爪哇伪枝藻胞外多糖诱导皮肤癌细胞(A431)凋亡的研究

为探讨爪哇伪枝藻胞外多糖(Extracellular polymeric substances of Scytonema javanicum, EPS)诱导人表皮癌A431细胞凋亡及其对凋亡相关基因caspase-3、bcl-2和bax表达的影响,本实验利用MTT法检测细胞生长抑制情况;HE染色法及透射电镜进行形态学观察;单细胞凝胶电泳法(SCGE/彗星电泳)分析DNA受损情况;免疫组织化学法检测细胞内caspase-3、bcl-2和bax表达水平。结果显示EPS能显著抑制A431细胞增殖,并呈时间和剂量依赖性,作用96h的半数抑制浓度IC50为4.25mg/mL,并出现细胞凋亡的形态学改变;彗星电泳结果与对照相比6mg/mL EPS作用48h能引起A431细胞DNA严重损伤;免疫组织化学检测发现6mg/mL EPS作用72h能显著上调A431细胞内凋亡相关基因caspase-3和bax的表达,而下调bcl-2的表达。     

Essential role of caspase-11 in activation-induced cell death of rat astrocytes

We have previously shown that rat astrocytes undergo apoptosis upon inflammatory activation. Nitric oxide (NO) produced by activated astrocytes was the major cytotoxic mediator in this type of autoregulatory apoptosis. However, an inhibitor of nitric oxide synthase did not completely block the apoptosis of activated astrocytes, suggesting the presence of other apoptotic pathways. Here, we present evidence that caspase-11 is an essential molecule in NO-independent apoptotic pathway of activated astrocytes. Inflammatory activation (lipopolysaccharide, interferon-gamma, and tumor necrosis factor-alpha treatment) of rat astrocyte cultures and C6 glioma cells led to the induction of caspase-11 followed by activation of caspases-11, -1, and -3. In contrast, NO donors induced activation of caspase-3 only. Inactivation of caspase-11 by the transfection of dominant negative mutant or treatment with the caspase inhibitors rendered the astrocytes partially resistant to the apoptosis following inflammatory activation, but not NO donor exposure. These results indicate that inflammatory stimuli not only induce the production of cytotoxic NO, but also initiate NO-independent apoptotic pathway through the induction of caspase-11 expression.     

Redistribution of cytochrome c is not an essential requirement in C2-ceramide induced apoptosis in HL-60 cells

bcl-2 has been shown to enhance cell survival by inhibiting apoptosis. The present study investigates the potential role of bcl-2 on apoptosis in HL-60 cells induced by different agents. HL-60/bcl-2 and control HL-60/neo cells were obtained by transfection of bcl-2 cDNA or the neomycin-resistant gene, respectively. Staurosporine (STS) promoted DNA fragmentation dose-dependently in the 6 h exposure assay while C2-ceramide was relatively slow in the induction of apoptosis (approximately 40% after 24 h) and required higher concentrations (> 20 microM). Caspases inhibitors, Ac-YVAD-cmk (100 microM) and zVAD-fmk (20 microM) had no effect on DNA fragmentation themselves. However, they blocked C2-ceramide-induced caspase-3 cleavage and apoptosis, but not the release of cytochrome c from the mitochondria. In addition, we found that both Ac-YVAD-cmk and zVAD-fmk failed to protect STS-induced apoptosis in HL-60 cells. Overexpression of bcl-2 inhibited STS and C2-ceramide induced cytochrome c redistribution, caspase-3 activation and apoptosis. These results suggest a protective role of bcl-2 in the regulation of apoptosis and cytochrome c release is unlikely to be involved in the final common pathway in apoptosis.     

Induction of apoptosis in rat C6 glioma cells by panaxydol

Panaxydol is a naturally occurring non-peptidyl small molecule isolated from the lipophilic fractions of Panax notoginseng, a well-known Chinese traditional medicine. Previous studies have shown that panaxydol inhibited the growth of various kinds of malignant cell lines. To date, there has been no report concerning the effect of panaxydol on cell growth inhibition in glioma cells. In this paper, we examined panaxydol's antiproliferation and proapoptotic effects on rat C6 glioma cells and investigated its mechanism. Cell growth inhibition of panaxydol was determined by MTT reduction assay. Apoptosis of cells was measured by both Hoechst 33258 staining and Annexin V analysis. It was found that panaxydol markedly inhibited proliferation of C6 cells in a dose-dependent manner with ID(50) of 40 microM. The cell apoptosis was observed at 48 h in the presence of panaxydol. In concert with these findings, Western blot analysis showed a decreased expression of bcl-2 and increased levels of Bax and caspase-3 in C6 cells treated by panaxydol. In conclusion, panaxydol has profound effects on growth and apoptosis of C6 cells, suggesting that panaxydol may be a potential candidate for the treatment of malignant gliomas.     

Alterations in the cellular distribution of bcl-2, bcl-x and bax in the adult rat substantia nigra following striatal 6-hydroxydopamine lesions

The proteins of the bcl-2 family play an important role during apoptosis and may also regulate cell death in response to oxidative stress, which has been implicated in Parkinson's disease. In this study we examined the localization of the pro-apoptotic protein bax, and the anti-apoptotic proteins bcl-2 and bcl-x_L in the substantia nigra (SN) of the adult rat and their response to oxidative stress caused by striatal injections of 6-hydroxydopamine (6-OHDA). Our data show that bcl-2, bcl-x and bax proteins are present in the SN. Bcl-2 and bax are localized primarily in neurons including all those positive for tyrosine hydroxylase (TH). The intraneuronal distribution of bcl-2 and bax were different. Bcl-2 was diffuse throughout the cell while bax was localized in well-defined structures around the nucleus and within processes. Bcl-x staining in neurons was weak, though it was strongly expressed in GFAP-positive astrocytes. 6-OHDA injections, which resulted in loss of dopamine neurons between 7–14 days post-lesion, altered the distribution of bax, bcl-2 and bcl-x proteins in the SN. Bcl-2 and bax were decreased in the TH-positive cells of the SN from 3 to 14 days post-lesion and many TH-positive neurons were bcl-2 negative. Neuronal bcl-x was initially unchanged after lesion, but increased in astrocytes between 3–7 days post-lesion before the increase in GFAP immunoreactivity, which was detectable at days 10–14. While the neuronal distribution of bcl-2 and bcl-x does not change following lesion, bax became evenly distributed thought the soma. Morphological features of apoptosis, including TUNEL labeling and chromatin condensation was not observed. These data suggest that striatal 6-OHDA lesions do not result in classical apoptosis in the SN of the adult rat, even though there are changes in the content and distribution of members of the bcl-2 family of proteins.     

Danthron Triggers ROS and Mitochondria-Mediated Apoptotic Death in C6 Rat Glioma Cells Through Caspase Cascades, Apoptosis-Inducing Factor and Endonuclease G Multiple Signaling

This research focused on the induction of cytotoxic effects by danthron, a natural anthraquinone derivative on C6 rat glioma cells through exploring the means of cell death and the effects on mitochondrial function. We found that danthron decreased the percentage of viable C6 cells and induced cell morphological changes in a dose-and time-dependent manner. The morphological and nuclei changes (DAPI staining) in C6 cells were observed using a contrast-microscope and fluorescence microscopy, respectively. The results suggest that cell death of C6 cells which are induced by danthron is closely related to apoptotic death. Danthron decreased the level of mitochondrial membrane potential (ΔΨ( m )), stimulated the release of cytochrome c from mitochondria to cytosol and promoted the levels of caspase-9 and caspase-3, or induced the release of AIF and Endo G from mitochondria. Based on both observations, we suggest that the danthron-provoked apoptotic death of C6 cells is mediated through the mitochondria-dependent pathway. Furthermore, our results also indicated that danthron triggered apoptosis through reactive oxygen species (ROS) production which were increased after 1 h exposure of danthron, which was reversed by the ROS scavenger N-acetyl-L: -cysteine (NAC). As a consequence, danthron-mediated cell death of C6 cells via ROS production, mitochondrial transmembrane potential collapse and releases of cytochrome c, AIF and Endo G. Taken together, danthron was demonstrated to be effective in killing C6 rat glioma cells via the ROS-promoted and mitochondria-dependent apoptotic pathways.     

A novel copper (II) complex activated both extrinsic and intrinsic apoptotic pathways in liver cancerous cells

Recent advances have put fundamental focus on the application of copper (II) (Cu [II]) complexes as agents for fighting against cancer. To determine whether [Cu(L)(2imi)] complex as a novel Cu complex can induce apoptosis in HepG2 as cancerous cells and L929 as normal cells via extrinsic or intrinsic apoptotic pathways, both cell lines were treated for 24 and 48 hours at IC₅₀ concentrations of [Cu(L)(2imi)] complex. Then, the expression of some apoptosis-related genes including p53, caspase-8, bcl-2, and bax were assayed by real-time polymerase chain reaction. The [Cu(L)(2imi)] complex seems to inhibit the expression of bcl-2 in complex-treated HepG2 cancerous cells following the 24- and 48-hour treatment. The complex upregulated the p53, bax, and caspase-8 genes, therefore treatment of HepG2 cancerous cells with [Cu(L)(2imi)] complex induces programmed cell death via the upregulation of relative bax/bcl-2 ratio. Finally, this copper complex triggered apoptosis in HepG2 cells via both intrinsic and extrinsic pathway, whereas treatment of normal L929 cells with this complex induce apoptosis only via intrinsic pathway with the upregulation of relative bax/bcl-2 ratio and does not affect the expression level of caspase-8 gene and does not trigger the extrinsic pathway. Finally, these results obtained from present study confirm the role of a novel Cu complex on the induction of apoptosis process in HepG2 and L929 cells by overexpression of bax, inhibition of bcl-2 and increase of the relative bax/bcl-2 ratio. These results support that the [Cu(L)(2imi)] complex is able to induce apoptosis in cancerous cells, therefore, it has a potential for development as a novel anticancer drug.     

Effect of progesterone on the expression of bax and bcl-2 and on caspase activity in bovine luteal cells

Bovine luteal cells from days 6-10 and 11-15 of the estrous cycle were exposed (6 h) to factors that support or disrupt steroidogenesis. The expression of bcl-2 and bax and level of active caspase-3 in cells was measured. Progesterone (P4) increased (P<0.01) while staurosporine decreased (P<0.01-P<0.001) bcl-2 expression at both stages of the estrous cycle studied. In cells from 11-15 days of the estrous cycle expression of bcl-2 was stimulated (P<0.05) by prostaglandin (PG)E2 and inhibited (P<0.01) by 3,3',4,4'-tertrachlorobiphenyl (PCB)-77. Treatment with aminoglutethimide (blocker of cytochrome P450scc; 1.5 x 10(-4)M), nitric oxide donor (spermine NONOate), and staurosporine increased bax expression in cells collected from both experimental periods. The influence of these factors was greater in cells from days 11-15 (P<0.001) than by cells on days 6-10 (P<0.05) of the estrous cycle. PCB-77 stimulated expression of bax in cells from 11-15 days of cycle (P<0.01) only. Treatment of luteal cells with P4 and PGE2 for 24 h decreased (P<0.05) level of active caspase-3 while aminoglutethimide (P<0.05), spermine NONOate (P<0.05), and staurosporine (P<0.001) increased caspase-3 activity in the cells. Moreover, P4 decreased (P<0.05) while staurosporine increased (P<0.01) the ratio of bax/bcl-2 at both stages of the cycle. Aminoglutethimide, spermine NONOate and PCB increased (0<0.05) this ratio in cells on days 11-15 of the cycle. These results suggest that P4 concentrations in luteal cells protects against apoptosis, while disruption of steroidogenesis and reduced ability of luteal cells to produce P4 can induce cell death.     

Connexin 43 confers resistance to hydrogen peroxide-mediated apoptosis

The current study aimed to understand the anti-apoptotic effect of overexpressed gap junction forming protein connexin (Cx) 43 in C6 glioma cells. C6 cells exposed to hydrogen peroxide (H2O2) or staurosporine demonstrated morphological and biochemical changes consistent with apoptosis, whereas C6 cells expressing Cx43 demonstrated relative resistance to H2O2, but not to staurosporine. This selective protection against H2O2 was due to inhibition of caspase-3 activation in Cx43 expressing cells. siRNA knockdown experiments in rat primary astrocytes confirmed the presence of endogenous Cx43-mediated anti-apoptotic effect. Cx43 interacts with the upstream apoptosis signal-regulating kinase 1 known to mediate H2O2-induced apoptosis providing a possible mechanism for protection. These findings provided new evidence for regulation of the mitogen activated protein kinase pathway and apoptosis by Cx43 implicating this protein in intracellular signaling beyond its role as a gap junction forming protein on the plasma membrane.     

Developmental patterns of caspase-3, bax and bcl-2 proteins expression in the human spinal ganglia

The distribution of the bcl-2, bax and caspase-3 proteins was investigated in the cells of developing human spinal ganglia. Paraffin sections of 10 human conceptuses between 5th and 9th gestational weeks were analysed morphologically, immunohistochemically and by TUNEL-method. Cells positive to caspase-3 had brown stained nuclei or nuclear fragmentations. At earliest stages, 6% of ganglion population were caspase-3 positive cells. Later on, a significant increase in number of caspase-3 positive cells appeared, particularly in the ventral part of ganglia (12%), and subsequently decreased to 6%. TUNEL-positive cells had the same distribution pattern as caspase-3 positive cells. Bax-positive cells followed the developmental pattern similar to caspase-3 cells, changing in range between 20% and 32%. There were 8% of bcl-2 positive cells at earliest stages. They increased significantly in dorsal part of the ganglion during the 7th week (28%), and than dropped to 15% by the end of the 8th week. These findings suggest a ventro-dorsal course of development in human spinal ganglia. Number of bcl-2, bax and caspase-3 positive cells changed in a temporally and spatially restricted manner, coincidently with ganglion differentiation. While apoptosis might control cell number, bcl-2 could act in suppression of apoptosis and enhancement of cell differentiation.     

Alkylphosphocholine-induced glioma cell death is BCL-X(L)-sensitive, caspase-independent and characterized by massive cytoplasmic vacuole formation

Alkylphosphocholines (APC) are candidate anticancer agents. We here report that APC induce the formation of large vacuoles and typical features of apoptosis in human glioma cell lines, but not in immortalized astrocytes. APC promote caspase activation, poly(ADP-ribose)-polymerase (PARP) processing and cytochrome c release from mitochondria. Adenoviral X-linked inhibitor of apoptosis (XIAP) gene transfer, or exposure to the caspase inhibitor, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoro-methylketone zVAD-fmk, blocks caspase-7 and PARP processing, but not cell death, whereas BCL-X(L) blocks not only caspase-7 and PARP processing but also cell death. APC induce changes in Delta Psi m in sensitive glioma cells, but not in resistant astrocytes. The changes in Delta Psi m are unaffected by crm-A (cowpox serpin-cytokine response modifier protein A), XIAP or zVAD-fmk, but blocked by BCL-X(L), and are thus a strong predictor of cell death in response to APC. Free radicals are induced, but not responsible for cell death. APC thus induce a characteristic morphological, BCL-X(L)-sensitive, apparently caspase-independent cell death involving mitochondrial alterations selectively in neoplastic astrocytic cells.     

Heat shock pretreatment inhibited the release of Smac/DIABLO from mitochondria and apoptosis induced by hydrogen peroxide in cardiomyocytes and C2C12 myogenic cells

Oxidative stress may cause apoptosis of cardiomyocytes in ischemia-reperfused myocardium, and heat shock pretreatment is thought to be protective against ischemic injury when cardiac myocytes are subjected to ischemia or simulated ischemia. However, the detailed mechanisms responsible for the protective effect of heat shock pretreatment are currently unclear. The aim of this study was to determine whether heat shock pretreatment exerts a protective effect against hydrogen peroxide(H2O2)-induced apoptotic cell death in neonatal rat cardiomyocytes and C2C12 myogenic cells and whether such protection is associated with decreased release of second mitochondria-derived activator of caspase-direct IAP binding protein with low pl (where IAP is inhibitor of apoptosis protein) (Smac/DIABLO) from mitochondria and the activation of caspase-9 and caspase-3. After heat shock pretreatment (42 +/- 0.3 degrees C for 1 hour, recovery for 12 hours), cardiomyocytes and C2C12 myogenic cells were exposed to H2O2 (0.5 mmol/L) for 6, 12, 24, and 36 hours. Apoptosis was evaluated by Hoechst 33258 staining and DNA laddering. Caspase-9 and caspase-3 activities were assayed by caspase colorimetric assay kit and Western analysis. Inducible heat shock proteins (Hsp) were detected using Western analysis. The release of Smac/DIABLO from mitochondria to cytoplasm was observed by Western blot and indirect immunofluorescence analysis. (1) H2O2 (0.5 mmol/L) exposure induced apoptosis in neonatal rat cardiomyocytes and C2C12 myogenic cells, with a marked release of Smac/DIABLO from mitochondria into cytoplasm and activation of caspase-9 and caspase-3, (2) heat shock pretreatment induced expression of Hsp70, Hsp90, and alphaB-crystallin and inhibited H2O2-mediated Smac/DIABLO release from mitochondria, the activation of caspase-9, caspase-3, and subsequent apoptosis. H2O2 can induce the release of Smac/DIABLO from mitochondria and apoptosis in cardiomyocytes and C2C12 myogenic cells. Heat shock pretreatment protects the cells against H2O2-induced apoptosis, and its mechanism appears to involve the inhibition of Smac release from mitochondria.     

No apoptosis is induced in rat cortical neurons exposed to GSM phone fields

The aim of this study was to investigate the radiofrequency (RF) electromagnetic fields (EMF) effects on neuronal apoptosis in vitro. Primary cultured neurons from cortices of embryonic Wistar rats were exposed to a 900-MHz global system for mobile communication (GSM) RF field for 24 h in a wire-patch cell. The average-specific absorption rate (SAR) used was 0.25 W/kg. Apoptosis rate was assessed immediately or 24 h after exposure using three methods: (i) DAPI staining; (ii) flow cytometry using double staining with TdT-mediated dUTP nick-end labeling (TUNEL) and propidium iodide (PI); and (iii) measurement of caspase-3 activity by fluorimetry. No statistically significant difference in the apoptosis rate was observed between controls and 24 h GSM-exposed neurons, either 0 h or 24 h post-exposure. All three methods used to assess apoptosis were concordant. These results showed that, under the conditions of experiment used, GSM-exposure does not significantly increase the apoptosis rate in rat primary neuronal cultures. This work is in accordance with other studies performed on cell lines and, to our knowledge, is the first one performed on cultured cortical neurons.     

Alterations in the content and physiological role of sphingomyelin in plasma membranes of cells cultured in three-dimensional matrix

Naringenin (NGEN), a naturally occurring citrus flavonone, has shown cytotoxicity in various human cancer cell lines as well as inhibitory effects on tumor growth. It has been also shown to access the brain and there is an increasing interest in its therapeutic applications. The up-regulated expression of Cx43 leads to the suppression of tumorigenicity with promoted apoptotic events. In this study, we investigated the in vivo effect of NGEN in fostering apoptosis in cerebrally implanted C6 glioma cells rat model. We analysed the expression of Cx43, caspase-3, caspase-9, Cyt C, Bcl-2 and Bax in vivo by immunoblot analysis and the ultra structure of brain cells by transmission electron microscopy. Supplementation of NGEN to experimental animals modulated Bcl-2/Bax ratio and up-regulation of caspase-3 and 9. NGEN was also found to up-regulate the expression of Cx43. These findings provide evidence that NGEN’s apoptotic effect, modulation of Bcl-2/Bax ratio leads to release of Cyt C from mitochondria, thereby activation of caspase-3 and caspase-9 is mediated by enhanced expression of Cx43. These observations were well supported by the transmission electron microscopic results which showed the characteristic apoptotic features. In conclusion, our findings demonstrate that NGEN promotes apoptosis in rat C6 glioma model.     

Decreased DNA repair rates and protection from heat induced apoptosis mediated by electromagnetic field exposure

In this study, we demonstrate that electromagnetic field (EMF) exposure results in protection from heat induced apoptosis in human cancer cell lines in a time dependent manner. Apoptosis protection was determined by growing HL-60, HL-60R, and Raji cell lines in a 0.15 mT 60 Hz sinusoidal EMF for time periods between 4 and 24 h. After induction of apoptosis, cells were analyzed by the neutral comet assay to determine the percentage of apoptotic cells. To discover the duration of this protection, cells were grown in the EMF for 24 h and then removed for 24 to 48 h before heat shock and neutral comet assays were performed. Our results demonstrate that EMF exposure offers significant protection from apoptosis (P<.0001 for HL-60 and HL-60R, P<.005 for Raji) after 12 h of exposure and that protection can last up to 48 h after removal from the EMF. In this study we further demonstrate the effect of the EMF on DNA repair rates. DNA repair data were gathered by exposing the same cell lines to the EMF for 24 h before damaging the exposed cells and non-exposed cells with H2O2. Cells were allowed to repair for time periods between 0 and 15 min before analysis using the alkaline comet assay. Results showed that EMF exposure significantly decreased DNA repair rates in HL-60 and HL-60R cell lines (P<.001 and P<.01 respectively), but not in the Raji cell line. Importantly, our apoptosis results show that a minimal time exposure to an EMF is needed before observed effects. This may explain previous studies showing no change in apoptosis susceptibility and repair rates when treatments and EMF exposure were administered concurrently. More research is necessary, however, before data from this in vitro study can be applied to in vivo systems.     

Epidermal growth factor and prostaglandin E(2) accelerate mucosal recovery from stress-induced gastric lesions via inhibition of apoptosis.

The repair of damaged gastric mucosa is a complex process involving prostaglandins (PG) and mucosal growth factors such as epidermal growth factor (EGF). Recently, we postulated that the increased occurrence of apoptosis in the gastric epithelium might be of pathophysiological importance in the development of stress lesions. The aim of the present study was to assess the effect of the pretreatment of rats, exposed to 3.5 h of water immersion and restraint stress (WRS), with EGF and PG (16,16 dmPGE(2)) on the number of stress lesions, recovery of gastric mucosa from stress and the expression of apoptosis related genes such as caspase-3 and antiapoptotic bcl-2. Rats were divided in following groups: (1) vehicle; (2) EGF 100 microg/kg i.p.; (3) 16,16 dm-PGE(2) (5 microg/kg i.g.) and caspase-1 inhibitor (ICE-I; 100 microg/kg i.p.). One hour later, the rats were exposed to 3.5 h of WRS and then sacrificed immediately (0 h) or at 6, 12, or 24 h after WRS. The number of acute gastric lesions was determined. Gastric epithelial apoptosis was assessed by TUNEL staining. In addition, mRNA expression of caspase-3, Bcl-2 and proinflammatory cytokines (IL-1 beta, TNFalpha) was assessed by RT-PCR. PGE(2) generation in gastric mucosa and luminal EGF were determined by RIA. Exposure to WRS resulted in the development of multiple acute stress erosions ( approximately 18) which almost completely healed during 24 h. The gastric blood flow was significantly reduced (approximately 70% of intact mucosa) immediately after WRS. The expression of mRNA for IL-1 beta and TNF alpha reached their peak at 12 h after stress exposure. The apoptosis rate was highest at 6 h after WRS and was accompanied by the highest caspase-3 expression. In rats pretreated with EGF or 16,16 dm-PGE(2), a significant decrease in caspase-3 mRNA and upregulation of bcl-2 mRNA as observed as compared to vehicle controls. Caspase-1 inhibitor significantly reduced the number of stress lesions. We conclude that EGF and PGE(2) accelerate healing of stress-induced lesions due to the attenuation of apoptosis via upregulation of bcl-2 in gastric mucosa. Inhibitors of apoptosis accelerate healing of stress lesions and may be potentially effective agents in the healing of damaged gastric mucosa.     

Peony Glycosides Protect Against Corticosterone-Induced Neurotoxicity in PC12 Cells

Preclinical and clinical investigations have shown the involvement of dysregulation of hypothalamic–pituitary–adrenal (HPA) axis in the pathogenesis of depression. Hypercortisolemia and the associated hippocampal atrophy were observed in patients with depression, which could be ameliorated by the treatment with antidepressants. Therefore, neuroprotection has been proposed to be one of the acting mechanisms of antidepressant. Previous studies in our laboratory have demonstrated the antidepressant-like activity of total glycosides of peony (TGP) in mice. This study aimed to examine the effect of TGP treatment on corticosterone-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. Treating the cells with corticosterone at 200 μM for 48 h caused apoptotic cell death. The cytotoxicity was associated with the activation of caspase-3 activity and the decrease in the mRNA ratio of bcl-2 to bax. TPG treatment at increasing doses (1–10 mg/l) protected against the corticosterone-induced toxicity in PC12 cells in a dose-dependent manner. The cytoprotection afforded by TGP treatment was associated with the inhibition of caspase-3 activity and the up-regulation of bcl-2/bax mRNA ratio. The anti-apoptotic effect of TGP is therefore likely mediated by the suppression of the mitochondrial pathway leading to apoptosis.     

Changes of mitochondrial pathway in hypoxia/reoxygenation induced cardiomyocytes apoptosis

The role of mitochondrial apoptotic pathway in cardiomyocytes subjected to hypoxia/reoxygenation(H/R) was studied. Cultured cardiomyocytes from neonatal Sprague-Dawley rats were exposed to hyoxia/reoxygenation, the apoptotic cardiomyocytes were stained with Annexin-V-FITC, Hoechst 33342 and TUNEL assay. Mitochondrial transmembrane potential of cardiomyocytes was assessed by JC-1 under fluorescence microscope, the expressions of bcl-2, bax, cytochrome c, apoptosis-induced factor (AIF), and caspase-3 were tested by western-blot. Our data showed apoptosis of cardiomyocytes was significantly increased during H/R, accompanied by translocation of bax to mitochondria, release of cytochrome c and AIF to cytosol. The results indicate that the mitochondrial-mediated apoptotic pathway is initiated as a result of H/R.     

Effect of 6 mT static magnetic field on the bcl-2, bax, p53 and hsp70 expression in freshly isolated and in vitro aged human lymphocytes

An increasing number of evidence indicates that static magnetic fields (SMFs) are capable of altering apoptosis, mainly through modulation of Ca²⁺ influx. Here we present data that suggest apoptotic-related gene expression as an alternative pathway, through which exposure to 6 milliTesla (mT) SMF can interfere with apoptosis. Exposure to 6 mT SMF affects the apoptotic rate (spontaneous and drug-induced) and [Ca²⁺]_i in isolated human lymphocytes; the aged cells are more susceptible to exposure than fresh ones. The exposure to 6 mT exerted a protective effect on chemical or physical-induced apoptosis, irrespective of the age of the cells.The investigation of the gene expression of bcl-2, bax, p53 and hsp70 in freshly isolated and in culture-aged human lymphocytes indicates that these genes are modulated by SMF exposure in the experimental conditions used, in a gene-, age- and time-dependent manner. The exposure of isolated lymphocytes to SMF for up to 24 h modulated increased bax and p53 and decreased hsp70, and bcl-2. The amount of increment and/or decrement of the proteins varied for each gene examined and was independent of the apoptotic inducers. Finally, the same stress applied to freshly isolated or aged lymphocytes resulted in different modulation of bcl-2, bax and hsp70.     

Epigenetic modification involved in benzene-induced apoptosis through regulating apoptosis-related genes expression

Benzene is an established haematotoxic and genotoxic carcinogen. DNA methyltransferase inhibitor, 5-aza (5-aza-2'-eoxycytidine) and histone deacetylase inhibitor, TSA (trichostatin A) are two kinds of key epigenetic modification reagents. Although apoptosis has been considered as the key cytotoxicity mechanism, the effects of these epigenetic reagents on benzene-induced apoptosis have not been reported. In this study, BMCs (bone marrow cells) from rats were incubated with benzene and then with either 5-aza, TSA alone or the combination of the two drugs. Apoptosis and mRNA expression were detected by annexin V/PI (propidium iodide) staining assay and real-time PCR, respectively. Results showed that benzene caused cell apoptosis accompanied with bcl-2 mRNA decrease, caspase-3 and bax mRNA increase. Moreover, benzene-induced apoptosis and the decrease of bcl-2 mRNA were both reversed by both 5-aza and TSA, but the role of TSA was significantly larger than 5-aza. More interestingly, these increases in benzene-induced caspase-3 and bax mRNA expression were obviously suppressed by 5-aza but not by TSA. In conclusion, 5-aza inhibited benzene-induced apoptosis through down-regulating of caspase-3 and bax and up-regulating bcl-2 mRNA expression, whereas the effect of TSA on apoptosis dominatingly affected bcl-2 mRNA expression, and 5-aza together with TSA had no synergic effect on benzene-induced apoptosis.