OBJECTIVETo investigate the effects of FTY720 on apoptosis in multiple myeloma cell line U266 and to clarify the molecular mechanism of apoptosis induced by FTY720.

METHODSU266 cells were treated with 2.5, 5, 10 and 20 µmol/L of FTY720 for 24 hours, the apoptotic rates were tested by flow cytometry with Annexin-V-FITC/PI staining. Then U266 cells were treated with 20 µmol/L FTY720 for 0, 6, 16 and 24 hours, the apoptotic rates were tested. U266 cells were treated with DMSO and FTY720 separately and then were stained with DAPI for 5 min. Drop the cells to the slides and cover the slide with the glass. The cells were observed by fluorescence microscopy. U266 cells were treated with 5 µmol/L FTY720 or together with different doses of Z-VAD-fmk (12.5, 25, 50 µmol/L), a pancaspase inhibitor, for 24 hours, then the cell viability was tested by CCK-8. U266 cells were treated with 2.5, 5, 10 and 20 µmol/L of FTY720 for 24 hours, the expression of cleaved caspase-3 was tested by Western blot. U266 cells were treated with 0, 5, 10 and 20 µmol/L of FTY720 for 24 hours, the expressions of MCL-1, survivin, BCL-2, BID, BAX, BAK, P-ERK were tested by Western blot.

RESULTSThe apoptotic rate increased in U266 cells treated with FTY720 and showed the characteristic of time-dependent and dose-dependent manner. Karyopyknosis and nuclearfragmentation could be observed in U266 cells treated with FTY720 after being stained with DAPI under fluorescent microscope. The same effect was not observed in the cells treated with DMSO. Z-VAD-fmk could rescue the apoptosis in U266 cells treated with FTY720 in dose-dependent manner. The expression of MCL-1, survivin and BCL-2 decreased in U266 cells treated with FTY720. The cleavage of BID could be observed in U266 cells treated with FTY720. FTY720 had no effect on the expression of BAX, BAK and P-ERK.

CONCLUSIONFTY720 can induce the apoptosis in U266 cells, the apoptosis was Caspase-3-depended. The apoptosis induced by FTY720 is due to the decrease of MCL-1, survivin and BCL-2, which are the inhibitors of apoptosis. Meanwhile, the apoptosis was also due to the activation of BID, which is pro-apoptotic protein.

Amino Acid Chloromethyl Ketones ; Apoptosis ; Caspase 3 ; Cell Line, Tumor ; Cell Survival ; Fingolimod Hydrochloride ; Humans ; Inhibitor of Apoptosis Proteins ; Multiple Myeloma

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 potent inhibitor of S-adenosylhomocysteine hydrolase, was previously proposed to induce intrinsic apoptosis in human leukemic cells. In the present study, we analyzed the mechanism underlying the DZA-induced intrinsic apoptotic pathway. DZA activated typical caspase-dependent apoptosis in HL-60 cells, as demonstrated by an accumulation of hypo-diploidic cells, the processing of multiple procaspases and an inhibitory effect of z-VAD-Fmk on this cell death. During DZA-induced apoptosis, cytochrome c (cyt c) was released into the cytosol. This was neither prevented by z-VAD-Fmk and nor was it associated with the dissipation of mitochondrial membrane potential (DeltaPsim). Prior to the release of cyt c, BAX was translocated from the cytosol to mitochondria and underwent oligomerization. Finally, the overexpression of BCL-XL protected HL-60 cells from apoptosis by blocking both the cyt c release and BAX oligomerization. Collectively, these findings suggest that DZA may activate intrinsic apoptosis by stimulating BAX activation and thereby the release of cyt c.
Adenosylhomocysteinase ; Amino Acid Chloromethyl Ketones ; Apoptosis ; bcl-2-Associated X Protein ; bcl-X Protein ; Cell Death ; Cytochromes c ; Cytosol ; HL-60 Cells ; Humans ; Membrane Potential, Mitochondrial ; Mitochondria ; Tubercidin

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 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

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 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

BACKGROUND/AIMS: Peroxisome proliferator-activated receptor (PPAR)-gamma ligand is known to inhibit the growth of several kinds of cancer cells, yet its effect on cholangiocarcinoma is indecisive. We investigated the effect of an endogenous ligand of PPAR-gamma, 15-deoxy-delta (12,14)-prostaglandin J2 (15-deoxy-PGJ2) on cholangiocarcinoma cells that were established from intrahepatic cholangiocarcinoma tissue of Korean patients. METHODS: Four cholangiocarcinoma cell lines, Cho-CK, Choi-CK, JCK and SCK, were studied. The mRNA expression of PPAR-gamma, bcl-2, and bax were examined by RT-PCR. Cell viability was determined by MTT assay. The cell cycle was analyzed by flow cytometry, and apoptosis by cell death detection ELISA kit. Caspase activity was measured by colorimetric assay. The effect of caspase inhibitors on 15-deoxy-PGJ2-induced apoptosis was determined by measuring cell viability using the MTT assay. RESULTS: PPAR-gamma mRNA was expressed in all cholangiocarcinoma cells. 15-deoxy-PGJ2 inhibited proliferation of all cells in a dose- and time-dependent manner. All cells treated with 15-deoxy-PGJ2 showed increased dose-dependent apoptosis. Caspase 3 was activated in all cells and caspase 9 was activated in all but JCK cells after 15-deoxy-PGJ2 treatment. Caspase 8 activity showed no significant change. The pan-caspase inhibitor, Z-VAD-FMK, and the caspase-3 inhibitor, Z-DEVD-FMK, blocked 15-deoxy-PGJ2-induced apoptosis in all cells dose-dependently. The expression of bcl-2 was decreased in Cho-CK, Choi-CK and SCK cells, and bax expression was not changed significantly after 15-deoxy-PGJ2 treatment. CONCLUSIONS: PPAR-gamma mRNA was expressed in all Korean cholangiocarcinoma cells. Our data suggest that 15-deoxy-PGJ2 exerts an antineoplastic effect against cholangiocarcinoma cells by inducing apoptosis through caspase activation.
Amino Acid Chloromethyl Ketones ; Apoptosis ; Caspase 3 ; Caspase 8 ; Caspase 9 ; Caspase Inhibitors ; Cell Cycle ; Cell Death ; Cell Line ; Cell Survival ; Cholangiocarcinoma ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Liver Neoplasms ; Oligopeptides ; Peroxisomes ; PPAR gamma ; Prostaglandin D2 ; RNA, Messenger