폐암세포주에서 IGF-1R 억제를이용한 TRAIL 및 gefitinib 에대한감수성증가를위한연구 서울대학교의과대학내과, 의학연구원폐연구소, 분당서울대학교병원내과, 폐센터이윤진, 박미영, 강영애, 권성연, 윤호일, 이재호, 이춘택 Enhancement of Sensitivity of Human Lung Cancer Cell Line to TRAIL and Gefitinib by IGF-1R Blockade Yoon-Jin Lee, M.S., Mi-Young Park, M.S., Young Ae Kang, M.D., Sung-Youn Kwon, M.D., Ho-Il Yoon, M.D., Jae-Ho Lee, M.D., Choon-Taek Lee, M.D. Department of Internal Medicine, Lung Institute of Medical Research Center, Seoul National University College of Medicine, Seoul, Korea, Department of Medicine, Respiratory Center, Seoul National University Bundang Hospital, Seongnam, Korea Background: TRAIL is a cytokine that selectively induces apoptosis in various cancer cell lines. Gefitinib is new targeted drug applied in lung cancer that selectively inhibits EGFR tyrosine kinase. However, lung cancers have shown an initial or acquired resistance to these drugs. This study examined the effect of IGF-1R and its blockade on enhancing the sensitivity of lung cancer cell lines to TRAIL and gefitinib. Methods: Two lung cancer cell lines were used in this study. NCI H46 is very sensitive to TRAIL and gefitinib. On the other hand, A549 shows moderate resistance to TRAIL and gefitinib. The IGF-1R blockade was performed using adenoviruses expressing the dominant negative IGF-1R and shrna to IGF-1R and AG124 (IGF-1R tyrosine kinase inhibitor). Results: The adenovirus expressing dominant negative IGF-1R(95st) induced the increased expression of defective IGF-1R on the lung cancer cell surface, and the adenovirus-shigf-1r effectively decreased the level of IGF-1R expression on cell surface. The genetic blockade of IGF-1R by the adenovirus-dnigf-1r and AG124 increased the sensitivity of A549 cells to TRAIL. The reduction of IGF-1R by transduction with ad-shigf-1r also increased the sensitivity of the A549 cells to gefitinib. Conclusion: The blockade of IGF-1R through various mechanisms increased the sensitivity of the lung cancer cell line that was resistant to TRAIL and gefitinib. However, further studies using other cell lines showing acquired resistance as well as in vivo animal experiments will be needed. (Tuberc Respir Dis 27; 63: 42-51) Key Words: TRAIL, Gefitinib, IGF-1R, Lung cancer. 서 Tumor necrosis factor related apoptosis inducing ligand( 이하 TRAIL) 은 TNF family에속하는사이토카인으로정상세포에는영향을주지않고암세포를포함한형질변형세포에만 apoptosis를유발한다고알 론 이연구는 24 년서울대학교신임교수정착연구비 (8-24-62) 보조로이루어졌음. Address for correspondence: Choon-Taek Lee, M.D. Department of Medicine, Respiratory Center, Seoul National University Bundang Hospital, 3 Gumi-dong, Bandang-gu, Seongnam 463-77, Korea Phone: 82-31-787-72, Fax: 82-31-787-452 E-mail: ctlee@snu.ac.kr Received: Apr. 2. 27 Accepted: Jul. 9. 27 려져있다 1. 이러한특성으로 TRAIL을암의치료에사용하려는연구가많이있어왔다. 그러나 TRAIL을임상에사용하기에는많은양이필요하고또한주사후 5시간이내에대부분이배설되는단점이있다. 이외에 TRAIL에대한문제점으로많은암세포가 TRAIL에대한저항성을보인다는점이다. TRAIL의작용기전은세포막에있는 death receptor 4, 5( 이하 DR4, DR5) 와결합하여일어난다. 현재까지알려진 TRAIL에대한저항성의기전으로는여러기전이제시되고있다 2. Decoy receptor 의발현 3, DR의변이또는발현감소 4, caspase 의소실 5, 및억제제 (cflip 등 ) 6 등이제시되고있다. 본연구자는단백질형태로투여하는 TRAIL(soluble form: strail) 대신에 TRAIL (full length) 을발현하는유전자재조합아데노바이러스 (ad-trail) 를이용하여 strail 에저항성을보이 42
Tuberculosis and Respiratory Diseases Vol. 63. No.1, Jul. 27 는암세포주 (A549, SKOV3, HT-29, LNCap) 에 apoptosis를유발할수있음을증명하여 TRAIL에세포내에서발현될때는저항성을극복할수있음을보고한바있다 7. Gefitinib(Iressa ) 은 epidermal growth factor I receptor(egfr) 의 tyrosine kinase를억제하는물질로최근비소세포폐암환자에새로운표적치료제로많이사용되고있다 8. 초기의기대와는달리대규모임상연구에서폐암환자의생존기간을유의하게증가시키는데는실패하였으나 9 일부의환자군에서획기적인효과를보이고있다. 즉비흡연자, 선암 ( 특히기관지폐포세포암 ), 여성및아시아인종에서는높은치료효과및생존기간의연장을보이고있다 1. 그러나 gefitinib 에초기부터저항성을보이거나혹은초기에는반응을보이다가저항성을보이는경우가많아지고있다. 많은연구에의해 gefitinib에대한폐암세포의감수성이특정 exon의변이에기인한다는사실이알려졌다 11,12. 그러나저항성발현의기전은아직확립되고있지않다. 새로운 EGFR의변이가저항성을유발한다는연구가있으나전부를설명하지는못하고있다. TRAIL 및 gefitinib 은모두폐암에사용될수있는새로운표적치료제로세포막의수용체에작용하여 apoptosis를유발한다는유사점이있다. 최근 TRAIL, gefitinib 등의항암물질에대한저항성의기전으로 antiapoptotic growth factor receptor 들의역할이주목을받고있다. 즉암세포의성장및형질유지에중요역할을하는성장인자및그수용체로 epidermal growth factor receptor(egfr), HER2/ neu 및 insulin-like growth factor-1 receptor(igf- 1R) 등이항암제, 방사선치료및여러항암 cytokine 의효과를억제하며이를억제하면그효과를증강시킬수있다. IGF-1 및그수용체인 IGF-1R 는대표적인성장인자의하나로암세포의성장및악성형질변화에중요한역할을하며세포에대한 apoptosis를유도하는자극 ( 항암치료, 방사선치료및그외에항암 cytokine) 에대한세포내의 phosphatidylinositol 3-kinase (PI3K)/Akt kinase의활성화, BAD의인산화및 cflip 을유도등을통해 antiapoptosis를유도한다고알려져있다 13-15. 갑상선암세포를 IGF-1 로자극하면 TRAIL에대한감수성이저하되고이는 DR의발현에대한영향없이 apoptosis의억제물질인 cflip, ciap2 등을증가시킴에기인한다는보고가있다 16. TRAIL은다발성골수종세포에서 NFκB의활성화와 IGF-1/Akt pathway 의활성화를이용하여 antiapoptotic protein 을상승시켜저항성을유도한다는보고가있다 17. 또한 IGF-1R 의 signaling을억제하면유방암, 전립선암에서 gefitinib 에대한 de novo 또는획득내성을억제한다는연구 18,19 들이보고되고있다. 또한간암세포에서도 gefitinib 에대한내성에서도 IGF-1/Akt pathway 가관여함이보고되었다 2. 본연구에서는 TRAIL에대한중등도의저항성을보이는폐암세포주 (A549) 를대상으로불완전형태의 IGF-1R 을발현하는 adenovirus-igf-1r/95과 soluble 형태의 482 aminoacid 크기의 IGF-1R 을세포밖으로분비하여 IGF-1과결합하여 IGF-1 pathway 를억제하는 adenovirus-igf-1r/482 21 및 IGF-1R 의 tyrosine kinase의억제제로알려진 AG124를이용하여 IGF-1 pathway 를차단한후 TRAIL에대한감수성의변화를관찰하고그기전을연구하였다. 또한 gefitinib 에중등도의저항성을보이는 A549를대상으로 IGF-1R 을 RNA 간섭 (interference) 22 을이용하여 IGF-1R의발현을억제하는 adenovirus-short hairpin(sh)igf-1r을이용하여 IGF-1 pathway 를차단후 gefitinib 에대한폐암세포주의감수성의변화를관찰하였다 23. 연구재료및방법 1. 폐암세포주 TRAIL, gefitinib에대해중등도의저항성을보이는사람기관지폐포세포암주 (A549) 및 TRAIL에감수성이높은사람폐대세포폐암주 (NCI H46) 을대상으로하였다. 위의세포주는한국세포주은행 ( 서울 ) 를통해구입하였으며 1% 우태혈청을보충한 RPMI-164 43
YJ Lee et al: Enhancement of sensitivity to TRAIL and gefitinib by IGF-1R blockade 배양액에서배양하였다. 2. 연구재료 1) TRAIL (soluble TRAIL:Apo2L; human) 은전 TRAIL의 114-281번 aminoacid를가진유전자재조합을통해생산된 soluble form의 TRAIL로 Biomol (Plymouth, PA, USA) 에서구입하였다. Gefitinib 은한국 Astra Zeneka사를통해제공받았다. 2) Adenovirus-IGF-1R/95 및 adenovirus-igf- 1R/482은본연구실에서제작하였다. 세포막에발현은할수있으나세포내 domain 의 tyrosine kinase domain 이없는불완전한형태의 IGF-1R(95 amino acid 크기 ) 를발현하는 adenovirus-igf-1r/95은불완전한형태의 IGF-1R을세포막에발현하여 IGF-1 과결합하여정상적인 IGF-1 의자극전달을차단한다. Adenovirus-IGF-1R/482은 soluble 형태의 482 아미노산크기의 IGF-1R(soluble IGF-1R) 을세포밖으로분비하여 IGF-1 과결합하여 IGF-1 pathway를억제한다. 즉위두종류의 recombinant adenovirus는 dominant negative 기전으로 IGF-1 pathway를억제하게된다 21. 3) Adenovirus-shIGF-1R은 IGF-1R을 RNA 간섭현상을이용하여전사단계에서 IGF-1R 의발현을억제한다. 즉 IGF-1R mrna 염기서열 (NM_875) 에서 Genscript corporation(scotch Plains, NJ, USA) 의 target finder program 및 pblast filtering 시행하여 1개의가능성이있는 sirna 염기서열을결정하였다. 이에대한 dsrna를제작한다음 (Genscript), 폐암세포주 (NCI H358) 에 FuGENE6를이용하여 transfection 한후 48시간후 IGF-1R 에대한 Western blot을시행하여가장억제가강한 sirna 을결정하였다. 위결과로부터 3425번부터의 19bp(ACG CCAATAAGTTCGTCCA) 을 sirna 염기서열로선정하였다. Adenovirus-shIGF-1R을제작하기위해 3425번부터 19bp의 DNA sequence(sense) 및 loop sequence와이어서 19bp의 antisense을가진 72bp의 oligonucleotide를제작하고이에대해상보적인 oligonucleotide를제작하였다 (Genscript). 위의 2 strand 의 64-bp DNA를 ligation 한후 H1 RNA promotor가진 adenoviral shuttle vector(pshuttle with H1 RNA promotor) 에 cloning한후 BD Adeno-X expression system(bd science) 을이용하여 E-coli에 transformation 한후 recombination이일어난 ad-shigf-1r 의 DNA를 293cell에 transfection 한후 adenovirus를생산하였다. Ad-shIGF-1R은세포내에감염된후 IGF-1R 에대한 sense-loop-antisense로이루어진 72bp의 RNA를전사한다. 이 RNA는 sense와 antisense 간의결합이일어나고한쪽에 loop 염기서열을가진 short hairpin 형태의 ds RNA를형성하고이는 DICER 에의해 loop부위가제거되어 ds sirna 가되어 RNA interference 현상을보이게된다 23,24. 4) AG124(Alexis Biochemicals, San Diego, CA, USA) 은 tyrphostin 유도체로 IGF-1R 와결합하여 autophosphorylation을억제하는 IGF-1R 의 tyrosine kinase inhibitor(tki) 물질이다 25. 3. Adenovirus-IGF-1R/95의감염에의한세포막에 IGF-1R 의발현의변화확인 Dominant negative 기전에의한 IGF-1 pathway 의억제를증명하기위해두종류의 adenovirus-igf- 1R(95, 482) 를 2 moi로 A549세포에 transduction 시킨후 48시간후 IGF-1R α chain(n2, Santa Cruz Biotechnology, Santa Cruz, CA, USA) 에대한항체로염색후 FACS를이용하여암세포의세포막에발현되는 IGF-1R의변화를관찰하였다. 4. Adenovirus-shIGF-1R 에의한 IGF-1R 의발현억제 IGF-1R 을억제하는 sirna 를발현하는 ad-shigf- 1R의효과를확인하기위해 A549세포에 ad-shigf- 1R을 5, 1, 2 moi로 transduction 후 48시간후 IGF-1R β chain 항체 (H-6, Santa Cruz Biotechnology, Santa Cruz, CA, USA) 를이용하여 Western blot을시행하여 IGF-1R의발현의변화를확인하였다 23. 44
Tuberculosis and Respiratory Diseases Vol. 63. No.1, Jul. 27 5. Adenovirus-IGF-1R(95, 482) 의전처치가 TRAIL 에대한폐암세포주의감수성에미치는영향 TRAIL에대한중등도의저항성을보이는 A549와감수성을보이는 NCI H46을 ad-igf-1r(95, 482) 를 2 moi로 transduction 하였다. 음성대조군으로 ad-null(target 유전자가없는 backbone만가진 adenovirus) 및양성대조군으로 NFκB의활성화를억제하여 TRAIL의감수성을높일수있음이증명된 NFκB의활성화를억제하기위해 26,27 adenovirus-iκb α를사용하였다. Transduction 후 24시간후 TRAIL 을, 1, 2, 4 nm로처리하였다. 72시간후세포수를측정하고각군의 TRAIL을처리하지않은군에대한 % 생존율을계산하였다. 고 72시간후세포수를측정하고각군의 TRAIL을처리하지않은 well의세포에대한 % 생존율을계산하였다. 7. Adenovirus-shIGF-1R 에의한 IGF-1R 의억제가폐암세포주의 Gefitinib에대한감수성에미치는영향 Gefitinib 에대해중등도의내성을보이는 A549 세포를, 1, 5, 1 및 2 moi의 ad-shigf-1r로 transduction시키고 24시간후 gefitinib을,.2, 2, 2 및 5 μm로처리하고 72시간후세포수를측정하고각군의 gefitinib 을처리하지않은 well의세포에대한 % 생존율을계산하였다. 6. IGF-1R 의 tyrosine kinase inhibitor (AG124) 가 TRAIL 감수성에미치는영향 TRAIL에중등도저항성을보이는 A549 세포에 AG 124(2 μm) 를 24시간투여후 ( 대조군은동량의 DMSO 투여 ) TRAIL을, 2 및 4 ng/ml 로처리하 8. 통계학적분석약제감수성비교는 one way analysis of variance (ANOVA) 로검증하였고 p<.5를유의한차이로정의하였다 (SPSS version 12.K, Chicago, IL, USA). A: control B: Ad-null C: Ad-IGF-1R/482 D: Ad-IGF-1R/95 Figure 1. Changes of IGF-1R expression on the cell surface of lung cancer cell lines after transduction with ad-igf-1r/482 and 95. (mean value of IGF-1R; Control: 2.6, ad-null: 18.7, ad-igf-1r/482: 17.8, ad-igf-1r/95: 138.5) 45
YJ Lee et al: Enhancement of sensitivity to TRAIL and gefitinib by IGF-1R blockade 2kDa 95kDa 결 1. Ad-IGF-1R95 및 ad-igf-1r/482 처리후세포막의 IGF-1R 의발현의변화불완전형태의 IGF-1R 를발현하는 ad-igf-1r (95, 482) 를폐암세포주 (A549) 에감염후세포막에발현되는 IGF-1R을측정한결과 ad-igf-1r/95으로감염시킨세포에서는평균치가 138.5로대조군 2.6, ad-null 18.7 및 ad-igf-1r/482의 17.8에비해현저히증가하여세포막에불완전형태의 IGF-1R 가발현하는것을확인할수있었다. Ad-IGF-1R/482은 482 aminoacid 크기의 IGF-1R 을발현하나이는세포막에발현되지못하고 soluble form으로세포밖으로배출되어세포막의 IGF-1R 의발현에는영향이없었다 (Figure 1). Ad-shIGF-1R(3425) 5 1 5 (moi) IGF-1Rβ(H-6) β-actin Figure 2. Suppression of IGF-1R expression by transduction with adenovirus-shigf-1r 과 2. Adenovirus-shIGF-1R 에의한 IGF-1R 의발현억제 ad-shigf-1r에의한 IGF-1R 의발현의변화를 western blot으로확인한결과 1, 5 moi의 ad-shigf-1r로감염시킨폐암세포주에서 IGF-1R 의 β chain의발현이현저히감소함을확인할수있었다 (Figure 2). FACS에의한 IGF-1R 의변화는본연구진의이전논문에서이미확인하였다 23. 3. Adenovirus-IGF-1R/482, 95에의한 IGF-1R 의 dominant negative억제가 TRAIL 에대한감수성의변화 TRAIL에저항성을보이는 A549 세포에 ad-igf- 1R/482, 95으로 IGF-1R를억제한결과 TRAIL에대한감수성이현저히증가하였다. 이현상은 ad-igf- 1R/95에서더강력하였다. Ad-IGF-1R/95이 ad- IGF-1R/482보다강력한이유는 ad-igf-1r/95은세포막에불완전한형태의 IGF-1R를발현하여 IGF-1 에대해강력하게경쟁적으로결합하여차단하나 ad-igf-1r/482는 soluble form의 inhibitor를 media 내로분비하여 media에서 IGF-1과결합하여억제하므로상대적으로억제능력이약하다. 이는이전의연구자의논문에서도확인된바있다 21. 또한이는 12 12 1 1 %survival 8 6 Ad-null Ad-IGF-1R/482 Ad-IGF-1R/95 Ad-IkBaSR %survival 8 6 Ad-null Ad-IGF-1R/482 Ad-IGF-1R/95 4 4 2 2 1 2 4 ng/ml(trail) 1 2 4 ng/ml(trail) A549 NCL H46 Figure 3. Enhancement of sensitivity of A549(resistant cell line to TRAIL) to TRAIL after transduction with ad-igf-1r/482 and 95(p<.1 compared with ad-null). No significant enhancement was found in NCI H46 (sensitive cell line to TRAIL). (Y-axis: relative survival) 46
Tuberculosis and Respiratory Diseases Vol. 63. No.1, Jul. 27 12 1 억제가 TRAIL에대한저항성을보이는폐암세포주에서 TRAIL의항암효과를높임을확인할수있었다. %survival 8 6 4 DMSO AG124 5. Adenovirus-shIGF-1R 을이용한 IGF-1R 의억제가폐암세포주의 gefitinib 에대한감수성에미치는영향 2 2 4 ng/ml(trail) Figure 4. Enhancement of sensitivity of A549(resistant cell line to TRAIL) to TRAIL after treatment with AG124 (IGF-1R tyrosine kinase inhibitor). (p<.5). (Y-axis: relative survival) Gefitinib 에중등도의저항성이있는 A549에 ad-shigf-1r을투여하여 IGF-1R 의발현을억제한결과 gefitinib 에대한감수성이증가하였다. 이는 5 moi 이상의감염에서현저하였다 (Figure 5). 고찰 % survival 1.2 1.8.6.4.2.2 2 2 5 um 1 5 1 2 Figure 5. Enhancement of sensitivity of A549(resistant cell line to gefitinib) to gefitinib after transduction with ad-shigf-1r. Significant enhancement was found at transduction with ad-shigf-1r over 5 moi (p<.5).(y-axis: relative survival) TRAIL에의한 NFκB의활성화를억제하여감수성을증가시킨다 28 고알려진 ad-iκbα 보다도강력하였다. 그러나 TRAIL에대한감수성이높은 NCI H46에서는이현상을확인할수없었다 (Figure 3). 4. IGF-1R tyrosine kinase inhibitor (AG124) 에의한 IGF-1R 의억제에의한 A549의 TRAIL 에대한감수성변화 AG124의투여로 IGF-1R의 tyrosine kinase를억제한결과 A549의 TRAIL에대한감수성이증가하였다 (Figure 4). 이상의결과로여러형태의 IGF-1R 의 IGF-1R는대표적인 antiapoptosis 역할을하는수용체로알려져있다. 즉암세포외부에서의독성자극에저항하는중요기전이라알려있다. IGF-1R 를억제하여암세포를사멸시키거나다른항암제또는방사선에대한감수성을높이려는연구가많이시도되고있으나아직임상에는적용되지않고있다 13,29. IGF-1R 를억제하는방법에는유전자적인방법과비유전자적인방법으로구분할수있다. 유전자적인억제방법으로는 antisense, ribozyme 및 sirna를이용한 RNA interference 등으로 mrna를억제하여 IGF-1R의발현을억제하는방법과 dominant negative 방식으로 truncated form의 IGF-1R 과또는 soluble form의 IGF-1R 를세포밖으로배출하여 IGF-1과결합하여 IGF-1R pathway를차단하는방법이있다. 비유전자적인방법에는 IGF-1R tyrosine kinase inhibitor(tki) 를사용하는방법과 IGF-1R 에대한 monoclonal antibody를사용하는방법이있다 (Figure 6) 13,24. 본연구진은이미 IGF-1R 에대한 antisense를발현하는 adenovirus(ad-asigf-1r) 3 와 truncated form 의 IGF-1R 을발현하는 ad-igf-1r/95 및 soluble form의 IGF-1R를발현하는 ad-igf-1r/482를제작하여폐암세포주 21 및여러암세포주에서항암효과를보이고또한항암제에대한감수성을증가시킴을보고한바있다 31,32. 또한최근 IGF-1R 에대한 sirna 를 47
YJ Lee et al: Enhancement of sensitivity to TRAIL and gefitinib by IGF-1R blockade IGF-I IGF-IR Ab Soluble IGF-IR Intact IGF-IR heterodimer Defective IGF-IR (no TK domain) TKI (small molecule) IGF-IR precursor Antisense, ribozyme, sirna Dominant negative inhibition IGF-IR mrna Helix IGF-IR gene : Block Figure 6. Blockade of dominant-acting genes such as IGF-1R using genetic and non-genetic inhibition (adopted from Lee CT and Carbone DP. Chapter 52 Gene Therapy in 'Lung Cancer' 3rd ed. Lippincott Williams & Wilkins with permission). 발현할수있는 ad-shigf-1r을제작하여폐암세포주에서항암제에대한감수성을증가시킴을보고하였다 23. 본연구에서 dominant negative inhibition 및 RNA interference 를이용한유전학적 IGF-1R 억제와 IGF-1R TKI인 AG124를이용하여 IGF-1R를억제하고 TRAIL 및 gefitinib 에대해서중등도의저항성을보이는페암세포주에서감수성의변화를관찰하였다. TRAIL에대해저항성을보이는폐암세포주 (A549) 에서는 IGF-1R 를억제하면 TRAIL에대한감수성이회복되었으나이미 TRAIL에대한감수성이높은 NCI H46에서는변화가없었다. 이로폐암세포의 TRAIL에대한저항성에 IGF-1R 가관여함을알수있었다. 특히 TRAIL에대한저항성의한기전으로 TRAIL 자극에대해 NFκB가활성화가증명되어있다 28. 이번실험에서는저항성극복의양성기준으로 NF κb의활성화를비가역적으로억제하는 ad-iκbα 를사용하였다. Ad-IGF-1R/482, 95과 ad-iκbα에전처치에의한 A549의 TRAIL에대한감수성의변화를비 교한결과 ad-igf-1r/95을이용한 IGF-1R의억제가 ad-iκbα에의한 NFκB의활성화억제보다강력함을확인하였다. EGFR tyrosine kinase inhibitor인 gefitinib에대한저항성이최근문제가되고있다. 일부에서는처음부터저항성을보이기도하고일부에서는초기에는감수성을보이다가치료중획득내성을보이기도한다. EGFR의점돌연변이및 deletion이그기전으로일부밝혀지고있으나전체를설명하지는못하고있다. 최근 EGFR과 IGF-1R 사이에 cross-talk의중요성이확인되면서 2,33 암의대표적인성장인자인 EGFR의억제와대표적인 antiapoptosis 인자인 IGF-1R 의이중억제가연구되고있다. 최근 EGFR tyrosine kinase inhibitor인 gefitinib과 erlotinib에저항성을보이는폐암세포주에서 IGF-1R tyrosine kinase inhibitor(ag124) 로 IGF-1R pathway를억제한결과 gefitinib에의한폐암성장억제효과가증가된다는본연구와비슷한연구결과가보고되었다 33. 같은연구 33 에서 EGFR과 IGF-1R 간의 cross-talk의기전으로 1) 폐암세포주의 IGF-1R 과 48
Tuberculosis and Respiratory Diseases Vol. 63. No.1, Jul. 27 pigf-1r의발현정도와 gefitinib에대한감수성과반비례하였고 2) IGF-1R 의발현이높은페암세포에 gefitinib 을처리하면 IGF-1R 및 downstream mediator의인산화를유도하고 3) gefitinib 은 EGFR과 IGF-1R 의 heterodimerization을유도하며 survivin 의발현을유도하여저항성을보임을보고하였다. 그러나 erlotinib에서는위의현상이발견되지않았다. 본연구에서는일부의폐암세포및체외실험의결과이지만 EGFR 억제와 IGF-1R 의억제를동시에시행하는새로운치료전략의기초자료가될수있으리라기대된다. 결론적으로여러기전을이용한 IGF-1R 의억제는 TRAIL 또는 gefitinib 에대해저항성을보이던폐암세포주의감수성을향상시킴을확인하였으며이는폐암의임상치료의한계를극복할수있는하나의단초를제공하였다. 향후여러종류의암세포주에서의검증및체내실험이필요하리라생각된다. 요약배경 : 폐암의새로운치료제로각광을받고있는 TRAIL은암에선택적으로 apoptosis를일으킨다고알려진 cytokine으로알려져있다. 또한 gefitinib (Iressa) 는폐암의적용된최초의표적치료제로각광을받고있다. 그러나일부의암세포에서는이에대한저항성을보이고있다. 본연구에서는암세포에서외부의 apoptotic 자극에저항성을보이는 IGF-1R 를억제함으로 TRAIL 및 gefitinib 의항암작용을증가시키고자실험을시행하였다. 방법 : 암세포주는 TRAIL 및 gefitinib에민감한 NCI H46와두약제에중등도의저항성을보이는 A549의폐암세포주로시행하였으며 IGF-1R의억제는본연구자가개발하였던 IGF-1 pathway 를 dominant negative inhibition을할수있는 adenovirus- IGF1R(482 ST, 95ST) 및 IGF-1R tyrosine kinase inhibitor인 Tyrphostin AG124를사용하였고 gefitinib에대한실험에서는 RNA interference 를이용하여 IGF-1R 의발현을억제하는 adenovirusshigf-1r을이용하였다. 결과 : TRAIL에저항성을보이는폐암세포주 (A549) 에서 adenovirus-igf1r(482st, 95ST) 및 AG124로 IGF-1R 를억제한후 TRAIL을투여한경우항암효과가증대되었다. A549에 adenovirusshigf-1r을감염시켜 IGF-1R 의발현을억제한결과 gefitinib에대한감수성이증가되었다. 결론 : 여러기전을이용한 IGF-1R 의억제는 TRAIL 및 gefitinib 에대해저항성을보이던폐암세포주의감수성을향상시킴을확인하였으며이는폐암의임상치료의한계를극복할수있는하나의단초를제공하였다. 향후여러종류의암세포주에서의검증및 in vivo 실험이필요하리라생각된다. 참고문헌 1.Griffith TS, Lynch DH. TRAIL: a molecule with multiple receptors and control mechanisms. Curr Opin Immunol 1998;1:559-63. 2. Zhang L, Fang B. Mechanisms of resistance to TRAIL-induced apoptosis in cancer. Cancer Gene Ther 25;12:228-37. 3. Pan G, Ni J, Wei YF, Yu G, Gentz R, Dixit VM. An antagonist decoy receptor and a death domaincontaining receptor for TRAIL. Science 1997;277: 815-8. 4. Lee SH, Shin MS, Kim HS, Lee HK, Park WS, Kim SY, et al. Alterations of the DR5/TRAIL receptor 2 gene in non-small cell lung cancers. Cancer Res 1999;59:5683-6. 5. Cheng J, Hylander BL, Baer MR, Chen X, Repasky EA. Multiple mechanisms underlie resistance of leukemia cells to Apo2 Ligand/TRAIL. Mol Cancer Ther 26;5:1844-53. 6. Brooks AD, Sayers TJ. Reduction of the antiapoptotic protein cflip enhances the susceptibility of human renal cancer cells to TRAIL apoptosis. Cancer Immunol Immunother 25;54:499-55. 7. Seol JY, Park KH, Hwang CI, Park WY, Yoo CG, Kim YW, et al. Adenovirus-TRAIL can overcome TRAIL resistance and induce a bystander effect. Cancer Gene Ther 23;1:54-8. 8. Herbst RS, Maddox AM, Rothenberg ML, Small EJ, Rubin EH, Baselga J, et al. Selective oral epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 is generally well-tolerated and has activity in non-small-cell lung cancer and other solid tumors: results of a phase I trial. J Clin Oncol 22;2: 49
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