N-Acetylphytosphingosine Enhances the Radiosensitivity of Lung Cancer Cell Line NCI-H460

  • Han, Youngsoo (Laboratory of Radiation Sensitization and Protection, Korea Institute of Radiological and Medical Sciences) ;
  • Kim, Kisung (Laboratory of Radiation Sensitization and Protection, Korea Institute of Radiological and Medical Sciences) ;
  • Shim, Ji-Young (Laboratory of Radiation Sensitization and Protection, Korea Institute of Radiological and Medical Sciences) ;
  • Park, Changsoe (Doosan Biotech BU) ;
  • Song, Jie-Young (Laboratory of Radiation Sensitization and Protection, Korea Institute of Radiological and Medical Sciences) ;
  • Yun, Yeon-Sook (Laboratory of Radiation Sensitization and Protection, Korea Institute of Radiological and Medical Sciences)
  • Received : 2007.07.16
  • Accepted : 2007.11.13
  • Published : 2008.04.30


Ceramides are well-known second messengers that induce apoptosis in various kinds of cancer cells, and their effects are closely related to radiation sensitivity. Phytoceramides, the yeast counterparts of the mammalian ceramides, are also reported to induce apoptosis. We investigated the effect of a novel ceramide derivative, N-acetylphytosphingosine (NAPS), on the radiosensitivity of NCI-H460 human lung carcinoma cells and its differential cytotoxicity in tumor and normal cells. The combination of NAPS with radiation significantly increased clonogenic cell death and caspase-dependent apoptosis. The combined treatment greatly increased Bax expression and Bid cleavage, but not Bcl-2 expression. However, there was no effect on radiosensitivity and apoptosis in BEAS2B cells, which derive from normal human bronchial epithelium. Cell proliferation and DNA synthesis were significantly inhibited by NAPS in both NCI-H460 and BEAS2B cells, but only the BEAS2B cells recovered by 48h after removal of the NAPS. Furthermore, the NCI-H460 cells underwent more DNA fragmentation than the BEAS2B cells in response to NAPS. Our results indicate that NAPS may be a potential radiosensitizing agent with differential effects on tumor vs. normal cells.


Apoptosis;Differential Effect;N-acetyl Phytosphingosine;Radiation;Sensitivity


Supported by : Korea Science and Engineering Foundation (KOSEF)


  1. Dagan, A., Wang, C., Fibach, E., and Gatt, S. (2003). Synthetic, non-natural sphingolipid analogs inhibit the biosynthesis of cellular sphingolipids, elevate ceramide and induce apoptotic cell death. Biochim. Biophys. Acta 1633, 161-169 https://doi.org/10.1016/S1388-1981(03)00122-7
  2. Ling, C.C., Chen, C.H., and Li, W.X. (1994). Apoptosis induced at different dose rates: implication for the shoulder region of cell survival curves. Radiother. Oncol. 32, 129-136 https://doi.org/10.1016/0167-8140(94)90099-X
  3. Mao, C., Saba, J.D., and Obeid, L.M. (1999). The dihydrosphingosine-1-phosphate phosphatases of Saccharomyces cerevisiae are important regulators of cell proliferation and heat stress responses. Biochem. J. 342, 667-675 https://doi.org/10.1042/0264-6021:3420667
  4. Rodriguez-Lafrasse, C., Alphonse, G., Aloy, M.T., Ardail, D., Gerard, J.P., Louisoot, P., and Rousson, R. (2002). Increasing endogenous ceramide using inhibitors of sphingolipid metabolism maximizes ionizing radiation-induced mitochondrial injury and apoptotic cell killing. Int. J. Cancer. 101, 589-598 https://doi.org/10.1002/ijc.10652
  5. Sheard, M.A., Uldrijan, S., and Vojtesek, B. (2003). Role of p53 in regulating constitutive and X-radiation-inducible CD95 expression and function in carcinoma cells. Cancer Res. 63, 7176-7184
  6. Radin, N.S. (2003). Killing tumours by ceramide-induced apoptosis: a critique of available drugs. Biochem. J. 371, 243-256 https://doi.org/10.1042/BJ20021878
  7. Szostak, M.J., and Kyprianou, N. (2000). Radiation-induced apoptosis: predictive and therapeutic significance in radiotherapy of prostate cancer (review). Oncol. Rep. 7, 699-706
  8. Woynarowska, B.A., Woynarowski, J.M., Herzig, M.C., Roberts, K., Higdon, A.L., and MacDonald, J.R. (2000). Differential cytotoxicity and induction of apoptosis in tumor and normal cells by hydroxymethylacylfulvene (HMAF). Biochem. Pharmacol. 59, 1217-1226 https://doi.org/10.1016/S0006-2952(00)00254-9
  9. Struckhoff, A.P., Bittman, R., Burow, M.E., Clejan, S., Elliott, S., Hammond, T., Tang, Y., and Beckman, B.S. (2004). Novel ceramide analogs as potential chemotherapeutic agents in breast cancer. J. Pharmacol. Exp. Ther. 309, 523-532 https://doi.org/10.1124/jpet.103.062760
  10. Nava, V.E., Cuvillier, O., Edsall, L.C., Kimura, K., Milstien, S., Gelmann, E.P., and Spiegel, S. (2000). Sphingosine enhances apoptosis of radiation-resistant prostate cancer cells. Cancer Res. 60, 4468-4474
  11. Wang, X.Z., Beebe, J.R., Pwiti, L., Bielawska, A., and Smyth, M.J. (1999). Aberrant sphingolipid signaling is involved in the resistance of prostate cancer cell lines to chemotherapy. Cancer Res. 59, 5842-5848
  12. Fan, T.J., Han, L.H., Cong, R.S., and Liang, J. (2005). Caspase family proteases and apoptosis. Acta Biochim. Biophys. Sin. (Shanghai) 37, 719-727 https://doi.org/10.1111/j.1745-7270.2005.00108.x
  13. Jaffrezou, J.P., Bruno, A.P., Moisand, A., Levade, T., and Laurent, G. (2001). Activation of a nuclear sphingomyelinase in radiation- induced apoptosis. FASEB J. 15, 123-133 https://doi.org/10.1096/fj.00-0305com
  14. Kim, H.J., Shin, W., Park, C.S., Kim, H.O., and Kim, T.Y. (2003). Differential regulation of cyclooxygenase-2 expres-sion by phytosphingosine derivatives, NAPS and TAPS, and its role in the NAPS or TAPS-mediated apoptosis. J. Invest. Dermatol. 121, 1126-1134 https://doi.org/10.1046/j.1523-1747.2003.12554.x
  15. Kolesnick, R. (2002). The therapeutic potential of modulating the ceramide/sphingomyelin pathway. J. Clin. Invest. 110, 3-8 https://doi.org/10.1172/JCI0216127
  16. Lee, J.S., Min, D.S., Park, C., Park, C.S., and Cho, N.J. (2001). Phytosphingosine and C2-phytoceramide induce cell death and inhibit carbachol-stimulated phospholipase D activation in Chinese hamster ovary cells expressing the Caenorhabditis elegans muscarinic acetylcholine receptor. FEBS Lett. 499, 82-86 https://doi.org/10.1016/S0014-5793(01)02527-3
  17. Woynarowski, J.M., Napier, C., Koester, S.K., Chen, S.F., Troyer, D., Chapman, W., and MacDonald, J.R. (1997). Effects on DNA integrity and apoptosis induction by a novel antitumor sesquiterpene drug, 6-hydroxymethylacylfulvene (HMAF, MGI 114). Biochem. Pharmacol. 54, 1181-1193 https://doi.org/10.1016/S0006-2952(97)00321-3
  18. Konopleva, M., Zhao, S., Xie, Z., Segall, H., Younes, A., Claxton, D.F., Estrov, Z., Kornblau, S.M., and Andreeff, M. (1999). Apoptosis. Molecules and mechanisms. Adv. Exp. Med. Biol. 457, 217-236
  19. Han, Y., Kim, Y., Kang, H., Hong, S.H., Kim, Y.H., Lim, D.S., Park, C., Yun, Y.S., and Song, J.Y. (2006). N-acetylphytosphingosine- induced apoptosis of Jurkat cells is mediated by the conformational change in Bak. Apoptosis 11, 581-588 https://doi.org/10.1007/s10495-006-4569-5
  20. Selzner, M., Bielawska, A., Morse, M.A., Rudiger, H.A., Sindram, D., Hannum, Y.A., and Clavien, P.A. (2001). Induction of apoptotic cell death and prevention of tumor growth by ceramide analogues in metastatic human colon cancer. Cancer Res. 61, 1233-1240
  21. Zhivotovsky, B., Joseph, B., and Orrenius, S. (1999). Tumor radiosensitivity and apoptosis. Exp. Cell Res. 248, 10-17 https://doi.org/10.1006/excr.1999.4452
  22. Mathias, S., Pena, L.A., and Kolesnick, R.N. (1998). Signal transduction of stress via ceramide. Biochem. J. 335, 465-480 https://doi.org/10.1042/bj3350465
  23. Thornborrow, E.C., and Manfredi, J.J. (2001). The tumor suppressor protein p53 requires a cofactor to activate transcriptionally the human BAX promoter. J. Biol. Chem. 276, 15598-15608 https://doi.org/10.1074/jbc.M011643200
  24. Skrzypek, M.S., Nagiec, M.M., Lester, R.L., and Dickson, R.C. (1999). Analysis of phosphorylated sphingolipid long-chain bases reveals potential roles in heat stress and growth control in Saccharomyces. J. Bacteriol. 181, 1134-1140
  25. von Haefen, C., Wieder, T., Gillissen, B., Starck, L., Graupner, V., Dorken, B., and Daniel, P.T. (2002). Ceramide induces mitochondrial activation and apoptosis via a Bax-dependent pathway in human carcinoma cells. Oncogene 21, 4009-4019 https://doi.org/10.1038/sj.onc.1205497
  26. Hwang, O., Kim, G., Jang, Y.J., Kim, S.W., Choi, G., Choi, H.J., Jeon, S.Y., Lee, D.G., and Lee, J.D. (2001). Synthetic phytoceramides induce apoptosis with higher potency than ceramides. Mol. Pharmacol. 59, 1249-1255 https://doi.org/10.1124/mol.59.5.1249
  27. Macchia, M., Barontini, S., Bertini, S., Di Bussolo, V., Fogli, S., Giovannetti, E., Grossi, E., Minutolo, F., and Danesi, R. (2001). Design, synthesis, and characterization of the antitumor activity of novel ceramide analogues. J. Med. Chem. 44, 3994-4000 https://doi.org/10.1021/jm010947r