• 대한한의학방제학회지
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• 제14권2호
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• pp.21-32
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• 2006

The purpose of this study was to investigate the effect of Imyosan on apoptosis in human colorectal cancer HCT116 cells. Phellodendron amurense Rupr. and Atratylodes lancea D.C. compose Imyosan. First of all, to study the cytotoxic effect of methanol extract of Imyosan (IMS-MeOH) on HCT116 cells, the cells were treated with various concentrations of IMS-MeOH and then cell viability was determined by XTT reduction method. IMS-MeOH reduced viability of HCT116 cells in a dose and time-dependent manner. To confirm the induction of apoptosis, the c1eavage of poly ADP-ribose polymerase (PARP), a substrate for caspase-3 and a typical sign of apoptosis, and the activation of caspase-3, procaspase-8 and procaspase-9 were examined by western blot analysis. IMS-MeOH decreased procaspase-3, procaspase-8 and procaspase-9 levels in a dose-dependent manner and induced the clevage of PARP. IMS-MeOH triggered the mitochondrial apoptotic signaling by increasing the release of cytochrome c from mitochondria to cytosol. Furthermore, IMS-MeOH also downregulated the anti-apoptotic Bcl-2 and upregulated the pro-apoptotic-Bax. Therefore, these results suggest that IMS-MeOH induced HCT1l6 cell death through the mitochondrial pathway. To explore whether the activities of caspases was required for induction of apoptosis by IMS-MeOH, caspase-3, -8, -9 activity measured by using substrates, respectively. IMS-MeOH increased caspase-3, -8, -9 activity. Co-treatment with inhibitors of caspase-3, -8, -9 and IMS-MeOH significantly blocked IMS-MeOH-triggered apoptosis in HCT1l6 cells. These results suggest that IMS-MeOH induces caspases-mediated apoptosis.

• 한국식품영양과학회지
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• 제41권7호
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• pp.901-906
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• 2012

본 연구에서는 넓패 메탄올 추출물의 농도별 처리가 인체 자궁암 세포 HeLa의 세포사멸에 미치는 영향을 확인하기 위하여 세포독성 측정, Hoechst 33258 staining, flow cytometry 분석을 통하여 세포사멸을 확인하였다. 넓패 메탄올 추출물 처리 시 HeLa 세포에서 농도 의존적으로 세포의 증식을 억제하였으며, 또한 넓패 메탄올 추출물은 농도 의존적으로 핵을 응축하고 apoptotic bodies을 생성하였다. 유세포 분석을 통하여 apoptosis를 측정한 결과, 넓패 메탄올 추출물의 농도가 증가함에 따라 유의적으로 apoptotic 세포가 증가하였다. Western blot을 통해 PARP 단백질의 절단 현상을 분석한 결과, 넓패 메탄올 추출물의 처리 농도와 시간에 따라 PARP 단백질의 절단 현상이 증가하였다. 또한 넓패 메탄올 추출물은 caspase-8, caspase-9 및 caspase-3 활성을 농도와 시간에 따라 증가시켰으며, caspase 저해제인 z-VAD-fmk로 처리 시 넓패 메탄올 추출물에 의한 세포사멸이 유의적으로 감소되어 넓패 메탄올 추출물에 의한 HeLa 세포의 apoptosis 유도에 caspase가 중요한 역할을 하고 있음을 확인하였다. 따라서 넓패 메탄올 추출물은 HeLa 자궁암 세포의 apoptosis를 유도하는 것으로 나타나 넓패의 항암효과 가능성을 제시하였다.

• 한국자원식물학회지
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• 제32권4호
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• pp.255-263
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• 2019

본 연구에서는 다양한 약리학적 활성을 가지는 것으로 알려진 감초 열수추출물(GRW)의 항암효능을 알아보기 위하여 인체 방광암 T24 세포에서 생존율 및 증식억제에 미치는 영향과 이와 연관된 apoptosis 유발 여부 및 관련 인자들의 발현 변화를 조사하였다. 먼저 GRW의 처리에 따른 증식억제 정도를 조사한 결과, GRW 처리 농도 의존적으로 생존율 및 증식억제 현상이 나타났으며, 핵의 형태 변화, DNA 단편화 및 apoptosis 유발에 관하여 조사한 결과 역시 GRW 처리 농도 의존적으로 증가됨을 확인할 수 있었다. 이는 GRW의 처리에 의한 암세포의 증식억제 및 형태적 변형이 암세포의 apoptosis 유발과 밀접한 관련이 있음을 시사하여 주는 것으로 사료된다. GRW 처리에 의한 apoptosis 유발에 관여하는 유전자의 탐색을 위하여 apoptosis와 연관성을 가지는 Bcl-2 family에 속하는 유전자의 발현을 조사한 결과 GRW 처리 농도 의존적으로 Bax 단백질의 발현증가와 더불어 Bcl-2 및 Bcl-xL 단백질의 발현감소가 관찰되었다(Fig. 3A). 이는 GRW에 의한 T24 세포의 apoptosis 유발에 Bcl-2 family에 속하는 유전자의 발현 조절이 중요한 역할을 하는 것으로 사료된다. 또한 GRW의 처리에 따른 MMP의 소실은 미트콘드리아 막의 교란이 유발되었음을 의미하는 것으로, 이러한 MMP 값의 변동은 Bcl-2 family 단백질의 발현 변화에 의한 것이라 추정된다. 한편 Apoptosis에 중요한 역할을 하는 것으로 알려진 caspase(-3/-8/-9)의 발현과 이들의 활성을 억제하는 IAP family (XIAP, cIAP-1, cIAP-2)의 발현에 GRW이 어떠한 영향을 미치는지를 조사한 결과, caspase-3, -8 및 -9의 활성형 단백질 발현 및 정량적 활성증가를 확인하였으며, IAP family 속한 3가지 단백질 모두 발현이 감소하는 것이 관찰되었다. 이상의 결과에서 GRW은 외인적 및 내인적 경로의 개시에 핵심적인 역할을 하는 caspase-8 및 -9의 활성을 모두 증가시켰으며, 이에 따른 caspase-3의 활성증가에 의하여 apoptosis가 유발되었음을 알 수 있었다. 이러한 두 경로의 동시 활성화에는 미트콘드리아의 기능 소실과 Bcl-2 및 IAP family의 발현 변화가 관여하고 있었으며, 특히 Bid의 발현 감소는 GRW에 의한 내인적 경로를 증폭시키는 효과로 작용했을 것이라 추정된다. 방광암의 치료에 보다 효과적인 생리활성을 갖는 물질을 발굴하고 그와 관련된 분자 및 세포수준에서의 기전을 밝히는 것이 중요하기에 본 연구의 결과는 향후 GRW로 수행될 추가 실험을 위한 기초자료로서 그 가치가 매우 높을 것으로 사료된다.

• Advances in Traditional Medicine
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• 제10권4호
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• pp.254-262
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• 2010

Artemisia capillaris (A. capillaries) is known to play roles in many cellular events, such as cell proliferation, differentiation, and apoptosis. We investigated the antifibrogenic efficacy of A. capillaris in the immortalized human hepatic stellate cell line LX2. Cell proliferation was determined by the MTT assay. Cell cycle was analyzed by the flow cytometry. Apoptotic cells were measured using a cell death detection ELISA. Caspase activity was detected by a colorimetric assay. The mRNA level of Bcl-2 and Bax mRNA were measured by real-time PCR. MEK and ERK protein were detected by Western blot analysis. We provide evidence that A. capillaris induces cell cycle arrest, apoptosis, and potently inhibits the mitogen-activated protein kinase pathway. A. capillaris inhibited cell proliferation of LX2 cells in a dose- and time-dependent manner, increased the apoptosis fraction at cell cycle analysis with an accompanying DNA fragmentation, and resulted in a significant decrease in Bcl-2 mRNA levels and an increase in Bax expression. Exposure of LX2 cells to A. capillaris induced caspase-3 activation, but co-treatment of A. capillaris with the pan-caspase inhibitor Z-VAD-FMK, and the caspase-3 inhibitor Z-DEVE-FMK, blocked apoptosis. A. capillaris down-regulated Mcl-1 protein levels and inhibited phosphorylation of MEK/ERK, suggesting that it mediates cell death in LX2 cells through the down-regulation of Mcl-1 protein via a MEK/ERK-independent pathway.

• 생명과학회지
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• 제23권9호
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• pp.1118-1125
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• 2013

감귤류에 많이 함유되어 있는 naringenin은 항암화학요법제로서 중요한 가능성을 가지고 있으나 항암활성에 대한 분자생물학적 기전에 대해서는 명확히 밝혀져 있지 않다. 본 연구에서는 인체 혈구암세포인 U937 세포에서 naringenin이 유발하는 항암효과 및 항암기전을 조사하였다. Naringenin 처리에 의한 U937 세포의 증식억제는 apoptosis 유발과 연관성이 있었으며, 이러한 현상은 caspases 활성화와 밀접한 관련이 있었다. 그러나 pan-caspase inhibitor인 z-VAD-fmk의 선처리에 의하여 U937 세포에서 naringenin이 유발하는 apoptosis가 억제되는 것으로 나타났으므로 caspases가 apoptosis 유발의 중요한 조절자라는 것을 알 수 있었다. 또한 U937 세포에 naringenin을 처리하였을 경우 pro-apoptotic Bcl-2 및 anti-apoptotic Bax의 발현에는 아무런 변화가 나타나지는 않았지만 Bcl-2가 과발현된 U937/Bcl-2 세포에서 naringenin에 의한 apoptosis가 억제되었다. 하지만 small-molecule Bcl-2 inhibitor인 HA14-1 및 naringenin을 같이 처리하였을 경우에는 XIAP 발현감소, Bid 단편화 및 caspase-3 활성화를 통하여 다시 apoptosis가 유발되었다. 따라서 HA14-1 및 naringenin에 의한 apoptosis 상승효과는 death receptor-mediated apoptosis pathway를 경유한다는 것을 제시하는 결과이다.

• Biomolecules & Therapeutics
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• 제22권3호
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• pp.184-192
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• 2014

${\beta}$-lapachone is a naturally occurring quinone that selectively induces apoptotic cell death in a variety of human cancer cells in vitro and in vivo; however, its mechanism of action needs to be further elaborated. In this study, we investigated the effects of ${\beta}$-lapachone on the induction of apoptosis in human gastric carcinoma AGS cells. ${\beta}$-lapachone significantly inhibited cellular proliferation, and some typical apoptotic characteristics such as chromatin condensation and an increase in the population of sub-G1 hypodiploid cells were observed in ${\beta}$-lapachone-treated AGS cells. Treatment with ${\beta}$-lapachone caused mitochondrial transmembrane potential dissipation, stimulated the mitochondria-mediated intrinsic apoptotic pathway, as indicated by caspase-9 activation, cytochrome c release, Bcl-2 downregulation and Bax upregulation, as well as death receptor-mediated extrinsic apoptotic pathway, as indicated by activation of caspase-8 and truncation of Bid. This process was accompanied by activation of caspase-3 and concomitant with cleavage of poly(ADP-ribose) polymerase. The general caspase inhibitor, z-VAD-fmk, significantly abolished ${\beta}$-lapachone-induced cell death and inhibited growth. Further analysis demonstrated that the induction of apoptosis by ${\beta}$-lapachone was accompanied by inactivation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. The PI3K inhibitor LY29004 significantly increased ${\beta}$-lapachone-induced apoptosis and growth inhibition. Taken together, these findings indicate that the apoptotic activity of ${\beta}$-lapachone is probably regulated by a caspase-dependent cascade through activation of both intrinsic and extrinsic signaling pathways, and that inhibition of the PI3K/Akt signaling may contribute to ${\beta}$-lapachone-mediated AGS cell growth inhibition and apoptosis induction.

• 생명과학회지
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• 제13권1호
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• pp.118-127
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• 2003

Phenylalanine의 구조유사체인 p-fluotophenylalanine (FPA)은 인체 급성백혈병세포주인 Jurkat T 세포의 세포자살을 유도한다. FPA에 의한 세포자살에 미치는 Bcl-2 또는 Bcl-xL의 영향을 조사하기 위해, Bcl-2 또는 Bcl-xL을 stable transfection하거나 empty vectors만을 Transfection한 Jurkat 세포를 이용하여 FPA의 세포독성과 FPA에 의한 세포내 세포자살 신호전달경로를 비교 분석하였다. Jurkt T 세포에 0.63∼3.0 mLf의 FPA를 처리하였을 때 세포의 생육도는 농도에 비례하여 감소하였다. 또한 세포자살관련 DNA fragmentation, caspase-8 activatoin, Bid cleavage, mitochondria로 부터의 cytochrome c 방출, caspase-9 및 -3 activation, PARP degradation 등이 유도되었다. 한편, FPA에 의해 유도되는 이러한 일련의 생화학적 현상들은 Bcl-2 또는 Bcl-xL의 overexpression에 의해 현저히 저해되었다. 이상의 결과들은 caspase-8 activation, Bid cleavage, mitochondnal cytochrome c 방출에 의해 활성화되는 casuase cascade 등의 현상이, Bcl-2 또는 Bcl-xL에 의해 억제됨을 나타내며 FPA에 의해 유도되는 세포자살에 필요한 과정임을 시사한다.

• Asian Pacific Journal of Cancer Prevention
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• 제15권15호
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• pp.6321-6326
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• 2014

${\alpha}$-Methyl-n-butylshikonin (MBS), one of the active components in the root extracts of Lithospermum erythrorhizon, posses antitumor activity. In this study, we assess the molecular mechanisms of MBS in causing apoptosis of SW620 cells. MBS reduced the cell viability of SW620 cells in a dose-and time-dependent manner and induced cell apoptosis. Treatment of SW620 cells with MBS down-regulated the expression of Bcl-2 and up-regulated the expression of Bak and caused the loss of mitochondrial membrane potential. Additionally, MBS treatment led to activation of caspase-9, caspase-8 and caspase-3, and cleavage of PARP, which was abolished by pretreatment with the pan-caspase inhibitor Z-VAD-FMK. MBS also induced significant elevation in the phosphorylation of JNK and p38. Pretreatment of SW620 cells with specific inhibitors of JNK (SP600125) and p38 (SB203580) abrogated MBS-induced apoptosis. Our results demonstrated that MBS inhibited growth of colorectal cancer SW620 cells by inducing JNK and p38 signaling pathway, and provided a clue for preclinical and clinical evaluation of MBS for colorectal cancer therapy.

• Molecules and Cells
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• 제22권2호
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• pp.168-174
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• 2006

Enhanced apoptosis has been observed in the placentas of women with preeclampsia, but few studies have examined changes at the molecular level. This study was designed to detect genes specifically expressed in full-term preeclamptic placentas. Tissue samples were collected immediately after cesarean delivery from 11 normal and 8 preeclamptic placentas at 35-40 weeks of gestation. Total RNAs were extracted and hybridized to a cDNA microarray. Results were confirmed by reverse-transcription polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. Hematoxylin and eosin and TUNEL staining were also performed to confirm apoptosis in preeclamptic placentas. Among 205 genes, three were up- or downregulated in preeclamptic placentas. The expression of caspase-10 and death receptor 3 (DR-3) was significantly increased, whereas insulin-like growth factor binding protein-3 (IGFBP-3) was strongly downregulated. RT-PCR analysis and Western blotting confirmed these effects. Immunohistochemical analysis showed that the DR-3, caspase-10 and IGFBP-3 proteins were localized in the syncytial membrane. Apoptosis in the trophoblast was also increased in term placentas from women with pregnancies complicated by preeclampsia. These results suggest that caspase-10, DR-3 and IGFBP-3 are involved in apoptosis in the preeclamptic placenta.

• BMB Reports
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• 제38권6호
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• pp.709-716
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• 2005

Apoptosis and necrosis are distinguished by modality primarily. Here we show an apoptosis occurred instantly, induced by $300\;{\mu}M$ W-7 ((N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride), inhibitor of calmodulin), which demonstrated necrotic modality. As early as 30 min after W-7 addition, apoptotic (sub-diploid) peak could be detected by fluorescence-activated cell sorter (FACS), “DNA ladders” began to emerge also at this time point, activity of caspase-3 elevated obviously within this period. Absence of mitochondrial membrane potential (MMP) reduction and cytochrome c, AIF (apoptosis inducing factor) release, verified that this rapid apoptosis did not proceed through mitochondria pathway. Activation of caspase-12 and changes of other endoplasmic reticulum (ER) located proteins ascertained that ER pathway mediated this necrosis-like apoptosis. Our findings suggest that it is not credible to judge apoptosis by modality. Elucidation of ER pathway is helpful to comprehend the pathology of diseases associated with ER stress, and may offer a new approach to the therapy of cancer and neurodegenerative diseases.