• 생명과학회지
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• 제23권11호
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• pp.1351-1359
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• 2013

${\beta}$-Ionone의 인체 비소폐암세포 A-549 cells (human non-small lung cancer A-549 cell)에 대한 anti-proliferative effect에 관한 연구를 수행하였다. A-549 cell에 다양한 농도의 ${\beta}$-ionone (1, 5, 10, and 15 ${\mu}M$)을 2, 4, 6일 간 처리하고, 배양 2일에 A-549 cell의 생육억제와 관련되는 biochemical marker를 측정하였다. ${\beta}$-Ionone은 A-549 cell 생육을 dose와 time 의존적으로 저해하였다. ${\beta}$-Ionone 배양 2일의 $IC_{50}$은 5.0 ${\mu}g/ml$이었다. ${\beta}$-Ionone은 농도 의존적으로 apoptosis를 유도하였다. ${\beta}$-Ionone은 p53, p21 및 Bax protein 수준을 증가시켰으나, Bcl-2 protein 발현은 억제시켰다. ${\beta}$-ionone은 cytosol cytochrome c 함량을 증가시켰고, caspase-9과 caspase-3 효소 활성증가를 유도하였다. 또한, ${\beta}$-ionone은 $cPLA_2$와 COX-2 protein level을 감소시켰다. 이와 같은 결과는 ${\beta}$-ionone의 A-549 cell에 대한 생육억제효과는 Bax와 Bcl-2 gene 발현을 reciprocal regulation하여 유도한 apoptosis와 $cPLA_2$ 및 COX-2 protein 발현 억제에 기인함을 의미한다.

• Molecular & Cellular Toxicology
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• 제3권4호
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• pp.215-221
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• 2007

Neuronal cell toxicity induced by decreased nitric oxide (NO) production may be caused by modulation of constitutive neuronal NO synthase (nNOS). We used lead acetate ($Pb^{2+}$) to modulate physiological NO release and the related pathways of protein kinases like PKC, CaM-KII, and PKA in CATH.a cells, a dopaminergic cell line that has constitutive nNOS activity. In the cells treated with $Pb^{2+}$, cell viability and modulation (phosphorylation) levels of nNOS were determined by MTT assay and Western blot analysis, respectively. nNOS reductase activity (cytochrome c) was also assessed to compare the phosphorylation site-specific nNOS activity. nNOS activity was also determined by NADPH consumption rates. $Pb^{2+}$ treatment alone increased the phosphorylation of nNOS with decreased reductase activity. The phosphorylation levels increased markedly with decreased nNOS reductase activity, when $Pb^{2+}$ was combined with inhibitors for two (PKC and CaM-KII) or three (PKA, PKC and CaM-KII) protein kinases. Interestingly, when the cells were exposed to $Pb^{2+}$ plus PKC or CaM-KII inhibitor, the nNOS was phosphorylated strongly with the lowest activity. However, the levels of phosphorylated nNOS following $Pb^{2+}$ treatment decreased significantly after combined treatment with the PKA inhibitor, and $Pb^{2+}$-induced suppression of reductase activity did not occur. These results demonstrate that physiological NO release in the neuronal cells exposed to $Pb^{2+}$ can be decreased by PKA-mediated nNOS phosphorylation that may be caused by interactions with PKC and/or CaM-KII.

• Journal of Ginseng Research
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• 제42권2호
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• pp.165-174
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• 2018

Background: Extended endoplasmic reticulum (ER) stress may initiate apoptotic pathways in cancer cells, and ER stress has been reported to possibly increase tumor death in cancer therapy. We previously reported that caspase-8 played an important role in compound K-induced apoptosis via activation of caspase-3 directly or indirectly through Bid cleavage, cytochrome c release, and caspase-9 activation in HL-60 human leukemia cells. The mechanisms leading to apoptosis in A549 and SK-MES-1 human lung cancer cells and the role of ER stress have not yet been understood. Methods: The apoptotic effects of compound K were analyzed using flow cytometry, and the changes in protein levels were determined using Western blot analysis. The intracellular calcium levels were monitored by staining with Fura-2/AM and Fluo-3/AM. Results: Compound K-induced ER stress was confirmed through increased phosphorylation of $eIF2{\alpha}$ and protein levels of GRP78/BiP, XBP-1S, and $IRE1{\alpha}$ in human lung cancer cells. Moreover, compound-K led to the accumulation of intracellular calcium and an increase in m-calpain activities that were both significantly inhibited by pretreatment either with BAPTA-AM (an intracellular $Ca^{2+}$ chelator) or dantrolene (an RyR channel antagonist). These results were correlated with the outcome that compound K induced ER stress-related apoptosis through caspase-12, as z-ATAD-fmk (a specific inhibitor of caspase-12) partially ameliorated this effect. Interestingly, 4-PBA (ER stress inhibitor) dramatically improved the compound K-induced apoptosis. Conclusion: Cell survival and intracellular $Ca^{2+}$ homeostasis during ER stress in human lung cancer cells are important factors in the induction of the compound K-induced apoptotic pathway.

• Biomolecules & Therapeutics
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• 제32권1호
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• pp.104-114
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• 2024

Licochalcone C (LCC; PubChem CID:9840805), a chalcone compound originating from the root of Glycyrrhiza inflata, has shown anticancer activity against skin cancer, esophageal squamous cell carcinoma, and oral squamous cell carcinoma. However, the therapeutic potential of LCC in treating colorectal cancer (CRC) and its underlying molecular mechanisms remain unclear. Chemotherapy for CRC is challenging because of the development of drug resistance. In this study, we examined the antiproliferative activity of LCC in human colorectal carcinoma HCT116 cells, oxaliplatin (Ox) sensitive and Ox-resistant HCT116 cells (HCT116-OxR). LCC significantly and selectively inhibited the growth of HCT116 and HCT116-OxR cells. An in vitro kinase assay showed that LCC inhibited the kinase activities of EGFR and AKT. Molecular docking simulations using AutoDock Vina indicated that LCC could be in ATP-binding pockets. Decreased phosphorylation of EGFR and AKT was observed in the LCC-treated cells. In addition, LCC induced cell cycle arrest by modulating the expression of cell cycle regulators p21, p27, cyclin B1, and cdc2. LCC treatment induced ROS generation in CRC cells, and the ROS induction was accompanied by the phosphorylation of JNK and p38 kinases. Moreover, LCC dysregulated mitochondrial membrane potential (MMP), and the disruption of MMP resulted in the release of cytochrome c into the cytoplasm and activation of caspases to execute apoptosis. Overall, LCC showed anticancer activity against both Ox-sensitive and Ox-resistant CRC cells by targeting EGFR and AKT, inducing ROS generation and disrupting MMP. Thus, LCC may be potential therapeutic agents for the treatment of Ox-resistant CRC cells.

• 생명과학회지
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• 제29권4호
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• pp.410-420
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• 2019

한의학에서는 진피(陳皮)라고 칭하는 귤의 껍질(Citrus unshiu peel) 추출물은 항산화, 항염증 및 항균 특성을 포함한 다양한 약리학적 효능을 갖는 것으로 알려져 있다. 최근 그들의 항암 활성에 대한 가능성이 보고되었지만 정확한 기전 연구는 여전히 미비한 실정이다. 본 연구에서는 인간 유방암 MDA-MB-231 세포를 대상으로 진피 에탄올(EECU, ethanol extract of C. unshiu peel) 추출물의 항암 효능을 평가하고 그에 따른 기전 연구를 수행하였다. 본 연구의 결과에 의하면 EECU에 의한 MDA-MB-231 세포의 증식억제는 세포사멸(apoptosis) 유도와 관련이 있었다. EECU에 의한 apoptosis는 caspase-8, -9 및 -3의 활성화와 IAPs 계열의 발현 감소에 따른 PARP의 분해와 Bax : Bcl-2 비율의 증가와 연관이 있었다. 또한 EECU는 Bid의 truncation과 함께 미토콘드리아 막 잠재력의 감소와 세포질로 cytochrome c의 이동을 촉진시켰다. 아울러 EECU는 AMPK 및 ACC의 인산화를 촉진시켰으나, AMPK 효소 활성의 저해제는 EECU에 의한 apoptosis 유도와 생존력 저하를 현저하게 억제하였다. 부가적으로 EECU는 AMPK 활성화의 상류 신호로 작용하는 활성산소종(ROS)의 생성을 촉진시켰으며, 강력한 항산화제인 NAC는 EECU에 의한 AMPK의 활성화와 apoptosis를 역전시켰다. 결론적으로 EECU는 ROS/AMPK 의존적인 내인성 및 외인성 apoptosis 경로를 활성화시킴으로써 MDA-MB-231 세포 증식을 억제하였음을 알 수 있었다.

• 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.

• 동의생리병리학회지
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• 제19권5호
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• pp.1281-1291
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• 2005

Chungpae-tang is suggested to have the antitumor activity on lung cancer. This study was peformed to investigate apoptotic effect in vitro and antitumor effect and immune response after injection of B16-F10 melanoma cells and Chungpae-tang into a tail vein of C57BL/6 mice and administratition of Chungpae-tang in A549 human lung cancer cell line in vivo, respectively. Experimental studies were obtained by measuring the median survival time and cytokine expression through RT-PCR, and ELISA assay. The results were summarized as follows: 5 mg/ml of Chungpae-tang causing DNA fragmentation, caspase-3 enzyme activation, PARP fragmentation, and cytochrome c release, suggested that Chungpae-tang has in vitro apoptotic effect in A549 human lung cancer cell line in the apoptosis-induced experiment. The median survival time of the Chungpae-tang treated group was 21 days and that of control group was 22 days, suggesting that the median survival time between the Chungpae-tang treated group and the control group was not significant. Cytokine expression between the Chungpae-fang treated group and the control group was noticeable, but was not significant in the RT-PCR. In the ELISA assay, IL-2 productivity in the Chungpae-tang treated group was to increase more than that in the normal group (p<0.05) and was no significant between the Chungpae-tang treated group and the control group. $INF-\gamma$ productivity of the control group decreased more than that of the normal group (p<0.05) and that of the Chungpae-tang-treated group increased more than that of the control group (p<0.05). IL-12 productivity of the control group increased more than that of the normal group (p<0.05) and that of the Chungpae-tang-treated group decreased more than that of the control group (p<0.05) and the normal group. IL-4 productivity of the Chungpae-tang-treated group increased more than that of the normal group and the control group (p<0.05). IL-10 productivity of the Chungpae-tang-treated group increased more than that of the normal group and the control group (p<0.05). Accordingly the results show Chungpae-tang could induce apoptosis in A549 human lung cancer cell line and bring to antitumor effect and immune response against injection of B16-F10 melanoma cells into a tail vein of C57BL/6 mice but it needs more research on the precise mechanism of such effects.

• Journal of Oral Medicine and Pain
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• 제34권3호
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• pp.261-276
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• 2009

Sreptomyces라는 세균에서 추출한 lactacystin은 선택적인 proteasome 억제제로서 많은 연구에서 사용되어져 왔다. Proteasome 억제제는 최근의 많은 연구를 통해서 암세포증식의 억제에 대한 효과가 증명되었으며, 특히 다른 항암제와 병용처리 시, 상호작용에 의한 상승효과가 있다고 알려져 있다. 현재 proteasome 억제제는 새로운 강력한 항암제로서 분류되어 있다. 본 연구는 사람혀편평세포암종세포(SCC25 cells)에서 lactacystin의 세포독성과 성장억제 효과, 그리고 세포자멸사의 유도에 대한 분자생물학적 기전을 밝히기 위해 실험을 시행하였다. SCC25 세포, 사람정상각화세포 (HaCaT cells) 그리고 사람치은섬유모세포(HGF-1 cells)의 생존율 측정은 MTT법을 시행하였고, SCC25 세포의 성장억제를 확인하기 위해서는 clonogenic assay를 사용하였다. lactcystin이 SCC25 세포에서 세포자멸사가 유도되는지를 확인하기 위해서 hoechst 염색법, hemacolor 염색법 그리고 TUNEL법을 시행하였다. 그리고 SCC25 세포에 lactacystin을 적용한 후, Western blot 분석, 세포면역화학염색, 공초점레이저주사현미경 검경, FACScan flow cytometry, 사립체막 전위변화, proteasome 활성도 측정 등을 시행하였다. Lactacystin으로 처리된 SCC25 세포는 시간 및 용량 의존적인 세포생존율의 감소, 용량의존적인 세포성장억제 그리고 세포자멸사에 의한 세포죽음을 보였다. 흥미롭게도 lactacytin은 정상세포인 HaCat 세포와 HGF-1 세포에서는 세포독성을 전혀 보이지 않았다. 그리고 lactacystin이 적용된 SCC25세포에서 핵 응축, DNA의 조각남, 사립체막전위와 proteasome 활성도의 감소, DNA 양의 감소, cytochrome c의 사립체에서의 세포질로의 유리, AIF와 DFF40 (CAD)의 핵으로의 이동, Bax의 증가, caspase-7, caspase-3, PARP, lamin A/C 그리고 DFF45 (ICAD)의 활성화 혹은 파괴와 같은 아주 다양한 세포자멸사 증거를 보였다. Flow cytometry 분석에서는 CDK 억제제인 $p21^{WAF1/CIP1}$와 $p27^{KIP1}$의 발현 증가와 관계있는 것으로 추정되어 지는 G1 세포주기 정지를 보였다. 이러한 결과는 lactacytin이 SCC25 세포에서 G1 세포주기정지와 proteasome, 사립체 및 caspase 경로의 연속반응을 통한 세포자멸사를 유도함을 명확하게 증명하고 있다. 이와 같은 세포주기 정지와 세포자멸사 유도능은 lactacytin이 사람혀편평상피세포암종의 새로운 치료전략으로서의 가능성을 제공한다고 생각한다.

• Journal of Oral Medicine and Pain
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• 제36권3호
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• pp.147-160
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• 2011

Chios gum mastic (CGM)은 그리이스 키오스 섬에서만 자생하는 Pistiacia lentiscus L. var. Chia. 의 잎과 줄기로부터 추출되어진 식물성 수지이며, 과거 수세기 동안 지중해와 중동 지역 국가들에서 음식 첨가물과 위궤양, 십이지장궤양 등의 민간 치료약재로서 사용되어져 왔다. 정향나무에서 추출하는 페놀화합물인 eugenol은 zinc oxide eugenol의 구성성분으로 치과치료를 위해 많이 사용되고 있다. 본 연구는 사람혀편평세포암종세포(SCC25 cells)에서 천연물질인 CGM과 eugenol을 병용처리한 후 세포자멸사 효과가 있는지를 알아보기 위해 수행하였다. CGM과 eugenol의 병용처리가 단독처리에 비해서 효과적인 세포생존율 감소가 있는지 확인하기 위하여 MTT법을 시행하였고, 세포자멸사의 유도와 증가를 알기 위하여 Hoechst 염색법, TUNEL 염색법, DNA hypoploidy법을 사용하였다. 그리고 세포자멸사에 관계하는 단백질의 발현 변화와 세포내에서의 이동을 밝혀내기 위하여 Western blot 분석과 면역형광염색법을 수행하였다. 본 연구에서는 CGM과 eugenol이 병용처리된 SCC25 세포에서 핵의 농축, DNA분절, Bax의 증가와 Bcl-2의 감소, DNA양의 감소, cytochrome c의 세포질로의 유리, AIF와 DFF40 (CAD)의 핵으로의 이동, caspase-3, caspase-6, caspase-7, caspase-9, PARP, Lamin A/C 그리고 DFF45 (ICAD)의 활성화와 같은 다양한 세포자멸사 증거를 보였다. 반면에 CGM과 eugenol이 각각 단독 처리된 SCC25 세포에서는 세포자멸사 현상이 미미하였다. 24시간 동안 40 ${\mu}g$/ml의 CGM과 0.5 mM의 eugenol을 각기 단독처리 한 결과에서는 세포자멸사를 거의 유도하지 못했으나, 병용처리 한 결과에는 아주 탁월한 세포자멸사의 유도를 보였다. 그러므로 본 실험결과는 사람구강편평세포암종 환자들에게 CGM과 eugenol의 병용요법이 새로운 치료전략으로서의 가능성을 줄 수 있다고 생각한다.

• The Korean Journal of Physiology and Pharmacology
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• 제11권2호
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• pp.57-64
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• 2007

Ischemic preconditioning (IPC) is known to protect the heart against ischemia/reperfusion (IR)-induced injuries, and regional differences in the mitochondrial antioxidant state during IR or IPC may promote the death or survival of viable and infarcted cardiac tissues under oxidative stress. To date, however, the interplay between the mitochondrial antioxidant enzyme system and the level of reactive oxygen species (ROS) in the body has not yet been resolved. In the present study, we examined the effects of IR- and IPC-induced oxidative stresses on mitochondrial function in viable and infarcted cardiac tissues. Our results showed that the mitochondria from viable areas in the IR-induced group were swollen and fused, whereas those in the infarcted area were heavily damaged. IPC protected the mitochondria, thus reducing cardiac injury. We also found that the activity of the mitochondrial antioxidant enzyme system, which includes manganese superoxide dismutase (Mn-SOD), was enhanced in the viable areas compared to the infarcted areas in proportion with decreasing levels of ROS and mitochondrial DNA (mtDNA) damage. These changes were also present between the IPC and IR groups. Regional differences in Mn-SOD expression were shown to be related to a reduction in mtDNA damage as well as to the release of mitochondrial cytochrome c (Cyt c). To the best of our knowledge, this might be the first study to explore the regional mitochondrial changes during IPC. The present findings are expected to help elucidate the molecular mechanism involved in IPC and helpful in the development of new clinical strategies against ischemic heart disease.