Tuberculosis and Respiratory Diseases

The Korean Academy of Tuberculosis and Respiratory Diseases (대한결핵및호흡기학회)
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Combination Treatment with Arsenic Trioxide and Sulindac Induces Apoptosis of NCI-H157 Human Lung Carcinoma Cells via ROS Generation with Mitochondrial Dysfunction

NCI-H157 폐암 세포주에서 활성산소종의 생성과 미토콘드리아 기능변화를 한 Arsenic Trioxide와 Sulindac 병합요법의 세포고사효과

  • Kim, Hak-Ryul (Department of Internal Medicine, College of Medicine Wonkwang University) ;
  • Yang, Sei-Hoon (Department of Internal Medicine, College of Medicine Wonkwang University) ;
  • Jeong, Eun-Taik (Department of Internal Medicine, College of Medicine Wonkwang University)
  • 김학렬 (원광대학교 의과대학 내과학교실 원광의과학연구소) ;
  • 양세훈 (원광대학교 의과대학 내과학교실 원광의과학연구소) ;
  • 정은택 (원광대학교 의과대학 내과학교실 원광의과학연구소)
  • Received : 2005.02.21
  • Accepted : 2005.06.09
  • Published : 2005.07.30
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Abstract

Background : Arsenic trioxide ($As_2O_3$) has been used to treat acute promyelocytic leukemia, and it induces apoptosis in a variety of solid tumor cell lines including non-small cell lung cancer cells. However, nonsteroidal antiinflammatory drugs (NSAID) can enhance tumor response to chemotherapeutic drugs or radiation. It was previously demonstrated that a combination treatment with $As_2O_3$ and sulindac induces the apoptosis of NCI-H157 human lung carcinoma cells by activating the caspase cascade. This study aimed to determine if a combination treatment augmented its apoptotic potential through other pathways except for the activation of the caspase cascade. Material and Methods : The NCI-H157 cells were treated with $As_2O_3$, sulindac and antioxidants such as glutathione (GSH) and N-acetylcysteine (NAC). The cell viability was measured by a MTT assay, and the level of intracellular hydrogen peroxide ($H_2O_2$) generation was monitored fluorimetrically using a scopoletin-horse radish peroxidase (HRP) assay. Western blotting and mitochondrial membrane potential transition analysis were performed in order to define the mechanical basis of apoptosis. Results : The viability of the cells was decreased by a combination treatment of $As_2O_3$ and sulindac, and the cells were protected using antioxidants in a dose-dependent manner. The increased $H_2O_2$ generation by the combination treatment was inhibited by antioxidants. The combination treatment induced changes in the mitochondrial transmembrane potential as well as the expression of the Bcl-2 family proteins, and increased cytochrome c release into the cytosol. However, the antioxidants inhibited the effects of the combination treatment. Conclusion : Combination treatment with $As_2O_3$ and sulindac induces apoptosis in NCI-H157 human lung carcinoma cells via ROS generation with a mitochondrial dysfunction.

연구배경 : Arsenic trioxide($As_2O_3$, 비소 삼산화물)는 급성전 골수성백혈병의 효과적인 치료제로 이용되고 있고, 비소세포폐암을 비롯한 여러 고형종양세포에서 세포 고사를 유도하나 백혈병에 비해 상대적으로 높은 농도가 필요하여 실제적인 치료에 제한이 있어왔다. Sulindac은 COX-2 억제, 암세포 성장의 억제 및 세포 고사 유도를 기전으로 하는 항암효과가 있고, 다른 화학요법이나 방사선치료의 반응성을 증가시키는 것으로 알려져 있다. 저자들은 NCI-H157 폐암세포주에서 $As_2O_3$와 sulindac의 병합요법이 Fas/FasL 신호전달계의 활성화와 caspase 단백질 활성화 의해 세포고사가 유도되었음을 보고한 바 있다. 이 연구의 목적은 이러한 경로 이외에 다른 기전유무를 밝히는데 있다. 방 법 : 세포 독성은 MTT 방법으로 구하였고, HRP 방법을 이용해 ROS를 직접 측정하였다. 세포고사의 형태학적 특성을 보기위해 핵산염색과 관련된 단백질의 발현을 확인하였다. 또한 미토콘드리아의 기능변화를 보기 위해 미토콘드리아의 막전위차를 측정하였고, anti-cytochrome c와 Bcl-2 family 단백질들의 발현 양상을 western blotting을 통해 관찰하였다. 결 과 : 단독요법에 비해 병합요법시에 생존율의 의의 있는 감소를 보였다. 이러한 생존율은 항산화제를 전처리 한 경우 농도 의존적으로 회복됨을 관찰할 수 있었다. ROS의 생성은 병합요법시에 대조군에 비해 의의 있게 증가하였고, 이러한 현상은 항산화제 전처리군에서 감소된 소견을 보였다. 또한 병합요법시 핵산염색에 의해 여러조각의 분절된 형광절편과 caspase 3, PARP의 활성을 통해 세포고사가 유도되었음을 확인하였고, 항산화제 전처리군에서 상쇄됨을 관찰하였다. JC-1 염색 후 형광현미경을 통한 미토콘드리아 막전위차에 미치는 영향은 병합처리군에서 녹색형광으로의 변화가 보였고 항산화제 전처리군에서 소실됨을 확인하였다. 또한 병합요법시의 cytochrome c의 세포 질내의 증가와 미토콘드리아내의 감소가 관찰되었고, Bax의 증가, Bid와 Bcl-xL의 발현감소를 확인할 수 있었으며 이러한 현상은 항산화제 전처리군에서 소실됨을 보였다. 결 론 : NCI-H157 폐암세포주에 $As_2O_3$와 sulindac의 병합 요법은 ROS생성과 미토콘드리아 기능변화를 통해 세포고사가 유도되었다.

Keywords

References

  1. Bae JM, Won YJ, Jung KW, Suh KA, Yun YH, Shin MH, et al. Survival of Korean cancer patients diagnosed in 1995. Cancer Res Treat 2002;34:319-25 https://doi.org/10.4143/crt.2002.34.5.319
  2. Sun HD, Ma L, Hu XC, Zhang TD. Treatment of acute promyelocytic leukemia by Ailing-1 therapy with use of syndrome differentiation of traditional Chinese medicine. Chin J Comb Trad Chin Med West Med 1992;12:170-1
  3. Soignet SL, Frankel SR, Douer D, Tallman MS, Kantarjian H, Calleja E, et al. United States multicenter study of arsenic trioxide in relapsed acute promyelocytic leukemia. J Clin Oncol 2001;19:3852-60 https://doi.org/10.1200/JCO.2001.19.18.3852
  4. Shen ZY, Tan LJ, Cai WJ, Shen J, Chen C, Tang XM, et al. Arsenic trioxide induces apoptosis of oesophageal carcinoma in vitro. Int J Mol Med 1999;4:33-7
  5. Akao Y, Nakagawa Y, Akiyama K. Arsenic trioxide induces apoptosis in neuroblastoma cell lines through the activation of caspase 3 in vitro. FEBS Lett 1999;455:59-62 https://doi.org/10.1016/S0014-5793(99)00841-8
  6. Zheng J, Deng YP, Lin C, Fu M, Xiao PG, Wu M. Arsenic trioxide induces apoptosis of HPV16 DNA-immortalized human cervical epithelial cells and selectively inhibits viral gene expression. Int J Cancer 1999;82:286-92 https://doi.org/10.1002/(SICI)1097-0215(19990719)82:2<286::AID-IJC21>3.0.CO;2-K
  7. Zhang TC, Cao EH, Li JF, Ma W, Qin JF. Induction of apoptosis and inhibition of human gastric cancer MGC-803 cell growth by arsenic trioxide. Eur J Cancer 1999;35:1258-63 https://doi.org/10.1016/S0959-8049(99)00106-9
  8. Giardiello FM, Spannhake EW, DuBois RN, Hylind LM, Robinson CR, Hubbard WC, et al. Prostaglandin levels in human colorectal mucosa: effects sulindac in patients with familial adenomatous polyposis. Dig Dis Sci 1998;43:311-6 https://doi.org/10.1023/A:1018898120673
  9. Smalley WE, DuBois RN. Colorectal cancer and non-steroidal anti-inflammatory drugs. Adv Pharmacol 1997;39:1-20 https://doi.org/10.1016/S1054-3589(08)60067-8
  10. Dannenberg AJ, Zakim D. Chemoprevention of colonrectal cancer through inhibition of cyclooxygenase-2. Semin Oncol 1999;26:499-504
  11. Shim H, Yang SH, Bak SM, Jeong ET. Sodium salicylate(NaSaL) induces poptosis of NCI-H1299 lung carcinoma cells via activation caspase-3 protease. Tuberc Respir Dis 2002:53:485-96 https://doi.org/10.4046/trd.2002.53.5.485
  12. Shiff SJ, Qiao L, Tsai LL, Rigas B. Sulindac sulfide, an aspirin-like compound, inhibits proliferation, causes cell cycle quiescence, and induces apoptosis in HT-29 colon adenocarcinoma cells. J Clin Invest 1995;96:491-503 https://doi.org/10.1172/JCI118060
  13. Chan TA, Morin PJ, Vogelstein B, Kinzler KW. Mechanism underlying nonsteroidal antiinflammatory drug-mediated apoptosis. Proc Natl Acad Sci U S A 1998;95:681-6
  14. Milas L. Cyclooxygenase-2 (COX-2) enzyme inhibitors as potential enhancers of tumor radioresponse. Semin Radiat Oncol 2001;11:290-9
  15. Koki AT, Leahy KM, Masferrer JL. Potential utility of COX-2 inhibitors in chemoprevention and chemotherapy. Expert Opin Investig Drugs 1999;8:1623-38 https://doi.org/10.1517/13543784.8.10.1623
  16. Milas L, Kishi K, Hunter N, Mason K, Masferrer JL, Tofilon PJ. Enhancement of tumor response to gamma-radiation by an inhibitor of cyclooxygenase-2 enzyme. J Natl Cancer Inst 1999;91:1501-4 https://doi.org/10.1093/jnci/91.17.1501
  17. Kim HR, Yang SH, Jeong ET. Induces apoptosis of NCI-H157 human lung carcinoma cells via activation of caspase cascade by combination treatment with arsenic trioxide and sulindac. Tuberc Respir Dis 2004:56:381-92 https://doi.org/10.4046/trd.2004.56.4.381
  18. Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science 1998:281:1322-6 https://doi.org/10.1126/science.281.5381.1322
  19. Antonsson B, Martinou JC. The Bcl-2 protein family. Exp Cell Res 2000:256:50-7 https://doi.org/10.1006/excr.2000.4839
  20. Vander Heiden MG, Chandel NS, Williamson EK, Schumacker PT, Thompson CB. Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria. Cell 1997:91:627-37 https://doi.org/10.1016/S0092-8674(00)80450-X
  21. Chen GQ, Shi XG, TangW, Xiong SM, Zhu J, Cai X, et al. Use of arsenic trioxide($As_{2}O_{3}$) in the treatment of acute promyelcytic leukemia(APL): I. $As_{2}O_{3}$ exerts dose-dependent dual effects on APL cells. Blood 1997;89:3345-53
  22. Chen GQ, Zhu J, Shi XG, Ni JH, Zhong HJ, Si GY, et al. In vitro studies on cellular and molecular mechanisms of arsenic trioxide in the treatment of acute promyelocytic leukemia: arsenic trioxide induces NB4 cell apoptosis with downregualation of Bcl-2 expression and modulation of PML-$RAR{\alpha}$/PML proteins. Blood 1996;88:1052-61
  23. Jung BH, Park C, Kim HR, Park MR. Arsenic trioxide induces apoptosis of HL-60 cells via activation of intrinsic caspase protease with mitochondrial dysfunction. Cancer Res Treat 2002;34:308-15 https://doi.org/10.4143/crt.2002.34.4.308
  24. Uslu R, Sanli UA, Sezgin C, Karabulut B, Terzioglu E, Omay SB, et al. Arsenic trioxide-mediated cytotoxicity and apoptosis in prostate and ovarian carcinoma cell lines. Clin Cancer Res 2000;6:4957-64
  25. Park WH, Seol JG, Kim ES, Hyun JM, Jung CW, Lee CC, et al. Arsenic trioxide-mediated growth inhibition in MC/CAR myeloma cells via cell cycle arrest in association with induction of cyclin-dependent kinase inhibitor, p21, and apoptosis. Cancer Res 2000;60:3065-71
  26. Larochette N, Decaudin D, Jacotot E, Brenner C, Marzo I, Susin SA, et al. Arsenite induces apoptosis via a direct effect on the mitochondrial permeability transition pore. Exp Cell Res 1999;249:413-21 https://doi.org/10.1006/excr.1999.4519
  27. Chen YC, Lin-Shiau SY, Lin JK. Involvement of reactive oxygen species and caspase 3 activation in arsenite-induced apoptosis. J Cell Physiol 1998;177:324-33 https://doi.org/10.1002/(SICI)1097-4652(199811)177:2<324::AID-JCP14>3.0.CO;2-9
  28. Torrance CJ, Jackson PE, Montgomery E, Kinzler KW, Vogelstein B, Wissner A, et al. Combinational chemoprevention of intestinal neoplasia. Nat Med 2000;6:1024-8 https://doi.org/10.1038/79534
  29. Duffy CP, Elliott CJ, O'Connor RA, Heenan MM, Coyle S, Cleary IM, et al. Enhancement of chemotherapeutic drug toxicity to human tumour cells in vitro by a subset of NSAIDs. Eur J Cancer 1998;34:1250-9 https://doi.org/10.1016/S0959-8049(98)00045-8
  30. Soriano AF, Helfrich B, Chan DC, Heasley LE, Bunn PA Jr, Chou TC. Synergistic effects of new chemopreventive agents and conventional cytotoxic agents against human lung cancer cell lines. Cancer Res 1999;59:6178-84
  31. Hida T, Kozaki K, Muramatsu H, Masuda A, Shimizu S, Mitsudomi T, et al. Cyclooxygenase-2 inhibitor induces apoptosis and enhances cytotoxicity of various anticancer agents in non-small cell lung cancer cell lines. Clin Cancer Res 2000;6:2006-11