• Molecules and Cells
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• v.41 no.5
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• pp.401-412
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• 2018

Oxymatrine (OMT) often used in treatment for chronic hepatitis B virus infection in clinic. However, OMT-induced liver injury has been reported. In this study, we aim to investigate the possible mechanism of OMT-induced hepatotoxicity in human normal liver cells (L02). Exposed cells to OMT, the cell viability was decreased and apoptosis rate increased, the intracellular markers of oxidative stress were changed. Simultaneously, OMT altered apoptotic related proteins levels, including Bcl-2, Bax and pro-caspase-8/-9/-3. In addition, OMT enhanced the protein levels of endoplasmic reticulum (ER) stress makers (GRP78/Bip, CHOP, and cleaved-Caspase-4) and phosphorylation of c-Jun N-terminal kinase (p-JNK), as well as the mRNA levels of GRP78/Bip, CHOP, caspase-4, and ER stress sensors (IREI, ATF6, and PERK). Pre-treatment with Z-VAD-fmk, JNK inhibitor SP600125 and N-acetyl-l-cysteine (NAC), a ROS scavenger, partly improved the survival rates and restored OMT-induced cellular damage, and reduced caspase-3 cleavage. SP600125 or NAC reduced OMT-induced p-JNK and NAC significantly lowered caspase-4. Furthermore, 4-PBA, the ER stress inhibitor, weakened inhibitory effect of OMT on cells, on the contrary, TM worsen. 4-PBA also reduced the levels of p-JNK and cleaved-caspase-3 proteins. Therefore, OMT-induced injury in L02 cells was related to ROS mediated p-JNK and ER stress induction. Antioxidant, by inhibition of p-JNK or ER stress, may be a feasible method to alleviate OMT-induced liver injury.

• Journal of Life Science
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• v.25 no.11
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• pp.1235-1243
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• 2015

Hwangheuk-san (HHS) is a Korean multi-herb formula comprising four medicinal herbs. HHS, which was recorded in “Dongeuibogam,” has been used to treat patients with inflammation syndromes and digestive tract cancer for hundreds of years. However, little is known about its anti-tumor efficacy. The present study investigated the pro-apoptotic effect and mode of action of HHS against AGS human gastric carcinoma cells. HHS inhibited the cell growth of AGS cells in a dose-dependent manner, which was associated with the induction of apoptotic cell death, as evidenced by the formation of apoptotic bodies, chromatin condensation, and an accumulation of cells in the sub-G1 phase. HHS-induced apoptotic cell death was associated with the up-regulation of pro-apoptotic Bax protein expression, down-regulation of antiapoptotic Bcl-2 protein, and the release of cytochrome c from mitochondria to the cytosol. The treatment of AGS cells with HHS significantly elevated the generation of reactive oxygen species (ROS). Additionally, apoptosis-inducing concentrations of HHS induced the activation of both caspase-9 and -8, initiator caspases of the mitochondrial-mediated intrinsic and death receptor-mediated extrinsic pathways, respectively, and caspase-3, accompanied by proteolytic degradation of poly (ADP-ribose)-polymerase. However, ROS scavenger and pan-caspases inhibitor significantly blocked HHS-induced growth inhibition and apoptosis. Taken together, these findings suggest that HHS induces apoptosis through ROS- and caspase-dependent mechanisms and that HHS may be a potential chemotherapeutic agent for the control of human gastric cancer.

• Journal of Life Science
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• v.28 no.11
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• pp.1268-1276
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• 2018

The cytidine analog decitabine (DEC) acts as a nucleic acid synthesis inhibitor, whereas ammonium pyrrolidine dithiocarbamate (PDTC) is an inhibitor of nuclear factor-${\kappa}B$. The aim of this study was to investigate the possible synergistic inhibitory effect of these two inhibitors on proliferation of human gastric cancer AGS cells. The inhibitory effect of PDTC on AGS cell proliferation was significantly increased by DEC in a concentration-dependent manner, and this inhibition was associated with cell cycle arrest at the G2/M phase and the induction of apoptosis. This induction of apoptosis by the co-treatment with PDTC and DEC was related to the induction of DNA damage, as assessed by H2AX phosphorylation. Further studies demonstrated that co-treatment with PDTC and DEC induced the disruption of mitochondrial membrane potential, increased the generation of intracellular reactive oxygen species (ROS) and the expression of pro-apoptotic Bax, and down-regulated the expression of anti-apoptotic Bcl-2, ultimately resulting in the release of cytochrome c from the mitochondria into the cytoplasm. Co-treatment with PDTC and DEC also activated caspase-8 and caspase-9, which are representative caspases of the extrinsic and intrinsic apoptosis pathways. Co-treatment also activated caspase-3, which was accompanied by proteolytic degradation of poly (ADP-ribose) polymerase. Taken together, these data clearly indicated that co-treatment with PDTC and DEC suppressed the proliferation of AGS cells by increasing DNA damage and activating the ROS-mediated extrinsic and intrinsic apoptosis pathways.

• Journal of Life Science
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• v.25 no.1
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• pp.93-100
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• 2015

Pachymic acid (PA) is a lanostane-type triterpenoid derived from the Poria cocos mushroom. Several beneficial biological features of PA provide medicine with a wide variety of valuable effects, such as anticancer and anti-inflammatory activity; it also has antioxidant effects against oxidative stress. Nonetheless, the biological properties and mechanisms that produce this anti-cancer action of PA remain largely undetermined. In this study, we investigated the pro-apoptotic effects of PA in T24 human bladder cancer cells. It was found that PA could inhibit the cell growth of T24 cells in a dose-dependent manner, which was associated with the induction of apoptotic cell death, as evidenced by the formation of apoptotic bodies and chromatin condensation and accumulation of cells in the sub-G1 phase. The induction of apoptotic cell death by PA was connected with an up-regulation of pro-apoptotic Bax and Bad protein expression and down-regulation of anti-apoptotic Bcl-2 and Bcl-xL proteins, and inhibition of apoptosis family proteins. In addition, apoptosis-inducing concentrations of PA induced the activation of caspase-9, an initiator caspase of the mitochondrial-mediated intrinsic pathway, and caspase-3, accompanied by proteolytic degradation of poly (ADP-ribose)-polymerase. PA also induced apoptosis via a death receptor-mediated extrinsic pathway by caspase-8 activation, resulting in the truncation of Bid and suggesting the existence of cross-talk between the extrinsic and intrinsic pathways. Taken together, the present results suggest that PA may be a potential chemotherapeutic agent for the control of human bladder cancer cells.

• Tuberculosis and Respiratory Diseases
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• v.53 no.5
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• pp.485-496
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• 2002

Background : Nonsteroidal anti-inflammatory drugs (NSAIDs) are useful in the chemoprevention of colon cancers. Continuous NSAID use results in a 40% to 50% reduction in the relative risk of colorectal cancer. The precise mechanism by which NSAIDs prevent and/or cause the regression of colorectal tumors is not known. Some investigators have reported that certain NSAIDs induce apoptosis and alter the expression of the cell cycle regulatory genes in some carcinoma cells when administered at a relatively high concentration. However, the possibility of NSAIDs application as chemopreventive agents in lung cancers remains to be elucidated. To address this question, the human lung cancer cell line NCI-H1299 was used to investigate whether or not NSAIDs might induce the apoptotic death of NCI-H1299 cells. Methods : A viability test was carried out using a MTT assay. Apoptosis was measured by flow cytometric analysis and unclear staining(DAPI). The talytic activity of the caspase family was measured by the fluirigenic cleavage of biosubstrates. To define the mechanical basis of apoptosis, western blot was performed to analyze the expression of the death substrates(PARP and ICAD). Results : NaSaL significantly decreased the viability of the NCI-H1299 cells, which was revealed as apoptosis characterized by an increase in the $subG_0/G_1$ population and unclear fragmentation. The catalytic activity of caspase-3 protease began to increase after 24 Hr and reached a peak 30 Hr after treatment with 10 mM NaSaL. In contrast, caspase-6, 8, and 9 proteases did not have a significantly altered enzymatic activity. Consistent with activation of caspase-3 protease, NaSaL induced the cleavage of the protease biosubstrate. Conclusion : NaSaL induces the apoptotic death of NCI-H1299 human lung cancer cells via the activation of caspase-3 protease.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.316.2-316.2
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• 2002

Manassantin B classified into dineolignans have been isolated from Saururus chinensis Manassantin B was found to induce apoptosis in human promyelocytic leukaemia HL -60 cells with characteristic apoptotic features like increase of nucleosomalladder. apoptotic body ormation. flipping of membrane phosphatidylserine. Manassantin B induced FAS and FAS ligand expression, and activated caspase 8 which cleaved bid to tbid in cytosol. The release of cytochrome c to sytosol was accompanied with decrease of bcl-2 protein and incresase of tbid and bax protein in mitochondria. Released xytochrome c activated caspase 9 and-3. but these effects were completely attenuated by the treatment of broad caspses ingibitor. Z-VAD fmk. These results indicate that manassatin B induce apoptosis through upregulation of FAS. caspase family and mitochondria-related proteins.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2003.11a
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• pp.67-67
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• 2003

Minocycline is known to protect neurons from microglia-mediated cell death in many experimental models of brain diseases including ischemic stroke, Huntingtons disease (HD), amyotrophic lateral sclerosis (ALS), traumatic brain injury, multiple sclerosis, and Parkinsons disease. When the activity of caspases was assessed using their fluorescent peptide substrates, activation of caspase-2, 3, 8, and 9 was evident within 2 8 hr following oxidative insult with 0.5 mM hydrogen peroxide in PC12 cells. Minocycline significantly attenuated activation of these caspases up to 18 hr, resulting a significant increase in the cell viability as assessed by MTT assay as well as trypan blue staining. However, cleavage of alpha-spectrin and a cdk5 activator p35, which are known to be substrates for calpain, remained unchanged in the presence of minocycline, suggesting that minocycline did not block caspase-3-independent cell death or necrosis. Moreover, co-treatment with minocycline and a calpain inhibitor calpeptin synergistically inhibited hydrogen peroxide-induced cell death. These data suggest that minocycline directly inhibited apoptosis, but not necrosis, after oxidative insult in PC12 cells.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.319.1-319.1
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• 2002

Yomogin. an eudesmane sesquiterpene isolated from Artemisia princeps, was found to induce apoptosis in human promyelocytic leukaemia, HL -60 cell with characteristic apoptotic features like nuclear condensation, apoptotic body formation, flipping of membrane phosphatidylserine, release of mitochondrial cytochrome c and caspase-8. -9. and -3 activation. Furthermore. early yomogin-induced cytochrome c release was not affected by the caspase inhibitor Z-VAd fmk and preceded loss of mitochondrial membrane potential. The results suggest that induction of apoptosis by yomogin may provide a pivotal mechanism for their cancer chemopreventive function.

• Journal of Periodontal and Implant Science
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• v.34 no.4
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• pp.807-818
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• 2004

산화질소는 생리적 농도에서 세포내 신호전달자로 작용하지만 높은 농도에서는 세포독성을 일으킨다. 최근 치은 섬유아세포와 치주인대 섬유아세포는 산화질소 합성효소를 가지고 있고 세균의 lipopolysaccharide나 cytokine에 의해 대량의 높은 농도의 산화질소가 합성된다는 보고가 있음에도 지금까지 치은 조직에서 산화질소의 세포독성에 대한 연구는 아직 이루어 지지않고 있다. 본 연구는 사람의 치은 섬유아세포에서, 산화질소유도세포 고사기전을 밝히는데 목적이 있다. 세포 생장력은 MTT 방법으로 측정하였고, 세포의 형태적 변화는 Diff-Quick 염색법으로 조사하였다. Bcl-2 famly와 Fas 발현 정도는 RT-PCR 방법에 의해 확인하였으며, caspase-3, -8 와 -9의 활성은 spectrophotometer로 reactive oxygen species (ROS)는 형광분광계에 의해 측정되었다. 미토콘드리아에서 세포질로 분비된 cytochrome c는 western blot으로 조사하였다. 산화질소 유리제인 sodium nitroprusside (SNP) 처리는 사람 섬유아세포의 생존률을 시간과 농도 의존적으로 감소시켰고, 세포용적축소, 염색사 용축, DNA 절편화를 일으켰다. 또한, SNP 처리로 미토콘드리아에서 세포질로 유리되는 cytochrome c 양이 증가되었고, caspase-9 과 caspase-3 의 활성이 증가되었다. 한편, SNP 처리에 의해 death receptor 구성요소인 Fas 발현이 증가되었고, caspase-8의 활성이 증가되었다. Bcl-2 family 에 대한 RT-PCR 분석결과, 세포고사를 억제하는 Bcl-2 발현은 감소되었으나 세포고사를 자극하는 Bax와 Bid의 발현은 증가되었다. Soluble guanylate cyclase 억제제인 ODQ는 SNP에 의한 세포 생존율 감소를 차단하지 못했다. 따라서, 본 실험의 결과들은 사람 섬유아세포에서 산화질소유도 세포고사에 Bcl-2 family나 ROS가 매개하는 미토콘드리아 의존 및 death receptor 의존 세포고사기전이 관여함을 시사하였다.

• Biomolecules & Therapeutics
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• v.15 no.3
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• pp.137-144
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• 2007

Although lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase, has been shown to have anti-cancer actions, the effect on human hepatoma cells was not investigated. Moreover, the exact mechanism of this action is not fully understood. In this study we investigated the mechanism by which lovastatin induces apoptosis using HepG2 human hepatoblastoma cells. Lovastatin induced apoptotic cell death in a dose-dependent manner in the cells, assessed by the flow cytometric analysis. Treatment with mevalonic acid, a precursor of cholesterol, did not significantly suppress the lovastatin-induced apoptosis. Lovastatin induced a rapid and sustained increase in intracellular $Ca^{2+}$ concentration. Treatment with EGTA, an extracellular $Ca^{2+}$ chelator did not significantly alter the lovastatin-induced intracellular $Ca^{2+}$ increase and apoptosis, whereas intracellular $Ca^{2+}$ reduction with BAPTA/AM and intracellular $Ca^{2+}$ release blockers (dantrolene and TMB-8) completely blocked these actions of lovastatin. In addition, the lovastatin-induced apoptosis was significantly reduced by a calpain inhibitor, a broad spectrum caspase inhibitor z-VAD-fmk and inhibitors specific for caspase-9 and caspase-3 (z-LEHD-fmk and z-DEVD-fmk, respectively), but not by an inhibitor specific for caspase-8 (z-IETD-fmk). Collectively, these results suggest that lovastatin induced apoptosis of HepG2 hepatoma cells through intracellular $Ca^{2+}$ release and calpain activation, leading to triggering mitochondrial apoptotic pathway. These results further suggest that lovastatin may be valuable for the therapeutic management of human hepatoma.