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

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

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

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

BACKGROUND: Cryopreservation of hematopoietic stem cells has become an important process due to the therapeutic protocol, which includes stem cell transplantation after chemotherapy, for many hematological malignancies. The conventional medium contains 10% dimethyl sulfoxide (DMSO) as a cryoprotectant, but this has been reported to be related with many complications. We analyzed the usefulness of trehalose, catalase and zVAD-fmk for cryopreservation along with using a reduced concentration of DMSO to 5%. METHODS: Peripheral blood stem cells were frozen in 10% DMSO as a control and also in 5% DMSO with trehalose and catalase. After 3 weeks of storage in a liquid nitrogen tank, the viability of the thawed hematopoietic stem cells was measured using Trypan blue staining and 7-AAD analysis via conducting flow cytometry. The colony forming potential was assessed using methylcellulose culture. We measured the viability of cells in 5% DMSO medium with or without addition of 30 uM zVAD-fmk right after thawing, and we also did this 6 and 24 hours after incubation. RESULTS: Cryopreserved cells in 5% DMSO with trehalose and catalase showed similar survival (50.42%) compared with the control (49.78%). The viability of cells that were also treated with added zVAD-fmk showed a better result (13.12%) than without it (5.5%) after 24 hours of incubation. Colony forming assay showed similar colony formation in 5% DMSO with the natural cryoprotectants. CONCLUSION: According to the results, lowering the DMSO concentration to 5% is significant and we can expect better cell viability and prevent many side effects of high dose DMSO when adding natural cryprotectants in the cryopreservation medium or by adding caspase-inhibitor right after thawing.
Amino Acid Chloromethyl Ketones ; Catalase ; Cell Survival ; Cryopreservation ; Dimethyl Sulfoxide ; Diminazene ; Flow Cytometry ; Hematologic Neoplasms ; Hematopoietic Stem Cells ; Methylcellulose ; Nitrogen ; Safrole ; Stem Cell Transplantation ; Stem Cells ; Trehalose ; Trypan Blue

Gambogic acid (GA) has powerful apoptotic actions. The authors investigated whether GA has apoptotic effects on aortic smooth muscle cells, and compared its potency with that of simvastatin. Smooth muscle cells were isolated from the aortas of Sprague-Dawley rats (4-6 week). Cell purities were confirmed by IF staining using alpha-smooth muscle actin antibody. The IC50 values for cell death by GA and simvastatin were determined using a MTT assay, and the apoptotic effects of 1 microM GA or 30 microM simvastatin (concentrations correspond to IC50 values) were determined after 24 h of treatment using live cell images and by FITC annexin-V and propidium iodide double-staining. In addition, western blotting was used to evaluate apoptosis by quantifying reductions in the expression levels of the PARP and procaspase-3 as well as cleavages of PARP and procaspase-3 after treatment with 1 microM GA or 30 microM simvastatin. The IC50 of GA (1 microM) was lower than that of simvastatin (30 microM). Cell numbers were markedly reduced by both drugs in live cell images. GA (1 microM) produced a higher level of apoptosis than 30 microM simvastatin (26.4+/-2.37% vs. 8.3+/-1.54%, respectively; P<0.05, n=3) by FITC annexin-V & PI double-staining. In addition, 1 microM GA reduced the expressions of PARP, procaspase-3, and Mcl-1 in cells, whereas 30 microM simvastatin did not. Pretreatment with z-VAD-fmk attenuated GA-induced apoptosis and the cleavages of PARP and procaspase-3. The decreased level of Mcl-1 protein induced by GA treatment was recovered by z-VAD-fmk. These results indicate that GA-induced apoptosis was mediated by a caspase-dependent pathway.
Actins ; Amino Acid Chloromethyl Ketones ; Aorta ; Apoptosis ; Blotting, Western ; Caspase 3 ; Cell Count ; Cell Death ; Fluorescein-5-isothiocyanate ; Inhibitory Concentration 50 ; Muscle, Smooth ; Muscles ; Myocytes, Smooth Muscle ; Propidium ; Rats, Sprague-Dawley ; Simvastatin ; Xanthones

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