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

• Development and Reproduction
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• v.1 no.1
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• pp.79-89
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• 1997

Recent studies have demonstrated that apoptotic cell death plays an important role in the mechanism underlying follicular atresia and luteolysis. However, the mechanisms responsible for initiating these processes have not been elucidated. In in vitro fertilization (IVF) programs, it is highly possible that continuous and repeated administration of FSH/hMG and GnRH agonists for the usage of ovarian hyperstimulation may induce apoptotic death of granulosa cells leading to atresia in the human ovarian follicles. The present study was performed to investigate whether FSH/hMG and GnRh agonists used for a longer period in controlled ovarian hyperstimulation has any effect on the apoptosis of granulosa-luteal (GL) cells obtained from hyperstimulated ovaries. To examine apoptotic cell death in the GL cells, cells were stained with acridie orange followed by observed in some of GL cells. Similar but distinct staining of apoptotic GL cells was observed when the cells were examined by using in situ TUNEL method. The healthy-looking cells with normal nuclear morphology were not stained, whereas cells with pyknotic nuclei or with apoptotic nuclei were intensively stained. After examining the ultrastructural features of GL cells by TEM, it was confirmed that the majority of cells seemed to have normal nuclei while GL cells undergoing apoptotic cel death were rarely found. The DNA extracted from GL cells showed a typical pattern of fragmentation following DNA electrophoretic analysis. We have confirmed that the apoptosis occurs in granulosa-luteal cells obtained from hyperstimulated ovaries. Technically, in situ apoptosis detection method is simple and reproducible and is well suited to identify the quality of oocytes retrieved from hyperstimulated ovaries.

• BMB Reports
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• v.39 no.1
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• pp.97-104
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• 2006

Endostatin is a tumor-derived angiogenesis inhibitor, and the endogenous 20 kDa carboxyl-terminal fragment of collagen XVIII. In addition to inhibiting angiogenesis, endostatin inhibits tumor growth and the induction of apoptosis in several endothelial cell types. However, the mechanisms that regulate endostatin-induced apoptotic cell death are unclear. Here, we investigated apoptotic cell death and the underlying regulatory mechanisms elicited of endostatin in human umbilical vein endothelial cells (HUVECs). Endostatin was found to induce typical apoptotic features, such as, chromatin condensation and DNA fragmentation in these cells. Thus, as the phosphoinositide 3-OH kinase (PI3K)/protein kinase B (PKB) signaling pathway has been shown to prevent apoptosis in various cell types, we investigated whether this pathway could protect cells against endostatin induced apoptosis. It was found that the inhibition of PI3K/PKB significantly increased endostatin-induced apoptosis, and that endostatin-induced cell death is physiologically linked to PKB-mediated cell survival through caspase-8.

• Journal of Pharmacopuncture
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• v.18 no.2
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• pp.26-32
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• 2015

Objectives: Rheum undulatum L. has traditionally been used for the treatment of many diseases in Asia. However, its anti-proliferative activity in cancer has still not been studied. In the present study, we investigated the anti-cancer effects of methanol extract of Rheum undulatum L. (MERL) on human adenocarcinoma gastric cell lines (AGS). Methods: To investigate the anti-cancer effect of MERL on AGS cells, we treated the AGS cells with varying concentrations of MERL and performed 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. Cell cycle analyses, measurements of the mitochondrial membrane potential (MMP), caspase activity assays and Western blots were conducted to determine whether AGS cell death occurred by apoptosis. Results: Treatment with MERL significantly inhibited growth of AGS cells in a concentration dependent manner. MERL treatment in AGS cells leaded to increased accumulation of apoptotic sub G1 phase cells in a concentration dependent manner. In control cultures, 5.38% of the cells were in the sub G1 phase. In MERL treated cells, however, this percentage was significantly increased (9.95% at $70{\mu}g/mL$, 15.94% at $140{\mu}g/mL$, 26.56% at $210{\mu}g/mL$ and 38.08% at $280{\mu}g/mL$). MERL treatment induced the decreased expression of pro-caspase-8 and -9 in a concentration dependent manner, whereas the expression of the active form of caspase-3 was increased. A subsequent Western blot analysis revealed increased cleaved levels of poly (ADP-ribose) polymerase (PARP) protein. Also, treatment with MERL increased the activities of caspase-3 and -9 compared with the control. MERL treatment increased the levels of the pro-apoptotic truncated Bid (tBid) and Bcl2 Antagonist X (Bax) proteins and decreased the levels of the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein, whose is the stabilization of mitochondria. However, inhibitions of p38, extracellular signal regulated kinases (ERKs) and C-Jun N-terminal kinases (JNK) by MERL treatment did not affect cell death. Conclusion: These results suggest that MERL mediated cell death is associated with an intrinsic apoptotic pathway in AGS cells.

• International Journal of Oral Biology
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• v.46 no.1
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• pp.23-29
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• 2021

Demethoxycurcumin (DMC), which is a curcuminoid found in turmeric, has anti-proliferative effects on cancer cells. However, the effect of DMC on osteosarcoma has not been established. The aim of this study was to examine the effects of DMC on cell growth and apoptosis induction in MG-63 human osteosarcoma cells. This study was investigated using 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromid assay, Live/Dead cell assay, 4', 6-diamidino-2-phenylindole staining, and immunoblotting in MG-63 cells. DMC induced MG-63 cell death in a dose-dependent manner, with an estimated IC50 value of 54.4 µM. DMC treatment resulted in nuclear condensation in MG-63 cells. DMC-induced apoptosis in MG-63 cells was mediated by the expression of Fas and activation of caspase-8, caspase-3, and poly (ADP-ribose) polymerase. Immunoblotting results showed that Bcl-2 and Bcl-xL were downregulated, while Bax and Bad were upregulated by DMC in MG-63 cells. These results indicated that DMC inhibits cell proliferation and induces apoptotic cell death in MG-63 human osteosarcoma cells via the death receptor-mediated extrinsic apoptotic pathway and mitochondria-mediated intrinsic apoptotic pathway.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.33 no.6
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• pp.617-624
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• 2007

Insulin-like growth factor(IGF) is the most abundant growth factor in bone matrix. Recent studies have shown that it can sensitize apoptotic cell death of osteoblasts. Thus, this study investigated whether IGF-II aggravates irradiation-induced cell death of osteoblasts. Cultured MC3T3 osteoblasts were irradiated and IGF-II was added at the concentration of 50 ng/ml immediately after the irradiation. Cell viability was measured by MTT assay. Changes in cell death and cell cycle were analyzed by flow cytometry. The expression of proapoptotic gene bax and antiapoptotic gene bcl-2 was quantified by real time RT-PCR and Western blot. A dose of 30 Gy caused G2/M arrest and increased cell death through both necrosis and apoptosis, while irradiation from 4 to 10 Gy little affected cell cycle and death. IGF-II treatment reduced cell viability without stimulating cell proliferation and changing cell cycle. Combined treatment of IGF-II with irradiation decreased cell viability and proliferation and increased cell death along with G2/M arrest. These effects were not different from those of irradiation only. At transcriptional and protein levels, IGF-II treatment did not affect bax and bcl-2 expression, whereas irradiation increased the expression ofbax without changes in bcl-2. IGF-II in combination with irradiation showed similar findings. These results suggest that IGF-II could modulate apoptotic cell death through mechanisms other than an imbalance between bax and bcl-2 gene expression, although its effect was overridden by irradiation.

• Animal cells and systems
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• v.11 no.1
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• pp.23-31
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• 2007

Programmed cell death was characterized in the silkworm thoracic ganglia TG1, TG2 and TG3 during postembryonic periods by TUNEL assay. Apoptotic cells were detected in the three TGs of all larval stages except for day-1, 2 1st instar larvae, in which no apoptotic cells were found. From day-7 5th larva, the numbers of apoptotic cells were dramatically increased and peaked on day-1 pupa and day-2 pupa and then abruptly decreased. Apoptotic cells finally disappeared in day-1 adult. In-vivo injection of 20-hydroxyecdysone (20E) into day-8 5th larva resulted in a striking decrease of apoptotic cells. Actinomycin D (Act D) or cycloheximide (CHX), injected into hemolymph of day-8 5th larva, resulted in a decrease of apoptotic cells in the three TGs. Injection of caspase-8 and -3 inhibitors also blocked cellular apoptosis. These results will provide valuable information for understanding of cellular changes in the three TGs during metamorphosis of the insect species.

• Journal of Life Science
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• v.29 no.12
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• pp.1393-1400
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• 2019

In the present study, we examined the effect of mitochondrial K⁺ channel opener diazoxide on the mitochondria-dependent apoptotic signaling in endothelial cells exposed to high glucose (HG) media. Endothelial cells derived from human umbilical veins were exposed to HG media containing 30 mM glucose, and the degree of apoptotic cell death associated with activation of the mitochondria-dependent apoptotic signaling pathway was determined. Exposure to HG media was seen to enhance apoptotic cell death in a time-dependent manner. In these cells, activation of caspases 3, 8, and 9 was observed, and while caspase-3 and -9 inhibitors suppressed the HG-induced apoptotic cell death, a caspase-8 inhibitor did not. The HG-treated cells exhibited disruption of mitochondrial membrane potential, formation of permeability transition pores, and cytosolic release of cytochrome c. Subsequently, diazoxide was seen to attenuate the HG-induced apoptotic cell death; caspase-9 activation was suppressed but caspase 8 was not. Diazoxide also suppressed the depolarization of mitochondrial membrane potential, the formation of mitochondrial permeability transition pores, and the release of cytochrome c. These effects were significantly inhibited by 5-hydroxydecanoate, a selective blocker of ATP-sensitive K⁺ channels (K_ATP). The present results demonstrate that diazoxide exhibits a beneficial effect to ameliorate HG-induced endothelial cell apoptosis. Opening the K_ATP could help preserve the functional integrity of mitochondria and provide an underlying mechanism to suppress HG-triggered apoptotic signaling.

• Korean Journal of Clinical Laboratory Science
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• v.52 no.3
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• pp.245-252
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• 2020

Macrophage cell death contributes to the formation of plaque, leading to the development of atherosclerosis. The accumulation of triglyceride (TG) is also associated with the pathogenesis of atherosclerosis. A previous study reported that TG induces the cell death of macrophages. This study examined whether the cytoplasmic release of cathepsin B from lysosome is associated with the TG-induced cell death of macrophage. The release of cathepsin B was increased in the TG-treated THP-1 macrophages, but the TG treatment did not affect cathepsin B expression. Furthermore, the inhibition of cathepsin B by its inhibitor, CA-074 Me, partially inhibited the TG-induced cell death of macrophage. TG-triggered macrophage cell death is mediated by the activation of caspase-1, -2, and apoptotic caspases. Therefore, this study investigated whether cathepsin B is implicated in the activation of these caspases. The inhibition of cathepsin B blocked the activation of caspase-7, -8, and -1 but did not affect the activity of caspase-3, -9, and -2. Overall, these results suggest that TG-induced cytoplasmic cathepsin B causes THP-1 macrophage cell death by activating caspase-1, leading to subsequent activation of the extrinsic apoptotic pathway.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.27 no.6
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• pp.511-518
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• 2001

The effect of mycolactone, a recently reported apoptosis-inducing factor, was investigated in SCC15 oral squamous cell carcinoma(OSCC) cell line. Mycolactone rapidly induced cell death in OSCC cells in 2days, which was similar to that found in apoptotic cell such as detaching from culture plate and rounding-up of cells. Apoptotic cells were increased 4hrs after mycolactone treatment and more than half of cells showed apoptosis after 72hrs. Caspase 3 activation a biochemical evidence of apoptosis, was determined by Western blotting. Caspase 3 activation was started at 2hrs that lasted until 8hrs after mycolactone treatment. The expression of bcl-2 family genes was determined to explain the mechanism of apoptosis found in OSCC cells. The expressions of bad, bak, and bax (pro-apoptotic genes) and bcl-w and bcl-2 genes (anti-apoptotic genes) were not changed by mycolactone treatment. The expression of bcl-xi was decreased 8 hrs after mycolactone treatment. Mcl-1 expression was initially increased at 2 hrs which was decreased 8 hrs after mycolactone treatment. The down-regulation of these two anti-apoptotic genes might explain the mycolactone-induced apoptosis in OSCC cells. In this study, mycholactone was revealed to induce cell death in OSCC cells apoptosis and the apoptosis mechanism of OSCC cells was shown to be down-regulation of anti-apoptotic genes, bcl-xi and mcl-1. These results suggested the applicability of mycolactone for the development of an anti-cancer drug candidate by inducing apoptosis of OSCC cancer cell.