• BMB Reports
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• 제42권11호
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• pp.719-724
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• 2009

Recent studies have revealed that endoplasmic reticulum (ER) disturbance is involved in the pathophysiology of neurodegenerative disorders, contributing to the activation of the ER stress-mediated apoptotic pathway. Therefore, we investigated here the molecular mechanisms underlying the ER-mitochondria axis, focusing on calcium as a potential mediator of cell death signals. Using NT2 cells treated with brefeldin A or tunicamycin, we observed that ER stress induces changes in the mitochondrial function, impairing mitochondrial membrane potential and distressing mitochondrial respiratory chain complex Moreover, stress stimuli at ER level evoked calcium fluxes between ER and mitochondria. Under these conditions, ER stress activated the unfolded protein response by an overexpression of GRP78, and also caspase-4 and-2, both involved upstream of caspase-9. Our findings show that ER and mitochondria interconnection plays a prominent role in the induction of neuronal cell death under particular stress circumstances.

• Animal cells and systems
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• 제16권1호
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• pp.20-26
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• 2012

Drug-induced parkinsonism has been associated with an increased risk for Parkinson's disease. Antipsychotic drugs have long been known to cause parkinsonian symptoms. However, it remains unclear whether antipsychotics can directly damage the nigrostriatal pathway. In the present study, we investigated the toxicity mechanism of two typical antipsychotics, perphenazine and trifluoperazine, in a human dopaminergic cell line, SH-SY5Y. Perphenazine and trifluoperazine induced mitochondrial damage as evidenced by fragmentation of mitochondria, activation of Bax, cytochrome c release and a decrease in cellular ATP level. In addition, activation of caspase-3 and apoptotic nuclei were observed following the drug treatment. However, pan-caspase inhibitor did not suppress the cell death induced by the antipsychotics, suggesting that the initiated apoptosis was possibly shifted to necrosis upon caspase inhibition. Damaged mitochondria may have induced oxidative stress since the drug-induced cell death was partially suppressed by an antioxidant. Taken together, our results suggest that perphenazine and trifluoperazine can induce apoptotic cell death in a dopaminergic cell line via mitochondrial damage accompanied by oxidative stress.

• Molecules and Cells
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• 제37권11호
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• pp.785-794
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• 2014

Mitophagy, a cellular process that selectively targets dysfunctional mitochondria for degradation, is currently a hot topic in research into the pathogenesis and treatment of many human diseases. Considering that hypoxia causes mitochondrial dysfunction, which results in cell death, we speculated that selective activation of mitophagy might promote cell survival under hypoxic conditions. In the present study, we introduced the Regulator of calcineurin 1-1L (Rcan1-1L) to initiate the mitophagy pathway and aimed to evaluate the effect of Rcan1-1L-induced mitophagy on cell survival under hypoxic conditions. Recombinant adenovirus vectors carrying Rcan1-1L were transfected into human umbilical vein endothelial cells and human adult cardiac myocytes. Using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay and Trypan blue exclusion assay, Rcan1-1L overexpression was found to markedly reverse cell growth inhibition induced by hypoxia. Additionally, Rcan1-1L overexpression inhibited cell apoptosis under hypoxic conditions, as detected by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis assay. Meanwhile, the mitochondria-mediated cell apoptotic pathway was inhibited by Rcan1-1L. In contrast, knockdown of Rcan1-1L accelerated hypoxia-induced cell apoptosis. Moreover, Rcan1-1L overexpression significantly reduced mitochondrial mass, decreased depolarized mitochondria, and downregulated ATP and reactive oxygen species production. We further delineated that the loss of mitochondrial mass was due to the activation of mitophagy induced by Rcan1-1L. Rcan1-1L overexpression activated autophagy flux and promoted translocation of the specific mitophagy receptor Parkin into mitochondria from the cytosol, whereas inhibition of autophagy flux resulted in the accumulation of Parkin-loaded mitochondria. Finally, we demonstrated that mitochondrial 1permeability transition pore opening was significantly increased by Rcan1-1L overexpression, which suggested that Rcan1-1L might evoke mitophagy through regulating mitochondrial permeability transition pores. Taken together, we provide evidence that Rcan1-1L overexpression induces mitophagy, which in turn contributes to cell survival under hypoxic conditions, revealing for the first time that Rcan1-1L-induced mitophagy may be used for cardioprotection.

• The Korean Journal of Physiology and Pharmacology
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• 제9권4호
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• pp.231-238
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• 2005

Neuronal apoptotic events, which result in cell death, are occurred in hypoxic/ischemic conditions. Estradiol is a female sex hormone with steroid structure known to provide neuroprotection through multiple mechanisms in the central nervous system. This study was aimed to investigate the signal transduction pathway of $CoCl_2$-induced neuronal cell death and the inhibitory effects of estradiol. Administration of $CoCl_2$ decreased cell viability in both a dose- and time-dependent manner in PC12 cells. $CoCl_2$-induced cell death produced genomic DNA fragmentation and morphologic changes such as cell shrinkage and condensed nuclei. It was found that $CoCl_2$-treated cells increased the reactive oxygen species (ROS) as well as caspase-8, -9 and -3 activities. However, pretreatment with estradiol before exposure to $CoCl_2$ prevented the reduction in cell viability reduction and attenuated DNA fragmentation and morphologic changes caused by $CoCl_2$. Furthermore, the $CoCl_2$-induced increases of ROS levels and caspases activities were attenuated by estradiol. Gene expression analysis revealed that estradiol blocked the underexpression of the Bcl-2 and ameliorated the increase in the release of cytochrome c from mitochondria into cytoplasm and Fas-ligand (Fas-L) upregulated by $CoCl_2$. These results suggest that $CoCl_2$ induce apoptosis in PC12 cells through both mitochondria- and death receptor-mediated cell death pathway. Estradiol was found to have a neuroprotective effect against $CoCl_2$-induced apoptosis through the inhibition of ROS production and by modulating apoptotic effectors associated with the mitochondria- and death-dependent pathway in PC12 cells.

• 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.

• 생명과학회지
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• 제30권4호
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• pp.402-409
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• 2020

신경괴사증바이러스(NNV)는 25 nm의 작은 입자 크기에 RNA1 (3.4 kb, RdRp), RNA2 (1.4 kb, capsid protein) 두 가닥의 RNA를 유전정보를 가진다. NNV는 1980년대 말 처음 보고된 이후 전 세계적으로 120여종의 어류에 감염을 일으키며 심각한 피해를 일으키고 있는 바이러스이다. NNV 감염에 의한 피해를 최소화하고 효율적인 백신들을 개발하기 위해서는 무엇보다 NNV 감염에 따른 세포내 신호전달체계를 이해할 필요가 있다. NNV는 세포내 감염 이후 숙주가 가진 바이러스 복제에 필요한 요소들을 이용할 수 있도록 숙주세포의 cell cycle arrest 등의 기작을 이용하는 것으로 알려졌다. 반면에 숙주 세포는 NNV와 감염된 세포를 제어하기 위해 RIG-1-like receptor signaling pathway 등을 통해 NNV 감염을 인지한 다음 IFN signaling pathway를 통해 항바이러스 작용에 필요한 ISG들을 발현시킨다. 또한 감염된 세포들을 사멸시키기 위해 ER stress를 통한 unfolded protein response (UPR), mitochondria-mediated cell death 작용을 통해 감염된 세포의 apoptosis를 유발한다. NNV 감염 기작에 대한 세포신호전달연구는 아직 초기단계이며 검증해야 할 pathway들이 아직도 많이 남아있는 상황이다. 따라서 NNV 감염과 연관된 다양한 세포신호전달체계를 탐색하고 질병 특이적인 세포신호전달체계를 이해함으로써 신속하고 정확한 진단법 및 백신 개발에 많은 도움이 될 것으로 생각된다.

• 동의생리병리학회지
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• 제26권2호
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• pp.199-205
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• 2012

An extract of Alnus japonica (Betulaceae) cortex has been traditionally used for purifying blood, and curing feces containing blood, enteritis, diarrhea, alcoholism and cut wounds. In the present study, we demonstrated that the ethanol extract of Alnus japonica (EAJ) exhibited significantly cytotoxicity in human colon adenocarcinoma HT-29 cells. The results showed that the induction of apoptosis in HT-29 cells by EAJ was characterized by chromatin condensation and activation of caspase-3. EAJ-induced activation of caspase-9 and -3 caused the cleavage of poly ADP-ribose polymerase (PARP) and the release of cytochrome c. The expressions of Bcl-2 and Bid were reduced by EAJ in HT-29 cells, whereas pro-apoptotic protein Bak was increased in the cells. EAJ-induced, dose-dependent induction of apoptosis was accompanied by sustained phosphorylation of MAP kinases (JNK and p38 MAPK), ASK1, and p53. NAC administration, a scavenger of ROS, reversed EAJ-induced cell death. In conclusion, these results indicated that EAJ can cause apoptosis through a ROS-mitochondria-caspases-dependent pathway in human HT-29 cells.

• The Korean Journal of Physiology and Pharmacology
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• 제16권5호
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• pp.321-326
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• 2012

Resveratrol, a natural compound, has been shown to possess anti-cancer, anti-aging, anti-inflammatory, anti-microbial, and neuroprotective activities. In this study, we examined the antiproliferative and cytotoxicity properties of resveratrol in Rat B103 neuroblastoma cells; although it's molecular mechanisms for the biological effects are not fully defined. Here, we examined the cellular cytotoxicity of resveratrol by cell viability assay, antiproliferation by BrdU assay, DNA fragmentation by DNA ladder assay, activation of caspases and Bcl-2 family proteins were detected by western blot analyses. The results of our investigation suggest that resveratrol increased cellular cytotoxicity of Rat B103 neuroblastoma cells in a dose-and time-dependent manner with $IC_{50}$ of 17.86 ${\mu}M$ at 48 h. On the other hand, incubation of neuroblastoma cells with resveratrol resulted in S-phase cell cycle arrests which dose-dependently and significantly reduced BrdU positive cells through the downregulation of cyclin D1 protein. In addition, resveratrol dose-dependently and significantly downregulated the expression of anti-apoptotic protein includes Bcl-2, Bcl-xL and Mcl-1 and also activates cleavage caspase-9 and-3 via the downregulation of procaspase-9 and -3 in a dose-dependent manner which indicates that involvement of intrinsic mitochondria-mediated apoptotic pathway. In conclusion, resveratrol increases cellular cytotoxicity and inhibits the proliferation of B103 neuroblastoma cells by inducing mitochondria-mediated intrinsic caspase dependent pathway which suggests this natural compound could be used as therapeutic purposes for neuroblastoma malignancies.

• 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.

• The Korean Journal of Physiology and Pharmacology
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• 제21권6호
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• pp.599-607
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• 2017

Most normal cells express L-type amino acid transporter 2 (LAT2). However, L-type amino acid transporter 1 (LAT1) is highly expressed in many tumor cells and presumed to support their increased growth and proliferation. This study examined the effects of JPH203, a selective LAT1 inhibitor, on cell growth and its mechanism for cell death in Saos2 human osteosarcoma cells. FOB human osteoblastic cells and Saos2 cells expressed LAT1 and LAT2 together with their associating protein 4F2 heavy chain, but the expression of LAT2 in the Saos2 cells was especially weak. JPH203 and BCH, a non-selective L-type amino acid transporter inhibitor, potently inhibited L-leucine uptake in Saos2 cells. As expected, the intrinsic ability of JPH203 to inhibit L-leucine uptake was far more efficient than that of BCH in Saos2 cells. Likewise, JPH203 and BCH inhibited Saos2 cell growth with JPH203 being superior to BCH in this regard. Furthermore, JPH203 increased apoptosis rates and formed DNA ladder in Saos2 cells. Moreover, JPH203 activated the mitochondria-dependent apoptotic signaling pathway by upregulating pro-apoptotic factors, such as Bad, Bax, and Bak, and the active form of caspase-9, and downregulating anti-apoptotic factors, such as Bcl-2 and Bcl-xL. These results suggest that the inhibition of LAT1 activity via JPH203, which may act as a potential novel anti-cancer agent, leads to apoptosis mediated by the mitochondria-dependent intrinsic apoptotic signaling pathway by inducing the intracellular depletion of neutral amino acids essential for cell growth in Saos2 human osteosarcoma cells.