The present study is to elucidate the mechanisms underlying Gleevec-induced apoptosis of chronic myeloid leukemia (CML) K562 cells in vitro. The apoptotic cell death and cell cycle distribution after Gleevec treatment and the effect of PDCD4 siRNA on Gleevec-induced apoptosis of K562 cells were analyzed by flow cytometry. The effect of Gleevec on p-Crkl, caspase-3, PARP and PDCD4 protein levels, and the knockdown efficacy of PDCD4 siRNA were detected by Western blotting. The results showed that Gleevec dramatically suppressed the phosphorylation level of Crkl in a dose-dependent manner and induced significant apoptosis and G0/G1 cell cycle arrest of K562 cells in time- and dose-dependent manners. In addition, Gleevec activated caspase-3 and its downstream substrates PARP, and the caspase pan inhibitor Z-VAD-FMK (50 micromol x L(-1)) markedly reduced Gleevec-induced apoptosis from 47.97% +/- 10.56% to 31.05% +/- 9.206% (P < 0.05). Moreover, Gleevec significantly increased the protein expression of programmed cell death 4 (PDCD4). PDCD4 knockdown by siRNA reduced Gleevec-induced apoptosis from 46.97% +/- 14.32% to 42.8% +/- 11.43%. In summary, Gleevec induced apoptosis in K562 cells via caspase-3 activation.
Amino Acid Chloromethyl Ketones ; Apoptosis ; drug effects ; Benzamides ; pharmacology ; Caspase 3 ; metabolism ; Cell Cycle ; drug effects ; Humans ; Imatinib Mesylate ; K562 Cells ; Phosphorylation ; Piperazines ; pharmacology ; Pyrimidines ; pharmacology

OBJECTIVE: To explore the exact mechanisms of programmed cell death (PCD) induced by Type II anti-CD20 mAb in CD20+ non-Hodgkin lymphoma (NHL) cells, and to provide theoretical basis for anti-tumor ability of new CD20 mAb.
 METHODS: After incubation with Rituximab (a Type I anti-CD20 mAb) and Tositumomab (a Type II anti-CD20 mAb), Raji cells were stained by annexin V & propidium iodide (PI). The ratio of programmed death cells were measured by two channel flow cytometry (FCM). Before the treatment of anti-CD20 mAbs, Raji cells was incubated with a caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Z-VAD-FMK) and a dihydroceramide synthase inhibitor fumonisin B1 (FB1) for 30 minutes to assess their inhibitory effect on PCD. High performance liquid chromatography (HPLC) was utilized to compare the ratio of programmed death cells between the pretreatment group (treated by Rituximab and Tositumomab) and the non-pretreatment group. The anti-CD20 mAbs-treated Raji cells were collected, and the ceramide levels in the Raji cells in the different pretreatment groups were also examined by HPLC, and the inhibitory effect of FB1 on the changes of ceramide levels in the Raji cells was measured. The Raji cells were incubated with different concentration C2-ceramide, C2-Ceramide-induced PCD was also evaluated by annexin V & PI staining after 16 hours. 
 RESULTS: Tositumomab (10 µg/mL) but not Rituximab (10 µg/mL) can induce significant PCD (28.6±4.2)% in Raji cells, with significant difference (t=26.48, P<0.01), which cannot be blocked by Z-VAD-FMK with a concentration range from 10 to 30 µmol/L (F=3.01, P>0.05). The cellular ceramide levels in Raji cells were significantly elevated after the treatment of Tositumomab (t=28.48, P<0.01). C2-ceramide can significantly induce PCD in Raji cells in a dose-dependent manner with a concentration range from 5 to 40 µmol/L (F=2.71, P>0.05). The dihydroceramide synthase inhibitor FB1 can significantly inhibit the elevated cellular ceramide levels (F=20.18, P<0.01) and cell programmed death induced by Tositumomab (F=17.02, P<0.01).
 CONCLUSION Type II but not Type I anti-CD20 mAbs can induce caspase independent PCD in CD20+ NHL cells through the elevation of cellular ceramide levels. The PCD is not associated with classic caspase pathway.
Amino Acid Chloromethyl Ketones ; Apoptosis ; drug effects ; Cell Line, Tumor ; drug effects ; Humans ; Lymphoma, Non-Hodgkin ; Rituximab ; pharmacology ; Sphingosine ; analogs & derivatives ; pharmacology

3-Deazaadenosine (DZA), a cellular methylation blocker was reported to induce the caspase-3-like activities-dependent apoptosis in U-937 cells. In this study, we analyzed the activation pathway of the caspase cascade involved in the DZA-induced apoptosis using specific inhibitors of caspases. In the U-937 cells treated with DZA, cytochrome c release from mitochondria and subsequent activation of caspase-9, -8 and -3 were observed before the induction of apoptosis. zDEVD-Fmk, a specific inhibitor of caspase-3, and zLEHD-Fmk, a specific inhibitor of caspase-9, prevented the activation of caspase-8 but neither caspase-3 nor caspase-9, indicating that caspase-8 is downstream of both caspase-3 and caspase-9, which are activated by independent pathways. zVAD-Fmk, a universal inhibitor of caspases, kept the caspase-3 from being activated but not caspase-9. Moreover, ZB4, an antagonistic Fas-antibody, exerted no effect on the activation of caspase-8 and induction of apoptosis by DZA. In addition, zVAD-Fmk and mitochondrial permeability transition pore (MPTP) inhibitors such as cyclosporin A (CsA) and bongkrekic acid (BA) did not block the release of cytochrome c from mitochondria. Taken together, these results suggest that in the DZA-induced apoptosis, caspase-8 may serve as an executioner caspase and be activated downstream of both caspase-3 and caspase-9, independently of Fas receptor-ligand interaction. And caspase-3 seems to be activated by other caspses including IETDase-like enzyme and caspse-9 seems to be activated by cytochrome c released from mitochondria without the involvement of caspases and CsA- and BA- inhibitory MPTP.
Amino Acid Chloromethyl Ketones/pharmacology ; Apoptosis/*drug effects ; Bongkrekic Acid/pharmacology ; Caspases/*metabolism ; Cell Line ; Cyclosporine/pharmacology ; Cytochrome c/drug effects/metabolism ; Enzyme Activation ; Human ; Leukocytes, Mononuclear/cytology ; Ligands ; Membrane Glycoproteins/metabolism ; Tubercidin/*pharmacology ; U937 Cells

3-Deazaadenosine (DZA), a cellular methylation blocker was reported to induce the caspase-3-like activities-dependent apoptosis in U-937 cells. In this study, we analyzed the activation pathway of the caspase cascade involved in the DZA-induced apoptosis using specific inhibitors of caspases. In the U-937 cells treated with DZA, cytochrome c release from mitochondria and subsequent activation of caspase-9, -8 and -3 were observed before the induction of apoptosis. zDEVD-Fmk, a specific inhibitor of caspase-3, and zLEHD-Fmk, a specific inhibitor of caspase-9, prevented the activation of caspase-8 but neither caspase-3 nor caspase-9, indicating that caspase-8 is downstream of both caspase-3 and caspase-9, which are activated by independent pathways. zVAD-Fmk, a universal inhibitor of caspases, kept the caspase-3 from being activated but not caspase-9. Moreover, ZB4, an antagonistic Fas-antibody, exerted no effect on the activation of caspase-8 and induction of apoptosis by DZA. In addition, zVAD-Fmk and mitochondrial permeability transition pore (MPTP) inhibitors such as cyclosporin A (CsA) and bongkrekic acid (BA) did not block the release of cytochrome c from mitochondria. Taken together, these results suggest that in the DZA-induced apoptosis, caspase-8 may serve as an executioner caspase and be activated downstream of both caspase-3 and caspase-9, independently of Fas receptor-ligand interaction. And caspase-3 seems to be activated by other caspses including IETDase-like enzyme and caspse-9 seems to be activated by cytochrome c released from mitochondria without the involvement of caspases and CsA- and BA- inhibitory MPTP.
Amino Acid Chloromethyl Ketones/pharmacology ; Apoptosis/*drug effects ; Bongkrekic Acid/pharmacology ; Caspases/*metabolism ; Cell Line ; Cyclosporine/pharmacology ; Cytochrome c/drug effects/metabolism ; Enzyme Activation ; Human ; Leukocytes, Mononuclear/cytology ; Ligands ; Membrane Glycoproteins/metabolism ; Tubercidin/*pharmacology ; U937 Cells

OBJECTIVETo investigate the effect of interleukin-6 (IL-6) on the apoptosis of annulus fibrosus (AF) cell induced by interleukin-1beta (IL-1beta).

METHODSCultured AF cells were divided into 6 groups and treated with no drug, 10 ng/mL IL-6, 10 ng/mL IL-1beta, 10 ng/mL IL-1beta and Z-VAD-FMK (a caspase-9 inhibitor), 10 ng/mL IL-1beta and 10 ng/mL IL-6, 10 ng/mL IL-1beta and 100 ng/mL IL-6, respectively. After three days of culture, the apoptosis rate, the positive rates of caspase-3, -8, and -9 of AF cells were detected with flow cytometry.

RESULTSThe apoptosis rates of cells in group 1 to 6 were 2.67% +/- 1.08%, 2.71% +/- 0.53%, 20.37% +/- 1.57%, 11.34% +/- 0.67%, 18.17% +/- 0.74%, and 9.42% +/- 1.08%, respectively. There was no significant difference between group 1 and 2, while the apoptosis rates of group 4, 5, and 6 were significantly lower than group 3 (P = 0.001, P = 0.172, and P = 0.001, respectively). Positive rates of caspase-3 in group 5 (12.35% +/- 0.64%) and 6 (9.26% +/- 0.36%) were significantly lower than group 3 (17.14% +/- 0.72%; P = 0.001 and P < 0.001, respectively). And positive rates of caspase-9 in group 5 (15.13% +/- 1.45%) and 6 (10.17% +/- 2.50%) were significantly lower than group 3 (19.4% +/- 0.98% ; P = 0.014 and P = 0.004, respectively). But there was not obvious change of caspase-8 activity after IL-6 was added.

CONCLUSIONIL-6 is capable of protecting AF cells from IL-1beta induced apoptosis in vitro. Mechanism of the protection is related with the inhibition of caspase-3 and -9 activities.

Amino Acid Chloromethyl Ketones ; pharmacology ; Animals ; Apoptosis ; drug effects ; Caspase Inhibitors ; Cells, Cultured ; Cysteine Proteinase Inhibitors ; pharmacology ; Interleukin-1beta ; pharmacology ; Interleukin-6 ; pharmacology ; Intervertebral Disc ; cytology ; drug effects ; enzymology ; Rabbits

AIMTo study the effects of caspases on cerebromicrovascular endothelial cell apoptosis induced by hypoxia in vitro.

METHODSThe cultured bovine cerebromicrovascular endothelial cells were exposed to NaCN in glucose-free medium. Cell viability was determined by trypan blue staining. Cell apoptosis was defined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and flow cytometry. The expression of caspase-3 was detected by immunocytochemical method. Four caspase inhibitors were used to validate the effect of caspases on cell apoptosis.

RESULTSNaCN in glucose-free medium initiated cerebromicrovascular endothelial cell injury markedly and typical apoptotic cells were found in this model. The expression of caspase-3 increased significantly. Four caspase inhibitors decreased the number of injured cells. Selective inhibitor of caspase-1 and -6 reduced expression of caspase-3 significantly.

CONCLUSIONThe results suggest that caspases family plays an important role in cerebromicrovascular endothelial cell apoptosis induced by NaCN and caspase-3 acts on the downstream of caspase-1 and -6 in protease cascade action to induce apoptosis.

Amino Acid Chloromethyl Ketones ; pharmacology ; Animals ; Apoptosis ; Brain ; blood supply ; Caspase 3 ; Caspase 6 ; Caspase Inhibitors ; Caspases ; metabolism ; Cattle ; Cell Hypoxia ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Microcirculation ; cytology ; Oligopeptides ; pharmacology ; Sodium Cyanide ; pharmacology

OBJECTIVETo investigate the role of autophagy in MnCl2-induced apoptosis in human bronchial epithelial 16HBE cells.

METHODSCell proliferation was measured by MTT assay. Mitochondrial membrane potential (MMP) and apoptosis were measured by flow cytometry. Autophagic vacuoles were detected by fluorescence microscopy. Cellular levels of apoptosis and autophagy-related proteins were measured by western blotting.

RESULTS16HBE cell proliferation was inhibited by MnCl2 in a dose- and time-dependent manner. MnCl2-induced 16HBE cell growth inhibition was related to MMP depolarization prior to the induction of apoptosis. Our data revealed that MnCl2-induced apoptosis in 16HBE cells was mediated by decreased expression of Bcl-2 and increased levels of cleaved caspase-3. It was observed that when we exposed 16HBE cells to MnCl2 in a dose-dependent manner, the formation of autophagic vacuoles and the levels of LC-3B-II were elevated. RNA interference of LC3B in these MnCl2-exposed cells demonstrated that MMP loss and apoptosis were enhanced. Additionally, the pan-caspase inhibitor Z-VAD-FMK increased the cellular levels of Bcl-2 and decreased apoptosis, but did not affect the cellular levels of LC3B in MnCl2-treated 16HBE cells.

CONCLUSIONMnCl2 dose- and time-dependently inhibits 16HBE cell proliferation and induces MMP loss and apoptosis. Autophagy acts in a protective role against MnCl2-induced apoptosis in 16HBE cells.

Amino Acid Chloromethyl Ketones ; pharmacology ; Apoptosis ; drug effects ; Autophagy ; drug effects ; physiology ; Bronchi ; Cell Line ; Chlorides ; pharmacology ; Down-Regulation ; Epithelial Cells ; drug effects ; Gene Expression Regulation ; drug effects ; Humans ; Manganese Compounds ; pharmacology

BACKGROUNDPyroptosis is the term for caspase-1-dependent cell death associated with pro-inflammatory cytokines. The role of alveolar macrophage (AM) pyroptosis in the pathogenesis of the acute lung injury and acute respiratory distress syndrome (ALI/ARDS) remains unclear.

METHODSC57BL/6 wild-type mice were assigned to sham, lipopolysaccharide (LPS) + vehicle, LPS + acetyl-tyrosyl-valyl- alanyl-aspartyl-chloromethylketone (Ac-YVAD-CMK) and LPS + Z-Asp-Glu-Val-Asp-fluoromethylketone groups. Mice were given intraperitoneal (IP) injections of LPS. Drugs were IP injected 1 h before LPS administration. Mice were sacrificed 16 h after LPS administration, and AMs were isolated. Western blot analysis for active caspase-1 and cleaved caspase-3, evaluation of lung injury and a cytokine release analysis were performed. AMs were treated with LPS and adenosine triphosphate (ATP); caspase-1-dependent cell death was evaluated using flow cytometry; the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) pyroptosomes were examined by immunofluorescence.

RESULTSThe expression of activated caspase-1 in AMs was enhanced following LPS challenge compared with the sham group. In the ex vivo study, the caspase-1/propidium iodide-positive cells, caspase-1 specks and ASC pyroptosomes were up-regulated in AMs following LPS/ATP stimulation. The specific caspase-1 inhibitor Ac-YVAD-CMK inhibited the activation of caspase-1 and pyroptotic cell death. Ac-YVAD-CMK also reduced the lung injury, pulmonary edema and total protein in bronchoalveolar lavage fluid (BALF). In addition, Ac-YVAD-CMK significantly inhibited interleukin-α2 (IL-1α2) release both in serum and BALF and reduced the levels of IL-18, tumor necrosis factor-α± (TNF-α±), High Mobility Group Box 1 (HMGB1) in BALF during LPS-induced ALI/ARDS.

CONCLUSIONSThis study reported AM pyroptosis during LPS-induced ALI/ARDS in mice and has demonstrated that Ac-YVAD-CMK can prevent AM-induced pyroptosis and lung injury. These preliminary findings may form the basis for further studies to evaluate this pathway as a target for prevention or reduction of ALI/ARDS.

Acute Lung Injury ; chemically induced ; prevention & control ; Amino Acid Chloromethyl Ketones ; pharmacology ; Animals ; Lipopolysaccharides ; toxicity ; Macrophages, Alveolar ; drug effects ; Male ; Mice ; Mice, Inbred C57BL ; Oligopeptides ; pharmacology ; Pyroptosis ; drug effects

In a preliminary study, we found that benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD- fmk), unlike Boc-aspartyl(OMe)-fluoromethylketone (BocD-fmk), at usual dosage could not prevent genistein-induced apoptosis of p815 mastocytoma cells. This study was undertaken to reveal the mechanism underlying the incapability of zVAD-fmk in preventing this type of apoptosis. We observed that 14-3-3 protein level was reduced in genistein-treated cells and that BocD-fmk but not zVAD-fmk prevented the reduction of 14-3-3 protein level and the release of Bad from 14-3-3. We also demonstrated that truncated Bad to Bcl-xL interaction in genistein- treated cells was prevented by BocD-fmk but not by zVAD-fmk treatment. Our data indicate that BocD- fmk, compared to zVAD-fmk, has a certain preference for inhibiting 14-3-3/Bad signalling pathway. We also elucidated that this differential efficacy of BocD-fmk and zVAD-fmk resulted from the different effect in inhibiting caspase-6 and that co-treatment of zVAD-fmk and caspase-6 specific inhibitor substantially prevented genistein-induced apoptosis. Our data shows that caspase-6 plays a role on Bad/14-3-3 pathway in genistein-induced apoptosis of p815 cells, and that the usual dose of zVAD-fmk, in contrast to BocD-fmk, did not prevent caspase-6 acting on 14-3-3/Bad-mediated event.
bcl-Associated Death Protein/*metabolism ; *Signal Transduction/drug effects ; Mitochondria/drug effects ; Mice ; Mastocytoma ; Hydrocarbons, Fluorinated/*pharmacology ; Genistein/pharmacology ; Enzyme Inhibitors/*pharmacology ; Cell Line, Tumor ; Caspase 6/antagonists & inhibitors/*metabolism ; Benzyl Compounds/*pharmacology ; Apoptosis/*drug effects ; Animals ; Amino Acid Chloromethyl Ketones/pharmacology ; 14-3-3 Proteins/*metabolism

BACKGROUND: Human telomerase reverse transcriptase (hTERT) is a catalytic enzyme that is required for telomerase activity (TA) and cancer progression. Telomerase inhibition or inactivation increases cellular sensitivity to UV irradiation, DNA-damaging agents, the tyrosine kinase inhibitor, imatinib, and pharmacological inhibitors, such as BIBR1532. hTERT is associated with apoptosis. Some patients show drug-resistance during anti-cancer drug treatment and the cancer cell acquire anti-apoptotic mechanism. Therefore, we attempted to study correlation between hTERT and drug-resistance. METHODS: To study the correlation between protein level and activity of hTERT and drug-resistance, Western blotting and telomerase repeat amplification protocol (TRAP) assays were performed. To investigate whether hTERT contributes to drug resistance in tumor cells, we transiently decreased hTERT levels using small interfering RNA (siRNA) in T24/R2 cells. RESULTS: hTERT knockdown increased Bax translocation into the mitochondria and cytochrome C release into the cytosol. Caspase inhibitors, especially Z-VAD-FMK, rescued this phenomenon, suggesting that the stability or expression of hTERT might be regulated by caspase activity. CONCLUSIONS: These data suggest that hTERT might be a target molecule for drug-resistant tumor therapy.
Amino Acid Chloromethyl Ketones/pharmacology ; Antineoplastic Agents/*pharmacology ; Caspases/antagonists & inhibitors/metabolism ; Cell Line, Tumor ; Cisplatin/*pharmacology ; Cysteine Proteinase Inhibitors/pharmacology ; Cytochrome c Group/metabolism ; Drug Resistance, Neoplasm/genetics ; Humans ; Neoplasms/therapy ; RNA, Small Interfering ; Telomerase/*antagonists & inhibitors/genetics/metabolism ; bcl-2-Associated X Protein/metabolism