PURPOSE: The present study investigated whether an autophagic process is involved in the apoptotic death of human tenon's capsule fibroblasts (HTCFs) caused by mitomycin-C. METHODS: An autophagic phenotype was tested using fluorescence microscopy and flow cytometry with specific biological staining dyes including monodansylcadaverine and acridine orange and microtubule-associated protein 1 light chain 3 (LC3). RESULTS: Treatment with mitomycin-C (0.4 mg/ml) increased the acidic vesicular organelles of tenon's capsule fibroblasts in a time dependent manner. Mitomycin-C induced both LC3-II cleavage and beclin-1 expression. 3-MA, a pharmacological inhibitor of autophagy, inhibited the mitomycin-C induced increase of acidic vesicular organelleS. CONCLUSIONS: Autophagy was induced with 0.4 mg/ml mitomycin-C in tenon's capsule fibroblasts. And, autophagic mechanisms may be involved in the early stage of apoptosis of fibroblasts.
Acridine Orange ; Apoptosis ; Autophagy ; Cadaverine ; Coloring Agents ; Fibroblasts ; Flow Cytometry ; Humans ; Light ; Microscopy, Fluorescence ; Microtubule-Associated Proteins ; Mitomycin ; Organelles ; Phenotype ; Tenon Capsule

Adriamycin (ADR) is a potent anticancer drug that causes often severe cardiomyopathy. Previous reports have demonstrated that zinc accumulation is shown in rat myocardial cells following ADR treatment. However, the mechanism and role of zinc accumulation in ADR-induced cardiomyopathy are not yet elucidated. Zinc may be one of the key executors in ADR-induced cardiomyopathy. To test this hypothesis, we examined the cytotoxic effects of zinc on various cell lines including H9c2 cardiomyoblast cells, HL-60, U937, and C(6)-glial cells. Zinc induced significant the death of H9c2 cells at 0.125 mM in a dose-dependent manner. However, zinc did not induce any cytotoxic effect on both promyelocytic leukemic HL-60 cells and monoblastoid U937 cells. The nuclear morphology of Zn(2+)-treated H9c2 cells displayed apparent chromatin condensation, but no formation of chromatin fragmentation. In addition, phosphatidylserine (PS) externalization was observed by annexin-V staining. Zinc markedly decreased the intracellular GSH level in a time-dependent manner. Exposure to 0.2 mM ZnCl(2) for 6 hr decreased the intracellular GSH content to 13% of control value. Zinc-induced death of H9c2 cells and the intracellular GSH depletion were completely prevented by the addition of exogenous GSH and NAC. These result suggests that intracellular GSH depletion is directly involved in zinc-induced cardiomyopathy.
Animals ; Cardiomyopathies ; Cell Death* ; Cell Line ; Chromatin ; Doxorubicin ; Free Radicals ; HL-60 Cells ; Humans ; Rats ; U937 Cells ; Zinc

Background: Diabetes mellitus is one of the most common chronic diseases worldwide, and cardiovascular disease is the leading cause of morbidity and mortality in diabetic patients. Diabetic cardiomyopathy (DCM) is a phenomenon characterized by a deterioration in cardiac function and structure, independent of vascular complications. Among many possible causes, the renin-angiotensin-aldosterone system and angiotensin II have been proposed as major drivers of DCM development. In the current study, we aimed to investigate the effects of pharmacological activation of angiotensin-converting enzyme 2 (ACE2) on DCM. Methods: The ACE2 activator diminazene aceturate (DIZE) was administered intraperitoneally to male db/db mice (8 weeks old) for 8 weeks. Transthoracic echocardiography was used to assess cardiac mass and function in mice. Cardiac structure and fibrotic changes were examined using histology and immunohistochemistry. Gene and protein expression levels were examined using quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. Additionally, RNA sequencing was performed to investigate the underlying mechanisms of the effects of DIZE and identify novel potential therapeutic targets for DCM. Results: Echocardiography revealed that in DCM, the administration of DIZE significantly improved cardiac function as well as reduced cardiac hypertrophy and fibrosis. Transcriptome analysis revealed that DIZE treatment suppresses oxidative stress and several pathways related to cardiac hypertrophy. Conclusion DIZE prevented the diabetes mellitus-mediated structural and functional deterioration of mouse hearts. Our findings suggest that the pharmacological activation of ACE2 could be a novel treatment strategy for DCM.

PURPOSE: In order to investigate the role of Fas on the chemosensitivity of cancer cells in regards to chemotherapeutic agents, the Fas/FasL signaling pathway of apoptosis was explored in human hepatoma cells. MATERIALS AND METHODS: Fas expression of hepatoma cells including Chang, Huh7, HepG2, and Hep3B cells, was determined by RT-PCR and flow cytometry analysis. Cell viability was measured by MTT assay and apoptosis was assessed by DNA fragmentation assay. The catalytic activity of the caspase-family proteases including caspase-3, 6, 8, and 9 proteases, was tested using fluorogenic biosubstrates. The expression of apoptotic mediators including cytochrome c, PARP, and Bcl2 family proteins were measured from cytosolic and mitochondrial compartments. Mitochondrial membrane potential was measured by fluorescence staining with JC-1, rhodamine 123. RESULTS: Fas mRNA was constitutively expressed in Chang and HepG2 as defined as Fas (+) cells, but not in Huh7 and Hep3B cells, defined as Fas (-) cells. Fas (+) cells were markedly sensitive to 5-FU whereas Fas (-) cells were resistant and able to survive. 5-FU increased Fas expression of Fas (+) HepG2 cells and simultaneously resulted in apoptotic death, characterized by the ladder-pattern fragmentation of genomic DNA. Moreover, it increased the catalytic activity of caspase-8 protease, which eventually cleaved the Bid into truncated Bid which translocated into mitochondria only in Fas (+) cells. It also increased the caspase-9 protease activity with Bax expression, cytosolic release of cytochrome c, and mytochondrial dysfunction only in Fas (+) HepG2 cells. Furthermore, 5-FU increased the enzymatic activity of caspase-3 protease with PARP digestion in HepG2 cells. CONCLUSION: 5-FU exerted cytotoxicity against hepatoma cells via activation of Fas-mediated apoptotic signaling including caspase cascades and mytochondrial dysfunction. Our data suggests that Fas may be an important modulator of the chemosensitivity of cancer cells vis- -vis anticancer chemotherapeutic agents.
Apoptosis* ; Carcinoma, Hepatocellular ; Caspase 3 ; Caspase 8 ; Caspase 9 ; Cell Survival ; Cytochromes c ; Cytosol ; Digestion ; DNA ; DNA Fragmentation ; Flow Cytometry ; Fluorescence ; Fluorouracil* ; Hep G2 Cells* ; Humans ; Membrane Potential, Mitochondrial ; Mitochondria* ; Peptide Hydrolases ; Rhodamine 123 ; RNA, Messenger

Gliotoxin, a fungal metabolite, is one of the epipolythiodioxopiperazine classes and has a variety of effects including imrnunomodulatory and apoptotic agents. This study is designed to evaluate the effect of zinc on gliotoxin-induced death of HL-60 cells. Here, we demonstrated that treatment of gliotoxin decreased cell viability in a dose and time-dependent manner. Gliotoxin-induced cell death was confirtned as apoptosis characterized by chromatin marginafion, fragmentation and ladder-pattern digestion of genomic DNA. Gliotoxin increased the proteolytic activities of caspase 3, 6, 8, and 9. Caspase-3 activation was further confirmed by the degradation of procaspase-3 and PARP in gliotoxin-treated HL-60 cells. Zinc compounds including ZnC12 and ZnSO4 markedly inhibited gliotoxin-induced apoptosis in HL-60 cells (from 30% to 90%). Consistent with anti- apoptotic effects, zinc also suppressed the enzymatic activities of caspase-3 and -9 proteases. In addition, cleavage of both PARP and procaspase 3 in gliotoxin-treated HL-60 cells was inhibited by the addition of zinc compounds. We further demonstrated that expression of Fas ligand by gliotoxin was suppressed by zinc compounds. These data suggest that zinc may prevent gliotoxin- induced apoptosis via inhibition of Fas ligand expression as well as suppression of caspase family cysteine proteases-3 and -9 in HL-60 cells.
Apoptosis* ; Caspase 3 ; Cell Death ; Cell Survival ; Chromatin ; Cysteine ; Digestion ; DNA ; Fas Ligand Protein ; Gliotoxin* ; HL-60 Cells ; Humans ; Peptide Hydrolases ; Zinc Compounds ; Zinc*

Trichostatin A (TSA), originally developed as an antifungal agent, is one of potent histone deacetylase (HDAC) inhibitors, which are known to cause growth arrest and apoptosis induction of transformed cells, including urinary bladder, breast, prostate, ovary, and colon cancers. However, the effect of HDAC inhibitors on human non-small cell lung cancer cells is not clearly known yet. Herein, we demonstrated that treatment of TSA resulted in a significant decrease of the viability of H157 cells in a dose-dependent manner, which was revealed as apoptosis accompanying with nuclear fragmentation and an increase in sub-G0/G1 fraction. In addition, it induced the expression of Fas/FasL, which further triggered the activation of caspase-8. Catalytic activation of caspase-9 and decreased expression of anti-aptototic Bcl-2 and Bcl-XL proteins were observed in TSA-treated cells. Catalytic activation of caspase-3 by TSA was further confirmed by cleavage of pro-caspase-3 and intracellular substrates, including poly (ADP-ribose) polymerase (PARP) and inhibitor of caspase-activated deoxyribonuclease (ICAD). In addition, a characteristic phenomenon of mitochondrial dysfunction, including mitochondrial membrane potential transition and release of mitochondrial cytochrome c into the cytosol was apparent in TSA-treated cells. Taken together, our data indicate that inhibition of HDAC by TSA induces the apoptosis of H157 cells through signaling cascade of Fas/FasL-mediated extrinsic and mitocondria-mediated intrinsic caspases pathway.
Signal Transduction ; Receptors, Death Domain/*metabolism ; Protein Isoforms/metabolism ; Mitochondria/*drug effects/*metabolism ; Lung Neoplasms/*metabolism/*pathology ; Hydroxamic Acids/*pharmacology ; Humans ; Histones/metabolism ; Enzyme Activation ; Cell Line, Tumor ; Catalysis ; Caspase 9/metabolism ; Caspase 8/metabolism ; Caspase 3/metabolism ; Apoptosis/*drug effects ; Acetylation

PURPOSE: Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL)/APO-2L is a member of the TNF family that can kill a wide variety of tumor cells, but not normal cells. This study was designed to investigate the down stream target proteins in TRAIL-mediated apoptosis of human liver, Chang cells. MATERIALS AND METHODS: The expressions of DR4/DR5 in hepatoma cells, including Chang, HepG2 and Hep3B cells, were determined by RT-PCR. Cell viability was measured by MTT assay and apoptosis was assessed by DNA fragmentation assay. The catalytic activity of caspase- family proteases, including caspase-3 and -9, was tested by using fluorogenic biosubstrates. Expression of apoptotic mediators, including procaspase-3 and PARP proteins, was measured by Western blotting. The expression profile of proteins in Chang cells by using two-dimensional (2-D) gel electrophoresis and MALDI-TOF. RESULTS: The results demonstrated that TRAIL (100 ng/ml) induced the apoptotic death of Chang cells, as characterized by the ladder-pattern fragmentation of genomic DNA. TRAIL increased the enzymatic activity of caspase- 3, corresponding to the time of appearance of cleaved PARP and caspase-9. In 2-D gel electrophoresis and MALDI- TOF analysis, the comparison of control versus apoptotic cells in the protein expressions revealed that signal intensity of 7 spots were decreased, whereas 6 spots were increased among 300 spots. These spots were resolved and identified as a protein information by MALDI-TOF. CONCLUSION: We suggested that TRAIL induces the apoptotic death of Chang cells via proteome alterations inducing caspase cascade.
Apoptosis* ; Blotting, Western ; Carcinoma, Hepatocellular ; Caspase 3 ; Caspase 9 ; Cell Survival ; DNA ; DNA Fragmentation ; Electrophoresis ; Electrophoresis, Gel, Two-Dimensional ; Humans* ; Liver* ; Peptide Hydrolases ; Proteome ; Rivers ; Tumor Necrosis Factor-alpha

The stress activated protein kinase, or Jun N-terminal kinase (SAPKs/JNKs), is activated in response to a variety of cellular stresses such as changes in osmolarity and metabolism, DNA damage, heat shock, ischemia, and inflammatory cytokines. Sek1 (JNKK/MKK4) is a direct activator of SAPKs/JNKs in response to environmental stresses or mitogenic factors. Thus, this study was conducted to investigate the role of Sek1 on nitric oxide (NO) induced apoptotic signaling pathway in H9c2 cell. The viability of SNP (Sodium Nitroprusside) treated inactive Sek1 kinase transfectants [Sek1/KI H9c2] is significantly decreased and SNP induce DNA fragmentation in Sek1/KI H9c2. Interestingly, concomitantly with SNP induced injuries, caspase 3-like activity is increased but caspase 1 like activity is not changed in Sek1/KI H9c2. Whereas wild type Sek1 kinase transfectants [Sek1/WT H9c2] is less susceptible to SNP induced apoptosis. In Sek1/KI H9c2, the injuries and DNA fragmentation by SNP is protected by adding Ac-DEVD-AMC, caspase 3 inhibitor. In conclusion, these results suggest that Sek1 plays a role in protecting NO-induced apoptosis and DNA fragmentaion in H9c2 cells by inhibiting caspase 3-like activation.
Apoptosis* ; Caspase 1 ; Caspase 3* ; Cytokines ; DNA ; DNA Damage ; DNA Fragmentation ; Hot Temperature ; Ischemia ; Metabolism ; Nitric Oxide ; Osmolar Concentration ; Phosphotransferases ; Protein Kinases ; Shock

BACKGROUND: Although many studies regarding several neurotransmitters and receptors have been conducted to define the mechanism involved in the development of dependence on opioids, definitive evidence has still not been presented. This study was designed to investigate the effect of morphine on glutamate-induced cytotoxicity of rat C6 glial cells. METHODS: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was used for cell viability. Morphology of nuclei was observed by fluorescent microscopy. Reduced glutathione (GSH) contents were measured in acid-soluble cell fractions. Generation of hydrogen peroxide (H2O2) was measured from the cultured supernatant of C6 glial cells using the scopoletin-horseradish peroxidase (HRP) assay. RESULTS: Glutamate induced the death of C6 glial cells in a time- and dose-dependent manner. Glutamate-induced cytotoxicity was protected by morphine and antioxidants, such as GSH and N-acetyl-L-cysteine (NAC). However, morphine antagonist, naloxone did not inhibit the protective effect of morphine on glutamate-induced cytotoxicity. In addition, the specific agonists, [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt (DAMGO), [D-Pen2,5]-Enkephalin (DPDPE) and U69593 did not protect C6 glial cells from glutamate-induced cytotoxicity. Furthermore, morphine recovered the depletion of GSH by glutamate and inhibited the generation of H2O2 by glutamate in C6 glial cells. CONCLUSIONS: We suggest that morphine protects C6 glial cells from glutamate-induced cytotoxicity via the inhibition of GSH depletion and the generation of H2O2 by glutamate.
Acetylcysteine ; Analgesics, Opioid ; Animals ; Antioxidants ; Cell Survival ; Glutamic Acid ; Glutathione ; Hydrogen Peroxide ; Microscopy ; Morphine* ; Naloxone ; Neuroglia* ; Neurotransmitter Agents ; Peroxidase ; Rats

We demonstrated previously that Coptidis rhizoma extract (CRE) prevented S-nitroso-N-acetylpenicillamine-induced apoptotic cell death via the inhibition of mitochondrial membrane potential disruption and cytochrome c release in RINm5F (RIN) rat insulinoma cells. In this study, the preventive effects of CRE against cytokine-induced beta-cell death was assessed. Cytokines generated by immune cells infiltrating pancreatic islets are crucial mediators of beta-cell destruction in insulin-dependent diabetes mellitus. The treatment of RIN cells with IL-1beta and IFN-gamma resulted in a reduction of cell viability. CRE completely protected IL-1beta and IFN-gamma-mediated cell death in a concentration-dependent manner. Incubation with CRE induced a significant suppression of IL-1beta and IFN-gamma-induced nitric oxide (NO) production, a finding which correlated well with reduced levels of the iNOS mRNA and protein. The molecular mechanism by which CRE inhibited iNOS gene expression appeared to involve the inhibition of NF-kappa B activation. The IL-1beta and IFN-gamma-stimulated RIN cells showed increases in NF-kappa B binding activity and p65 subunit levels in nucleus, and IkappaBalpha degradation in cytosol compared to unstimulated cells. Furthermore, the protective effects of CRE were verified via the observation of reduced NO generation and iNOS expression, and normal insulin-secretion responses to glucose in IL-1beta and IFN-gamma-treated islets.
Animals ; Cell Death/drug effects ; Cell Line ; Cell Nucleus/metabolism ; Cell Survival/drug effects ; Drugs, Chinese Herbal/*pharmacology ; Gene Expression Regulation, Enzymologic/drug effects ; Glucose/pharmacology ; I-kappa B Proteins/metabolism ; Insulin/secretion ; Insulin-Secreting Cells/*cytology/*drug effects/enzymology ; Interferon-gamma/*pharmacology ; Interleukin-1beta/*pharmacology ; Male ; NF-kappa B/*metabolism ; Nitric Oxide/biosynthesis ; Nitric Oxide Synthase Type II/genetics/metabolism ; Protein Transport/drug effects ; RNA, Messenger/genetics/metabolism ; Rats ; Rats, Sprague-Dawley