Increased Bax Protein Expression Research Articles

Aqueous fraction of Nephelium ramboutan-ake rind induces mitochondrial-mediated apoptosis in HT-29 human colorectal adenocarcinoma cells.

The aim of this study was to investigate the cytotoxic and apoptotic effects of Nephelium ramboutan-ake (pulasan) rind in selected human cancer cell lines. The crude ethanol extract and fractions (ethyl acetate and aqueous) of N. ramboutan-ake inhibited the growth of HT-29, HCT-116, MDA-MB-231, Ca Ski cells according to MTT assays. The N. ramboutan-ake aqueous fraction (NRAF) was found to exert the greatest cytotoxic effect against HT-29 in a dose-dependent manner. Evidence of apoptotic cell death was revealed by features such as chromatin condensation, nuclear fragmentation and apoptotic body formation. The result from a TUNEL assay strongly suggested that NRAF brings about DNA fragmentation in HT-29 cells. Phosphatidylserine (PS) externalization on the outer leaflet of plasma membranes was detected with annexin V-FITC/PI binding, confirming the early stage of apoptosis. The mitochondrial permeability transition is an important step in the induction of cellular apoptosis, and the results clearly suggested that NRAF led to collapse of mitochondrial transmembrane potential in HT-29 cells. This attenuation of mitochondrial membrane potential (Δψm) was accompanied by increased production of ROS and depletion of GSH, an increase of Bax protein expression, and induced-activation of caspase-3/7 and caspase-9. These combined results suggest that NRAF induces mitochondrial-mediated apoptosis.

Effect of Pingxian granules on protein expression of apoptosis regulatory genes in hippocampus of pentylenetetrazol-induced epileptic model rats

To observe the effect of Pingxian granules on the protein expression of apoptosis regulatory genes Bcl-2 and Bax in the hippocampus of epileptic model rats and study the molecular biological mechanism of the anti-epileptic effect of Pingxian granules. Totally 60 45-days-old Wistar rats were selected and then randomly assigned into 5 groups: the normal control group, the model group, the positive control group, the Pingxian high dose group and the Pingxian low dose group, with 12 in each group. Except the normal control group, all the groups were intraperitoneally injected with 35 mg x kg(-1) pentylenetetrazol to establish the models of epilepsy. The Pingxian high dose (1.66 g x mL(-1)) and low dose (0.42 g x mL(-1)) groups were intragastrically infused with Pingxian granules 2 mL x d(-1). The positive control group received 3.6 g x L(-1) phenobarbital suspension by gastric perfusion. The normal group and the model group were drenched with distilled water, 2 mL x d(-1), for 5 weeks. Bcl-2 and Bax protein positive cells were labeled with immunohistochemical SABC at 3, 5 w. (1) Rats in the model group appeared the epileptic behavior at the 1st week, and became serious with the kindle frequency; grade VIepileptic behavior appeared at the 4th week. The attack frequency and grade of the Pingxian group were less and lower, the highest grade were only IV, and there were no significant differences in the attack grade and frequency. (2) With the increase in kindle frequency, the model group showed a notable decrease in the Bcl-2 expression compared with the normal control group at the 3rd and 5th weekend (P < 0.01), but a significant increase in Bax protein expression (P <0. 01). The number of the Bcl-2 protein expression in Pingxian groups and the positive control group were obviously more than the model group (P < 0.01); and the number of the Bax protein expression in Pingxian groups and the positive control group were obviously less than the model group (P < 0.01). Pingxian granules may decrease neuronal cell apoptosis by improving the protein expression of apoptosis regulatory gene Bcl-2 and inhibiting the protein expression of apoptosis regulatory gene Bax with a view of anti-epilepsy.

STGC3 inhibits xenograft tumor growth of nasopharyngeal carcinoma cells by altering the expression of proteins associated with apoptosis

STGC3 is a potential tumor suppressor that inhibits the growth of the nasopharyngeal carcinoma cell line CNE2; the expression of this protein is reduced in nasopharyngeal carcinoma compared with normal nasopharyngeal tissue. In this study, we investigated the tumor-suppressing activity of STGC3 in nude mice injected subcutaneously with Tet/pTRE-STGC3/CNE2 cells. STGC3 expression was induced by the intraperitoneal injection of doxycycline (Dox). The volume mean of Tet/pTRE-STGC3/CNE2+Dox xenografts was smaller than that of Tet/pTRE/CNE2+Dox xenografts. In addition, Tet/pTRE-STGC3/CNE2+Dox xenografts showed an increase in the percentage of apoptotic cells, a decrease in Bcl-2 protein expression and an increase in Bax protein expression. A proteomic approach was used to assess the protein expression profile associated with STGC3-mediated apoptosis. Western blotting confirmed the differential up-regulation of prohibitin seen in proteomic analysis. These results indicate that overexpression of STGC3 inhibits xenograft growth in nude mice by enhancing apoptotic cell death through altered expression of apoptosis-related proteins such as Bcl-2, Bax and prohibitin. These data contribute to our understanding of the function of STGC3 in human nasopharyngeal carcinoma and provide new clues for investigating other STGC3-associated tumors.

Dehydrocorydaline Inhibits Breast Cancer Cells Proliferation by Inducing Apoptosis in MCF-7 Cells

Dehydrocorydaline is an alkaloid isolated from traditional Chinese herb Corydalis yanhusuo W.T. Wang. We discovered that it possessed anti-tumor potential during screening of anti-tumor natural products from Chinese medicine. In this study, its anti-tumor potential was investigated with breast cancer line cells MCF-7 in vitro. The anti-proliferative effect of dehydrocorydaline was determined by MTT assay and the mitochondrial membrane potential (Δ Ψ m) was monitored by JC-1 staining. DNA fragments were visualized by Hoechst 33342 staining and DNA ladder assay. Apoptotic related protein expressions were measured by Western blotting. Dehydrocorydaline significantly inhibited MCF-7 cell proliferation in a dose- dependent manner, which could be reversed by a caspase-8 inhibitor, Z-IETD-FMK. Dehydrocorydaline increased DNA fragments without affecting ΔΨm. Western blotting assay showed that dehydrocorydaline dose-dependently increased Bax protein expression and decreased Bcl-2 protein expression. Furthermore, dehydrocorydaline induced activation of caspase-7,-8 and the cleavage of PARP without affecting caspase-9. These results showed that dehydrocorydaline inhibits MCF-7 cell proliferation by inducing apoptosis mediated by regulating Bax/Bcl-2, activating caspases as well as cleaving PARP.

Epidermal growth factor receptor (EGFR) and neuregulin (Neu) activation in human airway epithelial cells exposed to nickel acetate

Nickel compounds are potential carcinogenic agents that produce a range of biological effects, including inhibition of cell death. Because suppression of apoptosis is thought to contribute to the initiation of carcinogenesis, we investigated the effects of nickel acetate (Ni2+) treatment on apoptosis in two different airway epithelial cell lines (A549 and Beas-2B, respectively). Furthermore, since both the epidermal growth factor receptor (EGFR) and neuregulin (Neu) are involved in neoplastic development, mRNAs and expression levels of total and phosphorylated proteins (p-EGFRTyr1173 and p-NeuTyr1248, respectively) were also measured. We found that exposure of A549 cells to Ni2+ resulted in significantly reduced cell viability, as well as increased apoptosis and DNA fragmentation at relatively low concentrations (0.1 and 0.5mM) after 24 and 48h. These changes were accompanied by reduced EGFR and Neu mRNAs and proteins, phosphorylated proteins as well as decreased Bcl-2 and increased BAX protein expression. Conversely, Beas-2B cells exposed to equivalent concentrations of Ni2+ did not show evident signs of apoptosis and DNA damage, hence showing increased expression and phosphorylation of both EGFR and Neu, increased Bcl-2 and reduced BAX expression. Altogether, our finding indicate that Ni2+ exposure differently affects apoptosis initiation either in non-tumorigenic (Beas-2B) and tumorigenic airway epithelial cells (A549), suggesting a potential involvement of EGFR/Neu receptors.

Mate ( Ilex paraguariensis St. Hilaire) saponins induce caspase-3-dependent apoptosis in human colon cancer cells in vitro

Saponins are naturally occurring metabolites associated with several health benefits. The objective was to quantify and purify saponins from mate dry leaves, and to assess their anti inflammatory and apoptotic mechanisms in human colon cancer cells in vitro. Matesaponins were extracted with methanol from dry leaves, partially purified and quantified. Leaves contained 10–15 mg/g dry weight total saponins, predominantly matesaponins 1 and 2. HPLC and LC/ESI-MS-MS identified saponins in six preparative chromatographic fractions (A, B, C, D, E, and F). Major matesaponins were identified as 1 [M–H] − = 911 and 2 [M–H] − = 1057, with trace amounts of 3 [M–H] − = 1073, 4 [M–H] − = 1219, and 5 [M–H] − = 1383. Fractions D, E, and F significantly inhibited iNOS (IC 35 = 36.3, 29.5, 43.7 μM), PGE 2 (IC 35 = 23.1, 22.3, 11.7 μM) and COX-2 (IC 35 = 45.7, 32.4, 17.0 μM). Fraction F reduced nuclear translocation of nuclear factor-κB subunits p50 (49.8%) and p65 (49.0%) and induced apoptosis through suppression of Bcl-2 and increased Bax protein expressions and activated caspase-3 activity. Saponins in leaves of mate prevent inflammation and colon cancer in vitro.

Effect of &lt;i&gt;Achyranthes bidentata&lt;/i&gt; polysaccharides on the expression of the BCL-2 and BAX in hepatic tissues of exhaustive exercise in rats

This study aims to assess the effects of Achyranthes bidentata polysaccharides (ABPS) on the expression of bcl-2 and bax in hepatic tissues after exhaustive exercise in order to provide theoretical support for the application of ABPS in the field of sports nutrition. Thirty male Sprague-Dawley rats were randomized into three groups, each consisting of 10 rats: Normal control group (NCG), Exhausting exercises control group (EECG), ABPS treated group (ATG). ABPS were fed orally by gastric intubation to rats of ABPS treated group (ATG) once daily for 7 days. Control animals (EECG and NCG) received the same amount of isotonic sodium chloride solution. Exhaustive exercise was performed on a rodent treadmill. The SP (streptavidin peroxidase) method for immunohistochemical staining was adopted to test the protein expression of bax and bcl-2 in the hepatic tissues of the rats. Exhausting exercises increased bax protein expression of hepatic tissues of rats and bax/bcl-2 ratio dramatically, but a decreased bcl-2 protein expression. In the rats fed ABPS orally by gastric intubation, the bax protein expression and bax/bcl-2 ratio obviously decreased, while bcl-2 protein expression increased. The result indicated that bax and bcl-2 co-regulated the exercise-induced hepatocyte apoptosis. Feeding ABPS orally by gastric intubation to rats can inhibit the hepatocyte apoptosis in exhaustive exercise.

Synergism of simvastatin with losartan prevents angiotensin II-induced cardiomyocyte apoptosis in vitro

Abstract Objectives Increasing evidence suggests that cardiomyocyte apoptosis has an important role in the transition from compensatory cardiac remodelling to heart failure. The synergistic effect of statins (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) and angiotensin II (Ang II) type 1 receptor antagonists reduces the incidence of cardiovascular events. However, the anti-apoptotic potential of the synergism between losartan and simvastatin in heart failure remains unexplored. Here, we demonstrate that Ang II-induced apoptosis is prevented by losartan and simvastatin in neonatal cardiomyocytes. Methods The in-vitro cardiomyocyte apoptosis model was established by co-culturing neonate rat cardiomyocytes with Ang II. Cell viability was analysed by the MTT assay. Cell apoptosis was evaluated using fluorescence microscopy and flow cytometry. Apoptosis-related proteins Bax and Bcl-2 expressions were measured by flow cytometry detection. Key findings Incubation with 10−7 m Ang II for 48 h increased cardiomyocyte apoptosis and decreased cell viability. Losartan (10−5 m) and simvastatin (10−5 m), either alone or in combination, significantly decreased Ang II-induced cardiomyocyte apoptosis and increased cell viability. The q values calculated by the probability sum test were 1.31 for cardiomyocyte apoptosis and 1.21 for cell viability. Ang II induced a significant increase in Bax protein expression, whereas Bcl-2 protein expression was decreased. Losartan alone or in combination with simvastatin blocked the increased Bax expression and increased Bcl-2 expression. However, simvastatin had no such effect. Conclusions Our data provide the first evidence that synergism of simvastatin with losartan prevents angiotensin II-induced cardiomyocyte apoptosis in vitro. Synergism between simvastatin and losartan may provide a new therapeutic approach to the prevention of cardiac remodelling.

Achyranthes bidentata polypeptides confer neuroprotection through inhibition of reactive oxygen species production, Bax expression, and mitochondrial dysfunction induced by overstimulation of N‐methyl‐D‐aspartate receptors

Achyranthes bidentata polypeptides (ABPP), the important constituents separated from the aqueous extract of Achyranthes bidentata, have been shown to attenuate N-methyl-D-aspartate (NMDA)-induced cell apoptosis in cultured hippocampal neurons through differential modulation of NR2A- and NR2B-containing NMDA receptors. The present study sought to investigate the possible mechanism underlying the neuroprotective effect of ABPP on NMDA-induced cell death. Western blot analysis and colorimetric enzymatic assay demonstrated that ABPP pretreatment inhibited NMDA-induced increase of Bax protein expression or caspase-3 activity in cultured hippocampal neurons. Fluorescence measurements after staining with 2,7-dichlorofluorescin diacetate and rhodamine 123 showed that ABPP treatment also reversed NMDA-induced intracellular radical oxygen species (ROS) elevation and mitochondrial membrane potential depression in cultured hippocampal neurons. Furthermore, the in vivo effects of ABPP on cerebral neuronal damage during focal ischemia-reperfusion were also investigated. In rat middle cerebral artery occlusion (MCAO) model, ABPP attenuated the increase in the neurological deficit and cerebral infarction induced by focal ischemia-reperfusion, showing in vivo neuroprotective effects. The results collectively suggest that ABPP might exert neuroprotective actions through inhibiting Bax protein expression, caspase-3 activity, ROS production, and mitochondrial dysfunction that are all caused by overstimulation of NMDA receptors.

Synergism of simvastatin with losartan prevents angiotensin II-induced cardiomyocyte apoptosis &lt;I&gt;in vitro&lt;/I&gt;

Increasing evidence suggests that cardiomyocyte apoptosis has an important role in the transition from compensatory cardiac remodelling to heart failure. The synergistic effect of statins (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) and angiotensin II (Ang II) type 1 receptor antagonists reduces the incidence of cardiovascular events. However, the anti-apoptotic potential of the synergism between losartan and simvastatin in heart failure remains unexplored. Here, we demonstrate that Ang II-induced apoptosis is prevented by losartan and simvastatin in neonatal cardiomyocytes. The in-vitro cardiomyocyte apoptosis model was established by co-culturing neonate rat cardiomyocytes with Ang II. Cell viability was analysed by the MTT assay. Cell apoptosis was evaluated using fluorescence microscopy and flow cytometry. Apoptosis-related proteins Bax and Bcl-2 expressions were measured by flow cytometry detection. Incubation with 10(-7) M Ang II for 48 h increased cardiomyocyte apoptosis and decreased cell viability. Losartan (10(-5) M) and simvastatin (10(-5) M), either alone or in combination, significantly decreased Ang II-induced cardiomyocyte apoptosis and increased cell viability. The q values calculated by the probability sum test were 1.31 for cardiomyocyte apoptosis and 1.21 for cell viability. Ang II induced a significant increase in Bax protein expression, whereas Bcl-2 protein expression was decreased. Losartan alone or in combination with simvastatin blocked the increased Bax expression and increased Bcl-2 expression. However, simvastatin had no such effect. Our data provide the first evidence that synergism of simvastatin with losartan prevents angiotensin II-induced cardiomyocyte apoptosis in vitro. Synergism between simvastatin and losartan may provide a new therapeutic approach to the prevention of cardiac remodelling.

Downregulation of Wip-1 phosphatase expression in MCF-7 breast cancer cells enhances doxorubicin-induced apoptosis through p53-mediated transcriptional activation of Bax

The human breast cancer cell line MCF-7 carries an amplified PPM1 D/Wip-1 gene and over expresses Wip-1 phosphatase protein. MCF-7 cells also harbor a wild type p53 gene. We established stable isogenic lines (MCF-Sp53 clones) which exhibit decreased levels of p53 protein. We show that although the PPM1 D gene is amplified in MCF-7 cells it is still expressed in a p53-dependent manner. Stable isogenic cell lines derived from MCF-7 cells (designated MCF-clones) were also established in which Wip-1 expression is significantly decreased by a plasmid-based PPM1D antisense RNA. Decreasing Wip-1 expression sensitized MCF-clones to doxorubicin-induced apoptosis. The enhanced apoptotic response was correlated with increased phosphorylation of N-terrninal p53-Ser15 and -Ser46 and increased expression of the pro-apoptotic Bax gene at both the mRNA and protein level. The enhanced apoptotic response was blocked by Bax-siRNA knock down suggesting that the increased response was a result of increased Bax protein expression. Moreover, reporter gene assays using the Waf-1 and Bax promoters to drive a luciferase gene revealed that luciferase activity driven by the Bax promoter was enhanced in MCF-clones while luciferase activity driven by the Waf-1 promoter was decreased relative to parental MCF-7 cells. The study reveals a novel molecular mechanism involving Wip-1 phosphatase, p53 phosphorylation and an enhanced apoptotic response mediated by transcriptional activation of the pro-apoptotic Bax gene.W. Edward Mercer Ph.D., who made great contributions to this paper, passed away on Thursday, October 30, 2008, after a brief illness. This paper is in memorial to his honorable attitude toward science and education.

Evaluation of insulin and ascorbic acid effects on expression of Bcl-2 family proteins and caspase-3 activity in hippocampus of STZ-induced diabetic rats.

Effects of insulin and ascorbic acid on expression of Bcl-2 family proteins and caspase-3 activity in hippocampus of diabetic rats were evaluated in this study. Diabetes was induced in Wistar male rats by streptozotocin (STZ). Six weeks after verification of diabetes, the animals were treated for 2 weeks with insulin or/and ascorbic acid in separate groups. Hippocampi of rats were removed and evaluation of Bcl-2, Bcl-x(L), and Bax proteins expression in frozen hippocampi tissues were done by SDS-PAGE electrophoresis and blotting. The Bcl-2, Bcl-x(L), and Bax proteins bands were visualized after incubation with specific antibodies using enhanced chemiluminescences method. Caspase-3 activity was determined using the caspase-3/CPP32 Fluorometric Assay Kit. Diabetic rats showed increase in Bax protein expression and decrease in Bcl-2 and Bcl-x(L) proteins expression. The Bax/Bcl-2 and Bax/Bcl-x(L) ratios were found higher compared with non-diabetic control group. Treatments with insulin and/or ascorbic acid were resulted in decrease in Bax protein expression and increase in Bcl-2 and Bcl-x(L) proteins expression. The Bcl-2/Bax and Bcl-x(L)/Bax ratios were found higher in treated groups than untreated diabetic group. Caspase-3 activity level was found higher in diabetic group compared with non-diabetic group. Treatment with insulin and ascorbic acid did downregulated caspase-3 activity. Our data provide supportive evidence to demonstrate the antiapoptotic effects of insulin and ascorbic acid on hippocampus of STZ-induced diabetic rats.

Apoptotic effect of citrus fruit extract nobiletin on lung cancer cell line A549 in vitro and in vivo

Lung cancer is the leading cause for cancer-related death worldwide and the effectiveness of current treatments is very limited. Here we reported that Nobiletin, an effective component of citrus fruit, has antiproliferative activity on lung cancer cells both in vitro and in vivo. Cell viability and clonogenic assay showed that Nobiletin dose-dependently suppressed the proliferation of human lung adenocarcinoma cell line A549 cells, while has having a minimal effect on human umbilical vein endothelial cell line ECV-304 cells. DNA fragment assay and comet assay demonstrated that Nobiletin induced A549 cell apoptosis. Nobiletin-induced cell cycle arrest at G2/M phase was detected by Flow cytometric analysis. In addition, western blot analysis revealed that A549 cells pretreated with Nobiletin showed decreased Bcl-2 and increased Bax protein expression, which were positively correlated with elevated expression of p53 compared to control. Furthermore, Nobiletin had overt inhibitory effect on the tumor growth in nude mice model was observed in vivo. Taken together, these results suggest that Nobiletin could induce p53-mediated cell cycle arrest and apoptosis via modulated the Bax:Bcl-2 protein ratio, is effective as a potent antitumor agent on lung tumors.

SENSITIZATION OF HUMAN GRANULOSA TUMOR CELL LINES TO TUMOR NECROSIS FACTOR-RELATED APOPTOSIS- INDUCING LIGAND (TRAIL)- INDUCED APOPTOSIS

The ability of TRAIL to target cancer cells for apoptosis without affecting normal cells has generated interest for its potential use in cancer therapy, however its efficacy in the treatment of ovarian granulosa cell tumors (GCT) has yet to be evaluated. Human ovarian cancers of granulosa cell origin currently represent approximately 3 to 5% of diagnosed cancers involving the ovary. Nevertheless, there is little known regarding either the etiology or selective treatment of GCTs as compared to those originating from surface epithelial cells. Recent studies have demonstrated that TRAIL treatment, in vitro, does not induce apoptosis in healthy ovarian granulosa cells, despite the expression of a functional TRAIL death receptor, DR5. Results obtained using two human GCT lines, COV434 and KGN (both of which were established to be p53 wildtype), demonstrate a slight, but significant, decrease in cell viability in response to TRAIL (100 ng/ml) treatment (KGN, fold vs control = 0.90 ± 0.02; COV434, fold vs control = 0.89 ± 0.015; p < 0.05). This loss of cell viability is not correlated with an increase in DR5 mRNA or protein expression. Rather, treatment with TRAIL significantly decreases DR5 mRNA in both GCT lines (fold-decrease vs control: KGN, 0.28 ± 0.21; COV434, 0.68 ± 0.04; p < 0.05). By comparison, treatment with the conventional chemotherapeutic, cisplatin (25 μM), significantly increases DR5 mRNA (mRNA fold-increase vs control: KGN, 2.02 ± 0.05; COV434, 1.55 ± 0.021; p < 0.05) and protein expression. Furthermore, pretreatment of KGN with cisplatin for 18 h, a time sufficient for upregulation of DR5 expression, led to a significant increase in TRAILinduced cell death upon both acute and long-term challenge (4 h: 22% increase in cell death vs cisplatin treatment alone; 18 h: 34% increase; p < 0.05). Treatment with cisplatin also significantly increases pro-apoptotic Bax protein expression, with similar high levels of expression maintained following co-treatment with TRAIL. To investigate whether the effects of cisplatin-induced sensitization to TRAIL are p53-dependent, transfection with a GFP construct (pEGFP-N1-p53) expressing nuclear-localized p53 was utilized. GCT cultures over-expressing p53 were found to be hypersensitive to treatment with TRAIL (cell death increased 33%, as compared to mock-transfected cells), cisplatin (cell death increased 45%), and TRAIL treatment combined with cisplatin (cell death increased 75%). These cells also show increased Bax protein expression as compared to cells transfected with pEGFP-N1 alone. Moreover, transfection with siRNA duplexes specifically targeting p53 results in a partial reversal of TRAIL plus cisplatin-induced cell death (by 34% compared to cells transfected with nonsense RNA). Taken together, these data indicate that, unlike normal granulosa cells, TRAIL treatment promotes a loss of cell viability in GCT, and that sensitivity to TRAIL is enhanced following treatment with cisplatin. Finally, p53 expression plays at least a partial role in the sensitization effect of cisplatin on TRAIL-induced apoptosis. This occurs by modulating both levels of DR5 and downstream proapoptotic mediators, including Bax. These results provide evidence that a greater efficacy in treating GCTs can be achieved with a regime of cisplatin followed by treatment with TRAIL. (Supported by DAMD17-03- 1-0206, and the Notre Dame Cancer Institute.) (platform)

Activation of Melanocortin 4 Receptors Reduces the Inflammatory Response and Prevents Apoptosis Induced by Lipopolysaccharide and Interferon-γ in Astrocytes

Alpha-MSH exerts an immunomodulatory action in the brain and may play a neuroprotective role acting through melanocortin 4 receptors (MC4Rs). In the present study, we show that MC4Rs are constitutively expressed in astrocytes as determined by immunocytochemistry, RT-PCR, and Western blot analysis. alpha-MSH (5 microm) reduced the nitric oxide production and the expression of inducible nitric oxide synthase (iNOS) induced by bacterial lipopolysaccharide (LPS, 1 microg/ml) plus interferon-gamma (IFN-gamma, 50 ng/ml) in cultured astrocytes after 24 h. alpha-MSH also attenuated the stimulatory effect of LPS/IFN-gamma on prostaglandin E(2) release and cyclooxygenase-2 (COX-2) expression. Treatment with HS024, a selective MC4R antagonist, blocked the antiinflammatory effects of alpha-MSH, suggesting a MC4R-mediated mechanism in the action of this melanocortin. In astrocytes, LPS/IFN-gamma treatment reduced cell viability, increased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells and activated caspase-3. alpha-MSH prevented these apoptotic events, and this cytoprotective effect was abolished by HS024. LPS/IFN-gamma decreased Bcl-2, whereas it increased Bax protein expression in astrocytes, thus increasing the Bax/Bcl-2 ratio. Alpha-MSH produced a shift in Bax/Bcl-2 ratio toward astrocyte survival because it increased Bcl-2 expression and also prevented the effect of LPS/IFN-gamma on Bax and Bcl-2 expression. In summary, these findings suggest that alpha-MSH, through MC4R activation, attenuates LPS/IFN-gamma-induced inflammation by decreasing iNOS and COX-2 expression and prevents LPS/IFN-gamma-induced apoptosis of astrocytes by modulating the expression of proteins of the Bcl-2 family.

Effect of nitric oxide synthase inhibition during post-hypoxic reoxygenation on Bax and Bcl-2 protein expression and DNA fragmentation in neuronal nuclei of newborn piglets

Previous studies have shown that cerebral tissue hypoxia results in increased generation of oxygen-free radicals including nitric oxide (NO), expression of the proapoptotic protein Bax and fragmentation of nuclear DNA. The present study tests the hypothesis that post-hypoxic reoxygenation for 6 h following hypoxia (FiO 2 = 0.06 for 1 h) results in continued hypoxia-induced, NO-mediated expression of the Bax protein and nuclear DNA fragmentation in the cerebral cortex of newborn piglets. Piglets were divided into normoxic (Nx), hypoxic (Hx, FiO 2 = 0.06 for 1 h), hypoxic with 6 h reoxygenation (Hx + reox) and hypoxic with 6 h reoxygenation injected with 7-nitroindazole sodium salt (7-NINA), a selective nNOS inhibitor, immediately after hypoxia (Hx + 7-NINA). Cerebral tissue hypoxia was documented by levels of ATP and phosphocreatine (PCr). Bax and Bcl-2 were analyzed by Western blot and DNA fragmentation was determined by agarose gel electrophoresis. ATP and PCr values in Hx, Hx + reox and Hx + 7-NINA were significantly different from Nx ( P < 0.05 vs. Nx). Bax protein (OD × mm 2) was 128.9 ± 38.7 in Nx; 223.6 ± 45.8 in Hx ( P < 0.05 vs. Nx); 340.5 ± 73.2 in Hx + reox ( P < 0.05 vs. Nx, Hx and Hx + 7-NINA); and 202.2 ± 34.8 in Hx + 7-NINA ( P = NS vs. Hx). Bcl-2 protein (OD × mm 2) was 14.9 ± 2.7 in Nx, 12.4 ± 2.1 in Hx, ( P < 0.05 vs. Nx), 15.7 ± 3.8 in Hx + reox, ( P < 0.05 vs. Hx) and 13.1 ± 2.2 in Hx + 7-NINA ( P = NS among groups). Nuclear DNA fragmentation (OD × mm 2) was 147 ± 15 in Nx; 797 ± 84 in Hx ( P < 0.05 vs. Nx); 1134 ± 127 in Hx + reox ( P < 0.05 vs. Nx, Hx and Hx + 7-NINA); and 778 ± 146 in Hx + 7-NINA ( P = NS vs. Hx, P < 0.05 vs. Hx + reox). The results show that post-hypoxic reoxygenation results in increased expression of Bax protein without affecting Bcl-2 protein and increased fragmentation of nuclear DNA, which are prevented by 7-NINA. We conclude that during post-hypoxic reoxygenation the increase in Bax protein expression and fragmentation of nuclear DNA are mediated by NO derived from nNOS. We propose that in addition to NO-mediated nuclear DNA damage, the hypoxia-induced increased ratio of Bax/Bcl-2 protein will lead to caspase-activated cascade of hypoxic neuronal death during post-hypoxic reoxygenation.

Protection from apoptotic cell death by cilostazol, phosphodiesterase type III inhibitor, via cAMP-dependent protein kinase activation

This study aimed to elucidate whether the effect of cilostazol to suppress apoptotic cell death is directly coupled to cAMP-dependent protein kinase activation in human umbilical vein endothelial cells (HUVECs). After exposure of HUVECs to LPS (1 μg ml −1) for 18 h, the endothelial cells irregularly aggregated with loss of cobblestone appearance, which was reversed by cilostazol (1–100 μM), as well as by cilostamide (cilostazol analog), and cilostazol metabolites (OPC-13015 and OPC-31213), respectively. LPS-stimulated production of reactive oxygen species (ROS) was significantly reduced by cilostazol (0.1–10 μM). In line with these, LPS (1 μg ml −1)- and TNF-α (200 ng ml −1)-induced DNA fragmentation, assessed by agarose gel electrophoresis, was significantly reduced by treatment with cilostazol (10 μM) as well as by dibutyryl cAMP (100 μM). This effect was reversed by cAMP-dependent protein kinase inhibitor, Rp-cAMPs (200 μM). Further, LPS (1 μg ml −1)-induced decrease in Bcl-2 and increase in Bax protein expression were fully reversed by cilostazol (10 μM) and dibutyryl cAMP (100 μM), all of which were antagonized by Rp-cAMPs (200 μM). Taken together, cilostazol effectively protected HUVECs from LPS- and TNF-α-induced cell death associated with oligonucleosomal DNA fragmentation via activation of cAMP-dependent protein kinase.

Estradiol treatment induces testicular oxidative stress and germ cell apoptosis in rats

In order to understand the pathogenesis of estradiol induced effects in the seminiferous epithelium, studies were undertaken in adult rats with estradiol-3-benzoate administered for different durations. After 30 d of treatment, a significant rise in lipid peroxidation with concomitant fall in the activities of superoxide dismutase and catalase was observed. Both, serum and intra-testicular testosterone levels were found severely depleted. Seminiferous epithelium was devoid of elongated spermatids and spermatozoa by 30 d of treatment. Number of spermatocytes and round spermatids were significantly (p < 0.001) reduced. Flowcytometric analysis confirmed a drastic reduction of the haploid cell population (1c peak). Beginning from day 10 of treatment, there was a consistent rise in the number of pyknotic/apoptotic germ cells in the seminiferous epithelium. A gradual increase in Bax protein expression was observed with the duration of treatment. The shift in Bax immunostaining from the cytoplasm and nucleus of germ cells (at 10 d of treatment) to only nuclei of cells by 30 d of treatment was also noticed. By this time testicular tissue showed three-fold increase in caspase-8 enzyme activity. Viable testicular cells isolated in vitro decreased drastically subsequent to different periods of estradiol treatment. The above findings substantiate the fact that the testicular pathogenesis of estradiol benzoate treatment may be primarily because of altered reproductive hormone levels and high oxidative stress leading to germ cell apoptosis and subsequent germ cell loss in the seminiferous epithelium.

Quercetin Induces p53-Independent Apoptosis in Human Prostate Cancer Cells by Modulating Bcl-2-Related Proteins: A Possible Mediation by IGFBP-3

Quercetin, a flavonoid found in onion, grapes, green vegetables, etc., has been shown to possess potent antiproliferative effects against various malignant cells. We report insulin-like growth factor-binding protein-3 (IGFBP-3) as an effector of quercetin-induced apoptosis in human prostate cancer cell lines in a p53-independent manner. We evaluated the production of IGFBP-3 in quercetin-treated cells. Apoptosis was studied in quercetin-treated cells to study the IGFBP-3-mediated role with flow cytometry and DNA fragmentation. Protein expressions of Bcl-2, Bcl-x(L), and Bax were studied by Western blot. Increased production of IGFBP-3 was associated with the increased ratio of proapoptotic to antiapoptotic members of the Bcl-2 family. In quercetin-treated PC-3 cells, an increase in Bax protein expression and a decrease in Bcl-x(L) protein and Bcl-2 protein were observed. As PC-3 is a p53-negative cell line, these modulations of proapoptotic proteins and induction of apoptosis were independent of p53. The level of IGFBP-3 on the response of PC-3 cells to quercetin was examined. There was a twofold increase in IGFBP-3 level in conditioned media of 100 microM quercetin-treated cells. Quercetin also brought a peak at sub-G1 in PC-3 cells. Thus, increased level of IGFBP-3 was associated with increased proapoptotic proteins and apoptosis in response to quercetin, suggesting it may be a p53-independent effector of apoptosis in prostate cancer cells via its modulation of the Bax/Bcl-2 protein ratio.

Epidermal Growth Factor Receptor Signaling Regulates Bax and Bcl-w Expression and Apoptotic Responses During Intestinal Adaptation in Mice

Normal intestinal adaptation to massive small-bowel resection requires intact epidermal growth factor receptor signaling and consists of increased enterocyte proliferation and apoptosis. Although emphasis has been placed on understanding the regulation of proliferation, few studies have evaluated the mechanism and contribution of apoptosis to the adaptation response. We sought to test the hypothesis that epidermal growth factor receptor signaling regulates specific Bcl-2 family members (Bax and Bcl-w) to direct apoptosis and adaptation after massive small-bowel resection. Laser capture microdissection microscopy permitted measurement of Bax and Bcl-w messenger RNA expression in crypt and villus enterocytes in control conditions and under epidermal growth factor receptor-inhibited (waved-2 mice) or stimulated (epidermal growth factor transgenic mice) conditions after a 50% small-bowel resection or sham operation. Resection-induced adaptation was then studied in Bax-null and Bcl-w-null mice under control circumstances and after epidermal growth factor receptor stimulation. When compared with Bcl-w, the most significant expression changes were observed with Bax and took place within crypt enterocytes. Epidermal growth factor receptor stimulation resulted in a decreased ratio of Bax to Bcl-w expression and decreased rates of apoptosis. Bax-null mice had no apoptosis response to small-bowel resection and displayed an amplified adaptation response to the administration of epidermal growth factor. Bcl-w-null mice had poor survival and impaired adaptation to small-bowel resection, an effect that was rescued by crossbreeding these mice with epidermal growth factor transgenic mice. The crypt expression of Bax and Bcl-w is influenced by epidermal growth factor receptor signaling and is key for the regulation of apoptosis. Epidermal growth factor receptor stimulation, coupled with apoptosis inhibition, may provide a novel strategy to amplify adaptation responses in patients after massive intestinal loss.