• 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.

• 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.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1998.11a
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• pp.127-127
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• 1998

Specific inhibitors of a calcium activated neutral protease calpain could be used for the treatment of neurodegenerative diseases, cataract and muscular dystrophy diseases because of their therapeutic effects. In the course of screening for potential calpain inhibitors from microorganisms, a new analogue of chymostatins named calmostinol was isolated from the culture filtrate of an actinomycete. The MW was determined to be 596 [(M + H)$\^$+/] by FAB-MS in glycerol matrix. The structure was elucidated to be N-[((S)-1-carboxy-2-phenylethyl)-carbamoyl]-${\alpha}$-[2- iminohexahydro-4(S)-pyrimidyl]-L-glycyl- L-valyl-phenylalaninol, by the spectroscopic methods such as NMR and MS fragmentation studies. Calmostinol exhibited strong activity against calpain while not against a Ca$\^$2+/ -independent cysteine protease papain.

• The Journal of Korean Obstetrics and Gynecology
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• v.19 no.2
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• pp.92-106
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• 2006

Purpose : To address the ability of Olibanum to induce cell death, we investigated the effect of olibanum on cell apoptosis. Twenty-four hours later, apoptosis occurred following olibanum exposure in a dose-dependent manner. Methods : We culture 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 : The treatment of BAPTA-AM regulated olibanum-induced apoptosis in HeLa human cervical carcinoma cells. The 24 hr-earlier -thapsigargin-pretreated cell showed the resistance against olibanum-induced apoptosis and the Ru360-mitochondrial uniporter-inhibited olibanum-induced apoptosis, too. It means that olibanum leads to the accumulation of calcium and the resultant apoptosis in HeLa cells. Immunoblotting data also shows that the expression of GRP78, ER stress marker protein, was induced by the olibanum. Bcl-2, anti-apototic protein, was decreased and that the expression of Bax, pro-apoptotic protein, was increased by the addition of olibanum. Interestingly, the olibanum increased the activity of caspase-8 as well as calpain cysteine pretense in HeLa cervical carcinoma cells. Calpain inhibitor-calpastatin as well as caspase-8C/A expression abrogated olibanum-induced apoptosis in the carcinoma cells. The inhibition of caspase-8 regulated olibanum-induced calpain activation but the inhibition of calpain did not have any effect on the caspase-8 activation in HeLa human cervical carcinoma cells. Conclusion : We conclude that olibanum induces the accumulation of calcium and the resultant apoptosis in which caspase-8 and calpain are involved.

• Parasites, Hosts and Diseases
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• v.49 no.2
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• pp.177-180
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• 2011

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that can cause amebic colitis and liver abscess in humans. E. histolytica has the capability to kill colon epithelial cells in vitro; however, information regarding the role of calpain in colon cell death induced by ameba is limited. In this study, we investigated whether calpains are involved in the E. histolytica-induced cell death of HT-29 colonic epithelial cells. When HT-29 cells were co-incubated with E. histolytica, the propidium iodide stained dead cells markedly increased compared to that in HT-29 cells incubated with medium alone. This pro-death effect induced by ameba was effectively blocked by pretreatment of HT-29 cells with the calpain inhibitor, calpeptin. Moreover, knockdown of m- and ${\mu}$-calpain by siRNA significantly reduced E. histolytica-induced HT-29 cell death. These results suggest that m- and ${\mu}$-calpain may be involved in colon epithelial cell death induced by E. histolytica.

• Food Science of Animal Resources
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• v.38 no.3
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• pp.515-529
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• 2018

The present study was designed to investigate the effects of calpain system on the formation of volatile flavor compounds in Hanwoo beef. In the first experiment (exp.1), Longissimus lumborum (LL) muscle samples were injected with solutions containing 50 mM $CaCl_2$ or 50 mM $ZnCl_2$ and 154 mM NaCl respectively, and aged for 7 d at $4^{\circ}C$. In the second experiment (exp.2), the ground LL muscle was incubated with the aforementioned solutions containing cathepsin inhibitor. The injection with $CaCl_2$ solution greatly elevated the calpain activity and concomitantly, significantly decreased the Warner-Bratzler shear force (p<0.05). The pH, meat color and cooking loss did not differ (p>0.05) between the treatment groups. A total of 51 volatile compounds were identified using the solid phase microextraction with gas chromatography (SPME-GC). Results on volatile analyses from the both experiments showed that the injection with calcium ions led to significant increase (p<0.05) concentrations of pyrazines and sulfuric compounds. These results coincide with a higher rate of protein degradation due to the $CaCl_2$ injection as compared to the control group. Significantly (p<0.05) higher levels of lipid oxidation derived-aldehydes were found in the samples with $ZnCl_2$. The exp.1 showed that cathepsin inhibitors had no effect on the formation of volatile flavor components after 7 d of aging. These results imply that the proteolytic activity of the calpain system is associated with generation of volatile compounds of chiller-aged beef, while the role of cathepsins is likely very limited.

• 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.

• Natural Product Sciences
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• v.18 no.2
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• pp.102-105
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• 2012

Marine organism extracts were prepared from 26 species of Korean indigenous marine organisms, including 25 species belonging in class Anthozoa of phylum Cnidaria and a species belonging to subphylum Urochordata of phylum Chordata, and screened their inhibitory effects against ${\mu}$-calpain. As a result, the thirteen extracts were found to be active in the criteria of $IC_{50}$ < 100 ${\mu}g/ml$. Among them, the MeOH extracts of Plexauroides praelonga and Alveopora japonica showed remarkable ${\mu}$-calpain inhibitory activity with $IC_{50}$ values of $4.62{\pm}0.22$ and $4.82{\pm}0.07{\mu}g/ml$, respectively. In addition, chemical investigation of A. japonica led to the isolation of an active compound, hexadecyl tetradecanoate, as a selective cathepsin B inhibitor ($IC_{50}=9.05{\pm}2.45{\mu}M$). This compound was isolated as constituent of A. japonica for the first time in the present study.

• Food Science of Animal Resources
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• v.25 no.3
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• pp.310-315
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• 2005

The changes in pH, temperature, R-values and ${\mu}-calpain$ and its inhibitor activity of M. longissimus from Hanwoo steer were investigated at 1, 3, 9 and 24h postmortem. The pH and temperature of M. longissimus were significantly (p<0.05) decreased during 24h postmortem time, and were 6.50 and $31.99^{\circ}C$, respectively, at 3h postmortem. $R_{248}\;and\;R_{250}$ were increased, but $R_{258}$ was decreased after 9h postmortem time (p<0.05). Calpain I and calpastatin activity were decreased after 3h and 9h postmortem time, respectively (p<0.05). pH and temperature showed high positive correlations with $R_{258}$ (r=0.967 and r=0.970, respectively), calpain I (r =0.956 and r=0.954, respectively) and calpastatin (r=0.978 and r=0.986, respectively) but had high negative correlations with $R_{248}$ (r=-0.982 and r=-0.973, respectively) and $R_{248}$ (r=-0.983 and r=-0.976, respectively). from these results, the change of postmortem metabolism of M. longissimus from Hanwoo steer likely occurred after 9h postmortem time. However, the further study on the establishment of metabolism from Hanwoo between postmortem 3h and 9h are necessary to produce Hanwoo beef with high acceptance in meat quality.

• The Korean Journal of Zoology
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• v.31 no.4
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• pp.295-299
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• 1988

An endogenous 17-6a inhibitor of Caa+_activated protease has been purified from neo-plastic tissues of human stomach using heat treahent and conventional chromatographir procedures. It appears to consist of a single polvpeptide since the same molecular weight was obtained by both gel fntration under nondenaturing condition and gel electrophoresis in the presence of SDS. Since the size of the inhibitor or is the smallest amens those reported so far, it may represent a functional unit for inhibiting Caa+_activated protease. This protein is also capable of inhibiting the protease isolated loom chick skeletal muscle. Thus, the functional unit of the inhibitor most well be conserved during evolution. Howrver, it remains unclear what may be the physiological significance of the presence of the Iow-molecular weight form of the inhibitor in neoplastic tissues of human stomach. 사람의 위암조직으로부터 Ca2)_activated protease을 저해하는 단백질을 열처리 및 여러 크로마토그라피 방법을 이용하여 순수분리 하였다. 이 저해단백질은SDS-전기영동카자1 filtra-tion의 방법에 의하여 그 분자량이 17 kDa으로 나타남으로 보아 단일 Polypeptide로 구성되어 있음을 알 수 있었다. 이 저해 단백질의 분자량은 지금까지 알려진 CaB+_activated protease의 저해단백질에 비하여 가장 작은 것이었다. 따라서, 이 단.백질은 Ca2)에 의하여 활성화되는 단백질 분해효소의 작용을 저해하는 기능적 단위로 추측되었다. 한편, 이 저해제는 계 골격근에서 추출한 Ca2+ _activated protease의 촹성 도 억제 하는 것으로 나타났다. 이러 한 결과는 이 저 해 단백질의 기능적 단위가 진화과정 동안 오래 보존되어 왔음을 시사한다. 그러나, 사람의 위암조직에서 이 저해제가 무슨 이유로 가장 작은 분자량의 단백질로 존재하는지에 대한 생리학적 중요성에 관한 문제는 앞으로 많이 연구되어져 야 할 것이다.