• Biomolecules & Therapeutics
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• v.25 no.2
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• pp.177-185
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• 2017

Auranofin has been developed as antirheumatic drugs, which is currently under clinical development for the treatment of chronic lymphocytic leukemia. Previous report showed that auranofin induced apoptosis by enhancement of annexin A5 expression in PC-3 cells. To understand the role of annexin A5 in auranofin-mediated apoptosis, we performed microarray data analysis to study annexin A5-controlled gene expression in annexin A5 knockdown PC-3 cells. Of differentially expressed genes, plasminogen activator inhibitor (PAI)-2 was increased by annexin A5 siRNA confirmed by qRT-PCR and western blot. Treatment with auranofin decreased PAI-2 and increased annexin A5 expression as well as promoting apoptosis. Furthermore, auranofin-induced apoptosis was recovered by annexin A5 siRNA but it was promoted by PAI-2 siRNA. Interestingly, knockdown of annexin A5 rescued PAI-2 expression suppressed by auranofin. Taken together, our study suggests that induction of annexin A5 by auranofin may enhance apoptosis through suppression of PAI-2 expression in PC-3 cells.

• Nutrition Research and Practice
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• v.14 no.5
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• pp.478-489
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• 2020

BACKGROUND/OBJECTIVES: Morusin, a marker component of Morus alba L., possesses anti-cancer activity. The objective of this study was to determine autophagy-inducing effect of morusin in non-small cell lung cancer (NSCLC) cells and investigate the underlying mechanism. SUBJECTS/METHODS: Autophagy induction and the expression of autophagy-related proteins were analyzed by LC3 immunofluorescence and western blot, respectively. The role of autophagy and AMP-activated protein kinase (AMPK) was determined by treating NSCLC cells with bafilomycin A1, an autophagy inhibitor, and compound C, an AMPK inhibitor. Cytotoxicity and apoptosis induction were determined by MTT assay, trypan blue exclusion assay, annexin V-propidium iodide (PI) double staining assay, and cell cycle analysis. RESULTS: Morusin increased the formation of LC3 puncta in the cytoplasm and upregulated the expression of autophagy-related 5 (Atg5), Atg12, beclin-1, and LC3II in NSCLC cells, demonstrating that morusin could induce autophagy. Treatment with bafilomycin A1 markedly reduced cell viability but increased proportions of sub-G1 phase cells and annexin V-positive cells in H460 cells. These results indicate that morusin can trigger autophagy in NSCLC cells as a defense mechanism against morusin-induced apoptosis. Furthermore, we found that AMPK and its downstream acetyl-CoA carboxylase (ACC) were phosphorylated, while mammalian target of rapamycin (mTOR) and its downstream p70S6 kinase (p70S6K) were dephosphorylated by morusin. Morusin-induced apoptosis was significantly increased by treatment with compound C in H460 cells. These results suggest that morusin-induced AMPK activation could protect NSCLC cells from apoptosis probably by inducing autophagy. CONCLUSIONS: Our findings suggest that combination treatment with morusin and autophagy inhibitor or AMPK inhibitor might enhance the clinical efficacy of morusin for NSCLC.

• 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.

• 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.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.12
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• pp.7095-7100
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• 2013

Histone deacetylase (HDAC) inhibitors of cyclic peptide have been proved to be the most complex but the most stable and relative efficient inhibitors because of their large cap region. In this paper, a series of studies were carried out to evaluate the efficacy of synthetic bicyclic tetrapeptide inhibitors 1-5 containing hydroxamic acid referring molecular docking, anti-proliferation, morphology and apoptosis. Docking analysis, together with enzyme inhibitory results, verified the selective capability of inhibitor 4 to HDAC4, which might closely related to haematological tumorigenesis, with Phe227, Asp115, Pro32, His198 and Ser114 participating into hydrophobic interactions and Van der Waals force which was familiar with former study. Moreover, inhibitor 4 inhibited K562 cell line at the $IC_{50}$ value of 1.22 ${\mu}M$ which was 51-67 times more efficient than that for U937 and HL60 cell lines. Inhibitor 4 exhibited the cell cycle-arrested capability to leukemia at S phase or G2/M phase as well as apoptosis-induced ability in different degrees. Finally, we considered that bicyclic tetrapeptide inhibitors were promising inhibitors used in cancer treatment and inhibitor 4 could prevent K562 cell line well from proliferation, arrest cell cycle and induce K562 towards apoptosis to achieve the goals of reversing cancer cells which could become a potential leukemia therapeutic agent in the future.

• Korean Journal of Head & Neck Oncology
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• v.18 no.2
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• pp.142-149
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• 2002

Objectve : Okadaic acid is a specific inhibitor of serine/threonine protein phosphatase 1 and 2A. In order to know the mechanism of apoptosis induced by okadaic acid, we treated FRTL-5 thyroid cells with okadaic acid and measured the changes of important proteins that are involved in apoptosis. Materials and Methods: We measured caspase 3 activity, $PLC-{\gamma}1$ degradation, the expression of XIAP, cIAP1, cIAP2, and cytochrome c release in okadaic acid-treated FRTL-5 thyroid cells. Results: Okadaic acid-induced caspase 3 activation and $PLC-{\gamma}1$ degradation and apoptosis were dose-dependent with a maximal effect at a concentration of 80 nmol and time-dependent with a maximal effect at 24 hours after treatment. The elevated caspase 3 activity in okadaic acid treated FRTL-5 thyroid cells are correlated with down-regulation of XIAP and cIAP1, but not cIAP2. General and potent inhibitor of caspases, z-VAD-fmk. abolished okadaic acid-induced caspase 3 activity and $PLC-{\gamma}1$ degradation. The release of cytochrome c in okadaic acid-induced FRTL-5 thyroid cells was dose-dependent with a maximal effect at a concentration of 80 nmol. Conclusions: These findings suggest that mechanism of okadaic acid-induced apoptosis is associated with cytochrome c release and increase of caspase 3 activation in FRTL-5 thyroid cells.

• BMB Reports
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• v.48 no.6
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• pp.330-335
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• 2015

Apoptosis inhibitor 5 (API5) has recently been identified as a tumor metastasis-regulating gene in cervical cancer cells.However, the precise mechanism of action for API5 is poorly understood. Here, we show that API5 increases the metastatic capacity of cervical cancer cells in vitro and in vivo via up-regulation of MMP-9. Interestingly, API5-mediated metastasis was strongly dependent on the Erk signaling pathway. Conversely, knock-down of API5 via siRNA technology decreased the level of phospho-Erk, the activity of the MMPs, in vitro invasion, and in vivo pulmonary metastasis. Moreover, the Erk-mediated metastatic action was abolished by the mutation of leucine into arginine within the heptad leucine repeat region, which affects protein-protein interactions. Thus, API5 increases the metastatic capacity of tumor cells by up-regulating MMP levels via activation of the Erk signaling pathway. [BMB Reports 2015; 48(6): 330-335]

• The Journal of Korean Obstetrics and Gynecology
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• v.19 no.2
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• pp.77-91
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• 2006

Purpose : To address the ability of Dohaekseungkitang (DST: a commonly used herb formulation in Korea, Japan and China to have anti-cancer effect on cervical carcinoma), we investigated the effects of DST on programmed cell death (apoptosis) in HeLa human cervical carcinoma cells. Methods : We cultured HeLa cell which is human metrocarcinoma cell in D-MEM included 10% fetal bovine serum(Hyclone Laboratories) below $37^{\circ}C$, 5% CO2. Then we observed apoptosis of log phage cell which is changed cultivation liquid 24 Hours periodically. Results : After the treatment of DST for 48 hours, apoptosis occurred in a dose-dependent manner. In this study, we have shown that DST induces calpain and the associated caspase-8 and -9 activations. Apoptosis was prevented by pre-incubation of the cells with the calcium cHeLator-BAPTA-AM, calcium channel blocker-Nif edipine or Ryonidine agonist-Ryonidine peptide, implicating calcium in the apoptotic process. Ubiquitous calpains (mu- and m-calpain) have been repeatedly implicated in apoptosis, especially in calcium-related apoptosis. However this study showed 1hat either calpain inhibitor-calpastin or caspase-3 inhibitor-DEVD- did not blocked the herb formulation-induced apoptosis in HeLa human cervical carcinoma cells. D ST initiates a cell death pathway that is partially dependent of caspases. DST-induced apoptosis requires caspase-independent mechanism. Conclusion : We conclude that DST-induced calpain activation triggers the intrinsic apoptotic pathway in which caspase-independent mechanism is also involved.

• Reproductive and Developmental Biology
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• v.37 no.4
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• pp.175-183
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• 2013

Cathepsin B is abundantly expressed peptidase of the papain family in the lysosomes, and closely related to the cell degradation system such as apoptosis, necrosis and autophagy. Abnormal degradation of organelles often occurs due to release of cathepsin B into the cytoplasm. Many studies have been reported that relationship between cathepsin B and intracellular mechanisms in various cell types, but porcine embryos has not yet been reported. Therefore, this study evaluated the effect of cathepsin B inhibitor (E-64) on preimplantation developmental competence and quality of porcine embryos focusing on apoptosis and oxidative stress. The expression of cathepsin B mRNA in porcine embryos was gradually decreased in inverse proportion to E-64 concentration by using real-time RT-PCR. When putative zygotes were cultured with E-64 for 24 h, the rates of early cleavage and blastocyst development were decreased by increasing E-64 concentration. However, the rate of blastocyst development in $5{\mu}M$ treated group was similar to the control. On the other hand, both the index of apoptotic and reactive oxygen species (ROS) of blastocysts were significantly decreased in the $5{\mu}M$ E-64 treated group compared with control. We also examined the mRNA expression levels of apoptosis related genes in the blastocysts derived from $5{\mu}M$ E-64 treated and non-treated groups. Expression of the pro-apoptotic Bax gene was shown to be decreased in the E-64 treated blastocyst group, whereas expression of the anti-apoptotic Bcl-xL gene was increased. Taken together, these results suggest that proper inhibition of cathepsin B at early development stage embryos improves the quality of blastocysts, which may be related to not only the apoptosis reduction but also the oxidative stress reduction in porcine embryos.

• Proceedings of the Korean Society of Toxicology Conference
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• 2001.10a
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• pp.151-152
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• 2001

MCS-5A, novel Cdk inhibitor, has been reported that it has exerted cell cycle arrest action and apoptotic effect to the human promyelocytic leukemias cell. The purpose of this study is to verify these effects of MCS-5A on human promyelocytic leukemia (HL-60) cells and to clarify the action of mechanism on MCS-5A-inducing apoptosis.(omitted)