• The Journal of Korean Medicine
• /
• v.22 no.2
• /
• pp.64-74
• /
• 2001

Objectives : The effects of aqueous extract of Backhapgogumtanggami-bang (BGTG, a newly devised herb medicine) on the induction of apoptotic cell death were investigated in human lymphoid origin leukemia cell lines, HL-60. Methods : Cells were treated with various concentrations and $400{\;}\mu\textrm{g}/ml$ BGTG for 12 hr. Genomic DNA was isolated and separated on 1.8% agarose gels. Lysates from the cells were used to measure the activity of caspase-2, -3, -8, and -9 protease by using fluorogenic peptide. Cells were preincubated with SB-203580 for 30 min. Nuclear protein from the cells was incubated with oliginucleotide probe of AP-l and NF-kB. Nuclear extracts from the cells were isolated and reacted with antibodies. Results : The viability of HL-60 cells were markedly decreased by BGTG extract in a dose- and time-dependent manner. BGTG extract induced the apoptotic death of HL-60 cells which was characterized by the DNA fragmentation. The activations of Caspase-2, 3, and 9 were induced by BGTG. However, selective inhibition of the p38 mitogen-activated protein kinase pathways by SB-203580 did not affect the extent of BGTG extract-induced cell death. Furthermore, we observed the transient activations of transcriptional factors such as AP-l and NF-kB. Conclusions : These results suggest that BGTG extract induced apoptotic death of HL-60 cells and caspase activations as well as the modulation of transcriptional factors such as AP-1 and NF-kB.

• Journal of Photoscience
• /
• v.9 no.2
• /
• pp.509-511
• /
• 2002

In this study, we investigated apoptotic cell death induced by photodynamic therapy using 5-aminolaevulinic acid (ALA-PDT) in human promyelocytic leukemia cells (HL-60). ALA-PDT induced apoptosis in HL-60 cells as confirmed by DNA agarose gel electrophoresis and nuclear staining with Hoechst 33342. The apoptotic cell death was inhibited by addition of broad-spectrum caspase inhibitor Z-Asp-CH$_2$-DCB, indicating that the apoptotic cell death was induced in a caspase-dependent manner. Actually, western blotting analysis revealed that caspase-3 was processed as early as 1.5 h after ALA-PDT. Cytoplasmic cytochrome c released from mitochondria was detected by western blotting. However, inhibitor of caspase-9, a cysteine protease located in the downstream of cytochrome c release, was not able to reduce the apoptotic cell death. Therefore, the mitochondrial apoptotic pathway was not involved in the ALA-PDT-induced apoptosis. On the other hand, it was found that ALA-PDT-induced apoptosis was clearly inhibited by pretreatment of caspase-8 inhibitor. These data suggest that caspase-8-mediated apoptotic pathway is important in ALA-PDT-induced cell death.

• Korean Journal of Veterinary Research
• /
• v.41 no.4
• /
• pp.571-578
• /
• 2001

To evaluate if the apoptotic fragment assay could be used to estimate the dose prediction after radiation exposure, we examined apoptotic mouse crypt cells per 1,000 cells after whole body $^{60}Co$ $\gamma$-rays and 50MeV ($p{\rightarrow}Be^+$) cyclotron fast neutron irradiation in the range of 0.25 to 1 Gy, respectively. The incidence of apoptotic cell death rose steeply at very low doses up to 1 Gy, and radiation at all doses tigger rapid changes in crypt cells in stem cell region. These data suggest that apoptosis may play an important role in homeostasis of damaged radiosensitive target organ by removing damaged cells. The curve of dose-effect relationship for the data of apoptotic fragments was obtained by the linear-quadratic model $y=0.18+(9.728{\pm}0.887)D+(-4.727{\pm}1.033)D^2$ ($r^2=0.984$) after $\gamma$-rays irradiation, while $y=0.18+(5.125{\pm}0.601)D+(-2.652{\pm}0.7000)D^2$ ($r^2=0.970$) after neutrons in mice. The dose-response curves were linear-quadratic, and a significant dose-response relationship was found between the frequency of apoptotic cell and dose. These data show a trend towards increase of the numbers of apoptotic crypt cells with increasing dose. Both the time course and the radiation dose-response curve for high and low linear energy transfer (LET) radiation modalities were similar. The relative biological effectiveness (RBE) value for crypt cells was 2.072. In addition, there were significant peaks on apoptosis induction at 4 and 6h after irradiation, and the morpholoigcal findings of the irradiated groups were typical apoptotic fragments in crypt cells that were hardly observed in the control group. Thus, apoptosis in crypt cells could be a useful in vivo model for studying radio-protective drug sensitivity or screening test, microdosimetric indicator and radiation-induced target organ injury. Since the apoptotic fragment assay is simple, rapid and reproducible in the range of 0.25 to 1 Gy, it will also be a good tool for evaluating the dose response of radiation-induced organ damage in vivo and provide a potentially valuable biodosimetry for the early dose prediction after accidental exposure.

• Proceedings of the Korean Society for Applied Microbiology Conference
• /
• 2004.06a
• /
• pp.297-301
• /
• 2004

The purpose of the present study was to investigate the anti-proliferative and apoptotic effects of MCS-C2, a novel analogue of toyocamycin and sangivamycin, in human promyelocytic leukemia (HL-60) cells. When treated with MCS-C2, inhibited proliferation associated with cell cycle arrest and apoptotic induction was found in the HL-60 cells in a concentration-dependent and time-dependent manner. This apoptotic induction was associated with the cleavage of Bid and a release of cytochrome c from mitochondria into the cytosol, followed by the activation of caspase-3 and inactivation of poly (ADP-ribose) polymerase (PARP). However, there was no significant change in any other mitochondrial membrane proteins, such as Bcl-2 and Bax. Consequently, the current findings suggest that the mitochondrial pathway was primarily involved in the MCS-C2-induced apoptosis in the human promyelocytic leukemia HL-60 cells.

• Journal of Acupuncture Research
• /
• v.31 no.2
• /
• pp.75-85
• /
• 2014

Objectives : We investigated whether bee venom(BV) inhibit cell growth through enhancement of death receptor expressions in the human cervix cancer C33A cells. Methods : BV($1{\sim}5{\mu}g/ml$) inhibited the growth of cervix cancer C33A cells by the induction of apoptotic cell death in a dose dependent manner. Results : Consistent with apoptotic cell death, expression of Fas, death receptor(DR) 3, 4, 5 and 6 was increased concentration dependently in the cells. Moreover, Fas, DR3 and DR6 revealed more sensitivity to BV. Thus, We reconfirmed whether they actually play a critical role in anti-proliferation of cervix cancer C33A cells. Consecutively, expression of DR downstream pro-apoptotic proteins including caspase-8, -3, -9 was upregulated and Bax was concomitantly overwhelmed the expression of Bcl-2. NF-${\kappa}B$ were also inhibited by treatment with BV in C33A cells. Conclusions : These results suggest that BV could exert anti-tumor effect through induction of apoptotic cell death in human cervix cancer C33A cells via enhancement of death receptor expression, and that BV could be a promising agent for preventing and treating cervix cancer.

• Development and Reproduction
• /
• v.1 no.2
• /
• pp.157-164
• /
• 1997

To study the regulation of porcine follicular cell apostosis by gonadotropin, steroid, and nitric oxide, we analyzed DNA fragmentation, the hallmark of apoptosis, and nitrite production of porcine granulosa cells. Dissected indiidual follicles from ovary were separated in size (small, 2-3 mm; medium, 5-6 mm; large, 7-8 mm) and isolated granulosa cells were classified morpholocally as atretic or nonatretic. Nitrite concentration was measured by mixing follicular fluids with an equal volume of Griess reagent. Follicular nitric oxide (NO) concentration of healthy follicles was higher than that of atretic follicles. Apoptotic DNA fragmentation was suppressed in non-apoptotic granulosa cells. Follicular apoptosis was induced by androgen but prevented by gonadotropin in vitro. Apoptosis was confined to the granulosa cells. But it was not clear whether apoptosis of granulosa cells were isolated, incubated with or without gonadotropin, androgen and sodium nitroprusside (SNP), respectively at $37^{\circ}C$ for 24 hrs. Cultured granulosa cells were used to extract genomic DNA and culture media was asssayed for nitrite concentration. Nitrite production of culture media was increased, while apoptotic DNA fragmentation was suppressed in PMSG, hCG, testosterone+SNP and SNP treated groups. Nitrite concentration in culture media was decreased, but apoptotic DNA fragmentation was induced in testosterone treated group. These data suggest that NO production and apoptosis may be involved of granulosa cell apoptosis induced by testosterone.

• Journal of Korean Ophthalmic Optics Society
• /
• v.8 no.1
• /
• pp.65-71
• /
• 2003

The corneal epithelium is constantly being shed. The mechanism of corneal desquamation is not fully understood. Apoptosis, programmed cell death, may play a role. Apoptosis can be induced by a number of factors and different mechanisms. The study was performed to examine the apoptotic index induced in human corneal epithelial cells maintained in tissue culture by various apoptotic inducers. Various inducers, recombinant human cytokines($INF{\gamma}$, $TNF{\alpha}$, FASAb), actinomycin D. camptothecin, cycloheximide, dexamethasone and etoposide, were purchased from commercial suppliers. Inducers at manufacturer-recommended concentration were added to the corneal epithelial cells for 48 hours. Cell viability was measured using MTT assay. The cells were then assessed for the level of apoptosis. Morphologic changes and quantification of apoptotic cells were determined and counted under fluorescence microscope after inducers-treated human corneal epithelial (HCE) cells for 48 hours with Hoechst 33342 staining. Annexin V-FITC/PI staining and DePsipher assay. The expression of Fas protein was studied by immunocytochemistry. All inducers induced apoptosis in HCE cells in a dose dependent manner. Actinomycin D. camptothecin and etoposide induced apoptosis at lower than manufacturer-recommended concentration, while cytokines, cycloheximide and dexamethasone induced apoptosis at higher concentrations at the end of 48 hours. All inducers elicited typical apoptotic morphologic changes (chromatin condensation, nucleus fragmentations non-orange-red colored mitochondria) and expresses Fas protein highly. Apoptotic index of HCE cells by these inducers was different from the other cell lines. RNA synthesis inhibitor and topoisomerase inhibitors induced apoptosis at lower concentration than manufacturer-recommended concentration. Cytokines, cycloheximide and dexamethasone were able to produce apoptosis at 10 times higher concentrations. RNA synthesis inhibitor and topoisomerase inhibitors are more sensitive than intracellular receptor-activators in apoptotic induction of HCE cells.

• Bulletin of the Korean Chemical Society
• /
• v.31 no.12
• /
• pp.3777-3781
• /
• 2010

The anticancer effect and apoptotic mechanism of a novel indole derivative AC-264, a lead derived from a chemical library, were investigated in human promyelocytic leukemia HL-60 cells. HL-60 cells treated with AC-264 at various concentrations showed the morphological features of apoptosis, such as plasma membrane blebbing and cell shrinkage. AC-264 exhibited cytotoxic effect in various cancer cell lines with different degrees of potency. Especially, AC-264 was effective on increasing the population of apoptotic cells in HL-60 cells, as detected by the number of cells stained with Annexin V and PI. Furthermore, AC-264 activated caspase-3 enzyme activity and induced internucleosomal DNA fragmentation. These results indicated that AC-264 produces anti-cancer effect via apoptotic cell death by activating caspase-3 and inducing internucleosomal DNA fragmentation in HL-60 cells.

• Korean Journal of Clinical Laboratory Science
• /
• v.42 no.1
• /
• pp.46-54
• /
• 2010

Ceramide induces cell death in a variety of cell types however, the underlying molecular mechanisms related to renal epithelial cells remain unclear. The present study was undertaken to determine the role of extracellular signal-regulated protein kinase (ERK) in ceramide-induced cell death in renal epithelial cells. An established renal proximal tubular cell line of opossum kidney (OK) cells was used for this research. Ceramide induced apoptotic cell death in these cells. Western blot analysis showed that ceramide induced activation of ERK. The ERK activation and cell death induced by ceramide were prevented by the ERK inhibitor PD98059. Ceramide caused cytochrome C release from mitochondria into the cytosol as well as activation of caspase-3. Both effects were prevented by PD98059. The ceramide-induced cell death was also prevented by a caspase inhibitor. These results suggest that ceramide induces cell death through an ERK-dependent mitochondrial apoptotic pathway in OK cells.

• The Journal of Internal Korean Medicine
• /
• v.32 no.4
• /
• pp.565-583
• /
• 2011

Objectives : This study investigated the biochemical mechanisms of anti-proliferative and pro-apoptotic effects of the water extract of Dan-Seon-Tang (DST) in human leukemia U937 cells. Methods : U937 cells were exposed to DST and growth inhibition was measured by MTT assay. Results : Exposure of U937 cells to DST resulted in the growth inhibition in a concentration-dependent manner. This inhibitory effect was associated with morphological changes and apoptotic cell death such as formation of apoptotic bodies, increased populations of apoptotic-sub G1 phase and induction of DNA fragmentation. The induction of apoptotic cell death in U937 cells by DST was associated with up-regulation of death receptor 4 (DR4) and down-regulation of Bid, surviving and cellular inhibition of apoptosis protein-2 (cIAP-2) expression. DST treatment also induced the proteolytic activation of caspase-3, caspase-8 and caspase-9, and a concomitant degradation of caspase-3 substrate proteins such as poly (ADP-ribose) polymerase (PARP), phospholipase (PLC)-${\gamma}1$, ${\beta}$-catenin and DNA fragmentation factor 45/inhibotor of caspase activated DNAse (DFF45/ICAD). Furthermore, apoptotic cell death by DST was significantly inhibited by caspase-3 specific inhibitor z-DEVD-fmk, demonstrating the important role of caspase-3. Conclusions : These findings suggest that herb prescription DST may be a potential chemotherapeutic agent for the control of human leukemia U937 cells; further study is needed to identify the active compounds.