[보고서]방광암에 대한 cisplatin 내성 기전으로서의 c-FLIP의 발현에 대한 연구

변석수

방광암에 대한 cisplatin 내성 기전으로서의 c-FLIP의 발현에 대한 연구
Study about c-FLIP expression as a cisplatin resistance mechanism in bladder cancer cells 원문보기

보고서 정보
주관연구기관	서울대학교 의과대학 Seoul National University
연구책임자	변석수
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2012-06
과제시작연도	2011
주관부처	교육과학기술부
사업 관리 기관	한국연구재단
등록번호	TRKO201300018542
과제고유번호	1345157197
사업명	기본연구지원사업
DB 구축일자	2013-09-21

초록 ▼

본 연구는 방광암에서 cisplatin 항암제 내성 기전으로서의 c-FLIP 및 관련경로의 역할을 규명하고 이를 기초로 방광암에 대한 c-FLIP 기반의 표적치료법의 개발 및 cisplatin 내성 극복에 필요한 기초자료를 마련하는 목적으로 계획되었다. 나아가 방광암세포주를 대상으로 한 관련 인자 발현을 유전자 및 단백 수준에서 다시 한번 분석함으로써 c-FLIP 기반 표적치료의 임상적 활용에 필요한 예후 예측인자를 발굴하기 위함이었다.
8개의 인체방광암 세포주 (T24, T24R2, J82, 253J, HTB5, HTB9, UMUC14, SW1710)에 대해 세포증식 (CCK-8), 클론원성 (clonogenic assay) 및 유세포 (flowcytometry) 분석을 시행하여 각각의 세포의 cisplatin에 대한 감수성을 비교 분석하였고, 각 세포의 cisplatin에 대한 감수성 차이를 c-FLIP 및 관련 인자의 발현의 차원에서 분석함과 동시에 anti-c-FLIP siRNA를 이용하여 c-FLIP 활성 변화에 따른 각 세포의 cisplatin에 대한 감수성 변화를 분석하였다. T24와 달리 cisplatin 내성 방광암 세포주인 T24R2에서 cisplatin 처치시에 c-FLIP이 높은 농도에서 발현되고, caspase 발현 및 Akt survival pathway가 억제됨을 확인하였다. siRNA targeting c-FLIP을 이용한 c-FLIP knockdown study 및 western blot 등의 연구를 통해서 c-FLIP이 방광암세포주에서 cisplatin 내성 획득 기전에서 permissive role을 확인하였다. Gene microarray study를 통해 microarray 상에서 확인된 cisplatin 내성 관련한 upregulated (19개) gene들을 찾아내었다. Upregulated gene들로는 SKP1, MCM7, LEF1, FN1, CUL2, PRKAR2A, PRKAR2B, CYCS, Bcl2, DFFB, CASP6, CDK6, CCNE1, STEAP3, MCM7, ORC5, ANAPC7, CDC7, CDC27이 관찰되었다. 이들 유전자들의 cellular component, molecular function, biological process, 그리고 각각의 알려진 gene ontology database의 경로를 기초로 방광암과 관련된 cisplatin 내성 유의 유전자들의 Fold change (> 2.0)와 통계학적 p-value (<0.05)를 기준으로 up regulatd gene들을 선별하여 이들의 발현 양상을 RT-PCR을 통해 확인하였다. Real time PCR 확인결과 대조군보다 실험군에서 MCM7 (F.C>4.7), ORC5 (F.C>2.7), CDC27 (F.C>3.9), CDK6 (F.C>2.5) 유전자들의 발현이 두드러지게 상승되어 나타났다. 그리고 다른 유전자들도 대조군보다 상승되는 양상을 보였다. 덧붙여 cisplatin내성 관련 경로에 대해 endoplasmic reticulum내의 protein processing과정을 분석된 경로를 기초로 도식화 하여 제시하였다.
이를 바탕으로 향후 방광암에서의 cisplatin 내성 기전으로서의 c-FLIP 및 관련 경로의 역할을 규명하고 내성 극복에 이용될 수 있는 c-FLIP 기반 표적치료의 개발에 필요한 기초 자료를 마련할 수 있을 것으로 기대된다 또한 새로운 치료법의 임상적 응용 가능성 및 활용의 극대화에 필요한 예후 예측 인자를 발굴할 수 있어 전이성 및 항암제 내성 방광암에 대한 치료 분야의 활성화에 도움을 줄 것으로 기대된다.

Abstract ▼

The ultimate goal of this study is to evaluate the cisplatin-resistance mechanism relating to the c-FLIP. Our study provides preliminary data to help develop prognostic factors and another further targeting therapy based on the c-FLIP in cisplatin resistant bladder cancer.
Cisplatin sensitivity of human bladder cancer cell lines (T24, T24R2, J82, 253J, HTB5, HTB9, UMUC14 and SW1710), assessed by the CCK-8 assay, clonogenic assay and flowcytometry. T24R2 cells were transfected with siRNA targeting c-FLIP (Si-c-FLIP). c-FLIP expression in Si-c-FLIP transfected cells at the mRNA and protein levels was evaluated by cell proliferation assay, RT-PCR and Western blot analysis. Si-c-FLIP transfected T24R2 cells demonstrated significantly lower survival compared with non-transfected T24R2 cells. c-FLIP was shown to play a permissive role in cisplatin-resistance mechanism in bladder cancer cells and c-FLIP modulation might partially resensitize cisplatin resistant bladder cancer cell line.
Through the analysis of microarray, 19 upregulated genes were identified candidates for cisplatin-resistant mechanism. The upregulated genes were SKP1, MCM7, LEF1, FN1, CUL2, PRKAR2A, PRKAR2B, CYCS, Bcl2, DFFB, CASP6, CDK6, CCNE1, STEAP3, MCM7, ORC5, ANAPC7, CDC7, and CDC27. After the 19 genes were classified into their cellular components, molecular functions, biological processes, and gene ontology databases, they were re-evaluated with the real time PCR to confirm their upregulated states and associations with the cisplatin resistance according to the fold change > 2.0 and statistical p-value< 0.05. The result in real time PCR showed that MCM7 (F.C> 4.7), ORC5 (F.C> 2.7), CDC27 (F.C> 3.9), and CDK6 (F.C> 2.5) genes distinctively upregulated in the experimental group(T24R2) cmparing to the control. And the rest of genes have a tendency to be upregulated, as well. In addition, a diagram of protein processing associated with cisplatin resistance in endoplasmic reticulum was proposed.
The results of this study, therefore, suggest another footstep to understand the cisplatin-resistance mechanism relating to the c-FLIP, and to develop another further targeting therapy based on the c-FLIP in cisplatin resitant bladder cancer. Furthermore, this study would help to develop a new treating plan and to investigate further prognostically predictive factors in order to maximize its usefulness of new plan in clinical fields, especially for the metastatic and cisplatin resistant bladder cancer.

목차 Contents

일반연구자지원사업 최종(결과)보고서 ... 1
목차 ... 3
Ⅰ. 연구결과 요약문 ... 4
Ⅱ. 연구내용 및 결과 ... 6
1. 연구과제의 개요 ... 6
2. 국내외 기술개발 현황 ... 7
3. 연구수행 내용 및 결과 ... 15
4. 목표 달성도 및 관련 분야에의 기여도 ... 36
5. 연구결과의 활용계획 ... 37
6. 연구과정에서 수집한 해외과학기술정보 ... 38
참고문헌 ... 38
Ⅲ. 연구성과 ... 41

표/그림 (25)

표 (A) T24 and T24R2 cells were treated with increasing dose of cisplatin (0.039 - 40.0uM) for 72 hours and cell viability was accessed by CCK-8 assay. T24R2 shows resistance to cisplatin up to 2ug/ml of cisplatin. (B) Cisplatin resistance of T24R2 was assessed in comparison of other human TCCs (HTB5 - grade 4 / HTB9 - grade 2 / J82 - grade 3 / SW1710 - grade 3 / UMUC14 - grade 4 / 253J - grade 4) by CCK-8 assay. T24R2 shows significantly higher resistance to cisplatin compared to other TCCs with various stage and differentiation.
표 T24 and T24R2 cells were treated with 0.5ug/ml of cisplatin for 48h and expression of c-FLIP was assessed by Western blotting. Compared to T24, T24R2 cell has higher expression of c-FLIP even before cisplatin treatment. Treatement of T24 with 5ug/ml of cisplatin completely abrogated c-FLIP expression while there was nearly no changes in c-FLIP expression in T24R2 after cisplatin treatment.
표 T24 and T24R2 cells were treated with increasing dose of cisplatin for 48h and expression of c-FLIP, p-Akt, t-Akt, caspase 3, 8, and 9 were analyzed by Western blot. Cisplatin treatment abrogated c-FLIP expression in both cell lines in a dose-dependent pattern which was accompanied by serial increase of caspase-3, -8, -9, p-Akt, and t-Akt. Compared to T24, T24R2 required higher dose of cisplatin to suppress c-FLIP expression and to induce caspases and Akt expression.
표 Five bladder cancer cell lines (HTB5, HTB9, 253J, SW1710, UMUC14) were treated with increasing dose of cisplatin for 48h and expression of c-FLIP was determined by Western blotting. In all cell lines tested, cisplatin treatment suppressed c-FLIP expression in a dos-dependent fashion
표 T24 and T24R2 cells were treated with increasing dose of TRAIL for 24h and cell viability was assessed by CCK-8 assay. TRAIL suppressed the proliferation of T24 in a dose-dependent manner while there was virtually no suppression of T24R2 proliferation by TRAILtreatment.
표 T24, T24R2, and MCF7 cells were treated with increasing dose of etoposide for 48h and cell viability was assessed by CCK-8 assay. T24R2 has nearly the same sensitivity to etoposide with MCF7 while T24 has higher sensitivity to etoposide compared with T24R2 and also MCF7.
표 (A) T24R2 was treated with increasing dose of cisplatin alone or in combination with NVP-BEZ235 for 48h and cell viablity was assessed with CCK-8 assay. (B) Synergism between two drugs was determined based on combination index. Treatment of T24R2 cells with cisplatin or NVP-BEZ235 alone didn' t cause any suppression of cell growth while combined treatment showed significant synergistic anti-tumor effect.
표 T24 and T24R2 cells were treated with BEZ235 and cisplatin alone or in combination for 72hr and c-FLIP, caspase 3, and 8 expressions were analayzed by Western blot. Treatment of T24 cells with cisplatin caused down-regulated expression of c-FLIP which was accompanied by increased expression of caspase 3 and 8. Compared to this treatemtn of T24R2 cells with cisplatin or NVP-BEZ235 alone didn' t result in any significant changes of c-FLIP or caspase 3 and 8 expression. When T24R2 cells were treated with cisplatin and NVP-BEZ235 together, there was significant decrease in c-FLIP expression and increased expression of caspased 3 and 8.
표 T24R2 cells was transfected with c-FLIP siRNA or control scrambled siRNA. 48hr after transfection, suppression of c-FLIP expression was analyzed by RT-PCR(A) and western blot(B).
표 Synergistic anti-tumor effect of gemcitabine and TSA in human bladder cancer cells. HTB5 (A ) and T24 (C ) cells were treated with an increasing dose of gemcitabine (0.005– 50.0 μM) and / or TSA (0.05– 1.0 μM) for 48 hours, and changes in cell proliferation was determined by the CCK-8 assay. The result was compared with that of HTB5 and T24 cells treated with the same dose of gemcitabine. Each daa point represents the mead ± SD of three independent experinents. Synergism between gemcitabine and TSA in HTB5 (B ) and T24 (D ) was determined by the combination index (CI), in which CI 1 (above dotted line) indicate synergistic, additive, and antagonistic effects between two drugs, respectively. (E ) Representative pictures of colonies of HTB5 and T24 cells formed after gemcitabine (GEM), TSA singe or combination (G+T) treatment.
$fa-CI plot. HTB5 and T24 cells were treated with increasing concentrations of gemcitabine or TSA either alone or in combination at a fixed ratio of 10:1 for 48 hours and fa – CI plot was generated based on a CI (combination index) of gemcitabine and TSA co-treatment at the corresponding fa which denotes fraction affected (e.g., fa 0.5 is equal to a 50% reduction in cell proliferation. CI > 1, CI = 1, and CI < 1 indicate antagonism, additivity, and synergism, respectively).$ 표 fa-CI plot. HTB5 and T24 cells were treated with increasing concentrations of gemcitabine or TSA either alone or in combination at a fixed ratio of 10:1 for 48 hours and fa – CI plot was generated based on a CI (combination index) of gemcitabine and TSA co-treatment at the corresponding fa which denotes fraction affected (e.g., fa 0.5 is equal to a 50% reduction in cell proliferation. CI > 1, CI = 1, and CI < 1 indicate antagonism, additivity, and synergism, respectively).
표 Representative flow cytometric DNA content histogram of HTB5 cells exposed to escalating doses of gemcitabine (A ), TSA (B ), and both drugs concomitantly (C ) for 48 hours and the quantitative analysis of the results (D ). Each data point represents the mean ± SD of duplicate experiments (* indicates p < 0.05; control vs. each treatment / CTR and GEM denote control and gemcitabine, respectively). After exposure to a suboptimal dose of gemcitabine (5.0 μM) and/or TSA (0.2 μM)for 48 hours, the chromatin condensation (arrow) in HTB5 cells, a characteristic feature of apoptosis, was examined after Hoechst 33342 staining (E , x400).
표 Western blot analysis of protein expression in HTB5 cells. HTB5 cells were exposed to gemcitabine (5.0 μM) and/or TSA (0.2 μM) for 48 hours and cell cycle (p21WAF1/CIP1, cyclin A, cyclin B1, cyclin D1, pCDC2, CDC2, pCDC25C, CDC25C, pRb), apoptosis (caspase-3, caspase-8, caspase-9, PARP, Bcl-2, Bad, Bax, cytochrome c), NF-κB (NF-κB, p-IkBα, IkBα, p-IKKα, IKKa, cIAP1, cIAP2, XIAP, cFLIP), survival (p-PI3K, PI3K, p-Akt, Akt, p-mTOR, mTOR, PTEN)-related protein expression was analyzed by Western blot analysis
표 T24 and T24R2 cells were exposed to escalating dose of cisplatin, carboplatin or orxaliplatin for 24 - 72 hours and cell viability was assessed by CCK-8 assay. While cisplatin-resistant T24R2 cells showed high resistance to carboplatin, it didn' t showed any discernible resistance to oxaloplatin.
표 IC50 and relative resistance of anti-cancer drugs
표 siRNA-mediated knockdown of c-FLIP in T24R2 cells. A) T24R2 cells were transfected with a control scrambled siRNA (Si-SCR) or an siRNA targeting c-FLIP (Si-c-FLIP). c-FLIP expression in indicated transfected cells at the mRNA and protein levels was determined by RT-PCR (left panel) and Western blot analysis (right panel). β-Actin and GAPDH were used as controls. B) Sensitivity of transfectant T24R2 cells to cisplatin. T24R2 cells transfected with scrambled siRNA or c-FLIP siRNA were exposed to increasing doses of cisplatin (1.25-20.0μg/ml) for 72 hours and viability of each cell was assessed by CCK-8 assay and compared with those of T24 and non-transfected T24R2 cells. Each data point represents for mean ± SD of at least three independent experiments and asterisks indicate significantly lower survival of c-FLIP transfected T24R2 cells compared with non-transfected T24R2 cells (p <0.05).
표 T24_CTR1 & T24R2 데이터의 히스토그램
표 마이크로어레이 데이터에서 검출된 유전자 발현 신호값의 box plot
표 Normalization 한 마이크로어레이 데이터 들간의 correlation matrix plot
표 MA Plot: T24_ vs T24_R2
표 분석 디자인에 따른 유의 유전자 및 GO, pathway 개수
표 Hierachy between T24-ctr and T24-cis
표 두 개의 군집 내 유전자의 발현 패턴 (T24 vs T24-R2 분석)
표 해당 GO category 에 통계적으로 의미 갖는 up-regulation 된 GO term 개수 및 Probe 개수
표 Pathway: Protein processing in endoplasmic reticulum

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연합인증

방광암에 대한 cisplatin 내성 기전으로서의 c-FLIP의 발현에 대한 연구
Study about c-FLIP expression as a cisplatin resistance mechanism in bladder cancer cells 원문보기