• 제목/요약/키워드: cell transfection

검색결과 493건 처리시간 0.022초

Effects of Down-regulation of HDAC6 Expression on Proliferation, Cell Cycling and Migration of Esophageal Squamous Cell Carcinoma Cells and Related Molecular Mechanisms

  • Li, Ning;Tie, Xiao-Jing;Liu, Pei-Jie;Zhang, Yan;Ren, Hong-Zheng;Gao, Xin;Xu, Zhi-Qiao
    • Asian Pacific Journal of Cancer Prevention
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    • 제14권2호
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    • pp.685-689
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    • 2013
  • Objective: To study the effects of down-regulation of HDAC6 expression on proliferation, cell cycling and migration of esophageal squamous cell carcinoma (ESCC) cells and related molecular mechanisms. Methods: ESCC cell line EC9706 cells were randomly divided into untreated (with no transfection), control siRNA (transfected with control siRNA) and HDAC6 siRNA (transfected with HDAC6 small interfering RNA) groups. Effects of HDAC6 siRNA interference on expression of HDAC6 mRNA and protein in EC9706 cells were investigated by semi-quantitative RT-PCR, Western blotting and immunocytochemistry methods. Effects of down-regulation of HDAC6 expression on cell proliferation, cell cycle, and cell migration were studied using a CCK-8 kit, flow cytometry and Boyden chambers, respectively. Changes of mRNA and protein expression levels of cell cycle related factor (p21) and cell migration related factor (E-cadherin) were investigated by semi-quantitative RT-PCR and Western blotting methods. Results: After transfection of HDAC6 siRNA, the expression of HDAC6 mRNA and protein in EC9706 cells was significantly downregulated. In the HDAC6 siRNA group, cell proliferation was markedly inhibited, the percentage of cells in G0/G1 phase evidently increased and the percentage of cells in S phase decreased, and the number of migrating cells significantly and obviously decreased. The mRNA and protein expression levels of p21 and E-cadherin in the HDAC6 siRNA group were significantly higher than those in the untreated group and the control siRNA group, respectively. Conclusions: HDAC6 siRNA can effectively downregulate the expression of HDAC6 mRNA and protein in EC9706 cells. Down-regulation of HDAC6 expression can obviously inhibit cell proliferation, arrest cell cycling in the G0/G1 phase and reduce cell migration. The latter two functions may be closely related with the elevation of mRNA and protein expression of p21 and E-cadherin.

Silencer 및 DNA methylation에 의한 JC virus early promoter의 뇌교세포 특이적인 조절 (Glial Cell-specific Regulation of the JC virus Early Promoter by Silencer and DNA Methylation)

  • 김희선;우문숙
    • 약학회지
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    • 제46권2호
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    • pp.143-148
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    • 2002
  • The human polyomavirus JC virus is the etiologic agent of progressive multifocal leukoencephalopathy (PML). The JC virus early promoter directs cell-specific expression of the viral replication factor large T antigen, thus transcriptional regulation constitutes a major mechanism of glial tropism in PML. Here we found that pentanucleotide sequence immediately upstream of the TATA sequence functions as a cell-specific silencer in the JC virus transcription. In vitro binding studies showed that synthetic oligonucleotides spanning a pentanucleotide sequence, designated "oligo 2", interacts with nuclear proteins from non-glial cells in a cell-specific manner. Furthermore, the sequence preferentially repressed the heterologous thymidine kinase promoter activity in non-glial cells. We also tested whether JC virus transcription is controlled by DNA methylation. Transient transfection of in vitro methylated JC virus promoter abolished transcription in both the glial and non-glial cells. The repression fold was much larger in glial cells than in non-glial cells. Taken together, this finding suggests that glial cell-specific expression of the JC virus is controlled by DNA methylation as well as cell-specific silencers.

siRNA-mediated Silencing of Survivin Inhibits Proliferation and Enhances Etoposide Chemosensitivity in Acute Myeloid Leukemia Cells

  • Karami, Hadi;Baradaran, Behzad;Esfahani, Ali;Estiar, Mehrdad Asghari;Naghavi-Behzad, Mohammad;Sakhinia, Masoud;Sakhinia, Ebrahim
    • Asian Pacific Journal of Cancer Prevention
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    • 제14권12호
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    • pp.7719-7724
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    • 2013
  • Background: Overexpression of survivin, a known inhibitor of apoptosis, is associated with tumor progression and drug resistance in numerous malignancies, including leukemias. The aim of this study was to investigate the effect of a specific survivin small interference RNA (siRNA) on proliferation and the sensitivity of HL-60 acute myeloid leukemia (AML) cells to the chemotherapeutic drug etoposide. Materials and Methods: The cells were transfected with siRNAs using Lipofectamine $^{TM}2000$ transfection reagent. Relative survivin mRNA and protein levels were measured by quantitative real-time PCR and Western blotting, respectively. Trypan blue exclusion assays were performed to monitor tumor cell proliferation after siRNA transfection. The cytotoxic effects of etoposide and survivin siRNA, alone and in combination, on leukemic cells were determined using MTT assay. Apoptosis was assessed by ELISA cell death assay. Results: Survivin siRNA markedly reduced both mRNA and protein expression levels in a time-dependent manner, leading to distinct inhibition of cell proliferation and increased spontaneous apoptosis. Surprisingly, survivin siRNA synergistically increased the cell toxic effects of etoposide. Moreover, survivin down-regulation significantly enhanced its induction of apoptosis. Conclusions: Our study suggests that down-regulation of survivin by siRNA can trigger apoptosis and overcome drug resistance of leukemia cells. Therefore, survivin siRNA may be an effective adjuvant in AML chemotherapy.

Inhibitory Effects of Syk Transfection on Lung Cancer Cell Invasion

  • Peng, Chuan-Liang;Zhang, Ying;Sun, Qi-Feng;Zhao, Yun-Peng;Hao, Ying-Tao;Zhao, Xiao-Gang;Cong, Bo
    • Asian Pacific Journal of Cancer Prevention
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    • 제14권5호
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    • pp.3001-3003
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    • 2013
  • Objective: Spleen tyrosine kinase (Syk) is closely related to tumor invasion and metastasis, and has been shown to have potential inhibitory effects in tumors. In this study, we constructed a eukaryotic expression vector for Syk and analyzed its effects on invasive ability of the A549 non-small cell lung cancer cell line in vitro. Methods: A fragment of Syk was obtained by RT-PCR from human lung cancer cells and cloned into the expression vector pLNCXSyk. After restriction endonuclease digestion, PCR and DNA sequencing confirmation, the recombinant Syk expression plasmid was transfected into A549 human lung cancer cells using lipofectamine protocols. After selection, the cells stably expressed Syk. Detection of Syk expression of the cells by RT-PCR, and invasive ability were examined. Results: The eukaryotic expression plamid pLNCXSyk was constructed and expressed stably in the A549 human lung cancer cells. The RT-PCR results showed that Syk mRNA expression was upregulated significantly (P<0.05). Lower invasion through a basal membrane were apparent after transfection (P<0.05). Conclusions: A eukaryotic expression plasmid to cause Syk expression in lung cancer cells can obviously inhibit their invasive ability in vitro.

Roles of MicroRNA-21 and MicroRNA-29a in Regulating Cell Adhesion Related Genes in Bone Metastasis Secondary to Prostate Cancer

  • Mohamad, Maisarah;Wahab, Norhazlina Abdul;Yunus, Rosna;Murad, Nor AzianAbdul;Zainuddin, Zulkifli Md;Sundaram, Murali;Mokhtar, Norfilza Mohd
    • Asian Pacific Journal of Cancer Prevention
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    • 제17권7호
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    • pp.3437-3445
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    • 2016
  • Background: There is an increasing concern in the role of microRNA (miRNA) in the pathogenesis of bone metastasis (BM) secondary to prostate cancer (CaP). In this exploratory study, we hypothesized that the expression of vinculin (VCL) and chemokine X3C ligand 1 (CX3CL1) might be down-regulated in clinical samples, most likely due to the post-transcriptional modification by microRNAs. Targeted genes would be up-regulated upon transfection of the bone metastatic prostate cancer cell line, PC3, with specific microRNA inhibitors. Materials and Methods: MicroRNA software predicted that miR-21 targets VCL while miR-29a targets CX3CL1. Twenty benign prostatic hyperplasia (BPH) and 16 high grade CaP formalin-fixed paraffin embedded (FFPE) specimens were analysed. From the bone scan results, high grade CaP samples were further classified into CaP with no BM and CaP with BM. Transient transfection with respective microRNA inhibitors was done in both RWPE-1 (normal) and PC3 cell lines. QPCR was performed in all FFPE samples and transfected cell lines to measure VCL and CX3CL1 levels. Results: QPCR confirmed that VCL messenger RNA (mRNA) was significantly down-regulated while CX3CL1 was up-regulated in all FFPE specimens. Transient transfection with microRNA inhibitors in PC3 cells followed by qPCR of the targeted genes showed that VCL mRNA was significantly upregulated while CX3CL1 mRNA was significantly down-regulated compared to the RWPE-1 case. Conclusions: The down-regulation of VCL in FFPE specimens is most likely regulated by miR-21 based on the in vitro evidence but the exact mechanism of how miR-21 can regulate VCL is unclear. Up-regulated in CaP, CX3CL1 was found not regulated by miR-29a. More microRNA screening is required to understand the regulation of this chemokine in CaP with bone metastasis. Understanding miRNA-mRNA interactions may provide additional knowledge for individualized study of cancers.

말초신경재생을 위한 hNGF-$\beta$ recombinant Adenovirus의 제작 및 수종세포주에서 신경성장인자의 발현 (CONSTRUCTION OF HNGF-$\beta$ RECOMBINANT ADENOVIRUS & SCREENING OF ITS EXPRESSION AFTER TRANSFECTION INTO VARIOUS CELL LINES)

  • 고은봉;정헌종;안강민;김윤태;박희정;성미애;김남열;유상배;명훈;황순정;김명진;김성민;장정원;이종호
    • Maxillofacial Plastic and Reconstructive Surgery
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    • 제27권5호
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    • pp.446-456
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    • 2005
  • Nerve growth factor(NGF) has a critical role in peripheral nerve regeneration. The aim of this study is to construct a well-functioning hNGF-$\beta$ recombinat adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with adenovirus mediated hNGF-$\beta$ gene transfection into Schwann cells. First PCR associated cloning of GFP-tagged hNGF-$\beta$ which was ligated into E1/E3 deleted adenoviral vector was performed and tranfected into E. coli to construct hNGF-$\beta$ recombinant adenovirus. After production of recombinat adenovirus in a large scale, its transfection efficiency, expression, and function were evaluated using cell lines or primarily cultured cells of HEK293 cells, Schwann cells, fibroblast(NIH3T3) and myocyte(CRH cells). GFP expression was observed in 90% of infected cells compared to uninfected cells. Total mRNA isolated from hNGF-$\beta$ recombinat adenoviru infected cells showed strong RT-PCR band, however, LacZ recombinant adenovirus infected or uninfected cells did not. NGF quantification by ELISA showed a maximal release of 18.865 +/- 0.31ng/mL at 4th day. PC-12 cells exposed to media with hNGF-$\beta$ recombinant adenovirus infected Schwann cell demonstrated higher levels of differentiation compared with controls. We generated hNGF-$\beta$ recombinant adenovirus and induced over expression of NGF successfully in nonneuronal and neuronal cells. Following these result, it is expected to develop an improved treatment strategy peripheral nerve regeneration using the hNGF-$\beta$ gene transfected cells.

hPDX1 유전자의 삽입에 의한 직접 췌도세포 분화 (Transdifferentiation of α-1,3-Galactosyltransferase Knock Out (GalT KO) Pig Derived Bone Marrow Mesenchymal Stromal Cells (BM-MSCs) into Pancreatic Cells by Transfection of hPDX1)

  • 옥선아;오건봉;황성수;김영임;권대진;임기순
    • 한국수정란이식학회지
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    • 제30권3호
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    • pp.249-255
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    • 2015
  • Diabetes mellitus, the most common metabolic disorder, is divided into two types: type 1 and type 2. The essential treatment of type 1 diabetes, caused by immune-mediated destruction of ${\beta}-cells$, is transplantation of the pancreas; however, this treatment is limited by issues such as the lack of donors for islet transplantation and immune rejection. As an alternative approach, stem cell therapy has been used as a new tool. The present study revealed that bone marrowderived mesenchymal stromal cells (BM-MSCs) could be transdifferentiated into pancreatic cells by the insertion of a key gene for embryonic development of the pancreas, the pancreatic and duodenal homeobox factor 1 (PDX1). To avoid immune rejection associated with xenotransplantation and to develop a new cell-based treatment, BM-MSCs from ${\alpha}$-1,3-galactosyltransferase knockout (GalT KO) pigs were used as the source of the cells. Transfection of the EGFP-hPDX1 gene into GalT KO pig-derived BM-MSCs was performed by electroporation. Cells were evaluated for hPDX1 expression by immunofluorescence and RT-PCR. Transdifferentiation into pancreatic cells was confirmed by morphological transformation, immunofluorescence, and endogenous pPDX1 gene expression. At 3~4 weeks after transduction, cell morphology changed from spindle-like shape to round shape, similar to that observed in cuboidal epithelium expressing EGFP. Results of RT-PCR confirmed the expression of both exogenous hPDX1 and endogenous pPDX1. Therefore, GalT KO pig-derived BM-MSCs transdifferentiated into pancreatic cells by transfection of hPDX1. The present results are indicative of the therapeutic potential of PDX1-expressing GalT KO pig-derived BM-MSCs in ${\beta}-cell$ replacement. This potential needs to be explored further by using in vivo studies to confirm these findings.

Down-regulation of SENP1 Expression Increases Apoptosis of Burkitt Lymphoma Cells

  • Huang, Bin-Bin;Gao, Qing-Mei;Liang, Wei;Xiu, Bing;Zhang, Wen-Jun;Liang, Ai-Bin
    • Asian Pacific Journal of Cancer Prevention
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    • 제13권5호
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    • pp.2045-2049
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    • 2012
  • Objective: To investigate the effect of down-regulation of Sentrin/SUMO-specific protease 1 (SENP1) expression on the apoptosis of human Burkitt lymphoma cells (Daudi cells) and potential mechanisms. Methods: Short hairpin RNA (shRNA) targeting SENP1 was designed and synthesized and then cloned into a lentiviral vector. A lentiviral packaging plasmid was used to transfect Daudi cells (sh-SENP1-Daudi group). Daudi cells without transfection (Daudi group) and Daudi cells transfected with blank plasmid (sh-NC-Daudi group) served as control groups. Flow cytometry was performed to screen GFP positive cells and semiquantitative PCR and Western blot assays were employed to detect the inference efficiency. The morphology of cells was observed under a microscope before and after transfection. Fluorescence quantitative PCR and Western blot assays were conducted to measure the mRNA and protein expression of apoptosis related molecules (caspase-3, 8 and 9). After treatment with $COCl_2$ for 24 h, the mRNA and protein expression of hypoxia inducible factor -$1{\alpha}$ (HIF-$1{\alpha}$) was determined. Results: Sequencing showed the expression vectors of shRNA targeting SENP1 to be successfully constructed. Following screening of GFP positive cells by FCM, semiqualitative PCR showed the interference efficiency was $79.2{\pm}0.026%$. At 48 h after transfection, the Daudi cells became shrunken, had irregular edges and presented apoptotic bodies. Western blot assay revealed increase in expression of caspase-3, 8 and 9 with prolongation of transfection (P<0.05). Following hypoxia treatment, mRNA expression of HIF-$1{\alpha}$ remained unchanged in three groups (P>0.05) but the protein expression of HIF-$1{\alpha}$ markedly increased (P<0.05). However, in the sh-SENP1-Daudi group, the protein expression of HIF-$1{\alpha}$ remained unchanged Conclusion: SENP1-shRNA can efficiently inhibit SENP1 expression in Daudi cells. SENP1 inhibition may promote cell apoptosis. These findings suggest that SENP1 may serve as an important target in the gene therapy of Burkitts lymphoma.

연 및 neferine의 암세포 항 성장 및 세포사멸 활성 (Anti-proliferative and Pro-apoptotic Activities of Nelumbo nucifera and Neferine in Human Colorectal HCT116 Cells)

  • 김용현;이은주;정정욱;손호용;박종이;김종식
    • 생명과학회지
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    • 제29권10호
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    • pp.1080-1085
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    • 2019
  • 본 연구에서는 연(Nelumbo nucifera)의 잎(leaf, NL), 자육(seed, NS), 자방(seedpod, NSP)의 에탄올 추출물을 제조하고 이들의 암세포 항 성장 활성과 세포사멸 활성을 연구하였다. 추출물 NL, NS, NSP는 농도의존적으로 세포 생존율을 감소시켰을 뿐 만 아니라 항암 유전자인 NAG-1 유전자와 단백질의 발현을 증가시켰다. 또한, NL은 NAG-1 단백질의 발현과 PARP cleavage를 시간 의존적으로 증가시켰다. PARP cleavage는 NAG-1 siRNA의 transfection에 의해 부분적으로 감소하였으며, 이러한 결과는 NAG-1이 NL에 의해 유도되는 apoptosis에 기여하는 유전자 중의 하나라는 것을 나타낸다. 그리고, 연근 유래의 순수물질인 neferine도 농도의존적으로 HCT116 세포 생존율을 감소시켰으며, NAG-1 단백질의 발현과 PARP cleavage를 농도의존적, 시간의존적으로 증가시켰다. Neferine에 의해 유도된 PARP cleavage는 NAG-1 siRNA의 transfection에 의해 부분적으로 회복되는 것을 확인하였으며, 이러한 결과는 NAG-1 단백질이 neferine에 의해 유도되는 apoptosis에도 기여하는 유전자 중의 하나라는 것을 시사한다. 종합적으로 본 연구 결과는 연근 부위별 추출물과 연근유래 순수물질인 neferine에 의한 암세포 항 성장 활성과 세포사멸 활성의 분자 생물학적 기전을 이해하는데 도움을 줄 것으로 생각된다.

A549 폐암세포주의 방사선-유도성 세포사에서 NF-${\kappa}B$ 활성화 및 cIAP 발현 (NF-${\kappa}B$ Activation and cIAP Expression in Radiation-induced Cell Death of A549 Lung Cancer Cells)

  • 이계영;곽상준
    • Tuberculosis and Respiratory Diseases
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    • 제55권5호
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    • pp.488-498
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    • 2003
  • 연구배경 : 세포에 방사선을 조사하면 세포사멸과 함께 AP-l, NF-${\kappa}B$와 같은 여러 전사인자가 활성화 되는 것으로 알려져 있다. 이중 염증과 면역반응의 중요한 전사인자인 NF-${\kappa}B$는 항아포프토시스의 기능이 있으며 NF-${\kappa}B$ 활성화를 차단함으로써 TNF-${\alpha}$나 daunorubicine 등에 의 한 항암효과를 상승시킬 수 있음이 밝혀져 있다. NF-${\kappa}B$의 항아포프토시스 기전은 NF-${\kappa}B$ 의존성 단백질인 cIAPI, 2의 전사발현 유도에 의한 것으로 cIAPl, 2는 caspase 3, 7, pro-caspase-9의 활성을 차단함으로써 아포프토시스를 억제하는 것으로 알려져 있다. 이에 저자들은 방사선 유도성 세포사멸에 비교적 내성을 보이는 A549 세포주에서 방사선에 의한 NF-${\kappa}B$ 활성화와 그에 따른 cIAP 발현유도를 조사하고 NF-${\kappa}B$ 활성화를 차단하여 방사선 유도성 세포사멸의 감작효과를 확인하기 위하여 본 연구를 시행하였다. 방 법 : 세포주는 A549 폐암세포주를 이용하였고, 방사선 조사는 Varian사의 Clinac 1800C 선형가속기를 이용하였으며 조사량은 10GY를 사용하였다. 세포독성 검사는 MTT Assay를 이용하였고, NF-${\kappa}B$ 활성화 검사는 luciferase reporter gene assay, electromobility shift assay, $I{\kappa}B-{\alpha}$ degradation에 대한 westem blot을 이용하였다. NF-${\kappa}B$활성을 차단하기 위하여 proteosome inhibitor인 MGI32와 $I{\kappa}B{\alpha}$-superrepressor plasmid를 transfection한 안정적 세포주 A549-$I{\kappa}B{\alpha}$-superrepressor를 이용하였다. cIAP의 발현은 RT-PCR을 이용하였고, cIAP2 promoter 활성은 NF-${\kappa}B$ site를 포함한 cIAP2 유전자 5' f1anking region(1.4kb)을 pGL2-Basic luciferase vector에 cloning 한 construct를 사용하여 transfection 후 luciferase assay를 시행하였다. 결 과 : A549 cell에서 10Gy 방사선 조사에 의한 세포독성은 24hr, 48hr에 각각 $10.82{\pm}.3%$, $17.7{\pm}6.4%$로 비교적 내성이 있음을 확인하였다. 방사선에 의한 NF-${\kappa}B$의 활성은 $I{\kappa}B-{\alpha}$ 분해에 대한 western blot과 EMSA로 확인하였으며 luciferase assay에서도 약 1.6 배 정도의 NF-${\kappa}B$ 활성화가 있었다. NF-${\kappa}B$활성을 차단하기 위해 사용한 MG132는 방사선 유도성 세포사멸에 영향을 주지 않았으며 A549-$I{\kappa}B{\alpha}$-superrepressor 세포주에서도 세포사멸의 감작효과는 없었다. 또한 RT-PCR 결과 방사선에 의한 cIAP1,2 mRNA 발현유도는 관찰되지 않았고 cIAP2 promoter luciferase assay에서도 cIAP2 전사활성 유도는 없었다. 결 론 : A549 폐암세포주에서 방사선에 의해 NF-${\kappa}B$의 활성화는 확인하였으나 활성화 정도가 미약하였고 NF-${\kappa}B$ 의존성 항아포프토시스 유전자인 cIAP가 방사선에 의해 발현유도 되지 않았으며, NF-${\kappa}B$ 활성을 차단함에도 세포독성에 감작효과가 없었으므로 A549 폐암세포주에서 방사선 유도성 세포사멸에 내성을 보이는 기전에는 NF-${\kappa}B$의 역할이 미미하리라고 사료된다.