• Journal of Applied Biological Chemistry
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• 제54권2호
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• pp.143-146
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• 2011

Novel pyrazoline (4) was synthesized from chalcone (3) which was prepared from 2'-hydroxy-l'acetonaphthone (1) and 4-methoxy benzaldehyde (2). Pyrazoline (4) forms resonance assisted hydrogen bond between naphthol hydroxyl group and imine nitrogen in a pyrazoline ring. Pyrazoline (4) shows Poly ADP-ribose Polymerase (PARP) cleavage ability as a proof of apoptosis in cancer cell, which reveals its anti-cancer property.

• Radiation Oncology Journal
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• 제20권4호
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• pp.367-374
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• 2002

목적 : 인체 대장암 세포주인 HT-29에 새로운 CDCA 합성유도체인 HS-1200을 처치하여 암세포의 증식에 미치는 영향과 아포토시스 유도 활성을 관찰하고 기전을 연구하고자 하였다. 대상 및 방법 : 지수증식기의 HT-29 세포에 다양한 농도의 CDCA 합성유도체인 HS-1200을 투약하여 $IC_{50}$를 구하였다. $IC_{50}$의 농도를 참조하여, 세포 생존능의 실험은 frypan blue exclusion assay를 이용하였고, 아포토시스 유도에 관한 관찰 실험은 agarose gel electrophoresis, TUNEL assay 및 Hoechst staining을 이용하였다. Western blotting을 통한 PARP [poly (ADP-ribose) polymerasel, caspase-3 및 DFF (DNA fragmentation factor)의 degradation 및 cleavage 등을 관찰하였다. Immunofluorescent method를 통한 cytochrome c 방출 측정 및 미토콘드리아 막전위 측정을 실시하였다. 결과 : HS-1200은 agarose gel electrophoresis 실험에서 DNA ladder의 관찰, TUNEL assay 및 Hoechst staining 에서 아포토시스 세포들이 대량으로 관찰되는 결과로 미루어 아포토시스에 의한 세포 사망을 유도하는 것으로 사료되었다. 아포토시스에 의한 세포 사망을 검증하기 위한 Western blotting을 통한 PARP cleavage, caspase-3 및 DFF 발현 관찰에서 공히 HS-1200 처치 후 4시간 째부터 PARP, caspase-3 및 DFF의 degradation 및 cleavage가 관찰되었다. HS-1200의 아포토시스 유도에 이르는 기전연구에서 미토콘드리아의 역할에 주목하고 시행한 cytochrome c 방출 측정 및 미토콘드리아막 전위 $(\Delta\Psi_m)$ 측정에서 공히 cytochrome c 방출 및 미토콘드리아막 전위 $(\Delta\Psi_m)$의 감소가 관찰되었다. 결론 : 인체 대장암 세포주(HT-29)에서 CDCA 합성유도체인 HS-1200을 이용한 세포 증식억제 및 세포사망 기전 연구에서, 아포토시스의 유도가 HS-1200의 세포 사망 기전에 중요한 역할을 하는 것을 확인하였다. 이러한 아포토시스의 유도에는 미토콘드리아의 역할이 중요하게 관련되는 것으로 관찰되었다. 상기 결과를 토대로 HS-1200의 항암 치료제로서의 역할에 관한 기초 자료로 서의 유용성을 제시할 수 있었다.

• 생명과학회지
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• 제29권3호
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• pp.318-324
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• 2019

두경부암은 전세계에서 발병률이 여섯 번째로 높은 암으로 그동안 수술적 치료를 선호하였으나 광범위한 절제에 따른 기능적 장애로 인해 항암치료에 대한 관심이 높아지고 있다. 두경부암에서 cisplatin이 가장 많이 사용되는 항암제이나 cisplatin 내성이 문제가 되고 있다. 따라서 부작용은 줄이고, 약제내성 기전에 대해 이해하여 암세포의 사멸은 증대시키는 새로운 항암제의 개발이 필요하다. Survivin은 inhibitor of apoptosis proteins (IAPs) family 중 하나로 두경부암에서 과발현되어 있다. YM155는 survivin을 억제하는 분자로 본 연구를 통해 YM155의 처리 후 두경부 암세포의 세포자멸사가 유도되며, 뇌암 세포와 신장암 세포에서도 세포자멸사가 유도됨을 확인할 수 있었다. 반면에 정상세포인 mesangial cells에는 YM155가 세포자멸사에 영향을 주지 않았다. YM155는 caspase의 활성화를 통해 세포자멸사를 촉진하며, anti-apoptotic protein인 c-FLIP, Mcl-1, survivin의 발현을 저해하는 것으로 확인되었다. YM155는 두경부 뿐만 아니라 다른 장기의 악성종양 치료법의 개발에 활용 될 수 있을 것으로 생각된다.

• Nutrition Research and Practice
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• 제3권3호
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• pp.185-191
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• 2009

The elevated level of circulating estradiol increases the risk of breast tumor development. To gain further insight into mechanisms involved in their actions, we investigated the molecular mechanisms of 4-hydroxyestradiol (4-$OHE_2$) to initiate and/or promote abnormal cell growth, and of $\alpha$- or $\gamma$-tocopherol to inhibit this process. MCF-10A, human breast epithelial cells were incubated with $0.1{\mu}M$ 4-$OHE_2$, either with or without $30{\mu}M$ tocopherols for 96 h. 4-$OHE_2$ caused the accumulation of intracellular ROS, while cellular GSH/GSSG ratio and MnSOD protein levels were decreased, indicating that there was an oxidative burden. 4-$OHE_2$ treatment also changed the levels of DNA repair proteins, BRCA1 and PARP-1. $\gamma$-Tocopherol suppressed the 4-$OHE_2$-induced increases in ROS, GSH/GSSG ratio, and MnSOD protein expression, while $\alpha$-tocopherol up-regulated BRCA1 and PARP-1 protein expression. In conclusion, 4-$OHE_2$ increases oxidative stress reducing the level of proteins related to DNA repair. Tocopherols suppressed oxidative stress by scavenging ROS or up-regulating DNA repair elements.

• The Korean Journal of Physiology and Pharmacology
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• 제7권6호
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• pp.325-331
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• 2003

3-Nitropropionic acid (3-NP) inhibits electron transport in mitochondria, leading to a metabolic failure. In order to elucidate the mechanism underlying this toxicity, we examined a few biochemical changes possibly involved in the process, such as metabolic inhibition, generation of reactive oxygen species (ROS), DNA strand breakage, and activation of Poly(ADP-ribose) polymerase (PARP). Exposure of SK-N-BE(2)C neuroblastoma cells to 3-NP for 48 h caused actual cell death, while inhibition of mitochondrial function was readily observed when exposed for 24 h to low concentrations (0.2${\sim}$2 mM) of 3-NP. The earliest biochemical change detected with low concentration of 3-NP was an accumulation of ROS (4 h after 3-NP exposure) followed by degradation of DNA. PARP activation by damaged DNA was also detectable, but at a later time. The accumulation of ROS and DNA strand breakage were suppressed by the addition of glutathione or N-acetyl-L-cysteine (NAC), which also partially restored mitochondrial function and cell viability. In addition, inhibition of PARP also reduced the 3-NP-induced DNA strand breakage and cytotoxicity. These results suggest that oxidative stress and activation of PARP are the major factors in 3-NP-induced cytotoxicity, and that the inhibition of these factors may be useful in protecting neuroblastoma cells from 3-NP-induced toxicity.

• Journal of Radiation Protection and Research
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• 제37권2호
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• pp.56-62
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• 2012

To determine the biological effects of low-dose-rate radiation ($^{137}Cs$, 2.95 mGy/h) on EL4 lymphoma cells during 24 h, we investigated the expression of genes related to apoptosis, cell cycle arrest, DNA repair, iron transport, and ribonucleotide reductase. EL4 cells were continuously exposed to low-dose-rate radiation (total dose: 70.8 mGy) for 24 h. We analyzed cell proliferation and apoptosis by trypan blue exclusion and flow cytometry, gene expression by real-time PCR, and protein levels with the apoptosis ELISA kit. Apoptosis increased in the Low-dose-rate irradiated cells, but cell number did not differ between non- (Non-IR) and Low-dose-rate irradiated (LDR-IR) cells. In concordance with apoptotic rate, the transcriptional activity of ATM, p53, p21, and Parp was upregulated in the LDR-IR cells. Similarly, Phospho-p53 (Ser15), cleaved caspase 3 (Asp175), and cleaved Parp (Asp214) expression was upregulated in the LDR-IR cells. No difference was observed in the mRNA expression of DNA repair-related genes (Msh2, Msh3, Wrn, Lig4, Neil3, ERCC8, and ERCC6) between Non-IR and LDR-IR cells. Interestingly, the mRNA of Trfc was upregulated in the LDR-IR cells. Therefore, we suggest that short-term Low-dose-rate radiation activates apoptosis in EL4 lymphoma cells.

• Toxicological Research
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• 제25권3호
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• pp.113-118
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• 2009

Cell death induced by menadione (vitamin K-3,2-methyl-1,4-naphthoquinone) has been investigated in human promyelocytic leukemia HL-60 cells. Menadione was found to induce both apoptosis and necrosis in HL-60 cells. Low concentration ($1{\sim}$50 ${\mu}$M) of menadione induced apoptotic cell death, which was demonstrated by typical DNA ladder patterns on agarose gel electrophoresis and flow cytometry analysis. In contrast, a high concentration of menadione (100 ${\mu}$M) induced necrotic cell death, which was demonstrated by DNA smear pattern in agarose gel electrophoresis. Necrotic cell death was accompanied with a great reduction of cell viability. Menadione activated caspase-3, as evidenced by both increased protease activity and proteolytic cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP) into 85 kDa cleavage product. Caspase-3 activity was maximum at 50 ${\mu}$M of menadione, and very low at 100 ${\mu}$M of menadione. Taken together, our results showed that menadione induced mixed types of cell death, apoptosis at low concentrations and necrosis at high concentrations in HL-60 cells.

• 생물정신의학
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• 제4권2호
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• pp.211-217
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• 1997

The Bcl-2 protein has been shown to block apoptosis induced by a variety of stimuli. We have performed the experiments which cell death can be blocked by the bcl-2 proto-oncogene under moderate(50-100mM) or high ethanol treatment(400-600mM). As a result of morphological changes, and MTT assay, cell death was blocked by Bcl-2 under 100mM ethanol. However, the results of DNA fragmentation and RT-PCR(ICE, and CPP32), immunoblotting(CPP32, and PARP) for SK-pcDNA3 cells(vector only) and SK-Bcl-2 cells(stably expressed bcl-2 gene) were showen to be no significant differences between two cell lines. These results suggested that cell death induced by ethanol was not followed by apoptosis mechanism, and was blocked by the bcl-2 proto-oncogene with moderate ethanol.

• 사상체질의학회지
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• 제25권4호
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• pp.359-372
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• 2013

Objectives The object of this study was to observe the effects of Hyeongbangjihwhang-tang (HB; Soyangin prescription) on the 5/6 NTX induced CRF rats. Methods Each of Hyeongbangjihwhang-tang aqueous extracts 200mg/kg were orally administered once a day for 35 days from 4 weeks after 5/6 NTX surgery. Four groups, each of 8 rats per group were used in this study, were sham group, CRF group, ${\alpha}$-Tocoperol group and HB group. Changes on the left remnant kidney weights, serum BUN, creatinine levels, caspase-3, PARP immunoreactivities were observed to nephroprotective effects, and relative immunomodulatory effects were monitored based on the changes of lymphatic organ weights and splenic cytokine contents. In addition, the changes on the kidney MDA, GSH contents and SOD, CAT activities were also calculated for antioxidant effects, and the effects on the CRF related cachexia were demonstrated based on the changes of body and epididymal fat pad weights, serum TG, TC, LDL and HDL levels. Results and Conclusions 1) HB was significantly decreased the left remnant kidney weights, serum BUN, creatinine levels and caspase-3, PARP immunoreactivities. 2) HB was significantly increased lymphatic organ weights and splenic cytokine contents. 3) HB was significantly increased body and epididymal fat pad weights, and was significantly decreased serum TG, TC, LDL and HDL levels. 4) HB was significantly decreased MDA contents, and was significantly increased GSH contents and SOD, CAT activities. The results obtained in this study suggest that HB significantly retarded immunosuppressions and cachexia related to the 5/6 NTX induced CRF through modulations of oxidative defense systems. Especially HB showed the highest favorable effects more than those of ${\alpha}$-tocoperol.

• 대한한방내과학회지
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• 제30권1호
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• pp.51-63
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• 2009

Background and Objective : The prospects for developing an anti-apoptotic natural component or a compound that exerts a neuroprotective effect with few or no side effects for the treatment of neurodegenerative disease appear favorable. In the present study, we evaluated the effects of the methanol extract of Phellodendri Cortex (PC extract) on 1-methyl-4-phenyl pyridinium($MPP^+$)-induced apoptosis in PC-12 cells. Materials and Methods : We used the methanol extract of Phellodendri Cortex (PC extract). PC-12 cells were cultured by RPMZ-1640. We found the PC extract's gene expression (Bax, Bcl-2) by using RT-PCR. We examined the PC extract's protein expression (Bcl-2, Bax, cytochrome c, poly (ADP-ribose) polymerase (PARP), caspase-3) by SDS-PAGE and Western blot. Results : Apoptosis in $MPP^+$-induced PC-12 cells was accompanied by an increased Bax/Bcl-2 ratio, release of cytochrome c to the cytosol and the activation of caspase-3. PC extract inhibited the down-regulation of Bcl-2 and the up-regulation of Bax, as well as the release of mitochondrial cytochrome c into the cytosol. In addition, PC extract attenuated caspase-3 activation and cleavage of poly (ADP-ribose) polymerase (PARP). Conclusion : These results suggest that the neuroprotective potentials of PC extract against $MPP^+$-induced apoptosis can be. at least partially, ascribed to its anti-apoptotic effects in PC-12 cells.