• Biomolecules & Therapeutics
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• 제22권6호
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• pp.519-524
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• 2014

We have previously shown that 2,4,3',5'-tetramethoxystilbene (TMS), a trans-stilbene analogue, induces apoptosis in human cancer cells. However, the detailed mechanisms of mitochondria-dependent apoptosis induced by TMS are not fully understood. In the present study, the possible roles of annexin A5 in TMS-mediated apoptosis were investigated in MCF7 human breast cancer cells. Quantitative real-time PCR analysis and Western blot analysis showed that the expression of annexin A5 was strongly increased in TMS-treated cells. TMS caused a strong translocation of annexin A5 from cytosol into mitochondria. Confocal laser scanning microscopic analysis clearly showed that TMS induced translocation of annexin A5 into mitochondria. TMS increased the expression and oligomerization of voltage-dependent anion channel (VDAC) 1, which may promote mitochondria-dependent apoptosis through disruption of mitochondrial membrane potential. When cells were treated with TMS, the levels of Bax, and Bak as well as annexin A5 were strongly enhanced. Moreover, we found that the cytosolic release of apoptogenic factors such as cytochrome c, or apoptosis-inducing factor (AIF) in mitochondria was markedly increased. Annexin A5 depletion by siRNA led to decreased proapoptotic factors such as Bax, Bak, and annexin A5. Taken together, our results indicate that annexin A5 may play an important role in TMS-mediated mitochondrial apoptosis through the regulation of proapoptotic proteins and VDAC1 expression.

• 대한한방소아과학회지
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• 제22권1호
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• pp.113-121
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• 2008

Objectives In human myeloid leukemia cells, there are no specific features of apoptosis compared with apoptosis in other cell types. Solanum nigrum L.(SNL) is a deciduous tree, which is widely distributed in Korea with reported anti-tumor, anti-inflammatory and non-specific immune-enhancing properties. Although the plant has been clinically used for treating a variety of diseases, its bioactive ingredients are unknown and its mode of action potential has never been investigated. Thus anti-tumor property of methanol extract was investigated. Methods In this study, anti-tumor property of methanol extract was investigated by determining its in vitro growth-inhibitory effects on human myeloid leukemia cells. XTT proliferation assay, DNA fragmentation, immunoblot analysis, densitometric analysis were used. Results 1. The methanol fraction of the extracts of SNL induced mitochondria-mediated apoptosis in human myeloid leukemia cells. 2. The methanol fraction exhibited relatively higher cytotoxic activity in a dose-dependent manner than chloroform, and hexane fraction. 3. Typical ladder profile of Oligonucleosomal fragments were appeared. 4. The secreted cytosolic cytochrome C level was increased by treatment of methanol fraction. Conclusions Methanol fraction of SNL is capable of inducing apoptosis in human myeloid leukemia cells.

• Biomolecules & Therapeutics
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• 제27권1호
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• pp.41-47
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• 2019

The apoptotic effects of shikonin (5,8-dihydroxy-2-[(1R)-1-hydroxy-4-methylpent-3-enyl]naphthalene-1,4-dione) on the human colon cancer cell line SNU-407 were investigated in this study. Shikonin showed dose-dependent cytotoxic activity against SNU-407 cells, with an estimated $IC_{50}$ value of $3{\mu}M$ after 48 h of treatment. Shikonin induced apoptosis, as evidenced by apoptotic body formation, sub-G_1$ phase cells, and DNA fragmentation. Shikonin induced apoptotic cell death by activating mitogen-activated protein kinase family members, and the apoptotic process was mediated by the activation of endoplasmic reticulum (ER) stress, leading to activation of the $PERK/elF2{\alpha}/CHOP$ apoptotic pathway, and mitochondrial $Ca^{2+}$ accumulation. Shikonin increased mitochondrial membrane depolarization and altered the levels of apoptosis-related proteins, with a decrease in B cell lymphoma (Bcl)-2 and an increase in Bcl-2-associated X protein, and subsequently, increased expression of cleaved forms of caspase-9 and -3. Taken together, we suggest that these mechanisms, including MAPK signaling and the ER- and mitochondria-mediated pathways, may underlie shikonin-induced apoptosis related to its anticancer effect.

• Journal of Microbiology and Biotechnology
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• 제23권11호
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• pp.1627-1635
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• 2013

Mitochondria often play central roles in apoptotic pathways, and disruption of the mitochondrial transmembrane potential (${\Delta}{\psi}m$) has been observed in various cells undergoing apoptosis. Human cytomegalovirus (HCMV) infection induces apoptosis in permissive cells; however, investigations of mitochondria-targeted apoptosis in HCMV-infected human foreskin fibroblast (HFF) cells have been limited. Here, we investigated the mitochondrial apoptosis pathway in HCMV-infected HFF cells. Flow cytometry analysis using JC-1 revealed that HCMV infection induces disruption of ${\Delta}{\psi}m$ in HFF cells when administered 24 h post-infection (hpi), and this disruption was maximized at 48 hpi. Moreover, cytochrome c, normally a mitochondrial inner membrane protein, was detected in cytoplasmic extracts of HCMV-infected cells, but not mock-infected cells, by western blot analysis at 24 hpi. A caspase activity assay based on fluorescence spectrophotometry using a fluorogenic substrate revealed an increase in caspase-3 activity at 48 hpi in HCMV-infected cells. Caspase-8 activity was increased at 72 hpi in HCMV-infected cells. These results imply that HCMV infection induces mitochondria-mediated apoptosis in HFF cells.

• BMB Reports
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• 제38권6호
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• pp.709-716
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• 2005

Apoptosis and necrosis are distinguished by modality primarily. Here we show an apoptosis occurred instantly, induced by $300\;{\mu}M$ W-7 ((N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride), inhibitor of calmodulin), which demonstrated necrotic modality. As early as 30 min after W-7 addition, apoptotic (sub-diploid) peak could be detected by fluorescence-activated cell sorter (FACS), “DNA ladders” began to emerge also at this time point, activity of caspase-3 elevated obviously within this period. Absence of mitochondrial membrane potential (MMP) reduction and cytochrome c, AIF (apoptosis inducing factor) release, verified that this rapid apoptosis did not proceed through mitochondria pathway. Activation of caspase-12 and changes of other endoplasmic reticulum (ER) located proteins ascertained that ER pathway mediated this necrosis-like apoptosis. Our findings suggest that it is not credible to judge apoptosis by modality. Elucidation of ER pathway is helpful to comprehend the pathology of diseases associated with ER stress, and may offer a new approach to the therapy of cancer and neurodegenerative diseases.

• Biomolecules & Therapeutics
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• 제30권2호
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• pp.137-144
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• 2022

Radiation resistance represents an imperative obstacle in the treatment of patients with colorectal cancer, which remains difficult to overcome. Here, we explored the anti-proliferative and migration-inhibiting properties of the natural product shikonin on a radiation-resistant human colon carcinoma cell line (SNU-C5RR). Shikonin reduced the viability of these cells in a dose-dependent manner; 38 µM of shikonin was determined as the half-maximal inhibitory concentration. Shikonin induced apoptotic cell death, as demonstrated by increased apoptotic body formation and the number of TUNEL-positive cells. Moreover, shikonin enhanced mitochondrial membrane depolarization and Bax expression and also decreased Bcl-2 expression with translocation of cytochrome c from mitochondria into the cytosol. In addition, shikonin activated mitogen-activated protein kinases, and their specific inhibitors reduced the cytotoxic effects of shikonin. Additionally, shikonin decreased the migration of SNU-C5RR cells via the upregulation of E-cadherin and downregulation of N-cadherin. Taken together, these results suggest that shikonin induces mitochondria-mediated apoptosis and attenuates epithelial-mesenchymal transition in SNU-C5RR cells.

• Journal of Microbiology and Biotechnology
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• 제21권5호
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• pp.525-528
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• 2011

The Raf-1 kinase inhibitory protein (RKIP) can regulate multiple key signaling pathways. Specifically, RKIP binds to Raf-1 kinase and inhibits the Ras-Raf-1-MEK1/2- ERK1/2 pathway. Additionally, Raf-1 has been shown to translocate to mitochondria and thereby protect cells from stress-mediated apoptosis. Recently, HBx was found to stimulate the mitochondrial translocation of Raf-1, contributing to the anti-apoptotic effect. We found that RKIP was downregulated during HBx-mediated hepatocarcinogenesis. In this study, we show that RKIP bound to Raf-1 and consequently inhibited the translocation of Raf-1 into mitochondria. This promoted the apoptosis of cells treated with apoptotic stimulus. Thus, the downregulation of RKIP increased the level of free Raf-1 and thereby elevated the mitochondrial translocation of Raf-1 during HBx-mediated hepatocarcinogenesis. The elevated Raf-1 mitochondrial translocation induced the increased anti-apoptotic effect and subsequently promoted HBx-mediated hepatocarcinogenesis.

• 동아시아식생활학회지
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• 제24권1호
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• pp.20-27
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• 2014

Our study is investigated the effects of Nelumbo nucifera root extract on HT-29 colon cancer cells. The anti-proliferative effect of 70% ethanol extract from Nelumbo nucifera root on HT-29 colon cancer cells was identified based on cell viability, Hoechst 33342 nuclear staining, apoptosis analysis, Western blotting and RT-PCR analyses. In our study, Nelumbo nucifera root extract inhibited the growth of HT-29 colon cancer cells in a dose-dependent manner. Concomitant activation of the mitochondria-dependent apoptotic pathway of HT-29 colon cancer cells by Nelumbo nucifera root extract occurred via modulation of Bax and Bcl-2 expressions, which activated cleavage of caspases-3 and -9. The findings of this study indicate that Nelumbo nucifera root extract induces apoptosis in HT-29 colon cancer cells, and this phenomenon is occurs via the death receptor-mediated and mitochondria-mediated apoptotic pathways.

• Molecules and Cells
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• 제27권5호
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• pp.533-538
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• 2009

Heat shock protein 27 (Hsp27) is a molecular chaperone protein which regulates cell apoptosis by interacting directly with the caspase activation components in the apoptotic pathways. With the assistance of the Tat protein transduction domain we directly delivered the Hsp27 into the myocardial cell line, H9c2 and demonstrate that this protein can reverse hypoxia-induced apoptosis of cells. In order to characterize the contribution of Hsp27 in blocking the two major apoptotic pathways operational within cells, we exposed H9c2 cells to staurosporine and cobalt chloride, agents that induce mitochondria-dependent (intrinsic) and -independent (extrinsic) pathways of apoptosis in cells respectively. The Tat-Hsp27 fusion protein showed a greater propensity to inhibit the effect induced by the cobalt chloride treatment. These data suggest that the Hsp27 predominantly exerts its protective effect by interfering with the components of the extrinsic pathway of apoptosis.

• Asian Pacific Journal of Cancer Prevention
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• 제15권23호
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• pp.10495-10500
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• 2015

Background: To evaluate the effects of bufalin in A549 human lung adenocarcinoma epithelial cells in vitro and assess the underlying mechanisms. Materials and Methods: Human A549 non-small cell lung cancer (NSCLC) cells were treated with various concentrations of bufalin. Cell proliferation was measured by CCK-8 assay, apoptotic cell percentage was calculated by flow cytometry and morphological change was observed by inverted phase contrast microscopy/transmission electron microscopy. In addition, the membrane potential of mitochondria was detected by JC-1 fluorescence microscopy assay, and the related protein expression of cytochrome C and caspase-3 was analyzed by Western blotting. Results: Bufalin could inhibit the proliferation of A549 cells via induction of apoptosis, with the evidence of characteristic morphological changes in the nucleus and mitochondria. Furthermore, bufalin decreased the mitochondrial membrane potential with up-regulation of cytochrome C in the cytosol, and activation of caspase-3. Conclusions: Bufalin inhibits the proliferation of A549 cells and triggers mitochondria-dependent apoptosis, pointing to therapeutic application for NSCLC.