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PubMed2004

Rat cerebellar granule cells are protected from glutamate-induced excitotoxicity by S-nitrosoglutathione but not glutathione.

In cultured rat cerebellar granule cells, glutamate or N-methyl-d-aspartate (NMDA) activation of the NMDA receptor caused a sustained increase in cytosolic Ca(2+) levels ([Ca(2+)](i)), reactive oxygen species (ROS) generation, and cell death (respective EC(50) values for glutamate were 12, 30, and 38 microM) but no increase in caspase-3 activity. Removal of extracellular Ca(2+) blocked all three glutamate-induced effects, whereas pretreatment with an ROS scavenger inhibited glutamate-induced cell death but had no effect on the [Ca(2+)](i) increase. This indicates that glutamate-induced cell death is attributable to [Ca(2+)](i) increase and ROS generation, and the [Ca(2+)](i) increase precedes ROS generation. Apoptotic cell death was not seen until 24 h after exposure of cells to glutamate. S-nitrosoglutathione abolished glutamate-induced ROS generation and cell death, and only a transient [Ca(2+)](i) increase was seen; similar results were observed with another nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine, but not with glutathione, which suggests that the effects were caused by NO. The transient [Ca(2+)](i) increase and the abolishment of ROS generation induced by glutamate and S-nitrosoglutathione were still seen in the presence of an ROS scavenger. Glial cells, which were present in the cultures used, showed no [Ca(2+)](i) increase in the presence of glutamate, and glutamate-induced granule cell death was independent of the percentage of glial cells. In conclusion, NO donors protect cultured cerebellar granule cells from glutamate-induced cell death, which is mediated by ROS generated by a sustained [Ca(2+)](i) increase, and glial cells provide negligible protection against glutamate-induced excitotoxicity.

Animals↗
PubMed2003

Apoptosis of bladder cancer cells induced by short-term and low-dose Mitomycin-C: potential molecular mechanism and clinical implication.

Adjuvant chemotherapies have been used to prevent recurrence of bladder transitional cell carcinoma (TCC), but their efficacies are variable due to the side effects of anti-cancer agents and the drug resistant property of the target cells. To provide experimental evidence for improving clinical management of TCCs, two human TCC cell lines, EJ and BIU, were treated for 1 or 2 h by 50, 100, 150 and 200 micro g/ml, respectively and their growth and death patterns were elucidated in 12-h intervals. The status of Fas, FasL and caspase-3 in the two cell lines were analyzed with immunocytochemical staining and Western blot hybridization, and their potential link to MMC-induced cell death was investigated by treating the cells with anti-Fas antibody (150 ng/ml) and by incubating the cells with an inhibitor of caspase-3 related proteases Ac-DEVO-CHO (250 micro M) 1 h before 100 micro g/ml MMC treatment. The results demonstrated that the lower dose (100 micro g/ml) and short-term (1 h) MMC treatment could induce sufficient apoptosis in EJ and BIU populations within 48 or 60 h. Constitutive soluble and membrane Fas and FasL were found in both cell lines, and caspase-3 could be upregulated after MMC treatment. Anti-Fas antibody could commit the target cells to die of apoptosis, while Ac-DEVD-CHO inhibited MMC-induced apoptosis. Our data thus suggest that MMC-induced apoptosis in EJ and BIU cells is mediated by Fas and upregulation and activation of caspase-3 is an essential element for the apoptotic process. Reduced dose and short-term MMC strategy would be of practical value either in determining the apoptotic susceptibility of individual TCC cases or in the clinical instillation of urothelial cancers.

Antibiotics, Antineoplastic↗
PubMed2002

Upregulation of the Fas receptor death-inducing signaling complex after traumatic brain injury in mice and humans.

Recent studies have implicated Fas in the pathogenesis of inflammatory, ischemic, and traumatic brain injury (TBI); however, a direct link between Fas activation and caspase-mediated cell death has not been established in injured brain. We detected Fas-Fas ligand binding and assembly of death-inducing signaling complexes (DISCs) [Fas, Fas-associated protein with death domain, and procaspase-8 or procaspase-10; receptor interacting protein (RIP)-RIP-associated interleukin-1beta converting enzyme and CED-3 homolog-1/Ced 3 homologous protein with a death domain-procaspase-2] by immunoprecipitation and immunoblotting within mouse parietal cortex after controlled cortical impact. At the time of DISC assembly, procaspase-8 was cleaved and the cleavage product appeared at 48 hr in terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive neurons. Cleavage of caspase-8 was accompanied by caspase-3 processing detected at 48 hr by immunohistochemistry, and by caspase-specific cleavage of poly(ADP-ribose) polymerase at 12 hr. Fas pathways were also stimulated by TBI in human brain, because Fas expression plus Fas-procaspase-8 interaction were robust in contused cortical tissue samples surgically removed between 2 and 30 hr after injury. To address whether Fas functions as a death receptor in brain cells, cultured embryonic day 17 cortical neurons were transfected with an adenoviral vector containing the gene encoding Fas ligand. After 48 hr in culture, Fas ligand expression and Fas-procaspase-8 DISC assembly increased, and by 72 hr, cell death was pronounced. Cell death was decreased by approximately 50% after pan-caspase inhibition (Z-Val-ALa-Asp(Ome)-fluoromethylketone). These data suggest that Fas-associated DISCs assemble in neurons overexpressing Fas ligand as well as within mouse and human contused brain after TBI. Therefore, Fas may function as a death receptor after brain injury.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Co-localized expression of FasL, Fas, Caspase-3 and apoptotic DNA fragmentation in mouse testis after oral exposure to di(2-ethylhexyl)phthalate.

Expression of apoptosis-related proteins FasL, Fas and Caspase-3, as well as DNA fragmentation were examined in mouse testis 12 h after exposure to 4-0.004 mg/g di(2-ethylhexyl)phthalate (DEHP). Immunocytochemical examination of the highest dose (4 mg/g DEHP) mouse revealed a distribution of FasL in Sertoli cell and Fas in nearby spermatocyte, and Fas and Caspase-3 in the same spermatocyte. Fas-positive spermatocytes had a DNA-fragmented nucleus detectable by terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL) method. After exposure to 4, 0.4, 0.04 or 0.004 mg/g DEHP, the maximum number of nuclei with fragmented DNA per 0.5 microm testis section was 22, 7, 5 and 3, respectively. In unexposed control the maximum number was 3. To further estimate total amount of the fragmented DNA in testis of the exposed mouse, the extracted DNA fragments were analyzed by agarose gel electrophoresis. The amount of fragments in the first three steps of the DNA ladder was estimated by a photo-densitometry. In the highest dose mouse (4 mg/g DEHP), the fragmented DNA was 2.2 times as much in the control. In lower dose mouse (0.4, 0.04 or 0.004 mg/g DEHP), it was 1.1 times as much in the control. Taken together, these observations suggest that a single oral exposure to DEHP as low as 0.04 mg/g might be effective to testicular DNA fragmentation and apoptosis.

Administration, Oral↗
PubMed2003

PPARgamma pathway activation results in apoptosis and COX-2 inhibition in HepG2 cells.

AIM: To investigate whether troglitazone (TGZ), the peroxisome proliferator-activated receptor (PPAR) gamma ligand, can induce apoptosis and inhibit cell proliferation in human liver cancer cell line HepG2 and to explore the molecular mechanisms. METHODS: (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), ((3)H) Thymidine incorporation, Hochest33258 staining, DNA ladder, enzyme-linked immunosorbent assay (ELISA), RT-PCR, Northern and Western blotting analyses were employed to investigate the effect of TGZ on HepG2 cells and related molecular mechanisms. RESULTS: TGZ was found to inhibit the growth of HepG2 cells and to induce apoptosis. During the process, the expression of COX-2 mRNA and protein and Bcl-2 protein was down-regulated, while that of Bax and Bak proteins was up-regulated, and the activity of caspase-3 was elevated. Furthermore, the level of PGE(2) was decreased transiently after 12 h of treatment with 30 microM troglitazone. CONCLUSION: TGZ inhibits cell proliferation and induces apoptosis in HepG2 cells, which may be associated with the activation of caspase-3-like proteases, down-regulation of the expression of COX-2 mRNA and protein, Bcl-2 protein, the elevation of PGE2 levels, and up-regulation of the expressions of Bax and Bak proteins.

Apoptosis↗
PubMed2000

Activity of the caspase-3/CPP32 enzyme is increased in "early stage" myelodysplastic syndromes with excessive apoptosis, but caspase inhibition does not enhance colony formation in vitro.

OBJECTIVE: Excessive apoptosis may have a role in the ineffective hematopoiesis and cytopenias observed in myelodysplastic syndromes. The goals of this study were 1) to quantify apoptosis in patients with "early stage" myelodysplasia [including patients with refractory anemia (RA), RA with ringed sideroblasts (RARS), RA with excess blasts and with less than 10% blasts (RAEB(<10))], and in patients with "late stage" myelodysplasia [including RAEB with more than 10% blasts (RAEB(>10)), RAEB in transformation (RAEB-t), and acute myeloid leukemia secondary to myelodysplasia (LAM2)]; 2) to study the activation of the caspase-3/CPP32 enzyme, a major "effector" caspase in hematopoiesis, in patients with "early stage" myelodysplasia, and 3) to evaluate the effect of caspase inhibition on the apoptotic phenotype and clonogenicity of hematopoietic progenitors in vitro in these patients. MATERIALS AND METHODS PATIENTS: Fifty-four patients with myelodysplastic syndromes, including 30 with "early stage" myelodysplasia and 24 with "late stage" myelodysplasia were studied. Study of apoptosis: TUNEL assay performed on bone marrow smears and/or quantification of annexin V positive bone marrow mononuclear cells by flow cytometric analysis. Caspacse-3/CPP32 activity: Quantitative measurement of caspase-3/CPP32 activity on total bone marrow mononuclear cells using a fluorogenic substrate. Effect of the caspase-inhibitor Z-VAD-FMK: 1) on the apoptotic phenotype of total bone marrow mononuclear cells and 2) on the clonogenicity of hematopoietic progenitor cells. RESULTS: The group of 30 patients with "early stage" myelodysplasia had statistically increased apoptosis compared to the group of 24 patients with "late stage" myelodysplasia (44.1% +/- 4.8 vs 21.8% +/- 3.6; p = 0.02) using the TDT-mediated dUTP nick-end labeling (TUNEL) assay. In the group of patients with RAEB, those with MDS(RAEB<10) had excessive apoptosis compared to those with MDS(RAEB>10) (44.0% +/- 3.5% vs 29.5% +/- 3.6%;p = 0.042) The median caspase-3 activity in 20 "early stage" myelodysplasia patients was 19,000 U (range 3,460-41,000) and significantly increased compared to normal individuals (4,256 U, range 3,200-5,200; p = 0.032) Bone marrow mononuclear cells from 12 "early stage" MDS patients (including 11 from the 20 studied for caspase-3 activity) were incubated with or without the broad-spectrum caspase inhibitor Z-VAD-FMK. In 4 of 9 evaluable patients (44.4%) with excessive apoptosis, the number of annexin V positive cells decreased in a dose-dependent manner in the presence of Z-VAD-FMK. However, in none of these patients was caspase inhibition with Z-VAD-FMK able to enhance colony formation in vitro. CONCLUSION: These results confirm that a major characteristic of patients with "early stage" myelodysplasia is increased apoptosis. The results also indicate that excessive apoptosis in these patients is accompanied by increased caspase-3/CPP32 activity. However, caspase inhibition with the broad-spectrum inhibitor Z-VAD-FMK cannot improve hematopoiesis in this group of patients, even when apoptosis is attenuated.

Amino Acid Chloromethyl Ketones↗
PubMed2001

Chondrocyte death in experimental osteoarthritis is mediated by MEK 1/2 and p38 pathways: role of cyclooxygenase-2 and inducible nitric oxide synthase.

OBJECTIVE: To explore the mechanisms responsible for in situ induction of chondrocyte death in experimental dog osteoarthritic (OA) cartilage. The roles of 2 mitogen activated protein kinases (MAPK), MEK 1/2 and p38, nuclear factor-kappaB (NF-kappaB), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and the caspase cascade were investigated. METHODS: OA knee cartilage was obtained from dogs that had received sectioning of the anterior cruciate ligament and were sacrificed 12 weeks after surgery. Cartilage explants were cultured in different inhibitors: Z-DEVD-FMK (caspase 3 inhibitor), Z-LEHD-FMK (caspase 9 inhibitor), PD 98059 (MEK 1/2 inhibitor). SB 202190 (p38 inhibitor), SN-50 (NF-kappaB inhibitor), NS-398 (COX-2 inhibitor), N-iminoethyl-l-lysine (L-NIL) (iNOS inhibitor). Cartilage specimens were stained for TUNEL reaction and immunostained using specific antibodies for caspase 3, COX-2, iNOS, and nitrotyrosine. Morphometric analyses were performed. RESULTS: The significant level of chondrocyte death in OA cartilage was markedly decreased by caspase 3 and caspase 9 inhibitors. The two MAPK inhibitors, but not the NF-kappaB inhibitor, decreased chondrocyte death concomitant with the levels of caspase 3 and iNOS. COX-2 level was reduced by all 3 inhibitors. Specific inhibition of either COX-2 or iNOS reduced the level of chondrocyte death and caspase 3. There was evidence of crosstalk between these 2 latter systems; specific inhibition of COX-2 reduced the iNOS level, and selective inhibition of iNOS reduced COX-2 expression. COX-2 and iNOS seem to function in a positive autoregulatory manner that triggers transcription of their own biosynthetic machinery, since the specific inhibition of each system downregulates its expression. CONCLUSION: This study shows that in the early lesions of experimental OA cartilage in situ, activation of the caspase cascade is responsible for induction of chondrocyte death. Marked inhibition of cell death by caspase inhibitors indicates a significant participation of apoptosis in the phenomenon. This phenomenon is linked to the activation of at least 2 major kinase pathways, MEK 1/2 and p38. These pathways are responsible for upregulating the expression of iNOS and COX-2, each of which seems essential for the induction of apoptosis. Data are provided about possible regulation and interregulation of the COX-2 and iNOS systems by prostaglandin E2 and NO.

Animals↗
PubMed2002

Arachidonic acid converts the glutathione depletion-induced apoptosis to necrosis by promoting lipid peroxidation and reducing caspase-3 activity in rat glioma cells.

Intracellular glutathione (GSH) depletion induced by buthionine sulfoximine (BSO) caused cell death that seemed to be apoptosis in C6 rat glioma cells. Arachidonic acid (AA) promoted BSO-induced cell death by accumulating reactive oxygen species (ROS) or hydroperoxides. AA inhibited caspase-3 activation and internucleosomal DNA fragmentation during the BSO-induced GSH depletion. Furthermore, AA reduced intracellular ATP content, induced dysfunction of mitochondrial membrane and enhanced 8-hydroxy-2'-deoxyguanosine (8-OH-dG) production. There was significant increase of 12-lipoxygenase activity in the presence of AA under the BSO-induced GSH depletion in C6 cells. These results suggest that AA promotes cell death by changing to necrosis from apoptosis through lipid peroxidation initiated by lipid hydroperoxides produced by 12-lipoxygenase under the GSH depletion in C6 cells. Some ROS such as hydroperoxide produced by unknown pathway make hydroxy radicals and induce 8-OH-dG formation in the cells. The conversion of apoptosis to necrosis may be a possible event under GSH depleted conditions.

Adenosine Triphosphate↗
PubMed2005

An autocrine or paracrine Fas-mediated counterattack: a potential mechanism for apoptosis of notochordal cells in intact rat nucleus pulposus.

STUDY DESIGN: Immunohistochemistry and in situ nick end-labeling (TUNEL) were performed in rat lumbar intervertebral discs. OBJECTIVES: To demonstrate the mechanism of notochordal cell death in the nucleus pulposus (NP). SUMMARY OF BACKGROUND DATA: With age, notochordal cells gradually disappear in the NP. We hypothesized that this phenomenon might be related to Fas-mediated apoptosis. MATERIALS AND METHODS: Expressions of Fas; Fas ligand (FasL); caspase 3, 8, 9, 10; Ki-67 protein; and TUNEL were examined in 4-week-, 6-month- and 12-month-old rat NPs. Apoptosis (TUNEL-positive) and proliferation potential (Ki-67-positive) indexes of notochordal cells were calculated and compared among age groups. RESULTS: Notochordal cells constitutively expressed both Fas and FasL. Among their downstream initiator (caspase 8, 9, and 10) and executioner (caspase 3) caspases tested, caspase 9 and 3 were expressed. Proliferation potential of the notochordal cells was the highest at 4 weeks (1.96 +/- 1.3%) and decreased to a significantly lower level at 6 (0.81 +/- 0.68%) and 12 months (0.8 +/- 0.37%; P = 0.03 and 0.01, respectively). In contrast, apoptosis of the notochordal cells was the lowest at 4 weeks (3.52 +/- 1.07%) and increased to a significantly higher level at 6 (19.38 +/- 10.99%) and 12 months (21.51 +/- 16.99%; P < 0.001 in both comparisons). CONCLUSIONS: Fas-mediated mitochondrial caspase 9 pathway is constitutively present in the rat notochordal cells. The constitutive expression of Fas, FasL and its downstream caspases, as well as the homogeneity ofnotochordal cell population suggests an autocrine or paracrine Fas-mediated counterattack to be a potential mechanism for apoptosis of rat notochordal cells. A regulated negative balance of notochordal cell proliferation against apoptosis is likely to involve the disappearance of notochordal cells in the rat NP. This information on the mechanism for apoptosis of notochordal cells could be important in the investigation of intervertebral disc development as well as aging and perhaps degeneration.

Aging↗
PubMed2006

[Diallyl trisulfide induces apoptosis of human gastric cancer cell line MGC803 through caspase-3 pathway].

BACKGROUND & OBJECTIVE: Garlic and the organosulfer compound from garlic have antitumor effects, but the mechanisms still remain unclear. This study was to investigate the changes and significance of caspase-3 activity in diallyl trisulfide (DATS)-induced apoptosis of human gastric cancer cell line MGC803. METHODS: Effects of DATS on the apoptosis of MGC803 cells and the change of activated caspase-3 were observed under a fluorescent and an electron microscopy, and detected by flow cytometry and Western blot. RESULTS: After incubation with DATS, MGC803 cells showed typical apoptotic morphologic changes, and the apoptosis rate increased significantly. DATS activated caspase-3 in a time-dependent manner: the positive rates of activated caspase-3 were 1.9%, 3.0%, 7.3%, 14.4%, and 27.6% respectively in MGC-803 cells treated with 12 mg/L DATS for 0, 4, 8, 12 and 24 h. When treated with 12 mg/L DATS for 24 h, the apoptosis rate was significantly lower in the cells with pretreatment of Ac-DEVD-CHO (a caspase-3 inhibitor) than in the cells without pretreatment (5.1% vs. 23.0%, P<0.01), indicating that Ac-DEVD-CHO efficiently attenuated DATS-induced apoptosis of MGC803 cells. Moreover, pro-caspase-3 was hydrolyzed and activated in DATS-treated MGC803 cells. CONCLUSION: DATS induces apoptosis in MGC803 cells which may be through the activation of caspase-3 pathway.

Adenocarcinoma, Mucinous↗
PubMed2004

Astilbic acid induced COLO 205 cell apoptosis by regulating Bcl-2 and Bax expression and activating caspase-3.

AIM: To investigate the effect of astilbic acid (3beta, 6beta-dihydroxyolean-12-en-27-oic acid, AA) on human colorectal carcinoma COLO 205 cell proliferation and apoptosis. METHODS: Proliferation of COLO 205 cells was measured by MTT assay. Content of DNA in COLO 205 cell was measured by modified diphenylamine assay. AA-induced morphological changes was observed with fluorescence microscope and transmission electron microscope. DNA fragmentation was visualized by agarose gel electrophoresis. Apoptosis rate and cell cycle distribution were determined by flow cytometric analysis. Expressions of Bcl-2 and Bax proteins were visioned by immunohistochemical analysis. The change of relative mitochondrial transmembrane potential (MTP) in COLO 205 cell was analyzed with FCM after rhodamine 123 staining. RESULTS: The IC50 (96 h) of AA for inhibiting COLO 205 cell proliferation was 61.56+/-0.34 micromol/L. AA induced a marked concentration- and time-dependent inhibition of COLO 205 cell proliferation and reduced the DNA content in COLO 205 cell. Cells treated with AA 64 micromol/L showed typical morphological changes of apoptosis and DNA ladder pattern. The cell cycle was arrested in G0/G1 phase, and the apoptosis rate was 28.25 % for COLO 205 cells treated with AA 64 micromol/L for 48 h. Meanwhile the expression of Bcl-2 protein was decreased while that of Bax was increased and relative MTP was decreased as well. DEVD-CHO 1 micromol/L could increase the viability of COLO 205 cells treated with AA for 48 h. CONCLUSION: AA showed potent inhibitory activity on COLO 205 cells proliferation, and could induce COLO 205 cells apoptosis through disturbing DNA replication, down-regulating Bcl-2 expression, and up-regulating Bax expression, lowering relative MTP, and activating caspase-3 pathway.

Antineoplastic Agents, Phytogenic↗
PubMed2003

Apoptosis in keratocytes caused by mitomycin C.

PURPOSE: The purpose of this study was to quantify the effect of mitomycin C on rabbit keratocytes, with a view to determining its potential in modulating corneal stromal wound healing. In addition, the pathway by which this regulation occurs was investigated. METHODS: Keratocytes were isolated from New Zealand White rabbits and cultured. Hoechst staining and flow cytometric analyses with annexin V were used to identify the nature of the keratocyte response to mitomycin C. The response of cultured keratocytes to 0.005%, 0.01%, 0.02%, 0.04%, and 0.06% mitomycin C was evaluated with the lactate dehydrogenase (LDH) assay. In addition, after exposure of keratocytes to 0.01% mitomycin C, the LDH assay was performed at different times of 6, 12, and 24 hours. Keratocytes were preincubated with various concentrations of CPP32-like protease inhibitor (Z-VAD-FMK), caspase-8 inhibitor (Z-IETD-FMK), and caspase-9 inhibitor (Z-LEHD-FMK) and treated with 0.01% mitomycin C. The LDH assay was performed after 12 hours. Cytochrome c immunostain was performed after exposure to 0.01% mitomycin C. RESULTS: Hoechst staining revealed shrinkage of the cytoplasm, formation of apoptotic bodies, and nuclear fragmentation. Apoptotic changes in cells were detected by flow cytometry. LDH activities increased significantly at concentrations of 0.005% mitomycin C or greater and were time dependent until 24 hours. Treatment with a CPP32-like protease inhibitor caused a decrease in LDH activity, although the results were not statistically significant. Specific inhibitors of caspase-8 and -9 significantly reduced the LDH activity induced by mitomycin C. Cytochrome c immunostaining of keratocytes pretreated with mitomycin C showed strongly positive findings. CONCLUSIONS: Mitomycin C induced apoptosis, not necrosis, in cultured corneal keratocytes through the caspase pathway-specifically, caspase-8 and -9-related to the mitochondrial pathway.

Animals↗
PubMed2006

Apoptosis induced by DNA primase inhibitor 3,3'-diethyl-9-methylthia-carbocyanine iodide in human leukemia HL-60 cells.

AIM: To investigate apoptosis induced by 3,3'-diethyl-9-methylthia-carbocyanine iodide (DMTCCI), an inhibitor of DNA primase found in our previous study, and the mechanism of DMTCCI in human myelogenous leukemia HL-60 cells. METHODS: HL-60 cells were cultured in RPMI-1640 medium and treated with different concentrations of DMTCCI. MTT assay was used to detect growth inhibition. Flow cytometry and DNA ladders were used to detect apoptosis. Western blotting was used to observe the expression of survivin, Bcl-xL, Bad, Bax, Bcl-2, caspase-9, caspase-3, caspase-6, PARP, DFF45 and lamin B protein. Caspase-3 activity was measured by ApoAlert Caspase-3 Assay Kit. RESULTS: DMTCCI inhibited proliferation of human leukemia HL-60 cells with IC50 value of 0.24 micromol x L(-1). The results of flow cytometry and DNA ladders showed that DMTCCI could induce apoptosis of HL-60 cells. The expression levels of protein survivin and Bcl-xL were down-regulated, Bad and Bax were up-regulated, while Bcl-2 protein had no change in response to DMTCCI treatment in HL-60 cells. Treatment of HL-60 cells with DMTCCI induced the proteolytic cleavage of caspase-9, caspase-3, caspase-6, PARP, DFF45 and lamin B protein. Caspase-3 activity apparently increased at 3 h and reached a peak at 12 h after exposure to 1 micromol x L(-1) of DMTCCI in HL-60 cells. CONCLUSION: DMTCCI inhibited proliferation and induced apoptosis of human leukemia HL-60 cells. Bcl-2 family proteins, survivin and caspases family proteins might play a role in the apoptosis process induced by DMTCCI.

Apoptosis↗
PubMed2005

Caspase-dependent regulation and subcellular redistribution of the transcriptional modulator YY1 during apoptosis.

The transcriptional regulator Yin Yang 1 (YY1) controls many aspects of cell behavior and is essential for development. We analyzed the fate of YY1 during apoptosis and studied the functional consequences. We observed that this factor is rapidly translocated into the cell nucleus in response to various apoptotic stimuli, including activation of Fas, stimulation by tumor necrosis factor, and staurosporine and etoposide treatment. Furthermore, YY1 is cleaved by caspases in vitro and in vivo at two distinct sites, IATD(12)G and DDSD(119)G, resulting in the deletion of the first 119 amino acids early in the apoptotic process. This activity generates an N-terminally truncated YY1 fragment (YY1Delta119) that has lost its transactivation domain but retains its DNA binding domain. Indeed, YY1Delta119 is no longer able to stimulate gene transcription but interacts with DNA. YY1Delta119 but not the wild-type protein or the caspase-resistant mutant YY1D12A/D119A enhances Fas-induced apoptosis, suggesting that YY1 is involved in a positive feedback loop during apoptosis. Our findings provide evidence for a new mode of regulation of YY1 and define a novel aspect of the involvement of YY1 in the apoptotic process.

Active Transport, Cell Nucleus↗
PubMed2002

Effects of the Bcr/abl kinase inhibitors STI571 and adaphostin (NSC 680410) on chronic myelogenous leukemia cells in vitro.

The adenosine triphosphate binding-site-directed agent STI571 and the tyrphostin adaphostin are undergoing evaluation as bcr/abl kinase inhibitors. The current study compared the effects of these agents on the survival of K562 cells, bcr/abl-transduced FDC-P1 cells, and myeloid progenitors from patients with chronic myelogenous leukemia (CML) compared with healthy donors. Treatment of K562 cells with 10 microM adaphostin resulted in decreased p210(bcr/abl) polypeptide levels in the first 6 hours, followed by caspase activation and accumulation of apoptotic cells in less than 12 hours. By 24 hours, 90% of the cells were apoptotic and unable to form colonies. In contrast, 20 microM STI571 caused rapid inhibition of bcr/abl autophosphorylation without p210(bcr/abl) degradation. Although this was followed by the inhibition of Stat5 phosphorylation and the down-regulation of Bcl-x(L) and Mcl-1, only 7% +/- 3% and 25% +/- 9% of cells were apoptotic at 16 and 24 hours, respectively. Instead, the cytotoxic effects of STI571 became more pronounced with prolonged exposure, with IC90 values greater than 20 microM and 1.0 +/- 0.6 microM after 24 and 48 hours, respectively. Consistent with these results, 24-hour adaphostin exposure inhibited CML granulocyte colony-forming units (CFU-G) (median IC50, 12 microM) but not normal CFU-G (median IC50, greater than 20 microM), whereas 24-hour STI571 treatment had no effect on CML or normal CFU-G. Additional experiments revealed that STI571-resistant K562 cells remained sensitive to adaphostin. Moreover, the combination of STI571 + adaphostin induced more cytotoxicity in K562 cells and in CML CFU-G than either agent alone did. Collectively, these results identify adaphostin as a mechanistically distinct CML-selective agent that retains activity in STI571-resistant cell lines.

Adamantane↗
PubMed2001

Inhalation anesthetics induce apoptosis in normal peripheral lymphocytes in vitro.

BACKGROUND: The authors hypothesized that perioperative lymphocytopenia is partially caused by apoptosis of lymphocytes induced by inhalation anesthetics. Therefore, they evaluated whether sevoflurane and isoflurane induce apoptosis of normal peripheral lymphocytes. METHODS: Normal peripheral blood mononuclear cells were exposed to sevoflurane and isoflurane, and the percentages of apoptotic lymphocytes was measured by Annexin V-fluorescein isothiocyanate-7-amino actinomycin D flow cytometry after 24 h of exposure (0.5, 1.0, and 1.5 mm) and after 6, 12, and 24 h of exposure (1.5 mm). The percentages of lymphocytes with caspase 3-like activity were also measured after 24 h of exposure (1.5 mm). RESULTS: The percentages of apoptotic lymhocytes were increased in a dose-dependent manner (controls: 5.1 +/- 1.4%; sevoflurane: 7.3 +/- 1.3% [0.5 mm], 9.1 +/- 1.5% [1.0 mm], 12.6 +/- 2.1% [1.5 mm]; isoflurane: 7.5 +/- 1.6% [0.5 mm], 10.5 +/- 1.5% [1.0 mm], 16.3 +/- 2.7% [1.5 mm]) after 24 h of exposure and in a time-dependent manner (controls: 1.2 +/- 0.4% [6 h], 3.4 +/- 0.7% [12 h], 5.6 +/- 1.2% [24 h]; sevoflurane: 1.8 +/- 0.4% [6 h], 6.4 +/- 1.2% [12 h], 11.3 +/- 2.2% [24 h]; isoflurane: 2.6 +/- 0.5% [6 h], 8.8 +/- 1.5% [12 h],16.0 +/- 1.9% [24 h]) at the concentration of 1.5 mm. The percentages of lymphocytes with caspase 3-like activity were increased (controls: 10.0 +/- 1.1%; sevoflurane: 13.8 +/- 1.2%; isoflurane: 17.0 +/- 1.3%). CONCLUSIONS: Both sevoflurane and isoflurane induced apoptosis in peripheral lymphocytes in dose-dependent and time-dependent manners in vitro.

Adult↗
PubMed2003

Suppression of extracellular signal-related kinase and activation of p38 MAPK are two critical events leading to caspase-8- and mitochondria-mediated cell death in phytosphingosine-treated human cancer cells.

We previously demonstrated that the phytosphingosine-induced apoptosis was accompanied by the concomitant induction of both the caspase-8-mediated and mitochondrial activation-mediated apoptosis pathways. In the present study, we investigated the role of mitogen-activated protein kinases (MAPKs) in the activation of these two distinct cell death pathways induced by phytosphingosine in human cancer cells. Phytosphingosine caused strong induction of caspase-8 activity and caspase-independent Bax translocation to the mitochondria. A rapid decrease of phosphorylated ERK1/2 and a marked increase of p38 MAPK phosphorylation were observed within 10 min after phytosphingosine treatment. Activation of ERK1/2 by pretreatment with phorbol 12-myristate 13-acetate or forced expression of ERK1/2 attenuated phytosphingosine-induced caspase-8 activation. However, Bax translocation and caspase-9 activation was unaffected, indicating that down-regulation of the ERK activity is specifically required for the phytosphingosine-induced caspase-8-dependent cell death pathway. On the other hand, treatment with SB203580, a p38 MAPK-specific inhibitor, or expression of a dominant negative form of p38 MAPK suppressed phytosphingosine-induced translocation of the proapoptotic protein, Bax, from the cytosol to mitochondria, cytochrome c release, and subsequent caspase-9 activation but did not affect caspase-8 activation, indicating that activation of p38 MAPK is involved in the mitochondrial activation-mediated cell death pathway. Our results suggest that phytosphingosine can utilize two different MAPK signaling pathways for amplifying the apoptosis cascade, enhancing the understanding of the molecular mechanisms utilized by naturally occurring metabolites to regulate cell death. Molecular dissection of the signaling pathways that activate the apoptotic cell death machinery is critical for both our understanding of cell death events and development of cancer therapeutic agents.

Apoptosis↗
PubMed2002

The mechanisms of lipoxygenase inhibitor-induced apoptosis in human breast cancer cells.

Previous experimental studies have shown that high dietary fat intake is associated with mammary carcinogenesis. In the current study, the effect of 5-LOX or 12-LOX inhibitors on human breast cancer cell proliferation and apoptosis, as well as the possible mechanisms were investigated. The LOX inhibitors, NDGA, Rev-5901, and baicalein all inhibited proliferation and induced apoptosis in MCF-7 (ER+) and MDA-MB-231 (ER-) breast cancer cell in vitro. In contrast, the LOX products, 5-HETE and 12-HETE had mitogenic effects, stimulating the proliferation of both cell lines. These inhibitors also induced cytochrome c release, caspase-9 activation, as well as downstream caspase-3, caspase-7 activation, and PARP cleavage. LOX inhibitor treatment also reduced the levels of anti-apoptotic proteins Bcl-2 and Mcl-1 and increased the levels of the pro-apoptotic protein bax. In conclusion, blockade of both 5-LOX and 12-LOX pathways induces apoptosis in breast cancer cells through the cytochrome c release and caspase-9 activation, with changes in the levels of Bcl-2 family proteins.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗