Upregulation Of Active Caspase-3 Research Articles

Involvement of Caspase-3 Activity and Survivin Downregulation in Cinobufocini-Induced Apoptosis in A 549 Cells

Cinobufocini injection is a preparation containing water-soluble components of the toad skin. The aim of the present study was to investigate the apoptosis of human lung adenocarcinoma cell line A 549 induced by cinobufocini. A 549 or HLF-1(human lung fibroblast) cells were treated with cinobufocini at different concentrations for 24 and 48 h, respectively. The proliferation of cells was detected with the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Morphology of cells was carried out with scanning electronic microscopy (SEM) and Hoechst 33258 staining. The apoptosis rate was examined by flow cytometry. The expression of survivin was examined with RT-PCR and Western blot assay. The caspase-3 and caspase-7 activities were detected with caspase colorimetric protease assay. We found that cinobufocini significantly inhibited tumor growth of A 549 cells in a dose- and time-dependent manner without damaging non-cancerous cells (HLF-1) and induced granular apoptotic bodies of A 549 cells. Next, cinobufocini increased the percentage of cells in G1 phase and decreased the percentage of cells in S phase in A 549 cells. Furthermore, cinobufocini downregulated the expression of survivin mRNA and protein. Finally, cinobufocini upregulated caspase-3 activity. We concluded that cinobufocini induces apoptosis of A 549 cells, which is associated with the decreasing expression of survivin mRNA and protein, increasing caspase-3 activity of A 549 cells.

Inhibition Effects of baicalin on Lymphoma Cell Line CA46 in Vitro and in Vivo

Baicalin is a traditional Chinese medicine with multiple biological effects. Some researches showed baicalin has anti-tumor effects in solid tumor, such as prostate cancer. In order to investigate its effects on proliferation inhibition and apoptosis induction in human lymphoma cell, we treated Burkitt lymphoma cell line CA46 with baicalin in vitro and in vivo of CA46 xenograft. Baicalin remarkably inhibited the cell proliferation, with an IC50 value of 10μM. Apoptosis was remarkably induced by baicalin in a dose-dependent manner, which was detected by Annexin V FITC/PI double staining analysis, TUNEL labeling method and DNA fragmentation respectively. Furthermore, RT-PCR showed that the mRNA expressions of c-myc and bcl-2 in treated CA46 cell decreased in a time-dependent manner. Western-Blot showed that the protein expressions of c-myc, bcl-2, procaspase-3 and PARP(116KD) in baicalin treated CA46 cell were down-regulated, while the expression of PARP(85KD) increased. Based on the results in vitro, we investigated in vivo efficacy of baicalin, alone or in combination with cytotoxic drug VP16, for treatment in CA46 nude mice xenograft. Baicalin with the dosage of 40mg/kg/d and 80kg/mg/d could remarkably inhibit the growth of the tumor compared with control group. Combination of baicalin and VP16 had better anti-tumor effects. Histological examination of tumor samples showed more necrotic cells in treated groups. And obvious apoptosis could be observed by electron microscope. No adverse events were found in treated groups. From above we could conclude that baicalin could efficiently induce proliferation inhibition and apoptosis of CA46 cells in vitro and in vivo, which may be related with the down-regulation of c-myc and bcl-2 expressions, as well as the up-regulation of caspase-3 activity.

Resistin induces rat insulinoma cell RINm5F apoptosis

Beta-cell apoptosis induced by adipokines may result in beta-cell dysfunction in type 2 diabetes. Resistin, an adipokine-linked obesity with type 2 diabetes, impairs glucose-stimulated insulin secretion (GSIS) in beta-cells. Presently, the effects of resistin on rat insulinoma cells RINm5F were examined. Treatment of RINm5F with resistin induced cell damage. Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) protected resistin-mediated cytotoxicity in RINm5F. Incubation with resistin up-regulated caspase-3 activity and induced the formation of a DNA ladder. TIMP-1 attenuated these effects. The molecular mechanism of TIMP-1 inhibition of resistin-mediated cytotoxicity appeared to involve Akt phosphorylation and activation of IkB-alpha phosphorylation. Resistin treatment suppressed Akt phosphorylation and activated IkB-alpha phosphorylation, which could be attenuated by TIMP-1. We conclude that resistin can induce beta-cell apoptosis and that resistin-related beta-cell apoptosis can be prevented by TIMP-1.

Chronic application of nonylphenol-induced apoptosis via suppression of bcl-2 transcription and up-regulation of active caspase-3 in mouse brain

Nonylphenol (NP) is an endocrine disruptor, which has been reported to have adverse effects on reproductive and immune systems. However, the influence of NP on the central nervous system (CNS) has not been extensively explored. The present study was performed to investigate the effects of chronic administration of NP on the apoptosis-related protein expression in mouse brain by in situ hybridization, RT-PCR and immunoblotting assays. The expression of bcl-2 mRNA was down-regulated by NP at the doses of 100 and 200mg/(kg day) (p<0.05), whereas the expression of bax mRNA was not affected in NP treated mice (p>0.05). Furthermore, as the main executor of apoptosis, the expression of active caspase-3 was up-regulated by 100 and 200mg/(kg day) NP (p<0.01), which is in accord with the results of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) (p<0.05). These results suggest that chronic application of NP could sensitize the mice brain to apoptosis.

Role of the WWOX gene, encompassing fragile region FRA16D, in suppression of pancreatic carcinoma cells

The WW-domain-containing oxidoreductase (WWOX) gene spans the common chromosomal fragile site FRA16D (16q23.2) and is believed to be a tumor suppressor in various human malignancies. We have previously shown frequent down-modulation of Wwox expression in pancreatic carcinoma (PC); however, biological function of Wwox in pancreatic duct carcinogenesis remains unknown. In PANC-1 (Wwox-negative) PC-derived cells, restoration of recombinant WWOX gene expression with adenoviral gene delivery (Ad-WWOX) effectively increased the number of cells with subG(1) DNA contents in a multiplicity of infection-dependent manners: Ad-WWOX infection up-regulated caspase-3 activity and reduced procaspase-3 and procaspase-8 levels. We also confirmed that restoration of WWOX gene suppressed cell growth in vitro and tumorigenicity in vivo. In addition, transduction of wild-type WWOX-expressing vector inhibited PANC-1 colony formation; however, substitution of Y33 of Wwox with arginine did not lead to inhibition of colony formation, suggesting the biological significance of the WW1 domain of Wwox for its tumor-suppressing activity. In PC tissue samples, abundant cytoplasmic Wwox expression was detected in the normal pancreatic duct epithelium, whereas Wwox expression was frequently reduced not only in a large fraction of PC but also in precancerous lesions in accord with the pancreatic intraepithelial neoplasia (PanIN) grade, which was closely correlated with patients' poorer outcome. Interestingly, the existence of Wwox expression was associated with elevated mothers against decapentaplegic homolog 4 (Smad4) protein levels in vitro and in vivo. These findings suggest that down-modulation of Wwox expression is an early event and may be associated with the down-regulation of Smad4 protein levels during pancreatic duct carcinogenesis.

Brain Derived Metastatic Prostate Cancer DU-145 Cells Are Effectively Inhibited In Vitro by Guava (Psidium gujava L.) Leaf Extracts

: The aqueous extract of Psidium guajava L. (PE) inhibited the cancer cell DU-145 in a dose- and time-dependent manner. At 1.0 mg/mL, PE reduced the viability of PCa DU-145 (the androgen independent PCa cells) to 36.1 and 3.59%, respectively after 48 h and 72 h of incubations. The absolute cell viability suppressing capability (VSC)AC could reach 262.5 cells-mL-h/mg on exposure to PE for 72 h, corresponding to the safe ranges, i.e. the percent viability suppressing rates (PVSR) of 2.72 and 2.41 folds for DU-145 comparing to PZ-HPV-7 cells when treated with PE at 0.5 and 1.0 mg/mL respectively for 72 h. In addition, the colony forming capability of DU-145 cells was apparently lowered. The suppressing rates of which reached 8.09 and 5.96 colony/mg/day for D-145 and PZ-HPV-7 cells, respectively within the concentration range of PE at 0.1 ∼ 0.25 mg/mL. Cell cycle arrests at G0/G1 phase in both cells were observed by TUNEL assay and flow cytometric analysis, yet more prominently evident in DU-145. In addition, suppression of the matrix metalloproteinases MMP-2 and MMP-9, and the upregulation of active caspase-3 at 0.10 to 1.0 mg/mL in DU-145 were also effected in a dose-dependent manner by PE at 0.25 to 1.0 mg/mL, implicating a potent anti-metastasis power of PE. Conclusively, we ascribe the anticancer activity of PE to its extraordinarily high polyphenolic (165.61 ± 10.39 mg/g) and flavonoid (82.85 ± 0.22 mg/g) contents. Furthermore, PE might be useful for treatment of brain derived metastatic cancers such as DU-145, acting simultaneously as both a chemopreventive and a chemotherapeutic.

Combination of non-viral connexin 43 gene therapy and docetaxel inhibits the growth of human prostate cancer in mice

Docetaxel (DTX) is used for the treatment of advanced hormone refractory prostate cancer. Connexin 43 (Cx43) is a tumor suppressor gene, and transfection of the Cx43 gene increases sensitivity to several chemotherapeutic agents. The objective of this study was to evaluate the effectiveness of combination therapy of Cx43-expressing plasmid DNA (pCMV-Cx43) and DTX both in vitro and in vivo using a non-viral vector in human prostate cancer PC-3 cells. Transfection of pCMV-Cx43 into the cells neither inhibited tumor growth nor increased gap junctional intercellular communication; however, combination therapy of pCMV-Cx43 and DTX significantly inhibited cell growth. Forced expression of Cx43 in the cells induced apoptotic cells by down-regulation of Bcl-2 expression and significantly more up-regulation of caspase-3 activity than either treatment alone. The combination of repeated intratumoral injection of pCMV-Cx43 (10 microg/tumor) with non-viral vector and a single intravenous injection of DTX (15 mg/kg) was compared with a repeated injection of Cx43 alone and a single injection of DTX alone on PC-3 tumor xenografts. Significant antitumoral effects were observed in mice receiving combined treatment, compared with DTX alone. The data presented here provide a rational strategy for treating patients with advanced hormone refractory prostate cancer.

Neuronal cell death induced by cystatin C in vivo and in cultured human CNS neurons is inhibited with cathepsin B

Cystatin C, a cysteine protease inhibitor, is implicated in pathogenesis of late-onset Alzheimer's disease and other neurological disorders. Our recent study showed that cystatin C injection into rat hippocampus induced neuronal cell death in granule cell layer of dentate gyrus in vivo. We further confirmed that cystatin C neurotoxicity was inhibited by simultaneous coapplication of cathepsin B, a cysteine protease. In vitro cytotoxicity was also studied in cultures of human CNS neurons, mixed cultures with astrocytes and A1 human hybrid neurons. Cystatin C induced neuronal cell death in a dose-dependent manner, which accompanied increased number of TUNEL (+) cells, up-regulation of active caspase-3 and DNA ladder. The results of the present study indicate that cystatin C participates in the process of apoptotic neuronal cell death in experimental conditions by means of inhibitory activity of cysteine proteases, and that cystatin C might be involved in the pathogenesis in human neurological disorders including Alzheimer's disease.

Polyamine Depletion Reduces TNFα/MG 132 ‐Induced Apoptosis in Bone Marrow Stromal Cells

Polyamines are powerful modulators of both growth and survival in mammalian cells. In this study, we investigated the possibility of attenuating the process of apoptosis in bone marrow stromal cells (BMSCs), which comprise mesenchymal stem cells, by reducing the intracellular levels of polyamines. BMSCs were isolated from rat femurs and expanded for 12 days. At this time, BMSCs were CD34neg, CD45neg, and mostly CD90pos. BMSCs were grown for an additional 2 days in the presence of 1 mM alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, which reduced the content of both putrescine and spermidine by nearly 90%. DFMO treatment progressively slowed down BMSC proliferation, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay, without arresting their growth completely. The effect of polyamine depletion on caspase-3 activity was evaluated in BMSCs after treatment with 500 U/ml tumor necrosis factor-alpha (TNFalpha) and 5 microM MG132, an inhibitor of proteasome. Caspase-3 activity increased linearly over a period of 24-hour stimulation (p<.01), but this augmentation was blunted by 50% after DFMO administration (p<.05). The effect of DFMO on TNFalpha/MG132-induced upregulation of caspase-3 activity was reversed by the addition of 100 microM putrescine, confirming that polyamines were really involved in the apoptotic process. Also, the number of apoptotic BMSCs after TNFalpha/MG132 treatment, as determined by terminal transferase-mediated dUTP nick end-labeling (TUNEL) assay, were threefold reduced after polyamine depletion (p<.05). On the contrary, DFMO did not affect the MG132-mediated increase in p53 abundance, nor its translocation to the nucleus. Thus, polyamine depletion can be considered a useful tool for counteracting programmed cell death in BMSCs without involving the p53 proapoptotic protein.

Differential inhibition of oxidized LDL-induced apoptosis in human endothelial cells treated with different flavonoids

High plasma level of cholesterol is a well-known risk factor for atherosclerotic diseases. Oxidized LDL induces cellular and nuclear damage that leads to apoptotic cell death. We tested the hypothesis that flavonoids may function as antioxidants with regard to LDL incubated with 5 microm-Cu(2+) alone or in combination with human umbilical vein endothelial cells (HUVEC). Cytotoxicity and formation of thiobarbituric acid-reactive substances induced by Cu(2+)-oxidized LDL were examined in the presence of various subtypes of flavonoid. Flavanols, flavonols and flavanones at a non-toxic dose of 50 microm markedly inhibited LDL oxidation by inhibiting the formation of peroxidative products. In contrast, the flavones luteolin and apigenin had no such effect, with >30 % of cells killed after exposure to 0.1 mg LDL/ml. Protective flavonoids, especially (-)-epigallocatechin gallate, quercetin, rutin and hesperetin, inhibited HUVEC nuclear condensation and fragmentation induced by Cu(2+)-oxidized LDL. In addition, immunochemical staining and Western blot analysis revealed that anti-apoptotic Bcl-2 expression was enhanced following treatment with these protective flavonoids. However, Bax expression and caspase-3 cleavage stimulated by 18 h incubation with oxidized LDL were reduced following treatment with these protective flavonoids. The down-regulation of Bcl-2 and up-regulation of caspase-3 activation were reversed by the cytoprotective flavonoids, (-)-epigallocatechin gallate, quercetin and hesperetin, at >/=10 microm. These results suggest that flavonoids may differentially prevent Cu(2+)-oxidized LDL-induced apoptosis and promote cell survival as potent antioxidants. Survival potentials of certain flavonoids against cytotoxic oxidized LDL appeared to stem from their disparate chemical structure. Furthermore, dietary flavonoids may have therapeutic potential for protecting the endothelium from oxidative stress and oxidized LDL-triggered atherogenesis.

Pectinesterase Inhibitor from Jelly Fig (Ficus awkeotsang Makino) Achene Induces Apoptosis of Human Leukemic U937 Cells

The antitumor activity of pectinesterase inhibitor (PEI), a group of cationic polypeptides, from jelly fig (Ficus awkeotsang Makino) achene was first examined as a treatment for leukemia in this study. PEI displayed strong growth inhibition against human leukemic U937 cells via induction of apoptosis in a dose- and time-dependent manner. At a level of 50 microg/mL, PEI inhibited 90% of cell growth, and the concentration of PEI required to induce 50% of cell viability (LC50) was about 180 microg/mL. Meanwhile, cell cycle arrest at G2/M phase was observed when cells were incubated with 100 microg PEI/mL for 24 h. PEI displayed a dose-dependent influence on mitochondria transmembrane potential (MTP, delta psi m) of cells when detected by a flow cytometry. MTP of more than 50% cells was reduced when cells were incubated with PEI at levels higher than 50 microg PEI/mL for 24 h. In addition, PEI upregulated caspase-3 activity. Taken together, PEI potently inhibited the proliferation of human leukemic U937 cells via cell cycle arrest and apoptosis in association with MTP reduction and caspase-3 activation, respectively, and showed therapy potential for U937 cells.

(-)Epigallocatechin Gallate and Quercetin Enhance Survival Signaling in Response to Oxidant-Induced Human Endothelial Apoptosis

We reported recently that (-)epigallocatechin gallate and quercetin inhibited H2O2-induced apoptosis through modulation of the expression of apoptosis-related Bcl-2 and Bax in endothelial cells. This study attempted to identify possible regulatory sites and mechanisms of antiapoptotic flavonoids, focusing on ROS-mediated signaling in HUVEC. The effects of apigenin on the signaling pathway downstream were compared. Submillimolar H2O2 caused >30% cell killing with intracellular oxidant generation. H2O2-induced oxidant generation markedly decreased total intracellular glutathione (GSH) levels. Micromolar (-)epigallocatechin gallate and quercetin partially eliminated the dichlorodihydrofluorescein (DCF) and phospho-p53 staining, suggesting that these flavonoids inhibited the accumulation of intracellular oxidants and nuclear transactivation of p53 in H2O2-exposed cells. In contrast, cells treated with apigenin remained DCF and phospho-p53 staining positive in response to H2O2. (-)Epigallocatechin gallate significantly raised the total GSH level that had been depleted by H2O2. Caspase-3 activity was enhanced by H2O2, and this increase was inhibited by (-)epigallocatechin gallate and quercetin. Additionally, the upregulation of caspase-3 activation was reversed by these flavonoids at > or =10 micromol/L; these inhibitory effects were dose dependent. Western blot data revealed that H2O2 upregulated phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), which was rapidly reversed by quercetin within 30 min; H2O2 activation of c-Jun was downregulated. (-)Epigallocatechin gallate inhibited H2O2-induced phosphorylation of JNK and p38 MAPK after 60 min. These results reveal that quercetin blocks JNK- and p38 MAPK-related signaling triggered by the oxidant and may regulate expression of apoptotic downstream genes, preventing apoptosis and promoting cell survival. (-)Epigallocatechin gallate may function as an antiapoptotic agent through other antiapoptotic pathways.

Doxycycline inhibits cell proliferation and invasive potential: combination therapy with cyclooxygenase-2 inhibitor in human colorectal cancer cells

Matrix metalloproteinases (MMPs) and cyclooxygenase (COX) enzymes play pivotal roles in the metastatic process of colorectal cancers. Inhibition of both MMPs and COX could be an attractive option for the inhibition of cell growth and invasion. Two human colorectal cancer cell lines, LS174T and HT29, were challenged with MMP inhibitor (doxycycline), selective COX-2 inhibitor (NS-398), or a combination of these agents to evaluate cancer cell proliferation and invasion. Dose-dependent growth inhibition was observed in both cell lines when they were treated with a single therapy. These effects were not related to MMP-2 or MMP-9 expression potential of the cell lines. Doxycycline (10 μg/mL) induced G 0/G 1 arrest, and 20 μg/mL provoked annexin V positivity and up-regulated caspase-3 activity in HT29 cells. However, 20 μg/mL doxycycline caused no distinct apoptotic change in LS174T cells. Although MMP expression was not inhibited by 5 to 10 μg/mL doxycycline or 50 to 100 μmol/L NS-398, MMPs' activities were down-regulated by these concentrations. Cellular invasion was noticed in LS174T cells, but their capacity for invasion was diminished by these inhibitors. The antiproliferative and antiinvasive effects of the combination therapy were more pronounced. Doxycycline (5 μg/mL) with 50 μmol/L NS-398 inhibited cell proliferation and doxycycline (5 μg/mL) with 100 μmol/L NS-398 attenuated MMP expression and activity, as well as capacity for invasion, compared with single therapy. These data suggest that combination therapy consisting of an MMP inhibitor with a COX-2 inhibitor is an attractive approach to the treatment of colorectal cancers. The use of this treatment regimen for chemoprevention or treatment of colorectal cancers should be considered in future clinical trials.

Thrombospondin-1 induces apoptosis in primary leukemia and cell lines mediated by CD36 and Caspase-3

Thrombospondin-1 (TSP-1) is a naturally occurring anti-angiogenic compound that induces apoptosis of endothelial and cancer cells via its receptor CD36. The objectives of our study were to investigate the in vitro effects of TSP-1 on the apoptosis of primary human leukemia cells as well as leukemia cell lines and the possible mechanism involving CD36. Our results demonstrated that TSP-1 induced apoptosis in CD36 positive cell lines CHRF-288-11, Meg-01 and HL-60, but not CD36 negative K562, at a dose-dependent manner as demonstrated by DNA ladder formation, Annexin V and propidium iodide (PI) stainings. The addition of anti-CD36 antibody FA6-152 or thrombopoietin (TPO) significantly nullified the effects of TSP-1. TSP-1-mediated apoptosis was consistently associated with the up-regulation of active Caspase-3. Responses of 2 CD36 positive primary AML samples to TSP-1 and FA6-152 were similar with those of leukemia cell lines. TSP-1 significantly induced apoptosis in B-ALL but the counter-effects of FA6-152 were less apparent. CD36 negative AML cells appeared less susceptible to TSP-1 and FA6-152. Our data provided strong evidence that TSP-1 exerted direct apoptotic effects on leukemia cells and could be developed as an adjunct to conventional therapy, particularly for leukemia cells that express CD36 or other TSP-1 receptors.

Hyperthermic preconditioning protects pig islet grafts from early inflammation but enhances rejection in immunocompetent mice.

The induction of heat shock proteins (HSP) protects isolated islet cells against the cytotoxicity of inflammatory mediators in vitro. Very little information is available about the effect of HSP overexpression on function of preconditioned islet grafts. The present study investigated the function of heat-exposed pig islets after transplantation into immunocompetent mice in comparison with in vitro resistance against inflammatory mediators. Pig islets were preconditioned at 43 degrees C or sham treated prior to subcapsular transplantation into diabetic C57/Bl6j mice. Nondiabetic mice simultaneously receiving preconditioned and control islets were subjected to bilateral nephrectomy for determination of pig insulin. Resistance against H2O2, NO, human Il-1beta, IFN-gamma, or TNF-alpha was assessed by trypan blue exclusion and insulin determination. Heat-induced protein expression was confirmed by Western blot analysis. Graft preconditioning increased resistance against H2O2, NO, or cytokines (p < 0.05) but decreased survival in nondiabetic mice (p < 0.05) and function in diabetic mice (p < 0.01). Upregulation of caspase-3 activity as well as Bax, Fas, FasL, and DFF expression (p < 0.05) indicated simultaneous induction of apoptosis. The coexpression of HSP and proapoptotic proteins reveals the dual character of the stress response simultaneously starting mechanisms for protection and apoptosis. In vitro assays seem to reflect only insufficiently the situation of islets after transplantation.

Ceramide induces neuronal apoptosis through the caspase-9/caspase-3 pathway

C 2-ceramide, a cell-permeable analog of ceramide, caused cell death in cultured rat cortical neuronal cells. C 2-ceramide-induced neuronal loss was accompanied by upregulation of caspase-3 activity, measured by cleavage of its fluorogenic substrate Ac-DEVD-AMC. Similar results were obtained when cortical neuronal cultures were treated with sphingomyelinase, an enzyme responsible for ceramide formation in the cell. Morphological evaluation of C 2-ceramide-treated cortical neurons showed nuclear condensation and fragmentation as visualized by Hoechst 33258 staining. Co-administration of the selective caspase-3 inhibitor z-DEVD-fmk or caspase-9 inhibitor z-LEHD-fmk significantly reduced C 2-ceramide-induced cell death, while co-application of the caspase-8, inhibitor z-IETD-fmk, was without effect. Immunoblot analysis of protein extracts from C 2-ceramide-treated cortical neuronal cultures revealed upregulation of active caspase-9 and caspase-3 protein levels, whereas presence of active caspase-8 immunoreactivity was undetectable in this system. Administration of C 2-ceramide to SH-SY5Y human neuroblastoma cells also caused apoptotic cell death. Moreover, ceramide-induced cell death was significantly decreased in caspase-9 dominant-negative SH-SY5Y cells, while both caspase-8 dominant-negative cultures and mock-transfected cells showed equally high levels of cell death following C 2-ceramide treatment. Taken together, these data suggest that neuronal death induced by ceramide may be linked to the caspase-9/caspase-3 regulated intrinsic pathway of cellular apoptosis.

A shift in the Bax/Bcl-2 balance may activate caspase-3 and modulate apoptosis in experimental glomerulonephritis

A shift in the Bax/Bcl-2 balance may activate caspase-3 and modulate apoptosis in experimental glomerulonephritis.BackgroundAlthough apoptosis has been linked to the renal cell deletion and ensuing renal fibrosis, its regulating mechanisms remain obscure. Of the known regulators of apoptosis, the best characterized is the Bax to Bcl-2 ratio. However, its importance in controlling apoptosis in glomerulonephritis is unclear. Here, using the nephrotoxic nephritis (NTN) model, we evaluated Bax/Bcl-2 in relation to changes in the apoptosis co-ordination enzyme, caspase-3.MethodsKidneys were harvested at days 7, 15, 30 and 45 post-injection of anti-glomerular basement membrane antibody into Wistar Kyoto rats. These were analyzed for apoptosis (in situ end labeling of fragmented DNA, light and electron microscopy), Bax/Bcl-2 protein (Western blotting), mRNA (Northern blotting) and distribution (immunohistochemistry), as well as caspase-3 activity (substrate cleavage assay), inflammation (ED1 staining), proliferation (proliferating cell nuclear antigen staining) and fibrosis (Masson's Trichrome staining).ResultsBax mRNA was significantly increased while that of Bcl-2 was decreased throughout the time course (+265% and -62% by day 45). Increased Bax and decreased Bcl-2 protein were noted, significantly so on day 7 (+177% and -21%) and day 45 (+363% and -17%). Bax protein was observed in dilated and atrophic tubules, sclerotic glomeruli and inflamed interstitium, while Bcl-2 was only visible in atrophic tubules. The ratios of Bax to Bcl-2 mRNA and protein were significantly increased at all time points. These correlated (P < 0.05) with up-regulated caspase-3 activity (r = 0.742 and 0.531), apoptosis (r = 0.712 and 0.540), proliferation (r = 0.611, mRNA only), inflammation (ED1+, r = 0.474 and 0.419) and fibrosis (r = 0.474 and 0.729).ConclusionsOur findings suggest that the changes in the ratio of Bax to Bcl-2 may contribute to the caspase-3 activation and the modulation of renal apoptosis associated with renal inflammation, tubular atrophy and renal fibrosis in experimental glomerulonephritis. A shift in the Bax/Bcl-2 balance may activate caspase-3 and modulate apoptosis in experimental glomerulonephritis. Although apoptosis has been linked to the renal cell deletion and ensuing renal fibrosis, its regulating mechanisms remain obscure. Of the known regulators of apoptosis, the best characterized is the Bax to Bcl-2 ratio. However, its importance in controlling apoptosis in glomerulonephritis is unclear. Here, using the nephrotoxic nephritis (NTN) model, we evaluated Bax/Bcl-2 in relation to changes in the apoptosis co-ordination enzyme, caspase-3. Kidneys were harvested at days 7, 15, 30 and 45 post-injection of anti-glomerular basement membrane antibody into Wistar Kyoto rats. These were analyzed for apoptosis (in situ end labeling of fragmented DNA, light and electron microscopy), Bax/Bcl-2 protein (Western blotting), mRNA (Northern blotting) and distribution (immunohistochemistry), as well as caspase-3 activity (substrate cleavage assay), inflammation (ED1 staining), proliferation (proliferating cell nuclear antigen staining) and fibrosis (Masson's Trichrome staining). Bax mRNA was significantly increased while that of Bcl-2 was decreased throughout the time course (+265% and -62% by day 45). Increased Bax and decreased Bcl-2 protein were noted, significantly so on day 7 (+177% and -21%) and day 45 (+363% and -17%). Bax protein was observed in dilated and atrophic tubules, sclerotic glomeruli and inflamed interstitium, while Bcl-2 was only visible in atrophic tubules. The ratios of Bax to Bcl-2 mRNA and protein were significantly increased at all time points. These correlated (P < 0.05) with up-regulated caspase-3 activity (r = 0.742 and 0.531), apoptosis (r = 0.712 and 0.540), proliferation (r = 0.611, mRNA only), inflammation (ED1+, r = 0.474 and 0.419) and fibrosis (r = 0.474 and 0.729). Our findings suggest that the changes in the ratio of Bax to Bcl-2 may contribute to the caspase-3 activation and the modulation of renal apoptosis associated with renal inflammation, tubular atrophy and renal fibrosis in experimental glomerulonephritis.

A shift in the Bax/Bcl-2 balance may activate caspase-3 and modulate apoptosis in experimental glomerulonephritis

Although apoptosis has been linked to the renal cell deletion and ensuing renal fibrosis, its regulating mechanisms remain obscure. Of the known regulators of apoptosis, the best characterized is the Bax to Bcl-2 ratio. However, its importance in controlling apoptosis in glomerulonephritis is unclear. Here, using the nephrotoxic nephritis (NTN) model, we evaluated Bax/Bcl-2 in relation to changes in the apoptosis co-ordination enzyme, caspase-3. Kidneys were harvested at days 7, 15, 30 and 45 post-injection of anti-glomerular basement membrane antibody into Wistar Kyoto rats. These were analyzed for apoptosis (in situ end labeling of fragmented DNA, light and electron microscopy), Bax/Bcl-2 protein (Western blotting), mRNA (Northern blotting) and distribution (immunohistochemistry), as well as caspase-3 activity (substrate cleavage assay), inflammation (ED1 staining), proliferation (proliferating cell nuclear antigen staining) and fibrosis (Masson's Trichrome staining). Bax mRNA was significantly increased while that of Bcl-2 was decreased throughout the time course (+265% and -62% by day 45). Increased Bax and decreased Bcl-2 protein were noted, significantly so on day 7 (+177% and -21%) and day 45 (+363% and -17%). Bax protein was observed in dilated and atrophic tubules, sclerotic glomeruli and inflamed interstitium, while Bcl-2 was only visible in atrophic tubules. The ratios of Bax to Bcl-2 mRNA and protein were significantly increased at all time points. These correlated (P < 0.05) with up-regulated caspase-3 activity (r = 0.742 and 0.531), apoptosis (r = 0.712 and 0.540), proliferation (r = 0.611, mRNA only), inflammation (ED1+, r = 0.474 and 0.419) and fibrosis (r = 0.474 and 0.729). Our findings suggest that the changes in the ratio of Bax to Bcl-2 may contribute to the caspase-3 activation and the modulation of renal apoptosis associated with renal inflammation, tubular atrophy and renal fibrosis in experimental glomerulonephritis.

CD 95-independent mechanisms of IL-2 deprivation-induced apoptosis in activated human lymphocytes.

Growth factor deprivation-induced apoptosis plays an important role in several cellular systems. However, knowledge of the molecular mechanisms involved are restricted to a few murine models or tumor cell lines. Therefore, we aimed studying signaling pathways leading to apoptosis in activated human peripheral T cells after IL-2 withdrawal. Lymphoblasts from patients with CD 95 (Fas/APO-1)-deficiency revealed that functional CD95 was not required to induce apoptosis after IL-2 withdrawal. Moreover, apoptosis induction in response to various cytotoxic stimuli was found to be mediated in the absence of functional CD95 but was affirmatorily influenced by IL-2 signaling. Immunoblots showed no downregulation of Bcl-2 or Bcl-xL and no upregulation of Bax, whereas decreased mitochondrial membrane potential was readily measurable 24 h after cytokine deprivation. Tetrapeptide inhibitors showed limited efficacy in preventing apoptosis whereas the caspase inhibitor zVAD-FMK potently blocked induction of apoptosis. Cleavage of different fluorogenic substrates revealed multiple caspase enzyme activities in lymphoblasts, which were not negatively affected by the fas mutation. Starting at 8 h after IL-2 withdrawal, upregulation of active caspase-3 but not of caspase-8 could be detected. Taken together, our data argue for molecular mechanisms of cytokine deprivation-induced apoptosis in activated human lymphocytes independent of CD95.

Zinc protects against apoptosis of endothelial cells induced by linoleic acid and tumor necrosis factor α

Zinc requirements of the vascular endothelium may be increased in inflammatory conditions, ie, atherosclerosis, in which apoptotic cell death is prevalent. We hypothesized that zinc deficiency may potentiate disruption of endothelial cell integrity mediated by fatty acids and inflammatory cytokines by enhancing pathways that lead to apoptosis and up-regulation of caspase genes. Endothelial cells were maintained in low-serum medium or grown in culture media containing selected chelators, ie, diethylenetriaminepentaacetate or N,N,N', N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), with or without zinc supplementation. Subsequently, cells were treated with linoleic acid, tumor necrosis factor alpha (TNF-alpha), or both. We studied the effect of zinc deficiency and supplementation on the induction of apoptosis by measuring caspase-3 activity, cell binding of annexin V, and DNA fragmentation. Our results indicated that linoleic acid and TNF-alpha independently, but more markedly in concert, up-regulated caspase-3 activity and induced annexin V binding and DNA fragmentation. Zinc deficiency, especially when induced by TPEN, dramatically increased apoptotic cell death induced by cytokines and lipids compared with control cultures. Supplementation of low-serum- or chelator-treated endothelial cells with physiologic amounts of zinc caused a marked attenuation of apoptosis induced by linoleic acid and TNF-alpha. Morphologic changes of cells observed during zinc deficiency were prevented by zinc supplementation. Media supplementation with other divalent cations (eg, calcium and magnesium) did not mimic the protective role of zinc against apoptosis. Our data indicate that zinc is vital to vascular endothelial cell integrity, possibly by regulating signaling events to inhibit apoptotic cell death.