DOI QR코드

DOI QR Code

SKOV-3 난소암 세포주에서 lysophosphatidic acid 유도 세포의 이동에 있어 활성산소의 역할

Reactive Oxygen Species Mediates Lysophosphatidic Acid-induced Migration of SKOV-3 Ovarian Cancer Cells

  • 김은경 (부산대학교 의학전문대학원 약리학교실) ;
  • 이혜선 (부산대학교 의학전문대학원 약리학교실) ;
  • 하홍구 (부산대학교병원 비뇨기과학교실) ;
  • 윤성지 (부산대학교 의학전문대학원 약리학교실) ;
  • 하정민 (부산대학교 의학전문대학원 약리학교실) ;
  • 김영환 (부산대학교 의학전문대학원 약리학교실) ;
  • 진인혜 (부산대학교 의학전문대학원 약리학교실) ;
  • 신화경 (부산대학교 한의학전문대학원 해부학교실) ;
  • 배순식 (부산대학교 의학전문대학원 약리학교실)
  • Kim, Eun Kyoung (MRC for Ischemic Tissue Regeneration, Medical Research Institute, Department of Pharmacology, Pusan National University School of Medicine) ;
  • Lee, Hye Sun (MRC for Ischemic Tissue Regeneration, Medical Research Institute, Department of Pharmacology, Pusan National University School of Medicine) ;
  • Ha, Hong Koo (Department of Urology, Pusan National University Hospital) ;
  • Yun, Sung Ji (MRC for Ischemic Tissue Regeneration, Medical Research Institute, Department of Pharmacology, Pusan National University School of Medicine) ;
  • Ha, Jung Min (MRC for Ischemic Tissue Regeneration, Medical Research Institute, Department of Pharmacology, Pusan National University School of Medicine) ;
  • Kim, Young Whan (MRC for Ischemic Tissue Regeneration, Medical Research Institute, Department of Pharmacology, Pusan National University School of Medicine) ;
  • Jin, In Hye (MRC for Ischemic Tissue Regeneration, Medical Research Institute, Department of Pharmacology, Pusan National University School of Medicine) ;
  • Shin, Hwa Kyoung (Department of Anatomy, Pusan National University School of Korean Medicine) ;
  • Bae, Sun Sik (MRC for Ischemic Tissue Regeneration, Medical Research Institute, Department of Pharmacology, Pusan National University School of Medicine)
  • 투고 : 2012.11.13
  • 심사 : 2012.12.04
  • 발행 : 2012.12.30

초록

세포의 이동은 성장, 면역 작용, 그리고 혈관 신생 등 많은 생리현상에 중요한 역할을 한다. 또한 염증 및 종양 세포 침윤 등의 다양한 병리적 현상과도 밀접한 연관이 있다. 본 연구에서는 lysophosphatidic acid (LPA)는 활성산소의 생성을 통해 SKOV-3 난소암세포의 이동을 조절한다는 것을 관찰하였다. 먼저, 난소 암세포인 SKOV-3에서 LPA에 의한 세포의 이동이 강하게 일어남을 확인하였다. LPA에 의한 SKOV-3 세포의 이동은 phosphatidylinositol 3-kinase (PI3K)/Akt 신호전달체계를 저해시키는 약물에 의해서 완벽히 억제됨을 확인하였으나 ERK 신호전달체계를 저해시키는 약물에 의해서는 전혀 영향을 받지 않았다. 그리고 SKOV-3 세포에서 LPA에 의한 활성산소 형성이 시간에 따라 강하게 일어남을 확인하였다. 더욱이 LPA에 의한 활성산소 형성도 PI3K 또는 Akt의 저해제에 의해서 완벽히 억제됨을 확인하였으나 ERK 신호전달을 억제하였을 때는 거의 영향을 받지 않았다. SKOV-3 세포에서 LPA에 의해 생성된 활성산소는 diphenylene idonium (DPI, $10{\mu}M$), apocyanin (Apo, $10{\mu}M$)과 같은 NADPH oxidase 억제제를 전 처리하였을 때 활성산소가 형성되지 못함을 관찰하였다. 그러나 xanthine oxidase (allopurinol, Allo, $10{\mu}M$), cyclooxygenase (indomethacin, Indo, $10{\mu}M$), 또는 mitochondrial respiratory chain complex I (rotenone, Rot, $10{\mu}M$)를 억제하였을 때는 LPA에 의한 활성산소 형성에 영향을 주지 못함을 확인하였다. 마지막으로 활성산소 억제제인 N-acetylcysteine (NAC, $10{\mu}M$)에 의해서 LPA에 의한 암세포의 이동이 억제됨을 관찰하였다. 이와 더불어 LPA에 의한 SKOV-3 세포의 이동도 NADPH oxidase 억제에 의해 저해가 됨을 확인하였다. 이러한 연구결과로 보아 LPA에 의한 활성산소의 형성에는 PI3K/Akt/NADPH oxidase 신호전달체계가 중추적인 역할을 하며 이를 통해 암세포의 이동을 조절한다는 것을 알 수 있었다.

Cell motility plays an essential role in many physiological responses, such as development, immune reaction, and angiogenesis. In the present study, we showed that lysophosphatidic acid (LPA) modulates cancer cell migration by regulation of generation of reactive oxygen species (ROS). Stimulation of SKOV-3 ovarian cancer cells with LPA strongly promoted migration. but this migration was completely blocked by pharmacological inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Inhibition of the ERK pathway had no effect on migration. Stimulation of SKOV-3 ovarian cancer cells with LPA significantly induced the generation of ROS in a time-dependent manner. LPA-induced generation of ROS was significantly blocked by pharmacological inhibition of PI3K or Akt, but inhibition of the ERK signaling pathway had little effect. LPA-induced generation of ROS was blocked by pretreatment of SKOV-3 ovarian cancer cells with an NADPH oxidase inhibitor, whereas inhibition of xanthine oxidase, cyclooxygenase, or mitochondrial respiratory chain complex I had no effect. Scavenging of ROS by N-acetylcysteine completely blocked LPA-induced migration of SKOV-3 ovarian cancer cells. Inhibition of NADPH oxidase blocked LPA-induced migration whereas inhibition of xanthine oxidase, cyclooxygenase, or mitochondrial respiratory chain complex I did not affect LPA-induced migration of SKOV-3 ovarian cancer cells. Given these results, we suggest that LPA induces ROS generation through the PI3K/Akt/NADPH oxidase signaling axis, thereby regulating cancer cell migration.

키워드

참고문헌

  1. Baumer, A. T., Ten Freyhaus, H., Sauer, H., Wartenberg, M., Kappert, K., Schnabel, P., Konkol, C., Hescheler, J., Vantler, M., and Rosenkranz, S. 2008. Phosphatidylinositol 3-kinase-dependent membrane recruitment of Rac-1 and p47phox is critical for ${\alpha}$-platelet-derived growth factor receptor-induced production of reactive oxygen species. J. Biol. Chem. 283, 7864-7876. https://doi.org/10.1074/jbc.M704997200
  2. Bian, D., Su, S., Mahanivong, C., Cheng, R. K., Han, Q., Pan, Z. K., Sun, P., and Huang, S. 2004. Lysophosphatidic Acid Stimulates Ovarian Cancer Cell Migration via a Ras-MEK Kinase 1 Pathway. Cancer Res. 64, 4209-4217. https://doi.org/10.1158/0008-5472.CAN-04-0060
  3. Chen, Q., Olashaw, N. and Wu, J. 1995. Participation of reactive oxygen species in the lysophosphatidic acid-stimulated mitogen-activated protein kinase kinase activation pathway. J. Biol. Chem. 270, 28499-28502. https://doi.org/10.1074/jbc.270.48.28499
  4. Chiarugi, P. 2008. Src redox regulation: there is more than meets the eye. Mol. Cells 26, 329-337.
  5. Contos, J. J., Ishii, I. and Chun, J. 2000. Lysophosphatidic acid receptors. Mol. Pharmacol. 58, 1188-1196.
  6. Dujardin, D. L., Barnhart, L. E., Stehman, S. A., Gomes, E. R., Gundersen, G. G. and Vallee, R. B. 2003. A role for cytoplasmic dynein and LIS1 in directed cell movement. J. Cell Biol. 163, 1205-1211. https://doi.org/10.1083/jcb.200310097
  7. Etienne-Manneville, S. and Hall, A. 2003. Cdc42 regulates GSK-3${\beta}$ and adenomatous polyposis coli to control cell polarity. Nature 421, 753-756. https://doi.org/10.1038/nature01423
  8. Fishman, D. A., Liu, Y., Ellerbroek, S. M. and Stack, M. S. 2001. Lysophosphatidic acid promotes matrix metalloproteinase (MMP) activation and MMP-dependent invasion in ovarian cancer cells. Cancer Res. 61, 3194-3199.
  9. Han, J., Luby-Phelps, K., Das, B., Shu, X., Xia, Y., Mosteller, R. D., Krishna, U. M., Falck, J. R., White, M. A. and Broek, D. 1998. Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav. Science 279, 558-560. https://doi.org/10.1126/science.279.5350.558
  10. Hernandez-Negrete, I., Carretero-Ortega, J., Rosenfeldt, H., Hernandez-Garcia, R., Calderon-Salinas, J. V., Reyes-Cruz, G., Gutkind, J. S., and Vazquez-Prado, J. 2007. P-Rex1 links mammalian target of rapamycin signaling to Rac activation and cell migration. J. Biol. Chem. 282, 23708-23715. https://doi.org/10.1074/jbc.M703771200
  11. Higuchi, M., Masuyama, N., Fukui, Y., Suzuki, A. and Gotoh, Y. 2001. Akt mediates Rac/Cdc42-regulated cell motility in growth factor-stimulated cells and in invasive PTEN knockout cells. Curr. Biol. 11, 1958-1962. https://doi.org/10.1016/S0960-9822(01)00599-1
  12. Hill, K., Krugmann, S., Andrews, S. R., Coadwell, W. J., Finan, P., Welch, H. C., Hawkins, P. T. and Stephens, L. R. 2005. Regulation of P-Rex1 by phosphatidylinositol (3,4,5)-trisphosphate and $G{\beta}{\gamma}$ subunits. J. Biol. Chem. 280, 4166-4173. https://doi.org/10.1074/jbc.M411262200
  13. Huo, Y., Qiu, W. Y., Pan, Q., Yao, Y. F., Xing, K. and Lou, M. F. 2009. Reactive oxygen species (ROS) are essential mediators in epidermal growth factor (EGF)-stimulated corneal epithelial cell proliferation, adhesion, migration, and wound healing. Exp. Eye Res. 89, 876-886. https://doi.org/10.1016/j.exer.2009.07.012
  14. Imamura, F., Mukai, M., Ayaki, M., Takemura, K., Horai, T., Shinkai, K., Nakamura, H. and Akedo, H. 1999. Involvement of small GTPases Rho and Rac in the invasion of rat ascites hepatoma cells. Clin. Exp. Metastasis 17, 141-148. https://doi.org/10.1023/A:1006598531238
  15. Kim, E. K., Yun, S. J., Ha, J. M., Kim, Y. W., Jin, I. H., Woo, D. H., Lee, H. S., Ha, H. K. and Bae, S. S. 2012. Synergistic induction of cancer cell migration regulated by $G{\beta}{\gamma}$ and phosphatidylinositol 3-kinase. Exp. Mol. Med. 44, 483-491. https://doi.org/10.3858/emm.2012.44.8.055
  16. Kim, E. K., Yun, S. J., Ha, J. M., Kim, Y. W., Jin, I. H., Yun, J., Shin, H. K., Song, S. H., Kim, J. H., Lee, J. S., Kim, C. D. and Bae, S. S. 2011. Selective activation of Akt1 by mammalian target of rapamycin complex 2 regulates cancer cell migration, invasion, and metastasis. Oncogene 30, 2954-2963. https://doi.org/10.1038/onc.2011.22
  17. Koh, J. S., Lieberthal, W., Heydrick, S. and Levine, J. S. 1998. Lysophosphatidic acid is a major serum noncytokine survival factor for murine macrophages which acts via the phosphatidylinositol 3-kinase signaling pathway. J. Clinic. Invest. 102, 716-727. https://doi.org/10.1172/JCI1002
  18. Lauffenburger, D. A. and Horwitz, A. F. 1996. Cell migration: a physically integrated molecular process. Cell 84, 359-369. https://doi.org/10.1016/S0092-8674(00)81280-5
  19. Liou, G. Y. and Storz, P. 2010. Reactive oxygen species in cancer. Free Radic. Res. 44, 479-496. https://doi.org/10.3109/10715761003667554
  20. Maffucci, T., Cooke, F. T., Foster, F. M., Traer, C. J., Fry, M. J. and Falasca, M. 2005. Class II phosphoinositide 3-kinase defines a novel signaling pathway in cell migration. J. Cell Biol. 169, 789-799. https://doi.org/10.1083/jcb.200408005
  21. Malchinkhuu, E., Sato, K., Horiuchi, Y., Mogi, C., Ohwada, S., Ishiuchi, S., Saito, N., Kurose, H., Tomura, H. and Okajima, F. 2005. Role of p38 mitogen-activated kinase and c-Jun terminal kinase in migration response to lysophosphatidic acid and sphingosine-1-phosphate in glioma cells. Oncogene 24, 6676-6688. https://doi.org/10.1038/sj.onc.1208805
  22. Meng, D., Lv, D. D. and Fang, J. 2008. Insulin-like growth factor-I induces reactive oxygen species production and cell migration through Nox4 and Rac1 in vascular smooth muscle cells. Cardiovasc. Res. 80, 299-308. https://doi.org/10.1093/cvr/cvn173
  23. Oikawa, T., Yamaguchi, H., Itoh, T., Kato, M., Ijuin, T., Yamazaki, D., Suetsugu, S. and Takenawa, T. 2004. PtdIns(3,4,5)P3 binding is necessary for WAVE2-induced formation of lamellipodia. Nat. Cell Biol. 6, 420-426. https://doi.org/10.1038/ncb1125
  24. Panetti, T. S., Nowlen, J. and Mosher, D. F. 2000. Sphingosine-1-phosphate and lysophosphatidic acid stimulate endothelial cell migration. Arterioscler. Thromb. Vasc. Biol. 20, 1013-1019. https://doi.org/10.1161/01.ATV.20.4.1013
  25. Pietruck, F., Busch, S., Virchow, S., Brockmeyer, N. and Siffert, W. 1997. Signalling properties of lysophosphatidic acid in primary human skin fibroblasts: role of pertussis toxin-sensitive GTP-binding proteins. Naunyn-Schmiedebergs Arch. Pharmacol. 355, 1-7.
  26. Raftopoulou, M. and Hall, A. 2004. Cell migration: Rho GTPases lead the way. Dev. Biol. 265, 23-32. https://doi.org/10.1016/j.ydbio.2003.06.003
  27. Rhee, S. G., Kang, S. W., Jeong, W., Chang, T. S., Yang, K. S. and Woo, H. A. 2005. Intracellular messenger function of hydrogen peroxide and its regulation by peroxiredoxins. Curr. Opin. Cell Biol. 17, 183-189. https://doi.org/10.1016/j.ceb.2005.02.004
  28. Ridley, A. J., Schwartz, M. A., Burridge, K., Firtel, R. A., Ginsberg, M. H., Borisy, G., Parsons, J. T. and Horwitz, A. R. 2003. Cell migration: integrating signals from front to back. Science 302, 1704-1709. https://doi.org/10.1126/science.1092053
  29. Russo, C., Gao, Y., Mancini, P., Vanni, C., Porotto, M., Falasca, M., Torrisi, M. R., Zheng, Y. and Eva, A. 2001. Modulation of oncogenic DBL activity by phosphoinositol phosphate binding to pleckstrin homology domain. J. Biol. Chem. 276, 19524-19531. https://doi.org/10.1074/jbc.M009742200
  30. Sasaki, A. T., Chun, C., Takeda, K. and Firtel, R. A. 2004. Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement. J. Cell Biol. 167, 505-518. https://doi.org/10.1083/jcb.200406177
  31. Saunders, J. A., Rogers, L. C., Klomsiri, C., Poole, L. B. and Daniel, L. W. 2010. Reactive oxygen species mediate lysophosphatidic acid induced signaling in ovarian cancer cells. Free Radic. Biol. Med. 49, 2058-2067. https://doi.org/10.1016/j.freeradbiomed.2010.10.663
  32. Shah, B. H., Neithardt, A., Chu, D. B., Shah, F. B. and Catt, K. J. 2006. Role of EGF receptor transactivation in phosphoinositide 3-kinase-dependent activation of MAP kinase by GPCRs. J. Cell. Physiol. 206, 47-57. https://doi.org/10.1002/jcp.20423
  33. Srinivasan, S., Wang, F., Glavas, S., Ott, A., Hofmann, F., Aktories, K., Kalman, D. and Bourne, H. R. 2003. Rac and Cdc42 play distinct roles in regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis. J. Cell Biol. 160, 375-385. https://doi.org/10.1083/jcb.200208179
  34. Weiner, O. D. 2002. Rac activation: P-Rex1 - a convergence point for PIP(3) and $G{\beta}{\gamma}$? Curr. Biol. 12, R429-431. https://doi.org/10.1016/S0960-9822(02)00917-X
  35. Weiner, O. D. 2002. Regulation of cell polarity during eukaryotic chemotaxis: the chemotactic compass. Curr. Opin. Cell Biol. 14, 196-202. https://doi.org/10.1016/S0955-0674(02)00310-1
  36. Welch, H. C., Coadwell, W. J., Ellson, C. D., Ferguson, G. J., Andrews, S. R., Erdjument-Bromage, H., Tempst, P., Hawkins, P. T. and Stephens, L. R. 2002. P-Rex1, a PtdIns(3,4,5)P3- and $G{\beta}{\gamma}$-regulated guanine-nucleotide exchange factor for Rac. Cell 108, 809-821. https://doi.org/10.1016/S0092-8674(02)00663-3
  37. Xu, Y., Shen, Z., Wiper, D. W., Wu, M., Morton, R. E., Elson, P., Kennedy, A. W., Belinson, J., Markman, M. and Casey, G. 1998. Lysophosphatidic acid as a potential biomarker for ovarian and other gynecologic cancers. JAMA 280, 719-723. https://doi.org/10.1001/jama.280.8.719