• Development and Reproduction
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• v.6 no.2
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• pp.131-139
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• 2002

Since GnRH and its receptor genes are expressed in the ovary, it has been suggested that ovarian GnRH might be involved in the regulation of ovarian function and the apoptosis of ovarian cells. However, it was not known well on the expression and function of GnRH and its receptor in the corpus luteum. The present study was undertaken to investigate whether GnRH and its receptor are expressed in luteal cells and GnRH has any effect on the apoptosis of luteal cells. Luteal cells obtained from the pregnant rats were cultured and stained for GnRH and its receptor proteins. Cultured luteal cells showed distinct immunoreactivity against both anti-GnRH and anti-GnRH receptor antibodies. In addition, the presence of GnRH receptor protein in cultured cells was confirmed by Western blot analysis. To investigate the effect of GnRH on the apoptosis of luteal cells, luteal cells were cultured in the presence of 10$^{-6}$ M GnRH-agonist(GnRH-Ag) for 3, 8, and 12h. TUNEL assay showed that the number of cells undergoing apoptosis increased 12h after culture(P<0.05). DNA fragmentation analysis confirmed the results such that the cells treated for 12h showed the greatest increase of fragmentation(p<0.05). Further, Western blot analysis of cytochrome c in the mitochondrial and cytoplasmic fractions of the luteal cells showed that GnRH-Ag treatment increased the content of cytochrome c in cytoplasm. These results demonstrate that the luteal cells express GnRH and its receptor and GnRH-Ag treatment induces apoptosis of the luteal cells via mitochondrial release of cytochrome c. The present study suggest that the releasing of cytochrome c from mitochondria might be involved in the luteal cell apoptosis induced by GnRH-Ag.

• Molecules and Cells
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• v.26 no.5
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• pp.436-442
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• 2008

Osteoclasts are multinucleated cells with the unique ability to resorb bone. Elevated activity of these cells under pathologic conditions leads to the progression of bone erosion that occurs in osteoporosis, periodontal disease, and rheumatoid arthritis. Thus, the regulation of osteoclast apoptosis is important for bone homeostasis. In this study, we examined the effects of the Janus tyrosine kinase 2 specific inhibitor AG490 on osteoclast apoptosis. We found that AG490 greatly inhibited osteoclast apoptosis. AG490 stimulated the phosphorylation of Akt and ERK. Adenovirus-mediated expression of dominant negative (DN)-Akt and DN-Ras in osteoclasts inhibited the survival of osteoclasts despite the presence of AG490. Cytochrome c release during osteoclast apoptosis was inhibited by AG490 treatment, but this effect was inhibited in the presence of LY294002 or U0126. AG490 suppressed the pro-apoptotic proteins Bad and Bim, which was inhibited in osteoclasts infected with DN-Akt and DN-Ras adenovirus. In addition, constitutively active MEK and myristoylated-Akt adenovirus suppressed the cleavage of pro-caspase-9 and -3 and inhibited osteoclast apoptosis induced by etoposide. Taken together, our results suggest that AG490 inhibited cytochrome c release into the cytosol at least partly by inhibiting the pro-apoptotic proteins Bad and Bim, which in turn suppressed caspase-9 and -3 activation, thereby inhibiting osteoclast apoptosis.

• BMB Reports
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• v.46 no.1
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• pp.47-52
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• 2013

Intestinal epithelial cell (IEC) apoptosis induced by hypoxia compromise intestinal epithelium barrier function. Both Akt and Hsp90 have cytoprotective function. However, the specific role of Akt and $Hsp90{\beta}$ in IEC apoptosis induced by hypoxia has not been explored. We confirmed that hypoxia-induced apoptosis was reduced by $Hsp90{\beta}$ overexpression but enhanced by decreasing $Hsp90{\beta}$ expression. $Hsp90{\beta}$ overexpression enhanced BAD phosphorylation and thus reduced mitochondrial release of cytochrome C. Reducing $Hsp90{\beta}$ expression had opposite effects. The protective effect of $Hsp90{\beta}$ against apoptosis was negated by LY294002, an Akt inhibitor. Further study showed that Akt phosphorylation was enhanced by $Hsp90{\beta}$, which was not due to the activation of upstream PI3K and PDK1 but because of stabilization of pAkt via direct interaction between $Hsp90{\beta}$ and pAkt. These results demonstrate that $Hsp90{\beta}$ may play a significant role in protecting IECs from hypoxia-induced apoptosis via stabilizing pAkt to phosphorylate BAD and reduce cytochrome C release.

• Journal of Physiology & Pathology in Korean Medicine
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• v.22 no.5
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• pp.1322-1329
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• 2008

Mulberry has been reported to contain wide range of polyphenols and have chemopreventive activity. However, little has been known regarding the effect of mulberry fruits on cell viability in human glioma cells. The present study was undertaken to examine the effect of mulberry fruit (Mar; Fructus) on cell viability and to determine its underlying mechanism in human glioma cells. Cell viability and cell death were estimated by MTT assay and trypanblue exclusion assay, respectively. Reactive oxygen species (ROS) generation was measured using the fluorescence probe DCFH-DA. The mitochondrial transmembrane potential was measured with $DiOC_6$(3). Bax expression and cytochrome c release were measured by Western blot analysis. Caspase activity was estimated using colorimetric kit. Mori Fructus resulted in apoptotic cell death in a dose- and time-dependent manner. Mori Fructus increased ROS generation and the Mori Fructus-induced cell death was also prevented by antioxidants, suggesting that ROS generation plays a critical role in Mari Fructus-induced cell death. Western blot analysis showed that Mori Fructus treatment caused an increase in Bax expression, which was inhibited by the antioxidant N-acetylcysteine (NAC). Mori Fructus induced depolarization of mitochondrial membrane potential and its effect was inhibited by the antioxidants NAC and catalase. Mori Fructus induced cytochrome c release, which was inhibited by NAC. Caspase activity was stimulated by Mori Fructus and caspase inhibitors prevented the Mori Fructus-induced cell death. These findings suggest that Mori Fructus results in human glioma cell death through ROS-dependent mitochondrial pathway in human glioma cells.

• Journal of Ginseng Research
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• v.44 no.1
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• pp.96-104
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• 2020

Objectives: Oleanolic acid, a minor element of ginsenosides, and its derivatives have been shown to have cytotoxicity against some tumor cells. The impact of cytotoxic effect of oleanolic acid 3-acetate on ovarian cancer SKOV3 cells and endometrial cancer HEC-1A cells were examined both in vivo and in vitro to explore the underlying mechanisms. Methods: Cytotoxic effects of oleanolic acid 3-acetate were assessed by cell viability, phosphatidylserine exposure on the cell surface, mitochondrial release of cytochrome C, nuclear translocation of apoptosis-inducing factor, depolarization of mitochondrial transmembrane potential (∆Ψ_m), and generation of reactive oxygen species (ROS). In vivo inhibition of tumor growth was also assessed with xenografts in immunocompromised mice. Results: Oleanolic acid 3-acetate exhibited potent cytotoxicity toward SKOV3 and HEC-1A cells by decreasing cell viability in a concentration-dependent manner. Importantly, oleanolic acid 3-acetate effectively suppressed the growth of SKOV3 cell tumor xenografts in immunocompromised mice. Furthermore, oleanolic acid 3-acetate induced apoptotic cell death as revealed by loss of ∆Ψ_m, release of cytochrome c, and nuclear translocation of apoptosis-inducing factor with a concomitant activation of many proapoptotic cellular components including poly(ADP-ribose) polymerase, Bcl-2, and caspases-8, caspase-3, and caspase-7. Oleanolic acid 3-acetate, however, caused a decrease in ROS production, suggesting the involvement of an ROS-independent pathway in oleanolic acid 3-acetate-induced apoptosis in SKOV3 and HEC-1A cells. Conclusion: These findings support the notion that oleanolic acid 3-acetate could be used as a potent anticancer supplementary agent against ovarian and endometrial cancer. Oleanolic acid 3-acetate exerts its proapoptotic effects through a rather unique molecular mechanism that involves an unconventional ROS-independent but mitochondria-mediated pathway.

• The Journal of Korean Obstetrics and Gynecology
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• v.18 no.1
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• pp.15-28
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• 2005

To address the ability of Kung-Kyung-Ilho-Jeon(KK) to induce cell death, we investigated the effect of KK on cell viability. Forty-eight hours later, loss of viability occurred following KK exposure in a dose-dependent manner. The treatment of KK, a commonly used herb formulation in Korea and China, caused a decrease in cell viability. KK also resulted in apoptotic morphology a brightly blue-fluorescent condensed nuclei by Hoechst 33258-staining, and reduction of cell volume. Our results show that KK induces caspase-3 and -9 activation in a time-dependent manner. In addtion, the translocation of cytochrome c release into cytoplasm has been observed under the presence of $5mg/m{\ell}$ KK. The subsequent loss of mitochondria membrane potential is collapsed by the addition of KK. Our immunoblotting data show that PARP, a well known caspase-3 and -6 substrate, is cleaved by KK. We show that a pro-apoptotic protein, Bax is increased in the presence of KK but that the amount of Bcl-2 is not changed. We suggest that Bax, a critical protein which can regulate channel of mitochondria to release cytochrome c, is a key protein in KK-induced apoptosis of Hela human cervical carcinoma cells

• Journal of Ginseng Research
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• v.37 no.4
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• pp.413-424
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• 2013

Korean Red Ginseng extract (KRGE) is a traditional herbal medicine utilized to prevent endothelium dysfunction in the cardiovascular system; however, its underlying mechanism has not been clearly elucidated. We here examined the pharmacological effect and molecular mechanism of KRGE on apoptosis of human umbilical vein endothelial cells (HUVECs) in a serum-deprived apoptosis model. KRGE protected HUVECs from serum-deprived apoptosis by inhibiting mitochondrial cytochrome c release and caspase-9/-3 activation. This protective effect was significantly higher than that of American ginseng extract. KRGE treatment increased antiapoptotic Bcl-2 and Bcl-$X_L$ protein expression and Akt-dependent Bad phosphorylation. Moreover, KRGE prevented serum deprivation-induced subcellular redistribution of these proteins between the mitochondrion and the cytosol, resulting in suppression of mitochondrial cytochrome c release. In addition, KRGE increased nitric oxide (NO) production via Akt-dependent activation of endothelial NO synthase (eNOS), as well as inhibited caspase-9/-3 activities. These increases were reversed by co-treatment of cells with inhibitors of eNOS and phosphoinositide 3-kinase (PI3K) and pre-incubation of cell lysates in dithiothreitol, indicating KRGE induces NO-mediated caspase modification. Indeed, KRGE inhibited caspase-3 activity via S-nitrosylation. These findings suggest that KRGE prevents serum deprivation-induced HUVEC apoptosis via increased Bcl-2 and Bcl-$X_L$ protein expression, PI3K/Akt-dependent Bad phosphorylation, and eNOS/NO-mediated S-nitrosylation of caspases. The cytoprotective property of KRGE may be valuable for developing new pharmaceutical means that limit endothelial cell death induced during the pathogenesis of vascular diseases.

• 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.

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

• Journal of Microbiology and Biotechnology
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• v.25 no.3
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• pp.418-422
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• 2015

In the course of screening for novel cell cycle inhibitors and apoptotic inducers, CR389, elucidated as 5-(1H-benzoimidazol-2-yl)-1H-pyridin-2-one, was generated as a new hit compound. Flow cytometric analysis and western blots of PA-1 cells treated with $60{\mu}M$ CR389 revealed an appreciable cell cycle arrest at the G2/M phase through direct inhibition of the CDK1 complex. In addition, activation of p53 via phosphorylation at Ser15 and subsequent up-regulation of p21^CIP1 showed that CR389 also induces p53-dependent-p21^CIP1-mediated cell cycle arrest. Furthermore, apoptotic induction in $60{\mu}M$ CR389-treated PA-1 cells is associated with the release of cytochrome c from mitochondria through up-regulation of the proapoptotic Bax protein, which results in the activation of procaspase-9 and -3, and the cleavage of poly(ADP-ribose) polymerase (PARP). Accordingly, CR389 seems to have multiple mechanisms of antiproliferative activity through p53-mediated pathways against human ovarian cancer cells. Therefore, we conclude that CR389 is a candidate therapeutic agent for the treatment of human ovarian cancer via the activation of p53.