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Apoptosis

, Volume 18, Issue 6, pp 739–750 | Cite as

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

  • Lenka Beranova
  • Antonio R. Pombinho
  • Jarmila Spegarova
  • Michal Koc
  • Magdalena Klanova
  • Jan Molinsky
  • Pavel Klener
  • Petr BartunekEmail author
  • Ladislav AnderaEmail author
Original Paper

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.

Keywords

Harringtonine Apoptosis Death receptor cFLIP Mcl-1 

Notes

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.

Supplementary material

10495_2013_823_MOESM1_ESM.doc (30 kb)
Supplementary material 1 (DOC 30 kb)
10495_2013_823_MOESM2_ESM.eps (640 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)
10495_2013_823_MOESM3_ESM.eps (4.3 mb)
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)
10495_2013_823_MOESM4_ESM.eps (1.2 mb)
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)
10495_2013_823_MOESM5_ESM.eps (621 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)
10495_2013_823_MOESM6_ESM.eps (1.2 mb)
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)
10495_2013_823_MOESM7_ESM.eps (1.9 mb)
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)

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Lenka Beranova
    • 1
  • Antonio R. Pombinho
    • 2
  • Jarmila Spegarova
    • 1
  • Michal Koc
    • 1
  • Magdalena Klanova
    • 3
  • Jan Molinsky
    • 3
  • Pavel Klener
    • 3
  • Petr Bartunek
    • 2
    Email author
  • Ladislav Andera
    • 1
    Email author
  1. 1.Department of Cell Signaling & ApoptosisInstitute of Molecular Genetics, Academy of Sciences of the Czech RepublicPraha 4Czech Republic
  2. 2.Center for Chemical Genetics, CZ-OPENSCREENInstitute of Molecular Genetics, Academy of Sciences of the Czech RepublicPraha 4Czech Republic
  3. 3.Department of Pathophysiology1st Medical Faculty, Charles University in PraguePragueCzech Republic

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