• IMMUNE NETWORK
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• 제12권6호
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• pp.284-290
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• 2012

Innate immune cells sense and respond to the cytoplasmic infection of bacterial pathogens through NLRP3, NLRC4 or AIM2 inflammasome depending on the unique molecular pattern of invading pathogens. The infection of flagellin- or type III secretion system (T3SS)-containing Gram-negative bacteria such as Salmonella enterica serovar Typhimurium (S. typhimurium) or Pseudomonas aeruginosa (P. aeruginosa) triggers NLRC4-dependent caspase-1 activation leading to the secretion of proinflammatory cytokines such as interleukin-1-beta (IL-$1{\beta}$) and IL-18. Previous studies have shown that apoptosis-associated speck-like protein containing a CARD (ASC) is also required for Salmonella-induced caspase-1 activation, but it is still unclear how ASC contributes to the activation of NLRC4 inflammasome in response to S. typhimurium infection. In this study, we demonstrate that S. typhimurium triggers the formation of ASC oligomer in a potassium depletion-independent manner as determined by in vitro crosslinking and in situ fluorescence imaging. Remarkably, inhibition of potassium efflux failed to block Salmonella-promoted caspase-1 activation and macrophage cell death. These results collectively suggest that ASC is substantially oligomerized to facilitate the activation of caspase-1 in response to S. typhimurium infection. Contrary to NLRP3 inflammasome, intracellular potassium depletion is not critical for NLRC4 inflammasome signaling by S. typhimurium.

• Toxicological Research
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• 제17권3호
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• pp.215-223
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• 2001

카드뮴의 주요한 표적장기이며 카드뮴이 만성 및 급성 폭로시 축적되는 가장 중요한 장기인 간의 세포독성을 Hepalclc7세포에서 caspases및 Bax단백질의 활성과 발현 그리고 미토콘드리아 세포막 전위 변화(MPT) 등을 조사하여 다음과 같은 결과를 얻었다. 1. 카드뮴은 농도의존적으로 간세포인 Hepalclc7 세포의 생존율을 감소시켰다. 2. 카드뮴을 농도별로 처리하였을 때 100 M 이상의 농도에서 세포사멸의 특징중의 하나인 DNA분절현상을 확인하였다. 3. 카드뮴 처리 후 caspase-3, caspase-8, caspase-9 의 활성변화를 조사한 결과 caspase-3,-9 pretease 활성이 시간이 경과함에 따라 증가하였다. 4. 카드뮴 처리 후 cytochrome c가 세포질내로 방출되었고 이는 caspase-9 proteas의 활성화를 유도하였다. 5. 카드뮴 처리 후 Bax가 세포질에서 미토콘드리아로 이동하여 cytochrome c의 세포질내로의 방출에 관여하였다. 6. 카드뮴 처리시 미토콘드리아 세포막 전위차의 감소를 JC-1 형광염색을 통하여 확인하였다. 이상의 결과는 카드뮴에 의한 Hepalclc7 세포사멸의 신호전달기전은 세포질내에 있는 Bax가 미토콘드리아로 이동, cytochrome c의 세포질내로의 방출, 그리고 caspase-3, 9 pretease 활성화를 의해서 매개되는 것으로 판단된다. 또한 Bax 단백질의 발현변화가 미토콘드리아의 기능변화에 기여하였으리라 사료된다.

• Journal of Photoscience
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• 제9권2호
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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.

• Asian Pacific Journal of Cancer Prevention
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• 제17권3호
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• pp.1499-1506
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• 2016

Background: Crocus sativus and its major constituent crocin are well established to have anti-cancer properties in breast cancer cells (MCF-7). However the role of C. sativus extract (CSE) and crocin on caspase signaling mediated MCF-7 cell death at molecular level is remains unclear. In this study, we tried to unravel role of CSE and crocin on caspase mediated MCF-7 cells death and their in vivo preclinical toxicity profiling and immune stimulatory effect. Materials and Methods: CSE extract was fractionated by HPLC and crocin was isolated and characterized by NMR, IR, and MS. MCF-7 cells were treated with both CSE and crocin and expression of Bcl-2 and Bax was assessed after 24 and 36 hours. Furthermore, caspase 3, caspase 8 and caspase 9 expression was determined by Western blotting after 24 hours of treatment. DNA fragmentation analysis was performed for genotoxicity of CSE and crocin in MCF-7 cells. The in vivo toxicity profile of CSE (300 mg/kg of b.wt) was investigated in normal Swiss albino mice. In addition, peritoneal macrophages were collected from crocin (1, 1.5 and 2 mg/kg body weight) treated mice and analyzed for ex vivo yeast phagocytosis. Results: Immunoblot analysis revealed that there was time dependent decline in anti-apoptotic Bcl-2 with simultaneous upregulation of Bax in CSE and crocin treated MCF-7 cells. Further CSE and crocin treatment downregulated caspase 8 and 9 and cleaved the caspase 3 after 24 hours. Both CSE and crocin elicited considerable DNA damage in MCF-7 cells at each concentration tested. In vivo toxicity profile by histological studies revealed no observable histopathologic differences in the liver, kidney, spleen, lungs and heart in CSE treated and untreated groups. Crocin treatment elicited significant dose and time dependent ex vivo yeast phagocytosis by peritoneal macrophages. Conclusions: Our study delineated involvement of pro-apoptotic and caspase mediated MCF-7 cell death by CSE and crocin at the molecular level accompanied with extensive DNA damage. Further we found that normal swiss albino mice can tolerate the maximum dose of CSE. Crocin enhanced ex vivo macrophage yeast phagocytic ability.

• 생명과학회지
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• 제17권8호통권88호
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• pp.1053-1062
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• 2007

유방암 치료제로 사용되는 탐옥시펜 (Tam)은 MCF-7 세포주에서 세포증식을 억제하며 세포사멸을 유도한다. 본 연구에서는 Tam의 세포독성 효과가 세포 내 칼슘이온 농도 증가에 따른 미토콘드리아-의존 기작에 의하여 일어난다는 것을 보여준다. Tam에 의해 유도된 세포 내 칼슘이온 농도 증가는 주로 외부로부터의 칼슘 유입에 의한 것으로 생각된다. 칼슘 채널 억제제를 이용한 실험 결과에 의하며, 칼슘 증가 초기 단계는 주로 전압의존 칼슘채널에 의한 것이며 후기에는 세포 내 저장된 칼슘의 유출, 혹은 다른 방법에 의한 칼슘 유입으로 생각된다. Tam에 의한 세포 내 칼슘 증가는 미토콘드리아로부터의 cytochrome c 방출과 미토콘드리아막의 탈분극에 의한 membrane potential 변화를 초래하였다. 세포사멸에 주도적인 역할을 하는 caspase의 확인에 있어서는, MCF-7 세포는 caspase-3이 결핍되어서 caspase-7이 중심적인 역할을 하는 것으로 이미 알려져 있다. 본 연구에서 확인한 결과 Tam 처리시 caspase-7이 활성화되었으며, 또한 세포사멸 조절 단백질인 Bcl-2 종류 단백질들의 발현을 조사 한 결과 세포사멸 억제 단백질인 Bcl-2의 발현에는 변화가 없었으나 촉진단백질인 Bax는 Tam 처리시 단백질 양이 2배로 증가되었다. 이상의 결과에 의하면, Tam에 의해 유도되는 세포사멸과정은 세포질 내 칼슘이온 농도증가에 의한 미토콘드리아의 변화가 주도적인 역할을 하는 것으로 생각된다.

• Korean Journal of Acupuncture
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• 제29권2호
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• pp.271-289
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• 2012

Objectives : Ganoderma lucidum(Ganoderma or lingzhi, 靈芝) is a well-known oriental medical mushroom containing many bioactive compounds. The possible mechanisms involved in its effects on cancer cells remain to be elucidated. In the present study, the anti-proliferative and apoptotic activities of the G. lucidum ethanol extract(GEE), in AGS human gastric cancer cells were investigated. Methods : It was found that exposure of AGS cells to GEE resulted in the growth inhibition in a dose and time dependent manner as measured by trypan blue count and MTT assay. The anti-proliferative effect of GEE treatment in AGS cells was associated with morphological changes and formation of apoptotic bodies, and the flow cytometry analysis confirmed that GEE treatment increased the populations of apoptotic-sub G1 phase. Growth inhibition and apoptosis of AGS cells by GEE were connected with a concentration and time-dependent up-regulation of tumour necrosis factor-related apoptosis-inducing ligand(TRAIL) expression. Results : The levels of XIAP and survivin expression, members of IAP family proteins, were gradually down-regulated by GEE treatment. However other members of IAP family proteins such as cIAP-1 and cIAP-2 remained unchanged in GEE-treated AGS cells. GEE treatment also induced the proteolytic activation of caspase-3, caspase-8 and caspase-9 and a concomitant degradation of poly(ADP-ribose) polymerase(PARP) protein, a caspase-3 substrate protein. Additionally, GEE-induced apoptosis was associated with the inhibition of Akt activation in a concentration and time-dependent manner, and pre-treatment with LY294002, a phosphoinositide 3-kinase(PI3K)/Akt inhibitor, significantly increased GEE-induced growth inhibition and apoptosis. Conclusions : Therefore, G. lucidum has a strong potential as a therapeutic agent for preventing cancers such as gastric cancer cells.

• Asian Pacific Journal of Cancer Prevention
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• 제14권11호
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• pp.6379-6384
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• 2013

S-benzyl-cysteine (SBC) is a structural analog of S-allylcysteine (SAC), which is one of the major water-soluble compounds in aged garlic extract. In this study, anticancer activities and the underlying mechanisms of SBC action were investigated and compared these with those of SAC using human gastric cancer SGC-7901 cells. SBC significantly suppressed the survival rate of SGC-7901 cells in a concentration- and time-dependent manner, and the inhibitory activities of SBC were stronger than those of SAC. Flow cytometry revealed that SBC induced G2-phase arrest and apoptosis in SGC-7901 cells. Typical apoptotic morphological changes were observed by Hoechst 33258 dye assay. SBC-treatment dramatically induced the dissipation of mitochondrial membrane potential (${\Delta}{\Psi}m$), and enhanced the enzymatic activities of caspase-9 and caspase-3 whilst hardly affecting caspase-8 activity. Furthermore, Western blotting indicated that SBC-induced apoptosis was accompanied by up-regulation of the expression of p53, Bax and the down-regulation of Bcl-2. Taken together, this study suggested that SBC exerts cytotoxic activity involving activation of mitochondrial-dependent apoptosis through p53 and Bax/Bcl-2 pathways in human gastric cancer SGC-7901 cells.

• 동의생리병리학회지
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• 제22권5호
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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.

• Environmental Analysis Health and Toxicology
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• 제23권4호
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• pp.325-335
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• 2008

The protective effects of the Naringin, on carbon tetrachloride ($CCl_4$)-induced hepatotoxicity and the possible mechanisms involved in this protection were investigated in mice. Pretreatment with Naringin prior to the administration of $CCl_4$ significantly prevented an increase in serum alanine, aspartate aminotransferase activity and hepatic lipid peroxidation in a dose-dependent manner. In addition, pretreatment with Naringin also significantly prevented the depletion of glutathione (GSH) content in the livers of $CCl_4$-induced mice. However, reduced hepatic glutathione levels was unaffected by treatment with Naringin alone. In addition, Naringin prevented $CCl_4$-induced apoptosis and necrosis, as indicated by a liver DNA laddering. To determine whether caspase-8,-3 pathway involved in $CCl_4$-induced acute liver injury, caspase-8, -3 activities were tested by ELISA. Naringin attenuated $CCl_4$induced caspase-8, -3 activities in mouse livers. $CCl_4$-induced hepatotoxicity was also prevented, as indicated by a liver histopathologic study. The effects of Naringin on the cytochrome P450 (CYP) 2E1, the major isozyme involved in $CCl_4$ were also investigated. Treatment of mice with Naringin resulted in a significant decrease of the CYP2E1-dependent hydroxyl at ion and aniline in a dose-dependent manner. These findings suggest that protective effects of Naringin against the $CCl_4$-induced hepatotoxicity may be due to its ability to block CYP2E1-mediated $CCl_4$ bioactivation and that is also protects against caspase-8, -3 pathway mediated apoptosis.

• Biomolecules & Therapeutics
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• 제29권2호
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• pp.175-183
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• 2021

The mitogen-activated protein kinase (MAPK) pathway controls intestinal epithelial barrier permeability by regulating tight junctions (TJs) and epithelial cells damage. Heme oxygenase-1 (HO-1) and carbon monoxide (CO) protect the intestinal epithelial barrier function, but the molecular mechanism is not yet clarified. MAPK activation and barrier permeability were studied using monolayers of Caco-2 cells treated with tissue necrosis factor α (TNF-α) transfected with FUGW-HO-1 or pLKO.1-sh-HO-1 plasmid. Intestinal mucosal barrier permeability and MAPK activation were also investigated using carbon tetrachloride (CCl4) administration with CoPP (a HO-1 inducer), ZnPP (a HO-1 inhibitor), CO releasing molecule 2 (CORM-2), or inactived-CORM-2-treated wild-type mice and mice with HO-1 deficiency in intestinal epithelial cells. TNF-α increased epithelial TJ disruption and cleaved caspase-3 expression, induced ERK, p38, and JNK phosphorylation. In addition, HO-1 blocked TNF-α-induced increase in epithelial TJs disruption, cleaved caspase-3 expression, as well as ERK, p38, and JNK phosphorylation in an HO-1-dependent manner. CoPP and CORM-2 directly ameliorated intestinal mucosal injury, attenuated TJ disruption and cleaved caspase-3 expression, and inhibited epithelial ERK, p38, and JNK phosphorylation after chronic CCl4 injection. Conversely, ZnPP completely reversed these effects. Furthermore, mice with intestinal epithelial HO-1 deficient exhibited a robust increase in mucosal TJs disruption, cleaved caspase-3 expression, and MAPKs activation as compared to the control group mice. These data demonstrated that HO-1-dependent MAPK signaling inhibition preserves the intestinal mucosal barrier integrity by abrogating TJ dysregulation and epithelial cell damage. The differential targeting of gut HO-1-MAPK axis leads to improved intestinal disease therapy.