Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Ceramide synthase 6 modulates TRAIL sensitivity and nuclear translocation of active caspase-3 in colon cancer cells

Oncogene volume 28, pages 1132–1141 (2009)Cite this article

Abstract

We have previously shown that the death receptor ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces an increase of intracellular C16-ceramide in sensitive SW480 but not in resistant SW620 cells. Resistance in SW620 cells was overcome by exogenous ceramide, leading us to propose that defective ceramide signaling contributes to TRAIL resistance. In this study we found that the increase in C16-ceramide in SW480 cells was inhibited by fumonisin B1, an inhibitor of ceramide synthases (CerS). Protein analysis revealed that TRAIL-resistant SW620 cells expressed lower levels of ceramide synthase 6 (CerS6, also known as longevity assurance homologue 6), which prompted us to investigate the effect of CerS6 modulation on TRAIL phenotype. RNAi against CerS6 resulted in a specific and significant decrease of the C16-ceramide species, which was sufficient to inhibit TRAIL-induced apoptosis. In cells with decreased levels of CerS6, caspase-3 was activated but failed to translocate into the nucleus. CerS6 localized primarily to the perinuclear region, suggesting this enzyme may be important in regulation of nuclear permeability. Moderate elevation in CerS6 expression was sufficient to reverse TRAIL resistance in SW620 cells. These results suggest that modulation of CerS6 expression may constitute a new therapeutic strategy to alter apoptotic susceptibility.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9

Similar content being viewed by others

References

  • Ashkenazi A, Herbst RS . (2008). To kill a tumor cell: the potential of proapoptotic receptor agonists. J Clin Invest 118: 1979–1990.

    Article  CAS  Google Scholar 

  • Barua S, Joshi A, Banerjee A, Matthews D, Sharfstein ST, Cramer SM et al. (2008). Parallel synthesis and screening of polymers for non-viral gene delivery. Mol Pharm (in press); doi:10.1021/mp80057j.

  • Basnakian AG, Ueda N, Hong X, Galitovsky VE, Yin X, Shah SV . (2005). Ceramide synthase is essential for endonuclease-mediated death of renal tubular epithelial cells induced by hypoxia-reoxygenation. Am J Physiol Renal Physiol 288: F308–F314.

    Article  CAS  Google Scholar 

  • Beckman M, Kihlmark M, Iverfeldt K, Hallberg E . (2004). Degradation of GFP-labelled POM121, a non-invasive sensor of nuclear apoptosis, precedes clustering of nuclear pores and externalisation of phosphatidylserine. Apoptosis 9: 363–368.

    Article  CAS  Google Scholar 

  • Birbes H, Luberto C, Hsu Y, El Bawab S, Hannun Y, Obeid L . (2005). A mitochondrial pool of sphingomyelin is involved in TNFalpha-induced Bax translocation to mitochondria. Biochem J 386: 445–451.

    Article  CAS  Google Scholar 

  • Bose R, Verheij M, Haimovitz-Friedman A, Scotto K, Fuks Z, Kolesnick R . (1995). Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals. Cell 82: 405–414.

    Article  CAS  Google Scholar 

  • Charles AG, Han TY, Liu YY, Hansen N, Giuliano AE, Cabot MC . (2001). Taxol-induced ceramide generation and apoptosis in human breast cancer cells. Cancer Chemother Pharmacol 47: 444–450.

    Article  CAS  Google Scholar 

  • Cremesti A, Paris F, Grassme H, Holler N, Tschopp J, Fuks Z et al. (2001). Ceramide enables fas to cap and kill. J Biol Chem 276: 23954–23961.

    Article  CAS  Google Scholar 

  • de Jong S, Timmer T, Heijenbrok FJ, de Vries EG . (2001). Death receptor ligands, in particular TRAIL, to overcome drug resistance. Cancer Metastasis Rev 20: 51–56.

    Article  CAS  Google Scholar 

  • Dohi T, Okada K, Xia F, Wilford CE, Samuel T, Welsh K et al. (2004). An IAP-IAP complex inhibits apoptosis. J Biol Chem 279: 34087–34090.

    Article  CAS  Google Scholar 

  • Eto M, Bennouna J, Hunter OC, Hershberger PA, Kanto T, Johnson CS et al. (2003). C16 ceramide accumulates following androgen ablation in LNCaP prostate cancer cells. Prostate 57: 66–79.

    Article  CAS  Google Scholar 

  • Faleiro L, Lazebnik Y . (2000). Caspases disrupt the nuclear-cytoplasmic barrier. J Cell Biol 151: 951–959.

    Article  CAS  Google Scholar 

  • Ferrando-May E, Cordes V, Biller-Ckovric I, Mirkovic J, Gorlich D, Nicotera P . (2001). Caspases mediate nucleoporin cleavage, but not early redistribution of nuclear transport factors and modulation of nuclear permeability in apoptosis. Cell Death Differ 8: 495–505.

    Article  CAS  Google Scholar 

  • Garzotto M, Haimovitz-Friedman A, Liao WC, White-Jones M, Huryk R, Heston WD et al. (1999). Reversal of radiation resistance in LNCaP cells by targeting apoptosis through ceramide synthase. Cancer Res 59: 5194–5201.

    CAS  PubMed  Google Scholar 

  • Gulbins E, Kolesnick R . (2003). Raft ceramide in molecular medicine. Oncogene 22: 7070–7077.

    Article  CAS  Google Scholar 

  • Huerta S, Heinzerling JH, Anguiano-Hernandez YM, Huerta-Yepez S, Lin J, Chen D et al. (2007). Modification of gene products involved in resistance to apoptosis in metastatic colon cancer cells: roles of Fas, Apaf-1, NFkappaB, IAPs, Smac/DIABLO, and AIF. J Surg Res 142: 184–194.

    Article  CAS  Google Scholar 

  • Jaattela M, Wissing D, Kokholm K, Kallunki T, Egeblad M . (1998). Hsp70 exerts its anti-apoptotic function downstream of caspase-3-like proteases. EMBO J 17: 6124–6134.

    Article  CAS  Google Scholar 

  • Kamada S, Kikkawa U, Tsujimoto Y, Hunter T . (2005a). A-kinase-anchoring protein 95 functions as a potential carrier for the nuclear translocation of active caspase 3 through an enzyme-substrate-like association. Mol Cell Biol 25: 9469–9477.

    Article  CAS  Google Scholar 

  • Kamada S, Kikkawa U, Tsujimoto Y, Hunter T . (2005b). Nuclear translocation of caspase-3 is dependent on its proteolytic activation and recognition of a substrate-like protein(s). J Biol Chem 280: 857–860.

    Article  CAS  Google Scholar 

  • Karahatay S, Thomas K, Koybasi S, Senkal CE, Elojeimy S, Liu X et al. (2007). Clinical relevance of ceramide metabolism in the pathogenesis of human head and neck squamous cell carcinoma (HNSCC): attenuation of C(18)-ceramide in HNSCC tumors correlates with lymphovascular invasion and nodal metastasis. Cancer Lett 256: 101–111.

    Article  CAS  Google Scholar 

  • Kelley SK, Harris LA, Xie D, Deforge L, Totpal K, Bussiere J et al. (2001). Preclinical studies to predict the disposition of Apo2L/tumor necrosis factor-related apoptosis-inducing ligand in humans: characterization of in vivo efficacy, pharmacokinetics, and safety. J Pharmacol Exp Ther 299: 31–38.

    CAS  PubMed  Google Scholar 

  • Kitatani K, Idkowiak-Baldys J, Bielawski J, Taha TA, Jenkins RW, Senkal CE et al. (2006). Protein kinase C-induced activation of a ceramide/protein phosphatase 1 pathway leading to dephosphorylation of p38 MAPK. J Biol Chem 281: 36793–36802.

    Article  CAS  Google Scholar 

  • Koschny R, Walczak H, Ganten TM . (2007). The promise of TRAIL—potential and risks of a novel anticancer therapy. J Mol Med 85: 923–935.

    Article  CAS  Google Scholar 

  • Koybasi S, Senkal CE, Sundararaj K, Spassieva S, Bielawski J, Osta W et al. (2004). Defects in cell growth regulation by C18:0-ceramide and longevity assurance gene 1 in human head and neck squamous cell carcinomas. J Biol Chem 279: 44311–44319.

    Article  CAS  Google Scholar 

  • Kroesen BJ, Jacobs S, Pettus BJ, Sietsma H, Kok JW, Hannun YA et al. (2003). BcR-induced apoptosis involves differential regulation of C16 and C24-ceramide formation and sphingolipid-dependent activation of the proteasome. J Biol Chem 278: 14723–14731.

    Article  CAS  Google Scholar 

  • MacFarlane M . (2003). TRAIL-induced signalling and apoptosis. Toxicol Lett 139: 89–97.

    Article  CAS  Google Scholar 

  • Mizutani Y, Kihara A, Igarashi Y . (2005). Mammalian Lass6 and its related family members regulate synthesis of specific ceramides. Biochem J 390: 263–271.

    Article  CAS  Google Scholar 

  • Moulin M, Carpentier S, Levade T, Arrigo AP . (2007). Potential roles of membrane fluidity and ceramide in hyperthermia and alcohol stimulation of TRAIL apoptosis. Apoptosis 12: 1703–1720.

    Article  CAS  Google Scholar 

  • Ndozangue-Touriguine O, Sebbagh M, Merino D, Micheau O, Bertoglio J, Breard J . (2008). A mitochondrial block and expression of XIAP lead to resistance to TRAIL-induced apoptosis during progression to metastasis of a colon carcinoma. Oncogene 27: 6012–6022.

    Article  CAS  Google Scholar 

  • Ogretmen B, Hannun YA . (2004). Biologically active sphingolipids in cancer pathogenesis and treatment. Nat Rev Cancer 4: 604–616.

    Article  CAS  Google Scholar 

  • Ogretmen B, Pettus BJ, Rossi MJ, Wood R, Usta J, Szulc Z et al. (2002). Biochemical mechanisms of the generation of endogenous long chain ceramide in response to exogenous short chain ceramide in the A549 human lung adenocarcinoma cell line. Role for endogenous ceramide in mediating the action of exogenous ceramide. J Biol Chem 277: 12960–12969.

    Article  CAS  Google Scholar 

  • Peter ME . (2004). The flip side of FLIP. Biochem J 382: e1–e3.

    Article  CAS  Google Scholar 

  • Pewzner-Jung Y, Ben-Dor S, Futerman AH . (2006). When do Lasses (longevity assurance genes) become CerS (ceramide synthases)? Insights into the regulation of ceramide synthesis. J Biol Chem 281: 25001–25005.

    Article  CAS  Google Scholar 

  • Provenzani A, Fronza R, Loreni F, Pascale A, Amadio M, Quattrone A . (2006). Global alterations in mRNA polysomal recruitment in a cell model of colorectal cancer progression to metastasis. Carcinogenesis 27: 1323–1333.

    Article  CAS  Google Scholar 

  • Riebeling C, Allegood JC, Wang E, Merrill Jr AH, Futerman AH . (2003). Two mammalian longevity assurance gene (LAG1) family members, trh1 and trh4, regulate dihydroceramide synthesis using different fatty acyl-CoA donors. J Biol Chem 278: 43452–43459.

    Article  CAS  Google Scholar 

  • Selzner M, Bielawska A, Morse MA, Rudiger HA, Sindram D, Hannun YA et al. (2001). Induction of apoptotic cell death and prevention of tumor growth by ceramide analogues in metastatic human colon cancer. Cancer Res 61: 1233–1240.

    CAS  PubMed  Google Scholar 

  • Senkal CE, Ponnusamy S, Rossi MJ, Bialewski J, Sinha D, Jiang JC et al. (2007). Role of human longevity assurance gene 1 and C18-ceramide in chemotherapy-induced cell death in human head and neck squamous cell carcinomas. Mol Cancer Ther 6: 712–722.

    Article  CAS  Google Scholar 

  • Sharpe J, Arnoult D, Youle R . (2004). Control of mitochondrial permeability by Bcl-2 family members. Biochimica et Biophysica Acta 1644: 107–113.

    Article  CAS  Google Scholar 

  • Siskind LJ, Colombini M . (2000). The lipids C2- and C16-ceramide form large stable channels. Implications for apoptosis. J Biol Chem 275: 38640–38644.

    Article  CAS  Google Scholar 

  • Stiban J, Caputo L, Colombini M . (2008). Ceramide synthesis in the endoplasmic reticulum can permeabilize mitochondria to proapoptotic proteins. J Lipid Res 49: 625–634.

    Article  CAS  Google Scholar 

  • Thomas Jr RL, Matsko CM, Lotze MT, Amoscato AA . (1999). Mass spectrometric identification of increased C16 ceramide levels during apoptosis. J Biol Chem 274: 30580–30588.

    Article  CAS  Google Scholar 

  • Tran EJ, Wente SR . (2006). Dynamic nuclear pore complexes: life on the edge. Cell 125: 1041–1053.

    Article  CAS  Google Scholar 

  • Ueda N, Camargo SM, Hong X, Basnakian AG, Walker PD, Shah SV . (2001). Role of ceramide synthase in oxidant injury to renal tubular epithelial cells. J Am Soc Nephrol 12: 2384–2391.

    CAS  PubMed  Google Scholar 

  • Venkataraman K, Riebeling C, Bodennec J, Riezman H, Allegood JC, Sullards MC et al. (2002). Upstream of growth and differentiation factor 1 (uog1), a mammalian homolog of the yeast longevity assurance gene 1 (LAG1), regulates N-stearoyl-sphinganine (C18-(dihydro)ceramide) synthesis in a fumonisin B1-independent manner in mammalian cells. J Biol Chem 277: 35642–35649.

    Article  CAS  Google Scholar 

  • Voelkel-Johnson C, Hannun YA, El-Zawahry A . (2005). Resistance to TRAIL is associated with defects in ceramide signaling that can be overcome by exogenous C6-ceramide without requiring down-regulation of cellular FLICE inhibitory protein. Mol Cancer Ther 4: 1320–1327.

    Article  CAS  Google Scholar 

  • Widlak P, Garrard WT . (2005). Discovery, regulation, and action of the major apoptotic nucleases DFF40/CAD and endonuclease G. J Cell Biochem 94: 1078–1087.

    Article  CAS  Google Scholar 

  • Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK et al. (1995). Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3: 673–682.

    Article  CAS  Google Scholar 

  • Wilson MR . (1998). Apoptosis: unmasking the executioner. Cell Death Differ 5: 646–652.

    Article  CAS  Google Scholar 

  • Xu Z, Zhou J, McCoy DM, Mallampalli RK . (2005). LASS5 is the predominant ceramide synthase isoform involved in de novo sphingolipid synthesis in lung epithelia. J Lipid Res 46: 1229–1238.

    Article  CAS  Google Scholar 

  • Yasuhara N, Eguchi Y, Tachibana T, Imamoto N, Yoneda Y, Tsujimoto Y . (1997). Essential role of active nuclear transport in apoptosis. Genes Cells 2: 55–64.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Rick Peppler and Kylie Martin for assistance with flow cytometry experiments, Dr Jacek Bielawski of the Lipidomics Core Facility for assistance with the mass spectrophotometric characterization of ceramide profiles, Dr Kaushal Rege for providing the EDGE3′3 polymer and Tejas Tirodkar for technical assistance. We also thank Dr Yusuf Hannun for reviewing our data and making insightful suggestions.

This work was supported by 1P20-RR17698 NIH COBRE award to CVJ; R01 NIH/NIA AG016583 and NIH/NCI P01CA097132 awards to LMO; CA-088932, DE016572 and CA-097132 awards to BO; NIH/NGA: 1T32ES012878 ‘Training Program in Environmental Stress Signaling’ to TM and by a scholarship from the Abney Foundation to SWG. The MUSC Lipidomics Core Facility (NIH C06 RR018823) and the Flow Cytometry and Cell Sorting Shared Resource Facility were in part supported by MUSC and the Hollings Cancer Center.

Author information

Authors and Affiliations

  1. Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC, USA

    S White-Gilbertson, P Lu & C Voelkel-Johnson

  2. Department of Medicine, Medical University of South Carolina, Charleston, SC, USA

    T Mullen & L Obeid

  3. Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA

    C Senkal & B Ogretmen

  4. Ralph H Johnson Veterans Affairs Medical Center, Charleston, SC, USA

    L Obeid

Authors
  1. S White-Gilbertson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. T Mullen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C Senkal
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. P Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. B Ogretmen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. L Obeid
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. C Voelkel-Johnson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to C Voelkel-Johnson.

Additional information

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

White-Gilbertson, S., Mullen, T., Senkal, C. et al. Ceramide synthase 6 modulates TRAIL sensitivity and nuclear translocation of active caspase-3 in colon cancer cells. Oncogene 28, 1132–1141 (2009). https://doi.org/10.1038/onc.2008.468

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2008.468

Keywords

This article is cited by

Search

Advanced search

Quick links