Skip to main content

Advertisement

SpringerLink
Log in

The plant alkaloid and anti-leukemia drug homoharringtonine sensitizes resistant human colorectal carcinoma cells to TRAIL-induced apoptosis via multiple mechanisms

  • Original Paper
  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

TNF-related apoptosis-inducing ligand (TRAIL) is a pro-apoptotic ligand from the TNF-alpha family that is under consideration, along with agonistic anti-TRAIL receptor antibodies, as a potential anti-tumor agent. However, most primary human tumors are resistant to monotherapy with TRAIL apoptogens, and thus the potential applicability of TRAIL in anti-tumor therapy ultimately depends on its rational combination with drugs targeting these resistances. In our high-throughput screening for novel agents/drugs that could sensitize TRAIL-resistant colorectal cancer cells to TRAIL-induced apoptosis, we found homoharringtonine (HHT), a cephalotaxus alkaloid and tested anti-leukemia drug, to be a very effective, low nanomolar enhancer of TRAIL-mediated apoptosis/growth suppression of these resistant cells. Co-treatment of TRAIL-resistant RKO or HT-29 cells with HHT and TRAIL led to the effective induction of apoptosis and the complete elimination of the treated cells. HHT suppressed the expression of the anti-apoptotic proteins Mcl-1 and cFLIP and enhanced the TRAIL-triggered activation of JNK and p38 kinases. The shRNA-mediated down-regulation of cFLIP or Mcl-1 in HT-29 or RKO cells variably enhanced their TRAIL-induced apoptosis but it did not markedly sensitize them to TRAIL-mediated growth suppression. However, with the notable exception of RKO/sh cFLIP cells, the downregulation of cFLIP or Mcl-1 significantly lowered the effective concentration of HHT in HHT + TRAIL co-treatment. Combined HHT + TRAIL therapy also led to the strong suppression of HT-29 tumors implanted into immunodeficient mice. Thus, HHT represents a very efficient enhancer of TRAIL-induced apoptosis with potential application in TRAIL-based, anti-cancer combination therapy.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Venugopal B, Evans TR (2011) Developing histone deacetylase inhibitors as anti-cancer therapeutics. Curr Med Chem 18:1658–1671

    Article  PubMed  CAS  Google Scholar 

  2. Schumacher M, Kelkel M, Dicato M, Diederich M (2011) A survey of marine natural compounds and their derivatives with anti-cancer activity reported in 2010. Molecules 16:5629–5646

    Article  PubMed  CAS  Google Scholar 

  3. Scheinberg DA, Villa CH, Escorcia FE, McDevitt MR (2010) Conscripts of the infinite armada: systemic cancer therapy using nanomaterials. Nat Rev Clin Oncol 7:266–276

    Article  PubMed  CAS  Google Scholar 

  4. Biasutto L, Dong LF, Zoratti M, Neuzil J (2010) Mitochondrially targeted anti-cancer agents. Mitochondrion 10:670–681

    Article  PubMed  CAS  Google Scholar 

  5. Deckert PM (2009) Current constructs and targets in clinical development for antibody-based cancer therapy. Curr Drug Targets 10:158–175

    Article  PubMed  CAS  Google Scholar 

  6. Yerbes R, Palacios C, Lopez-Rivas A (2011) The therapeutic potential of TRAIL receptor signalling in cancer cells. Clin Transl Oncol 13:839–847

    Article  PubMed  CAS  Google Scholar 

  7. Bernardi S, Secchiero P, Zauli G (2012) State of art and recent developments of anti-cancer strategies based on TRAIL. Recent Pat Anticancer Drug Discov 7:207–217

    Article  PubMed  CAS  Google Scholar 

  8. Abdulghani J, El-Deiry WS (2010) TRAIL receptor signaling and therapeutics. Expert Opin Ther Targets 14:1091–1108

    Article  PubMed  CAS  Google Scholar 

  9. Pennarun B, Meijer A, de Vries EG, Kleibeuker JH, Kruyt F, de Jong S (2010) Playing the DISC: turning on TRAIL death receptor-mediated apoptosis in cancer. Biochim Biophys Acta 1805:123–140

    PubMed  CAS  Google Scholar 

  10. Gonzalvez F, Ashkenazi A (2010) New insights into apoptosis signaling by Apo2L/TRAIL. Oncogene 29:4752–4765

    Article  PubMed  CAS  Google Scholar 

  11. Fu K, Ren H, Wang Y, Fei E, Wang H, Wang G (2011) DJ-1 inhibits TRAIL-induced apoptosis by blocking pro-caspase-8 recruitment to FADD. Oncogene 31:1311–1322

    Article  PubMed  Google Scholar 

  12. Cao X, Pobezinskaya YL, Morgan MJ, Liu ZG (2011) The role of TRADD in TRAIL-induced apoptosis and signaling. Faseb J 25:1353–1358

    Article  PubMed  CAS  Google Scholar 

  13. Sun M, Song L, Li Y, Zhou T, Jope RS (2008) Identification of an antiapoptotic protein complex at death receptors. Cell Death Differ 15:1887–1900

    Article  PubMed  CAS  Google Scholar 

  14. Mulherkar N, Prasad KV, Prabhakar BS (2007) MADD/DENN splice variant of the IG20 gene is a negative regulator of caspase-8 activation. Knockdown enhances TRAIL-induced apoptosis of cancer cells. J Biol Chem 282:11715–11721

    Article  PubMed  CAS  Google Scholar 

  15. Xiao C, Yang BF, Asadi N, Beguinot F, Hao C (2002) Tumor necrosis factor-related apoptosis-inducing ligand-induced death-inducing signaling complex and its modulation by c-FLIP and PED/PEA-15 in glioma cells. J Biol Chem 277:25020–25025

    Article  PubMed  CAS  Google Scholar 

  16. Castro Alves C, Terziyska N, Grunert M et al (2012) Leukemia-initiating cells of patient-derived acute lymphoblastic leukemia xenografts are sensitive towards TRAIL. Blood 119(18):4224–4227

    Article  PubMed  Google Scholar 

  17. Piggott L, Omidvar N, Perez SM, Eberl M, Clarkson RW (2011) Suppression of apoptosis inhibitor c-FLIP selectively eliminates breast cancer stem cell activity in response to the anti-cancer agent, TRAIL. Breast Cancer Res 13:R88

    Article  PubMed  CAS  Google Scholar 

  18. Zhang L, Fang B (2005) Mechanisms of resistance to TRAIL-induced apoptosis in cancer. Cancer Gene Ther 12:228–237

    Article  PubMed  CAS  Google Scholar 

  19. Ehrhardt H, Fulda S, Schmid I, Hiscott J, Debatin KM, Jeremias I (2003) TRAIL induced survival and proliferation in cancer cells resistant towards TRAIL-induced apoptosis mediated by NF-kappaB. Oncogene 22:3842–3852

    Article  PubMed  CAS  Google Scholar 

  20. Amm HM, Oliver PG, Lee CH, Li Y, Buchsbaum DJ (2011) Combined modality therapy with TRAIL or agonistic death receptor antibodies. Cancer Biol Ther 11:431–449

    Article  PubMed  CAS  Google Scholar 

  21. Fulda S (2012) Histone deacetylase (HDAC) inhibitors and regulation of TRAIL-induced apoptosis. Exp Cell Res 318(11):1208–1212

    Article  PubMed  CAS  Google Scholar 

  22. Sayers TJ (2011) Targeting the extrinsic apoptosis signaling pathway for cancer therapy. Cancer Immunol Immunother 60:1173–1180

    Article  PubMed  CAS  Google Scholar 

  23. Ding J, Polier G, Kohler R, Giaisi M, Krammer PH, Li-Weber M (2012) Wogonin and related natural flavones overcome tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein resistance of tumors by down-regulation of c-FLIP protein and up-regulation of TRAIL receptor 2 expression. J Biol Chem 287:641–649

    Article  PubMed  CAS  Google Scholar 

  24. Gupta SC, Reuter S, Phromnoi K et al (2011) Nimbolide sensitizes human colon cancer cells to TRAIL through reactive oxygen species- and ERK-dependent up-regulation of death receptors, p53, and Bax. J Biol Chem 286:1134–1146

    Article  PubMed  CAS  Google Scholar 

  25. Whitson EL, Sun H, Thomas CL et al (2012) Synergistic TRAIL sensitizers from barleria alluaudii and diospyros maritima. J Nat Prod 75:394–399

    Article  PubMed  CAS  Google Scholar 

  26. Kim TD, Frick M, le Coutre P (2011) Omacetaxine mepesuccinate for the treatment of leukemia. Expert Opin Pharmacother 12:2381–2392

    Article  PubMed  CAS  Google Scholar 

  27. Klag T, Hartel N, Erben P et al (2012) Omacetaxine mepesuccinate prevents cytokine-dependent resistance to nilotinib in vitro: potential role of the common beta-subunit c of cytokine receptors. Leukemia 26:1321–1328

    Article  PubMed  CAS  Google Scholar 

  28. Meng H, Yang C, Jin J, Zhou Y, Qian W (2008) Homoharringtonine inhibits the AKT pathway and induces in vitro and in vivo cytotoxicity in human multiple myeloma cells. Leuk Lymphoma 49:1954–1962

    Article  PubMed  CAS  Google Scholar 

  29. Eckelbarger JD, Wilmot JT, Epperson MT et al (2008) Synthesis of antiproliferative Cephalotaxus esters and their evaluation against several human hematopoietic and solid tumor cell lines: uncovering differential susceptibilities to multidrug resistance. Chemistry 14:4293–4306

    Article  PubMed  CAS  Google Scholar 

  30. Efferth T, Sauerbrey A, Halatsch ME, Ross DD, Gebhart E (2003) Molecular modes of action of cephalotaxine and homoharringtonine from the coniferous tree Cephalotaxus hainanensis in human tumor cell lines. Naunyn Schmiedebergs Arch Pharmacol 367:56–67

    Article  PubMed  CAS  Google Scholar 

  31. Dimberg LY, Anderson CK, Camidge R, Behbakht K, Thorburn A, Ford HL (2012) On the TRAIL to successful cancer therapy? Predicting and counteracting resistance against TRAIL-based therapeutics. Oncogene. doi:10.1038/onc.2012.164

  32. Siegemund M, Pollak N, Seifert O et al (2012) Superior antitumoral activity of dimerized targeted single-chain TRAIL fusion proteins under retention of tumor selectivity. Cell Death Dis 3:e295

    Article  PubMed  CAS  Google Scholar 

  33. Frese S, Schuller A, Frese-Schaper M, Gugger M, Schmid RA (2009) Cytotoxic effects of camptothecin and cisplatin combined with tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) in a model of primary culture of non-small cell lung cancer. Anticancer Res 29:2905–2911

    PubMed  Google Scholar 

  34. Chen LH, Jiang CC, Kiejda KA et al (2007) Thapsigargin sensitizes human melanoma cells to TRAIL-induced apoptosis by up-regulation of TRAIL-R2 through the unfolded protein response. Carcinogenesis 28:2328–2336

    Article  PubMed  CAS  Google Scholar 

  35. Chen R, Guo L, Chen Y, Jiang Y, Wierda WG, Plunkett W (2011) Homoharringtonine reduced Mcl-1 expression and induced apoptosis in chronic lymphocytic leukemia. Blood 117:156–164

    Article  PubMed  CAS  Google Scholar 

  36. Sah NK, Munshi A, Kurland JF, McDonnell TJ, Su B, Meyn RE (2003) Translation inhibitors sensitize prostate cancer cells to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by activating c-Jun N-terminal kinase. J Biol Chem 278:20593–20602

    Article  PubMed  CAS  Google Scholar 

  37. Kantarjian HM, Talpaz M, Santini V, Murgo A, Cheson B, O’Brien SM (2001) Homoharringtonine: history, current research, and future direction. Cancer 92:1591–1605

    Article  PubMed  CAS  Google Scholar 

  38. Kim SH, Ricci MS, El-Deiry WS (2008) Mcl-1: a gateway to TRAIL sensitization. Cancer Res 68:2062–2064

    Article  PubMed  CAS  Google Scholar 

  39. Bagnoli M, Canevari S, Mezzanzanica D (2011) Cellular FLICE-inhibitory protein (c-FLIP) signalling: a key regulator of receptor-mediated apoptosis in physiologic context and in cancer. Int J Biochem Cell Biol 42:210–213

    Article  Google Scholar 

  40. Sharp DA, Lawrence DA, Ashkenazi A (2005) Selective knockdown of the long variant of cellular FLICE inhibitory protein augments death receptor-mediated caspase-8 activation and apoptosis. J Biol Chem 280:19401–19409

    Article  PubMed  CAS  Google Scholar 

  41. Haag C, Stadel D, Zhou S et al (2011) Identification of c-FLIP(L) and c-FLIP(S) as critical regulators of death receptor-induced apoptosis in pancreatic cancer cells. Gut 60:225–237

    Article  PubMed  CAS  Google Scholar 

  42. Thome M, Schneider P, Hofmann K et al (1997) Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature 386:517–521

    Article  PubMed  CAS  Google Scholar 

  43. Pop C, Oberst A, Drag M et al (2011) FLIP(L) induces caspase 8 activity in the absence of interdomain caspase 8 cleavage and alters substrate specificity. Biochem J 433:447–457

    Article  PubMed  CAS  Google Scholar 

  44. Fricker N, Beaudouin J, Richter P, Eils R, Krammer PH, Lavrik IN (2011) Model-based dissection of CD95 signaling dynamics reveals both a pro- and antiapoptotic role of c-FLIPL. J Cell Biol 190:377–389

    Article  Google Scholar 

  45. Micheau O, Thome M, Schneider P et al (2002) The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signaling complex. J Biol Chem 277:45162–45171

    Article  PubMed  CAS  Google Scholar 

  46. Tromp JM, Geest CR, Breij EC et al (2012) Tipping the Noxa/Mcl-1 balance overcomes ABT-737 resistance in chronic lymphocytic leukemia. Clin Cancer Res 18:487–498

    Article  PubMed  CAS  Google Scholar 

  47. Jacquemin G, Granci V, Gallouet AS et al (2012) Quercetin-mediated Mcl-1 and survivin downregulation restores TRAIL-induced apoptosis in non-Hodgkin’s lymphoma B cells. Haematologica 97:38–46

    Article  PubMed  Google Scholar 

  48. Leitch AE, Riley NA, Sheldrake TA et al (2010) The cyclin-dependent kinase inhibitor R-roscovitine down-regulates Mcl-1 to override pro-inflammatory signalling and drive neutrophil apoptosis. Eur J Immunol 40:1127–1138

    Article  PubMed  CAS  Google Scholar 

  49. Pennarun B, Kleibeuker JH, Boersma-van Ek W et al (2012) Targeting FLIP and Mcl-1 using a combination of aspirin and sorafenib sensitizes colon cancer cells to TRAIL. J Pathol 229(3):410–421

    Article  Google Scholar 

  50. Ricci MS, Kim SH, Ogi K et al (2007) Reduction of TRAIL-induced Mcl-1 and cIAP2 by c-Myc or sorafenib sensitizes resistant human cancer cells to TRAIL-induced death. Cancer Cell 12:66–80

    Article  PubMed  CAS  Google Scholar 

  51. Jennewein C, Karl S, Baumann B, Micheau O, Debatin KM, Fulda S (2012) Identification of a novel pro-apoptotic role of NF-kappaB in the regulation of TRAIL- and CD95-mediated apoptosis of glioblastoma cells. Oncogene 31:1468–1474

    Article  PubMed  CAS  Google Scholar 

  52. Koschny R, Ganten TM, Sykora J et al (2007) TRAIL/bortezomib cotreatment is potentially hepatotoxic but induces cancer-specific apoptosis within a therapeutic window. Hepatology 45:649–658

    Article  PubMed  CAS  Google Scholar 

  53. Lecis D, Drago C, Manzoni L et al (2010) Novel SMAC-mimetics synergistically stimulate melanoma cell death in combination with TRAIL and bortezomib. Br J Cancer 102:1707–1716

    Article  PubMed  CAS  Google Scholar 

  54. Menke C, Bin L, Thorburn J, Behbakht K, Ford HL, Thorburn A (2011) Distinct TRAIL resistance mechanisms can be overcome by proteasome inhibition but not generally by synergizing agents. Cancer Res 71:1883–1892

    Article  PubMed  CAS  Google Scholar 

  55. Unterkircher T, Cristofanon S, Vellanki SH et al (2011) Bortezomib primes glioblastoma, including glioblastoma stem cells, for TRAIL by increasing tBid stability and mitochondrial apoptosis. Clin Cancer Res 17:4019–4030

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The work on this project was mainly supported by grants from the Czech Science Foundation No. P301-10-1971 and the Ministry of Education No. LH12202 to LA, then by IGA-MZ: NT13201-4/2012, PRVOUK-P24/LF1/3, and UNCE 204021 to PK, GA-UK 259211/110709 to JM, the Ministry of Education grants No. LC06077 and LO2011022 to PB and by the institutional grant RVO 68378050. We are grateful to James Dutt for proofreading the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Cell Signaling & Apoptosis, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, 14220, Praha 4, Czech Republic

    Lenka Beranova, Jarmila Spegarova, Michal Koc & Ladislav Andera

  2. Center for Chemical Genetics, CZ-OPENSCREEN, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, 14220, Praha 4, Czech Republic

    Antonio R. Pombinho & Petr Bartunek

  3. Department of Pathophysiology, 1st Medical Faculty, Charles University in Prague, Prague, Czech Republic

    Magdalena Klanova, Jan Molinsky & Pavel Klener

Authors
  1. Lenka Beranova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Antonio R. Pombinho
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jarmila Spegarova
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michal Koc
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Magdalena Klanova
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jan Molinsky
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Pavel Klener
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Petr Bartunek
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Ladislav Andera
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Petr Bartunek or Ladislav Andera.

Additional information

L. Beranova and A.R. Pombinho contributed equally to this study.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 30 kb)

Suppl. Fig. 1

High-throughput screening validation identifies Homoharringtonine as a potent, low nanomolar sensitizer of HT-29 cells to TRAIL-induced apoptosis. HT-29 cells (2500 cells/25 μl/well) were plated in white 384-well plates and the primary hits resulting from high-throughput screening were titrated in the presence of 1 μg/ml TRAIL. After incubating for 6 hours, apoptosis was measured using the Caspase 3/7 GLO assay (EPS 640 kb)

Suppl. Fig. 2

The combinatorial effect of homoharringtonine and TRAIL suppresses the viability only of tumor, but not normal, colon epithelial cells. RKO or CCD 841 cells were cultivated with TRAIL (100 ng/ml), 20 or 50 nM HHT or their combination and analyzed for their survival. RKO (A.) or CCD 841 (B.) cells were grown in 96-well plates to 50% confluency, then the indicated reagents were added and cells cultivated for an additional 48 hours. Subsequently, their viability was analyzed using the WST-1 assay (Roche). C. Phase-contrast microscopy images of RKO cells cultivated for 48 hours with HHT, TRAIL or their combinations. The data in A and B are shown as the means of three independent experiments with standard deviations and statistical significance (* = < 0.05, ** = < 0.01) (EPS 4362 kb)

Suppl. Fig. 3

Cell surface expression of death receptors in HHT-treated cells. RKO or HT-29 cells were either untreated or treated with 50 nM HHT for 3 hrs and then stained for on the cell surface expressed DR4, DR5 and Fas/CD95 with corresponding monoclonal antibodies. Representative histograms of three independent experiments are shown (EPS 1197 kb)

Suppl. Fig. 4

Blocking NFkB activation with IKKa inhibitor Bay 11-7082 attenuates HHT-mediated sensitization of RKO cell to TRAIL-induced apoptosis. RKO cells were pre-treated with none, 10 mM or 20 mM Bay 11-7082 for 1 hr and then treated HHT (12.5 nM to 50 nM), TRAIL (100 ng/ml) and their combinations for 5 hrs (A) or 3 hrs (B). Cells were then stained with Annexin V-FITC and Hoechst 33258 proportion of apoptotic/dead cells was analyzed by flow cytometry (A) or the activity of effector caspases 3/7 was determined by Caspase-Glo 3/7 assay (Promega). The data in A and B represent means of four independent experiments with standard deviations (EPS 621 kb)

Suppl. Fig. 5

The downregulation of cFLIP or Mcl-1 expression affects the TRAIL-induced activation of caspases and the relative viability of RKO and HT-29 cells. RKO (A) or HT-29 (B) cells (none – mock transduced with pLKO1 lentivirus, two different cFLIP and Mcl-1 shRNAs) were cultivated in triplicate in a 384-well plate. Upon reaching 50% confluency, the cells were treated with 25 nM HHT, 20 ng/ml TRAIL or their combination for 3 hours (activation of caspase-3 using the Caspase-3/7 GLO assay) or for 48 hours (cell viability using the CellTiter-Blue assay). The average values and standard deviations from three independent experiments are shown with marked statistical significance for the casapase-3/7 activity assays (NS-non-significant, * = < 0.05, ** =< 0.01 and ***=<0.001) (EPS 1211 kb)

Suppl. Fig. 6

The proliferation of treated RKO cells is variably affected by the downregulation of cFLIP or Mcl-1 expression. RKO cells (transduced with pLKO1 lentivirus – none, cFLIP shRNA – shFLIP1 and Mcl-1 shRNA - shMcl-1 5/4) were cultivated in triplicate in a 96-well xCELLigence E-plate and treated with TRAIL (20 ng/ml), 25 nM HHT or their combination. The average values of the readings (only the hourly values are plotted) and standard deviations are shown. The data shown are representative of three independent experiments (EPS 1909 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Beranova, L., Pombinho, A.R., Spegarova, J. et al. The plant alkaloid and anti-leukemia drug homoharringtonine sensitizes resistant human colorectal carcinoma cells to TRAIL-induced apoptosis via multiple mechanisms. Apoptosis 18, 739–750 (2013). https://doi.org/10.1007/s10495-013-0823-9

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-013-0823-9

Keywords

Search

Navigation