J Lung Cancer 2010;9(2):64-71 Inhibitory Effect of Epidermal Growth Factor on the Proliferation of Lung Cancer Cell Lines Purpose: Epidermal growth factor (EGF) is known to cause cellular proliferation, differentiation and survival. However, there are a few articles that have reported on the cell killing effect of EGF. We evaluated the effects of EGF on the survival of some lung cancer cell lines and we tried to determine the mechanism of action. Materials and Methods: We examined various lung cancer cell lines. The cultured cells were exposed to radiation alone (0, 2, 5, and 10 Gy), EGF alone (0 1,000 nm) or a combination of radiation and EGF (10 nm). Survival was measured using a clonogenic assay and the expressions of epidermal growth factor receptor (EGFR), Ki-67 and cleaved- PARP were detected by western blot analysis. K-ras mutations were detected using polymerase chain reaction and sequencing. Results: Treatment of EGF induced cell death in the lung cancer cell lines. The addition of EGF (10 nm) to radiation (0, 2, 5, and 10 Gy) resulted in an increased cell killing effect of radiation. The EGFR expression decreased with the addition of EGF. EGF increased the expression of cleaved-parp, but it did not increase the expression of Ki-67. The effects of EGF were not correlated with EGFR mutation or K-ras mutation. Conclusion: In our study, EGF inhibited the proliferation of lung cancer cell lines and it induced apoptosis. EGF enhanced the radiosensitivity of lung cancer cells when EGF was combined with radiation. It is suggested that EGF seem to be one of the cytotoxic agents for lung cancer cell lines. (J Lung Cancer 2010;9(2):64 71) Key Words: Epidermal growth factor, Radiation, Apoptosis, Lung cancer cell lines Seung-Hee Lee, M.S. and Hong-Gyun Wu, M.D., Ph.D. Department of Radiation Oncology & Cancer Research Institute, Seoul National University College of Medicine and Institute of Radiation Medicine, Medical Research Center, Seoul National University, Seoul, Korea Received: July 29, 2010 Revised: August 9, 2010 Accepted: August 19, 2010 Address for correspondence Hong-Gyun Wu, M.D., Ph.D. Department of Radiation Oncology, Seoul National University College of Medicine, 28, Yeongeon-dong, Jongnogu, Seoul 110-744, Korea Tel: 82-2-2072-3177 Fax: 82-2-765-3317 E-mail: wuhg@snu.ac.kr This work supported by research grant from Korean Association for the Study of Lung Cancer in 2008 and grant No. 2009-0083886 from the Korea Science and Engineering Foundation grant funded by the Korea government (MEST). 서론상피세포성장인자 (epidermal growth factor, EGF) 는미국의스탠리코헨이바로태어난쥐등에서눈꺼풀이열리는효과를갖는단백질로처음발견하였고, 53개의아미노산과 3개의분자내이황화결합을가진분자량 6,045-Da이다 (1-3). EGF는세포막에있는 EGF수용체 (epidermal growth factor receptor, EGFR) 와결합함으로써 tyrosine kinase를활성화시켜신호전달을통해세포의분열을유도하고증식을촉진하는것으로알려져있다. 따라서상처가발생하게되면혈액으로부터공급되어상피세포를증식시키고신생혈관을형성하여상처의회복을향상시키기때문에 (4,5) 당뇨 성족부궤양이나방사선치료시의부작용으로나타나는구강점막염과손상된각막등의치료에좋은영향을미친다고보고되고있다 (6-8). EGFR의과발현은여러상피성암종에서관찰되어암의발생, 진행, 표준치료법에대한내성, 불량한예후등과관련이있는것으로알려져있다 (9,10). 최근에는다양한 EGFR 억제제가개발되어암의치료에이용되고있다 (11-13). 그러나일반적으로 EGF가세포를증식시킨다는개념과반대로 EGFR이과발현하는암세포주에서는세포의성장을억제시킨다는보고가있으며 (14-16), EGF의세포분화작용으로인해항암제나방사선치료와 EGF를병행한연구도보고되고있다. 여러종류의암세포주에항암제와함께 64
Inhibitory Effect of Epidermal Growth Factor on Proliferation of Lung Cancer Cell Lines 65 EGF를병행했을때항암효과가높게나타났고 (17), 방사선조사시 EGF와병행함으로써효과가상승하였다 (18,19). 본연구실에서도 EGF가정상섬유모세포의증식을촉진시키지만두경부암세포주인 AMC-HN3에서현저하게세포사를일으키고다른암세포주에서도방사선과병용시방사선효과를저해하지않으며오히려방사선효과를증가시키는것을확인하였다 (20). 따라서본연구에서는 EGFR을발현하는폐암세포주에서 EGF의작용을평가하고작용기전을알아보고자하였다. 또한방사선과병행시의효과도평가하고자하였다. 대상및방법 1) 세포배양 EGF의효과를알아보기위하여여러가지폐암세포주를이용하였다. A549는 American Type Culture Collection (ATCC) 에서구입하였고, HCC-827, HCC-1438, NCI-H1437, NCI- H157, NCI-H1650은분당서울대학교병원의이춘택교수로부터공급받았다. 10% fetal bovine serum (Sigma, St. Louis, MO, USA) 을첨가한 RPMI 1640 (Gibco BRL, Gaithersburg, MD, USA) 배지에서 37 o C, 5% 이산화탄소하에서배양하였다. 2) EGF 처리및방사선조사 EGF는대웅제약 (Seoul, Korea) 에서무상제공받았으며세포배양액에원하는농도로희석하여투여하였다. 세포증식을보기위한실험에서는 0 1,000 nm의농도로처리하여배양액을교환하지않은상태로세포집락이형성될때까지배양하였고, 방사선과병용처치시에는예비실험에서구한최적의농도 10 nm을사용하여 EGF 처리후바로방사선을조사하였다. Western blot을위한실험에서도 10 nm로사용하였다. EGFR과 Ki-67, cleaved-parp 발현을평가하기위하여 4시간, 24시간, 48시간동안 EGF를처리한후단백질을추출하였다. 방사선은선형가속기 (Clinac 4/100; Varian, Palo Alto, CA, USA) 를이용하여 0, 2, 5, 10 Gy 선량으로조사하였다. 3) Clonogenic assay 6 well 배양접시에 10 2 10 3 개의세포를심고 1일후에 EGF를처치또는방사선을조사하고 10 15일간세포집락이형성되도록배양하여차가운 100% methanol로고정시킨후 0.5% crystal violet 용액으로 10분염색하여광학현미경하에서집락의수를계측하였다. 집락을형성한세포수가 50개이상일때하나의집락으로인정하였다. 4) 단백질추출및 western blotting 세포를모은후 cell lysis buffer (intron Biotechnology, Seoul, Korea) 에초음파파쇄기로용해한뒤 13,000 rpm으로 20분간원심분리하여상층액을취해단백질을정량하였다. 적절한양의단백질을 6 8% SDS-PAGE로전기영동한다음, PVDF (Millipore Co., Bedford, MA, USA) 로단백을이동시켰다. 이를 5% 탈지유와 0.1% Tween-20을포함하는 Tris-buffered saline (TBST) 에 1시간동안처리하고 1차항체를 4 o C에서하룻밤처리하였다. TBST로세척후상온에서 2차항체를 1시간동안반응시킨다음 WEST-ONE TM chemiluminescent substrate (intron Biotechnology) 로발색시킨후 LAS-3000 (FujiFilm, Tokyo, Japan) 으로 band를관찰하였다. 사용한 1차항체는 EGFR (Cell Signaling Technology, Beverly, MA, USA), Ki-67 (Abcam, Cambridge, UK), cleaved- PARP (Cell Signaling Technology) 이었다. 5) Mutation 평가세포에트립신처리후 QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) 을이용한 proteinase K 숙성방법을사용하여 DNA를추출하였다. K-ras 변이를검색하기위한중합효소연쇄반응 (PCR) 의시발체는엑손 1번의코돈 12, 13과엑손 2번의코돈 61을포함하도록제작하였다 (codon12,13- forward: 5 -GAGATGTTCTAATATAGT-3, reverse: 5 -CTG- TATCAAAGAATGGTCCT-3 ; codon61-forward: 5 -GACTG- TGTTTCTCCCTTCT-3, reverse: 5 -ACTATAATTACTCCT- TAATGTC-3 ). 제작된시발체는 10 pmole/μl의농도로 Premix Taq (TaKaRa, Tokyo, Japan) 과혼합하여 94 o C에서 5 분간전변성시킨후 94 o C에서 30초, 50 o C에서 45초, 73 o C에서 45초로 35 cycle을증폭하고 73 o C에서 5분간반응시켰다. 증폭된 PCR 산물은 2% agarose gel에서전기영동으로확인하고마크로젠 (Macrogen Co., Seoul, Korea) 에 DNA 정제및염기서열분석을의뢰하였다. 결과 1) EGF에의한세포증식억제효과 Clonogenic assay를이용하여 0 1,000 nm의 EGF 농도에따른폐암세포주의세포집락수를비교하였다. HCC-827에서는 EGF를처리하였을때농도의증가와비례하는세포증식억제효과가나타났으며, 다른세포주에서도농도와비례하는양상은관찰되지않았으나 EGF를처리하지않은대
66 J Lung Cancer 2010;9(2):64-71 조군에비하여처치군에서세포집락수가감소했다 (Fig. 1). 혈청에는다양한성장인자와성장과관련된다양한호르몬이존재하기때문에 EGF에의한효과를확인하기위하여 0.05% 혈청을첨가한배지에서 24시간동안 serum starvation시킨후같은실험을반복했으나혈청유무에관계없이같은결과를보였다 (Fig. 2). 2) EGF가방사선효과에미치는영향 EGF와방사선을병행했을때효과를알아보기위하여 10 nm의 EGF와 0, 2, 5, 10 Gy의방사선을함께처리하였다. A549 세포주에서는효과가적었으나, 다른모든폐암세포주에서방사선단독처리시보다 EGF를병용처리하였을때방사선의세포성장억제효과가증가하는결과를보여주 Fig. 1. The effect of epidermal growth factor (EGF) on the proliferation of the lung cancer cell lines. The cells were cultured for 24 hours prior to treatment with various concenrations (0, 0.01, 0.1, 1, 10, 100, and 1,000 nm) of EGF. The surviving cells were counted on day 10 using a clonogenic assay.
Inhibitory Effect of Epidermal Growth Factor on Proliferation of Lung Cancer Cell Lines 67 Fig. 2. The effect of epidermal growth factor (EGF) on the proliferation of lung cancer cell lines. The cells were growth-arrested by incubation in medium containing 0.05% serum for 1 day. The cells were cultured for 12 days with various concenrations (0, 0.01, 0.1, 1, 10, 100, and 1,000 nm) of EGF. 었다 (Fig. 3). 3) EGFR, Ki-67, cleaved-parp 발현의변화단백면역분석으로여러종류의세포주에서 EGFR이발현되는것을확인하였다 (Fig. 4). 모든세포주에서 EGF 처치후 4시간, 24시간, 48시간으로시간이경과함에따라 EGFR 의발현이뚜렷하게감소하였다. 세포증식을나타내는지 표인 Ki-67은 EGF 처리와상관없이변화가없었다. 세포고사와관련이있는 cleaved-parp는 HCC-1438, NCI-H1437, NCI-H1650에서 EGF 처리 24시간후현저히증가하였고, HCC-827과 NCI-H157에서는 48시간후에증가하였다 (Fig. 5).
68 J Lung Cancer 2010;9(2):64-71 Fig. 3. Clonogenic survival of the lung cancer cells treated with epidermal growth factor (EGF) followed by irradiation with a range of radiation doses. Although the radiation sensitizing effect was minimal in the A549 cell line, the rest of lung cancer cell lines showed the radiation sensitizing effect of EGF. 4) EGF 와 EGFR 및 K-ras mutation 과의연관성 Fig. 4. Expression of epidermal growth factor receptor (EGFR) in various cancer cell lines. EGF의효과가 EGFR 변이나 K-ras 변이와관계가있는지알아보고자하였다. HCC-827, NCI-H1650은 EGFR결실변이 (dele746-a750) 를가지고있으며 NCI-H1437, NCI-H157, A549는 EGFR wild type (wt) 인것으로알려져있고 (18,19), A549와 H157에서는코돈 12, 13의 K-ras 변이가발견되었다
Inhibitory Effect of Epidermal Growth Factor on Proliferation of Lung Cancer Cell Lines 69 Fig. 5. The epidermal growth factor receptor (EGFR), Ki-67 and cleaved PARP expressions in response to epidermal growth factor (EGF) treatment on the lung cancer cell lines. Treatment with EGF decreased the expression of EGFR and it increased the expression of cleaved PARP. Fig. 6. Mutation analysis of K-ras in the lung cancer cell lines. (Fig. 6). 그러나 EGFR 변이나 K-ras 변이가있는세포주들과변이가없는 H1437을비교했을때 EGF의성장억제효과에서차이점을보이지않았다 (Figs. 1, 3). 고안및결론 EGFR은 erb-b2 (HER2/neu), erb-b3, erb-b4와함께인체내상피성수용체인 tyrosine kinase에속하는세포막당단백으로, 자체 tyrosine kinase 활성을가지고있는데, EGF와결 합하여활성화되면유전자발현, 세포증식, 주변조직에대한침범, 세포고사억제, 혈관신생등의효과를유발하여암을일으키거나더욱악화시킬수있다 (21,22). EGFR은비소세포폐암을포함한많은상피세포종양에서비정상적으로과발현되는데 (23), 최근 EGFR 억제제인 gefitinib, erlotinib, cetuximab 등이표적치료제로개발되어일부암의치료에사용되고있다 (11-13). 그러나 EGF는세포의성장을촉진하는기능외에높은농도로암세포에처리하였을때암세포의성장을억제하고세포고사를촉진한다는연구결과들이보고되었다 (24,25). 특히 EGFR의발현이적은세포에서는 EGF 처리시성장이촉진되지만 EGFR이과발현하는세포에서는 EGF가성장을저해하고결국세포고사를유도하는것으로나타났다 (26-28). 세포고사의기전은 caspase라고하는단백질분해효소에의해세포내단백질이분해되면서신호가전달되는과정으로여러종류의 caspase들이세포사멸에관여하며 caspase의활성을통해세포고사의정도를파악할수있다 (29). 최종적으로활성화된 caspase-3는 PARP를활성화시키고활성화된 PARP는 DNA 분절화현상을유도하면서세포고사를일으키는것으로보고되고있다 (30). 또한방사선조사와 EGF를병행하였을때그효과가상승된다는보고가
70 J Lung Cancer 2010;9(2):64-71 있다 (18,19). 본연구에서도여러가지폐암세포주를대상으로 EGF를처리하였을때세포사가일어나는것을확인하였고, 시간에따른 EGFR 발현의감소와 cleaved-parp의발현증가로그기전이세포고사의증가에의한것임을알수있었다. 방사선조사와 EGF를병행하여처리했을때는 A549에서그효과가미약하기는하였지만다른폐암세포주에서는방사선조사단독처리결과보다효과가있는것으로나타났다. Ki-67은세포증식을나타내는지표로서세포주기의 G0기와초기 G1기를제외한모든세포의핵내에존재하고, 활발하게분열하고있는세포들의분포를반영하며종양의재발및악성도와연관이있다 (31). 본연구결과에서 Ki-67은모든세포주에서변화를보이지않았다. 이는 EGF 가폐암세포주를증식시키지않는다는것을보여주었다. 본연구자들은이전실험에서 EGF가정상섬유아세포의증식을촉진시키고방사선조사에의한세포사를억제시키는반면두경부암세포주인 AMC-HN3에서는세포사를증가시키는것을확인하였다 (20). 또한 EGFR 변이와 K-ras 변이는상호배타적이지만폐암에있어서기능적으로같은역할을한다 (32,33). EGFR 변이는선암, 비흡연자, 동양인, 여성에게서많이발견되는데 EGFR 표적치료제의임상결과에효과적이며 K-ras 변이는약물저항성의예측인자라고할수있다. 항암제의효과가 EGFR 변이또는 K-ras 변이와밀접한관련이있는것으로알려져있기때문에 EGF의암세포증식억제효능이변이에의한것인지확인하였다. 결과적으로변이의유무에따른차이는나타나지않았으므로 EGF는변이와무관하게효과가있는것으로생각된다. 본연구에서 EGF의세포고사를통한폐암세포주증식억제효과를확인하였다. 또한방사선과병용시방사선단독처리보다그효과가증가되었으므로방사선민감제로서의역할도기대할수있을것으로생각된다. 향후추가적인실험을통하여세포고사를일으키는기전과 EGF가정상조직에대한최소한의부작용을보이면서암조직의성장을억제시킬수있는가능성에대한확인이필요하다. REFERENCES 1. Carpenter G, Cohen S. Epidermal growth factor. J Biol Chem 1990;265:7709-7712. 2. Carpenter G, Cohen S. Epidermal growth factor. Annu Rev Biochem 1979;48:193-216. 3. Cohen S. Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal. J Biol Chem 1962;237:1555-1562. 4. Servold SA. Growth factor impact on wound healing. Clin Podiatr Med Surg 1991;8:937-953. 5. Brown GL, Curtsinger L 3rd, Brightwell JR, et al. Enhancement of epidermal regeneration by biosynthetic epidermal growth factor. J Exp Med 1986;163:1319-1324. 6. Hong JP, Jung HD, Kim YW. Recombinant human epidermal growth factor (EGF) to enhance healing for diabetic foot ulcers. Ann Plast Surg 2006;56:394-398. 7. Lee SW, Jung KI, Kim YW, Jung HD, Kim HS, Hong JP. Effect of epidermal growth factor against radiotherapy-induced oral mucositis in rats. Int J Radiat Oncol Biol Phys 2007;67: 1172-1178. 8. Mishima H, Nakamura M, Murakami J, Nishida T, Otori T. Transforming growth factor-beta modulates effects of epidermal growth factor on corneal epithelial cells. Curr Eye Res 1992;11:691-696. 9. Bianco R, Gelardi T, Damiano V, Ciardiello F, Tortora G. Rational bases for the development of EGFR inhibitors for cancer treatment. Int J Biochem Cell Biol 2007;39:1416-1431. 10. Arteaga CL. Epidermal growth factor receptor dependence in human tumors: more than just expression? Oncologist 2002;7 (Suppl 4):31-39. 11. Paez JG, Jänne PA, Lee JC, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004;304:1497-1500. 12. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129-2139. 13. Pao W, Miller V, Zakowski M, et al. EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci U S A 2004;101:13306-13311. 14. Gill GN, Lazar CS. Increased phosphotyrosine content and inhibition of proliferation in EGF-treated A431 cells. Nature 1981;293:305-307. 15. Kamata N, Chida K, Rikimaru K, Horikoshi M, Enomoto S, Kuroki T. Growth-inhibitory effects of epidermal growth factor and overexpression of its receptors on human squamous cell carcinomas in culture. Cancer Res 1986;46:1648-1653. 16. MacLeod CL, Luk A, Castagnola J, Cronin M, Mendelsohn J. EGF induces cell cycle arrest of A431 human epidermoid carcinoma cells. J Cell Physiol 1986;127:175-182. 17. Kröning R, Jones JA, Hom DK, et al. Enhancement of drug sensitivity of human malignancies by epidermal growth factor. Br J Cancer 1995;72:615-619. 18. Christen RD, Hom DK, Eastman A, Howell SB. Epidermal growth factor regulates the ability of human ovarian carcinoma cells to repair DNA damage. Proc Am Assoc Cancer Res 1991;32:430. 19. Kwok TT, Sutherland RM. Enhancement of sensitivity of human squamous carcinoma cells to radiation by epidermal growth factor. J Natl Cancer Inst 1989;81:1020-1024. 20. Kwon EK, Lee SH, Kim KB, Wu HG, Lee SW. Differential effect of recombinant human EGF on proliferation and radiation survival of normal fibroblast and cancer cell lines.
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