent assay(ELISA),the expression of PDGF-BB mRNA and protein was detected,and detected proliferation of hRPE cells by MTT.The cells were divided into 6 groups,different siRNAs were transfected into PDGF-BB siRNA1 group,PDGF-BB siRNA2 group,PDGF-BB siRNA3 group (because PDGF-BB has three different siRNAs,one of which is a valid siRNA),β-actin siRNA group,unrelated sequence siRNA group,only Lip2000 was plused in the blank control group.After transfection of 4-6 hours,except for blank control group,the other five groups were added to PDGF-BB mRNA and protein and its effect on the proliferation of hRPE cells obviously CoCl2 concentrations (15 μmol/L) simulated hypoxia environment of 24 hours,the detection of PDGF-BB mRNA and protein and hRPE cell proliferation rate.Results Neither PDGF-BB mRNA or protein was found in the blank control group.The production of PDGF-BB mRNA and protein in hRPE cells cultured by different concentrations were different,with significant differences among them (F=43.737,P＜0.01;F=54.612,P＜0.05),and the expressions of PDGF-BB mRNA and protein of 15 μmol/L CoCl2 group were more than other groups,with significant differences between the two groups (all at P＜0.05).MTT showed that PDGF-BB protein and hRPE cell proliferation rate in different concentions of CoCl2 groups were different,with significant differences among them(F=95.379,P＜0.01 ; F =63.375,P＜0.05),the expression of PDGF-BB protein was more and hRPE cell proliferation rate was higher in 15 μmol/L CoCl2group than other groups,with significant differences between the two groups (all at P＜0.05),postive correlation was found between PDGF-BB protein and hRPE cell proliferation rate (r =0.994,P＜0.05).The production of PDGF-BB mRNA and protein in the different siRNA transfected groups were different,with significant differences among them (F =156.330,125.650,both at P＜0.01),and the expressions of PDGF-BB mRNA and protein of PDGF-BB siRNA2 group were more than other groups,with significant differences between them(all at P＜0.05).MTT showed that PDGF-BB protein and hRPE cell proliferation rate in different siRNA transfectedgroups were different,with significant differences among them (F =73.131,98.564,both at P＜ 0.01).The expression of PDGF-BB protein was less and hRPE cell proliferation rate was lower in PDGF-BB siRNA2 group than other groups,with significant differences between them (all at P ＜ 0.05),postive correlation was found between PDGF-BB protein and hRPE cell proliferation rate (r =0.996,P＜0.05).Conclusions Proper hypoxia can induce the expression of PDGF-BB,PDGF-BB expression can significantly promote the proliferation of hRPE cells.In the transfection targeting PDGF-BB siRNA,PDGF-BB expression is suppressed,can effectively reduce the value of hRPE cell hyperplasia.

Effect of BNIP3-induced autophagy in gastric carcinoma / 中国药理学通报

Aim To study the effect of BNIP3 on hy-poxia-induced autophagic cell death in gastric carcino-ma.Methods The protein levels of BNIP3 and LC3-Ⅱ of 1 8 cases of gastric carcinoma were studied with immunocytochemistry and Western blot analysis be-tween normal and tumor tissues.The expression of LC3-Ⅱ in SGC-7901 cells was assessed while knock-down of BNIP3 by siRNA in CoCl2-induced hypoxia, and the effect of 3-MA was also studied.Cell viability in hypoxia treatment for 48 h was estimated with cell counting kit-8 (CCK8).Results Compared with nor-mal tissues,the protein levels of BNIP3 and LC3-Ⅱwere increased in tumor tissues.BNIP3 siRNA could decrease the expression of LC3-Ⅱ and enhance the cell viability,while inhibition of autophagy could also in-crease the cell viability in SGC-7901 cells.Conclu-sions BNIP3 may play a role in hypoxia-induced cell death in gastric carcinoma,and the possible mecha-nism may be related to the regulation of autophagy.

Cobalt Chloride-induced Hypoxia Ameliorates NLRP3-Mediated Caspase-1 Activation in Mixed Glial Cultures

Hypoxia has been shown to promote inflammation, including the release of proinflammatory cytokines, but it is poorly investigated how hypoxia directly affects inflammasome signaling pathways. To explore whether hypoxic stress modulates inflammasome activity, we examined the effect of cobalt chloride (CoCl2)-induced hypoxia on caspase-1 activation in primary mixed glial cultures of the neonatal mouse brain. Unexpectedly, hypoxia induced by oxygen-glucose deprivation or CoCl2 treatment failed to activate caspase-1 in microglial BV-2 cells and primary mixed glial cultures. Of particular interest, CoCl2-induced hypoxic condition considerably inhibited NLRP3-dependent caspase-1 activation in mixed glial cells, but not in bone marrow-derived macrophages. CoCl2-mediated inhibition of NLRP3 inflammasome activity was also observed in the isolated brain microglial cells, but CoCl2 did not affect poly dA:dT-triggered AIM2 inflammasome activity in mixed glial cells. Our results collectively demonstrate that CoCl2-induced hypoxia may negatively regulate NLRP3 inflammasome signaling in brain glial cells, but its physiological significance remains to be determined.

Correlation between hypoxia inducible factor -1α and renin expression in rats kidney induced by cobalt chloride.

Background: Cobalt chloride can be used as an agent to stabilize hypoxia inducible factor-1α (HIF-1α) and to imitate hypoxia without low levels of oxygen inside the body. We intended to investigate if there was any regulation of renin expression by HIF-1α. Therefore, we conducted several studies to clarify this possibility starting with the induction of hypoxic mimicry in rats by intra-peritoneal (IP) injection of cobalt chloride (CoCl2) to obtain the levels and pattern of HIF-1α and renin mRNA and protein expression. Methods: Twenty-four rats were randomly divided into four groups, control group and incubation groups 2, 8, and 24 hours after intra-peritoneal injection of 30 mg CoCl2 per kg BW. After the rats were sacrificed, kidneys were excised, weighed and kidney weight compared to BW. Tissue parameters were measured such as RNA concentration, HIF-1α protein by ELISA, and renin mRNA by RT-PCR. Results: Differences between the groups in the ratios of kidney weight to BW and in the concentrations of HIF-1α protein were statistically not significant (p > 0.05). Relative expression of renin mRNA increased markedly starting 8 hours after CoCl2 IP injection (30 times over controls) and further rising until 24 hours (2465 times over controls). Correlation between HIF-1α and renin mRNA by Pearson analysis was strongly positive, but not significant (R = 0.91; p = 0.09). Conclusion: Renin gene regulation in renal hypoxic mimicry strongly correlates with HIF-1α.

Effect of hypoxia on expressions of stromal cell-derived factor-1 and integrin-linked kinase in retinal pigment epithelium cell in vitro / 中华实验眼科杂志

Background Hypoxia is a crucial factor of neovascularization.Many researches found that stromal cell-derived factor-1 (SDF-1) and integrin-linked kinase (ILK) play an important role in the neovascular disease.However,effect of SDF-1 and ILK in eye neovascular disease is below understood.Objective The aim of this study was to investigate the effect of hypoxia on the expressions of SDF-1 and ILK in cultured retinal pigment epithelium(RPE) cells in vitro.Methods RPE tissue was isolated from 4-week-old C57BL/6 mouse and was digested and cultured in DMEM/F12 with 10％ fetal bovine serum (FBS).The cells with 80％ confluence were collected and passaged.The third generation of cells were identified with cytokeratin 18 (CK18) antibody by immunochemistry.The cells were inoculated at the density of 5×104 cells/ml to free-serum DMEM/F12 for 24 hours and then were cultured in regular medium in the normoxic control group.RPE cells were cultured for 1 hour and 3,6,12,24,48,72 hours with 200 μmol/L CoCl2 in the hypoxia group.Reverse transcription-PCR(RT-PCR) was used to evaluate the expressing change of SDF-1 mRNA and ILK mRNA in RPE cells,and Western blot was used to assay the expressing change of SDF-1 protein and ILK protein in RPE cells in different time points.The detected outcomes were represented as the ratio of target gene A value/β-actin A value.Results Cultured cells showed the polygon in shape with the black pigment granules in cytoplasm.Over 90％ cells were positive response for CK18.Expressions of the SDF-1 mRNA and ILK mRNA were increased in different time points after CoCl2 co-cultured(SDF-1 mRNA:F=281.875,P=0.000 ;ILK mRNA: F=187.566,P=0.000),with the highest expressing value in hypoxia at 12 hours.No significant change in the expression of SDF-1 mRNA and protein was found 1 hour after CoCl2 co-cultured,but expressions of SDF-1 mRNA and ILK mRNA were significantly higher in 3,6,12,24,48 and 72 hours than the normoxic control group(P＜0.01).The expressions of SDF-1 protein and ILK protein were gradually ascended with the time increase of CoCl2 co-culture,showing a significant difference among different time points(SDF-1: F=44.719,P =0.000 ; ILK: F =144.481,P =0.000),and the up-regulation of SDF-1 protein and ILK protein expression was seen mainly in 3,6,12,24,48 and 72 hours after CoCl2 co-cultured in comparison with the normoxic control group (P＜0.01).Conclusions SDF-1 and ILK are involved in the hypoxic response of RPE cells and may play a potential role in ischemic/hypoxic retinopathy.

Up-regulation of monocyte chemoattractant protein-1 in 3T3-L1 adipocytes by CoCl2 induced hypoxia / 中华内分泌代谢杂志

To investigate the effects of hypoxia chemically induced by CoCl2 on the expression of monocyte chemoattractant protein-1 ( MCP-1 ) in mouse 3T3-L1 adipocytes.3T3-L1 preadipocytes were cultured in vitro and differentiated into the matured adipocytes.Cell differentiation and lipid accumulation was determined by Oil Red O staining.CoCl2 was used as a chemical hypoxia-inducible reagent to mimic hypoxic microenvironment.The effect of CoCl2 on cell viability was estimated by MTT assay.Hypoxia-inducible factor-1α(HIF-1α) expression under hypoxia was detected by realtime fluorescent PCR and Western blot,while MCP-1 expression was detected by real-time fluorescent PCR and ELISA.CoCl2 induced hypoxia led to a marked recruitment of HIF-1α in mouse 3T3-L1adipocytes.Similarly,both mRNA and protein levels of MCP-1 were up-regulated.Exposure of 3T3-L1 adipocytes to CoCl2 induced hypoxic microenvironment in vitro,and hypoxic induction of MCP-1 expression and secretion may be mediated by HIF-1 α and may contribute to chronic low grade inflammation in adipose tissue.

Study on the expression change of HIF-1α and VEGF in protein levels and the correlation of them in QBC939 cell line under chemical hypoxia induced by CoCl2 / 肿瘤研究与临床

Objective To examine the expression change of HIF-1α and VEGF in protein levels and the corelation of them in Q8C939 cell line under chemical hypoxia induced by CoCl2 in vitro. Methods ELISA and immunohistochemical technique were used to examine the change of VEGF and HIF-1α protein level in QBC-939 line in different concentrations (200, 150, 100, 50, 0 μmol/L) of COCl2 and different periods (6 h, 12 h, 24 h, 48 h) in hypoxia. Results CoCl2 can up-regulate the expression of HIF-1α and VEGF at least partly in a dose and time dependent pattern. ELISA demonstrated that the level of VEGF protein in different concentrations and different hypoxia periods, the result showed that the VEGF protein was in different concentration groups over contrast group (P<0.05). The VEGF protein also increased continuously with the growing of hypoxia time (P<0.05). Immunohistochemical detection of concentrations of different groups, HIF-1α, VEGF protein in the average gray compared with the control group was different(P<0.05). Different time periods of hypoxia HIF-1α, VEGF protein in the average gray, any two group comparisons have difference (P<0.05). Different concentrations, the normal control group, different time period HIF-1α and VEGF protein expression of relevance were analyzed. The correlation coefficient was 0.830, P <0.01, 0.909, P <0.01. Conclusion In QBC939 cell line of hypoxia, there is up-regulation HIF-1α and VEGF.HIF-1α regulated the expression of VEGF.

The effect of cobalt chloride on adenovirus gene expression / 肿瘤

Objective: To investigate the effect of cobalt chloride (CoCl2) on transgene expression and viral particle titers in tumor cells infected by conditionally replicating adenovirus expression vector with hypoxia response elements(HRE)-regulated E1AE1B expression (Ad-5HRE-E1AE1B-RFP) and non-HRE regulated replication-deficient adenovirus expression vector (Ad-EGFP, Ad-Luc) in vitro and in vivo. Methods: Ad-5HRE-E1AE1B-RFP had five duplicated HRE and mini CMV acted as a promoter to drive E1AE1B expression and constitutive expression of RFP as reporter. The hypoxia model was optimized by exposing tumor cells to different concentrations of CoCl2. The hypoxia-inducible factor 1 alpha (HIF-1α) protein expression was determined by Western blotting. Under the optimized hypoxia model, the positive expression of exogenous gene and virus replication of Ad-5HRE-E1AE1B-RFP or Ad-EGFP-infected tumor cells were examined by conversed microscopic observation, FACS analysis and plaques formation test. Furthermore, transgene expression induced by combined application of hypoxia-inducible replicative adenovirus and replication deficient adenovirus (Ad-Luc) was also evaluated by examining the lucifererse activity in xenografted tumor models in nude mice by micro PET. Results: Western blotting results showed that CoCl2 at 0.4 and 0.08 μg/mL could stabilize and acumulate HIF-1α protein in gastric cancer SGC7901 cells, which could better mimic hypoxia condition. The microscopic observation and FACS analysis showed that CoCl2 at 0.4 μg/mL could remarkably increase the transduction efficacy of Ad-5HRE-E1AE1B-RFP, which was verified by significant increase in the percentage of positive expression of exogenous gene RFP and fluorescence intensity. But plaques formation test showed that Ad-5HRE-E1AE1B-RFP had no replication. CoCl2 0.4 μg/mL augmented the tranduction efficacy and expression levels of non-HRE regulated replication deficient adenovirus Ad-EGFP and Ad-Luc. Combined intratumoral injection of Ad-5HRE-E1AE1B-RFP and Ad-Luc significantly increased the expression of Ad-Luc in nude mice.Conclusion: CoCl2 markedly enhances transgene expression of recombinant adenovirus. However, the underlying mechanism is not only related to the CoCl2-induced hypoxia, but also probably related to regulation of gene transcription.

The Effect of Pretreatment of CoCl2 on the Prevention of Noise-Induced Hearing Loss / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: The study was designed to investigate the changes in the expression of hypoxia inducible factor-1alpha (HIF-1alpha) according to time after being exposed to noise trauma and find out the effect of HIF-1 alpha in the prevention of noiseinduced hearing loss by pre-treatment with cobalt chloride (CoCl2). SUBJECTS AND METHOD: BALB/c hybrid mice with 25 dB HL or less ABR were used in this study. In the study group, subjects were exposed to 120 dB SPL broad white band noise for 3 hours per day for 3 days. The changes in their hearing were documented before and after 1, 2, 3, 4, and 9 days of the first noise exposure. CoCl2 was injected into the peritoneum 2 hours prior to each noise exposure to see the effect of induced HIF-1alpha on noise-induced hearing loss. For the control, injection with distilled water was performed and hearing thresholds were measured in the same manner. Cochlea from each group was obtained in order to observe the morphological changes in the inner ear and the expression of the HIF-1alpha using immunohistochemial staining and immuno-fluorescein staining along with quantification of the hair cell loss. RESULTS: Mice exposed to the noise for 3 days, showed permanent threshold shift and the expression of HIF-1alpha was increased. When HIF-1alpha was induced by pre-treatment of CoCl2 prior to the noise exposure, however, hearing recovery was observed to some degree. And hair cell survival rate was also higher when treated with CoCl2 compared to the distilled water treated group. CONCLUSION: When pre-treated with CoCl2, inducing HIF-1alpha before the noise trauma, it allowed for a less stereocilia loss in the hair cells in the organ of Corti. HIF-1alpha may play an important role in the prevention of noise-induced hearing loss.

The impact of hypoxia-inducible transcription factor-1? on ovarian carcinoma cell / 中国癌症杂志

Background and purpose:HIF-1? (Hypoxia-inducible factor-1?) is an important transcription factor under hypoxia condition. It plays the role of dominating the expressions of correlative genes. It also promotes tumor deterioration, tumor metastasis and tumor invasion. The molecular role of HIF-1? has been intensively studied in cancer basic research. This article is to investigate the expression of HIF-1? under hypoxic and reoxygenation induced by CoCl_ 2 and the impact of hypoxia-inducible transcription factor-1? on human ovarian carcinoma cell line HO-8910PM. Methods:Semi quantitative reverse transcription-polymerase chain reaction (RT-PCR) is performed to detect the expression of HIF-1? mRNA in human ovarian carcinoma cell HO-8910PM exposed during the phase of hypoxia and reoxygenation. The relations of the quantity-efficiency and the time-efficiency were analyzed. The effects of HIF-1? gene on the proliferation, invasion and adhesion of HO-8910PM cell were estimated by either MTT, Boyden cell or cell adhesion tests.Results:There was endogenous expression of HIF-1? in human ovarian carcinoma cell line HO-8910PM. RT-PCR show that over-expression of HIF-1? mRNA could be measured under hypoxia induced by CoCl_ 2 (P

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

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.

Inhibition of Hypoxia-induced Apoptosis in PC12 Cells by Estradiol

Neuronal apoptotic events, which result in cell death, are occurred in hypoxic/ischemic conditions. 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 of CoCl2-induced neuronal cell death and the inhibitory effects of estradiol. Administration of CoCl2 decreased cell viability in both a dose- and time-dependent manner in PC12 cells. CoCl2-induced cell death produced genomic DNA fragmentation and morphologic changes such as cell shrinkage and condensed nuclei. It was found that CoCl2-treated cells increased the reactive oxygen species (ROS) as well as caspase-8, -9 and -3 activities. However, pretreatment with estradiol before exposure to CoCl2 prevented the reduction in cell viability reduction and attenuated DNA fragmentation and morphologic changes caused by CoCl2. Furthermore, the CoCl2-induced increases of ROS levels and caspases activities were attenuated by estradiol. Gene expression analysis revealed that estradiol blocked the underexpression of the Bcl-2 and ameliorated the increase in the release of cytochrome c from mitochondria into cytoplasm and Fas-ligand (Fas-L) upregulated by CoCl2. These results suggest that CoCl2 induce apoptosis in PC12 cells through both mitochondria- and death receptor-mediated cell death pathway. Estradiol was found to have a neuroprotective effect against CoCl2-induced apoptosis through the inhibition of ROS production and by modulating apoptotic effectors associated with the mitochondria- and death-dependent pathway in PC12 cells.