• Asian Pacific Journal of Cancer Prevention
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• 제16권18호
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• pp.8313-8319
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• 2016

We have recently shown that thymoquinone (TQ) has a potent cytotoxic effect and induces apoptosis via caspase-3 activation with down-regulation of XIAP in mouse neuroblastoma (Neuro-2a) cells. Interestingly, our results showed that TQ was significantly more cytotoxic towards Neuro-2a cells when compared with primary normal neuronal cells. In this study, the effects of TQ on cell-cycle regulation and the mechanisms that contribute to this effect were investigated using Neuro-2a cells. Cell-cycle analysis performed by flow cytometry revealed cell-cycle arrest at G2/M phase and a significant increase in the accumulation of TQ-treated cells at sub-G1 phase, indicating induction of apoptosis by the compound. Moreover, TQ increased the expression of p53, p21 mRNA and protein levels, whereas it decreased the protein expression of PCNA, cyclin B1 and Cdc2 in a dose-dependent manner. Our finding suggests that TQ could suppress cell growth and cell survival via arresting the cell-cycle in the G2/M phase and inducing apoptosis of neuroblastoma cells.

• 동의생리병리학회지
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• 제19권4호
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• pp.1062-1067
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• 2005

The purpose of this study was to examine the inhibition effect of Zizania latifolia that has been used heart disease, Diabetes Mellitus and Skin disease for a long time on apoptosis induced by $H_2O_2$ in Neuro2A cell. Neuro2A cells were cultivated in RPMI(GibcoBRL) with $5\%$, FBS and treated with $H_2O_2$, and Zizania latifolia. We measured the cell viability and analyzed DNA fragmentation. Activity of PARP, Cytochrome C, caspase-9, caspase-3, p53, p21, Bax and Bcl-2 in the cell was examined by using western blot. The cell viability in Zizania latifolia treatment (60ug/ml<) decreased significantly compared with that of none treatment. (p<0.001) Zizania latifolia increased cell viability about twice as much as that being injury by $H_2O_2$. (Zizania Latifolia 20ug/ml, $H_2O_2$ 200uM, P<0.001) DNA fragmentation developed by $H_2O_2$, but was not developed in Zizania latifolia treatment. PARP, Cytochrome C, caspase-9 and caspase-3 activated all by $H_2O_2$ but were not activated in Zizania latifolia treatment.. P53, P2l and Bu activated by $H_2O_2$, and Bcl-2 got into inactivation. But the opposite results appeared in Zizania latifolia treatment. In conclusion, these results suggest that Zizania latifolia inhibit the development of DNA fragmentation and apoptosis by $H_2O_2$ and the antioxidant action of Zizania latifolia is effective. More researches about effect of Zizania latifolia are considered to need.

• 동의생리병리학회지
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• 제20권1호
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• pp.149-155
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• 2006

To prevent human body injury from oxidative stress, antioxidants are very important and many research about antioxidants are generally being conducted. Hydrogen peroxide($H_2O_2$) that is one of vitality oxygen species has been seen that cause various diseases, DNA damage and gene change. The purpose of this study was to examine the inhibition effect of Zizania latifolia Rhizoma on apoptosis induced by $H_2O_2$ in Neuro2A cell. Neuro2A cells were cultivated in RPMI(GibcoBRL) with 5% FBS and treated with $H_2O_2$ and Zizania latifolia Rhizoma. We measured the cell viability and analyzed DNA fragmentation. Activity of PARP, Cytochrome C, caspase-9, caspase-3, p53, p21, Bax and Bcl-2 in the cell was examined dy using western blot. The results obtained were as Follows: The cell viability in Zizania latifolia Rhizoma treatment (60ug/ml<) decreased significantly compared with that of none treatment. (P<0.001) Zizania latifolia Rhizoma increased cell viability about twice as much as that being injury by $H_2O_2$. (Zizania Latifolia Rhizoma 20ug/ml, $H_2O_2$ 200uM, P<0.001) DNA fragmentation developed by $H_2O_2$, but was not developed in Zizania latifolia Rhizoma treatment. PARP, Cytochrome C, caspase-9 and caspase-3 activated all by $H_2O_2$ but were not activated in Zizania latifolia Rhizoma treatment. P53, P2l and Bax activated dy $H_2O_2$, and Bcl-2 got into inactivation. But the opposite results appeared in Zizania latifolia Rhizoma treatment. In conclusion, these results suggest that Zizania latifolia Rhizoma inhibit the development of DNA fragmentation and apoptosis by $H_2O_2$ and the antioxidant action of Zizania latifolia Rhizoma is effective. More researches about effect of Zizania latifolia Rhizoma are considered to need.

• 대한한의학회지
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• 제31권2호
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• pp.1-18
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• 2010

Objective: The water extract of Chilgi-tang (CGT) has been traditionally used in treatment of heart diseases caused by stress in Oriental Medicine. However, little is known about the mechanism by which CGT rescues neuronal cells from injury damage. Therefore, this study was designed to evaluate the protective effects of CGT on Neuro-2A cells by glucose deprivation-induced cell death. Methods: We investigated how cell death induced by glucose deprivation was associated with increased reactive oxygen species (ROS) generation. Result: The CGT treatment prior to glucose deprivation insult significantly reduced the number of cell deaths and the glucose deprivation-induced increase in ROS. Nitric oxide (NO) was also attenuated by CGT treatment. In addition, we demonstrated that the anti-cell death effect of CGT was blocked by heme oxygenase-1 (HO-1) activation. Finally, pretreatment of cells with a hemin, HO-1 inducer, reduced glucose deprivation-induced cell death. In contrast, pretreatment of cells with a ZnPP, HO-1 activity inhibitor, attenuated CGT-induced inhibition of cell death. Conclusions: These findings indicate that ROS plays an important role in glucose deprivation-induced cell death and that CGT may prevent glucose deprivation-induced cell death by inhibiting the ROS generation through HO-1 activation in Neuro-2A cells.

• 동의생리병리학회지
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• 제20권4호
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• pp.936-941
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• 2006

To prevent human body injury from oxidative stress, antioxidants are very important and many research about antioxidants are generally being conducted. Hydrogen peroxide$(H_20_2)$ that is one of vitality oxygen species has been seen that cause various diseases, DNA damage and gene change. We have already known that the inhibition effect of Zizania latifolia Radix, Rhizoma on apoptosis induced by $H_2O_2$ in Neuro2A cell. And the purpose of this study was that we made a comparative study on the inhibition effect of apoptosis in Neuro2A cell on the region of Zizania latifolia(Radix, Rhizoma, Herba). Neuro2A cells were cultivated in RPMI(GibcoBRL) with 5% FBS and treated with $H_2O_2$ and Zizania latifolia(Radix, Rhizoma, Herba). Separately we measured the cell viability and analyzed DNA fragmentation. Activity of PARP, Cytochrome C, caspase-9, caspase-3, p53, p21, Bax and Bcl-2 in the cell was examined by using western blot. The results obtained were as Follows: The cell viability in all of Zizania latifolia (Radix, Rhizoma, Herba) treatment (60ug/m1<) decreased significantly compared with that of none treatment(p<0.001). Zizania latifolia Radix increased cell viability was most effective of three regions. But we had no significant difference among three regions. All of Zizania latifolia (Radix, Rhizoma, Herba) increased cell viability about twice as much as that being injury by $H_2O_2$,(Zizania Latifolia (Radix, nhizoma, Herba) 20ug/m1, $H_2O_2$ 200uM, p<0.001). DNA fragmentation developed by $H_2O_2$, but was not developed in all of Zizania latifolia (Radix, Rhizoma, Herba) treatment. PARP, Cytochrome C, caspase-9 and caspase-3 activated all by $H_2O_2$ but were not activated in all of Zizania latifolia (Radix, Rhizoma, Herba) treatment. P53, P2l and Bax activated by $H_2O_2$, and Bcl-2 got into inactivation. But the opposite results appeared in all of Zizania latifolia (Radix, Rhizoma, Herba) treatment. In conclusion, these results suggest that all of Zizania latifolia (Radix, Rhizoma, Herba) inhibit the development of DNA fragmentation and apoptosis by $H_2O_2$and the antioridant action of all of Zizania latifolia (Radix, Rhizoma, Herba) is effective.

• 한국해양생명과학회지
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• 제9권1호
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• pp.9-21
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• 2024

마비성 패류 독소(Paralytic shellfish poisoning, PSP)는 유해 조류에 의해 생성되며, 독소에 노출된 수산물을 섭취하였을 때 중독이 발생한다. 수산물 중 PSP를 검출하는 표준 시험법인 Mouse bioassay (MBA)는 낮은 검출한계와 동물 윤리 문제로 대체 시험법의 개발 필요성이 대두되고 있다. 이러한 대체 시험법 중, PSP가 신경 세포막의 Na⁺ 채널을 차단하는 기전을 이용한 마우스 뇌신경 모세포종 세포 기반 시험법(Neuro-2a assay)의 표준화를 위한 노력이 대두되고 있다. Neuro-2a assay의 원리는 Neuro-2a 세포주에 Na⁺/K⁺ ATPase 억제제인 Ouabain(O)과 Na⁺ 채널 활성화제인 Veratridine (V)을 처리하여 과도한 Na⁺ 유입으로 인한 세포사멸을 유도한 상태에서, Na⁺ 채널 억제제인 PSP를 처리하게 되면 Na⁺ 유입이 차단되어 세포가 생존하는 것을 측정하는 것이다. 본 연구에서는 PSP 검출을 위한 Neuro-2a assay를 국내 연구 환경에 맞게 다양한 매개변수를 개선하여 최적 시험법을 확립하고자 하였다. 고려한 매개변수들은 세포밀도, 배양 조건 및 PSP 처리 조건 등으로, 그 결과는 아래와 같다. 초기 세포밀도는 40,000 cells/well로, 세포 배양시간 및 처리시간은 각각 24시간으로 설정하였다. 또한 최적 O/V 농도는 500/50 μM로 설정하였다. 본 연구에서 PSP 중 Saxitoxin (STX)에 대해서 O/V 처리가 된 상태에서 S자형 용량-반응 그래프가 도출되는 8가지 농도(368~47,056 fg/μl)를 확인하였고, Neuro-2a assay의 실험실 간 변동성 비교를 통해, 실험의 적정성 확인을 위한 5가지 Quality Control Criteria와 실험 데이터의 신뢰가능 범위(Data Criteria) 6가지를 설정하였다. 확 립된 조건으로 Neuro-2a assay를 진행한 결과 반수영향농도(EC₅₀) 값은 약 1,800~3,500 fg/μl로 나타났다. 실험실 간 변동성 비교 결과, Quality Control Criteria 값 및 Data criteria 값의 변동계수(coefficients of variation (CVs))가 1.98~29.15% 범위로 산출되어 실험의 적정성 및 재현성이 확인되었다. 본 연구를 통해 우리나라에서 활용할 수 있는 PSP 검출용 Neuro-2a assay 시험법의 최적 조건 및 5가지 Quality control 기준을 제시하였고, PSP 중 대표적인 독소인 STX을 대상으로 Neuro-2a assay를 실시한 결과 유의한 EC₅₀ 값을 산출할 수 있었으며, 향후 국내 수산물을 대상으로 MBA를 대체할 수 있는 PSP 검출법으로 활용될 것으로 기대된다.

• Molecules and Cells
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• 제41권12호
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• pp.1061-1071
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• 2018

From Xenopus embryo studies, the BMP4/Smad1-targeted gene circuit is a key signaling pathway for specifying the cell fate between the ectoderm and neuro-ectoderm as well as the ventral and dorsal mesoderm. In this context, several BMP4/Smad1 target transcriptional factors have been identified as repressors of the neuro-ectoderm. However, none of these direct target transcription factors in this pathway, including GATA1b, Msx1 and Ventx1.1 have yet been proven as direct repressors of early neuro-ectodermal gene expression. In order to demonstrate that Ventx1.1 is a direct repressor of neuro-ectoderm genes, a genome-wide Xenopus ChIP-Seq of Ventx1.1 was performed. In this study, we demonstrated that Ventx1.1 bound to the Ventx1.1 response cis-acting element 1 and 2 (VRE1 and VRE2) on the promoter for zic3, which is a key early neuro-ectoderm gene, and this Ventx1.1 binding led to repression of zic3 transcription. Site-directed mutagenesis of VRE1 and VRE2 within zic3 promoter completely abolished the repression caused by Ventx1.1. In addition, we found both the positive and negative regulation of zic3 promoter activity by FoxD5b and Xcad2, respectively, and that these occur through the VREs and via modulation of Ventx1.1 levels. Taken together, the results demonstrate that the BMP4/Smad1 target gene, Ventx1.1, is a direct repressor of neuro-ectodermal gene zic3 during early Xenopus embryogenesis.

• 대한한의학회지
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• 제26권1호
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• pp.161-173
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• 2005

Objective : The water extract of Seokchangpowonji-san (SWS) has traditionally been used for treatment of dementia in oriental medicine. However, little is known about the mechanism by which the water extract of SWS rescues cells from neurodegenerative disease such as Alzheimer's disease. Methods & Results: This study was designed to investigate the protective mechanisms of SWS on $\beta-amyloid$ or $H_2O_2$-induced$ cytotoxicity in neuro 2A cells. $H_2O_2$ markedly decreased the viability of neuro 2A cells, which was characterized by apparent apoptotic features such as membrane blebbing as well as fragmentation of genomic DNA and nuclei. However, the water extract of SWS significantly reduced $H_2O_2-induced$ cell death and apoptotic characteristics through reduction of intracellular peroxide generation. Also, the. extract prevented the mitochondrial dysfunction including the disruption of mitochondria membrane permeability transition (MPT) and the modulation in expression of Bcl-2 family proteins in $H_2O_2­treated$ neuro 2A cells. Furthermore, pretreatment with SWS inhibited the activation of caspase-3, and in turn, degradation of ICAD/DFF45 was completely abolished in $H_2O_2-treated$ cells. Conclusion: Taken together, the data suggest that the protective effects of the water extract of SWS against $\beta-amyloid$ induced oxidative injuries may be achieved through modulation of mitochondrial dysfunction.

• Clinical and Experimental Pediatrics
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• 제45권3호
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• pp.354-361
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• 2002

목 적: 본 연구에서는 in vitro 및 in vivo에서 HSV-TK 유전자 치료의 bystander 효과 및 기전을 관찰하고, 싸이토카인 유전자의 병합 치료가 bystander 효과를 증폭시킬 수 있는지 조사하여 그 결과를 향후 신경모세포종 치료에 적용하고자 본 연구를 시행하였다. 방 법: A/J 마우스 신경모세포종 모델에서, neuro-2a/TK 세포와 unmodified neuro-2a세포를 여러 비율로 혼합하여 접종한 후 GCV를 투여하여 종양의 크기 변화 및 생존 유무를 관찰하였다. 마우스 신경모 세포종에서 bystander 효과의 기전을 알아보기 위해 neuro-2a/TK 세포를 주입한 마우스의 조직을 절개하여 connexin 43, CD4+ 및 CD8+ 세포 침윤을 관찰 하였다. 10% 및 25% HSV-TK 투여 군에서 neuro-2a/IL-2 세포의 투여가 bystander 효과를 증폭시키는 지 조사하였다. 결 과 : 1) in vitro 및 in vivo에서 bystander 효과는 뚜렷하게 관찰되었다. 2) 마우스 신경모세포종의 면역 조직 화학 염색 검사에서 connexin 43 발현은 관찰되지 않았으나 많은 수의 CD4+ 및 CD8+ T 세포의 침윤이 관찰되었다. 3) 10% 및 25% HSV-TK 투여군의 마우스에서 neuro-2a/IL-2 세포의 투여는 대조군과 비교하여 종양의 성장을 더 억제 시켰다. 결 론: In vitro 및 in vivo에서 HSV-TK/GCV 유전자 치료의 bystander 효과는 뚜렷하였으며, 그 기전으로 면역 세포가 중요한 역할을 할 것으로 추측된다. 그리고 bystander 효과는 IL-2 투여에 의해 증폭됨을 확인하였다. 이는 향후 신경모세포종의 수술 후 잔존암 치료에 HSV-TK/GCV 유전자 치료가 이용 가능할 것으로 생각된다.

• Journal of Microbiology and Biotechnology
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• 제11권2호
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• pp.234-241
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• 2001

The C-terminal 393 bp region of the human interleukin-16 (IL-16) gene was cloned and expressed in E. coli along with mammalian cell lines. Recombinant IL-16 expressed from E. coli was 22 kDa on SDS-PAGE and showed 260% of chemoattractant activity at a concentration of $0.1\;{\mu}g/ml$. HeLa, COS, and Neuro-2a cells were transduced by recombinant retrovirus vector pLNC/IL-16/IRES/TK and the intracellular and secreted amounts of IL-16 produced by HeLa/IL-16/TK, COS/IL-16/TK, and Neuro-2a/IL-16/TK cells were determined by enzyme-linked immunosorbent assay (ELISA). HeLa/IL-16/TK $(1{\times}10^5)$ and COS/IL-16/TK $(1{\times}10^5)$ cells secreted 36.1 and 13.3 ng of IL-16 for 48 h, respectively. Forty-nine ng and 86.4 ng of IL-16 remained in the cell lysates of HeLa/IL-16/TK and COS/IL-16/TK. Intracellular and secreted amounts of IL-16 from Neuro-2a/IL-16/TK $(5{\times}10^5)$ cells during 24 h cultivation were 50 ng and 3.3 ng, respectively. Also, HeLa and COS cells wee stably transfected with mammalian expression vector pCRIII/IL-16. Both culture media and cell lysates prepared from HeLa/IL-16 cells and COS/IL-16 cells showed chemoattractant activity ranging from 190% to 460% as compared to the control experiment. Expression of the herpes simplex virus thymidine kinase (HSV0tk) gene in pLNC/IL-16/ IRES/TK bicistronic retroviral expression vector was verified by performing a genciclovir (GCV) sensitivity assay. Finally, IL-16 repressed Tat-transactivated human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) promoter activity.