Cellular Oncology

, Volume 41, Issue 2, pp 201–212 | Cite as

Synergistic anticancer effect of panobinostat and topoisomerase inhibitors through ROS generation and intrinsic apoptotic pathway induction in cervical cancer cells

Original Paper
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Abstract

Purpose

Various combinations of drugs may be effective in the treatment of different types of cancer. Previously, we have shown that combinations of the histone deacetylase inhibitor panobinostat and the topoisomerase inhibitors topotecan or etoposide act synergistically, but the underlying mode of action has remained unknown. Here, we aimed at uncovering the mechanisms underlying this synergism.

Methods

The effects of (combinations of) panobinostat and topotecan or etoposide on cervical cancer-derived HeLa and SiHa cells were assessed using morphological evaluations, scratch wound healing assays, cell cycle analyses, AO/EB staining assays, Annexin V/PI staining assays, reactive oxygen species (ROS) and mitochondrial membrane potential measurements and Western blotting.

Results

We found that combinations of panobinostat and the topoisomerase inhibitors topotecan or etoposide synergistically enhanced the induction of apoptosis in both HeLa and SiHa cells. This enhanced apoptosis induction was found to be mediated through increased ROS production and induction of the mitochondrial apoptotic pathway. We also found that the combination treatment resulted in inhibition of the PI3K/AKT and NF-κB pro-survival pathways and in activation of the ERK pathway, which is associated with intrinsic apoptosis.

Conclusions

From our data we conclude that combinations of panobinostat and the topoisomerase inhibitors topotecan or etoposide provoke strong cell death responses in cervical cancer-derived cells via induction of the intrinsic apoptotic pathway. Since this drug combination may potentially be effective in the treatment of cervical cancer, further preclinical investigations are warranted.

Keywords

Combination therapy Panobinostat Topoisomerase inhibitors ROS Apoptosis AKT/NF-κB ERK 
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Notes

Acknowledgements

The authors wish to thank the University of Delhi, India, for providing funds in the form of a DUR&D grant and a UGC-SAP grant and UGC, New Delhi, India, for providing a fellowship to LW. We also wish to thank Mr. Prateek Arora for helping in FCM.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

13402_2017_366_MOESM1_ESM.docx (2.5 mb)
ESM 1 (DOCX 2.54 mb)

References

  1. 1.
    Y. Sun, Z. Sheng, C. Ma, K. Tang, R. Zhu, Z. Wu, R. Shen, J. Feng, D. Wu, D. Huang, D. Huang, J. Fei, Q. Liu, Z. Cao, Combining genomic and network characteristics for extended capability in predicting synergistic drugs for cancer. Nat Commun 6, 8481 (2015).  https://doi.org/10.1038/ncomms9481 CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    S. Grant, Is the focus moving toward a combination of targeted drugs? Best Pract Res Clin Haematol 21, 629–637 (2008).  https://doi.org/10.1016/j.beha.2008.08.003 CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    M. Bots, R.W. Johnstone, Rational combinations using HDAC inhibitors. Clin Cancer Res 15, 3970–3977 (2009).  https://doi.org/10.1158/1078-0432.CCR-08-2786 CrossRefPubMedGoogle Scholar
  4. 4.
    J. Gray, C.L. Cubitt, S. Zhang, A. Chiappori, Combination of HDAC and topoisomerase inhibitors in small cell lung cancer. Cancer Biol. Ther. 13, 614–622 (2012).  https://doi.org/10.4161/cbt.19848 CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    A. Ferraro, Altered primary chromatin structures and their implications in cancer development. Cell Oncol 39, 195–210 (2016).  https://doi.org/10.1007/s13402-016-0276-6 CrossRefGoogle Scholar
  6. 6.
    L. Nolan, P.W. Johnson, A. Ganesan, G. Packham, S.J. Crabb, Will histone deacetylase inhibitors require combination with other agents to fulfil their therapeutic potential? Br J Cancer 99, 689–694 (2008).  https://doi.org/10.1038/sj.bjc.6604557 CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    M.S. Kim, M. Blake, J.H. Baek, G. Kohlhagen, Y. Pommier, F. Carrier, Inhibition of histone deacetylase increases cytotoxicity to anticancer drugs targeting DNA. Cancer Res 63, 7291–7300 (2003)PubMedGoogle Scholar
  8. 8.
    K. Ozaki, F. Kishikawa, M. Tanaka, T. Sakamoto, S. Tanimura, M. Kohno, Histone deacetylase inhibitors enhance the chemosensitivity of tumor cells with cross-resistance to a wide range of DNA-damaging drug. Cancer Sci 99, 376–384 (2008).  https://doi.org/10.1111/j.1349-7006.2007.00669.x CrossRefPubMedGoogle Scholar
  9. 9.
    L. Wasim, M. Chopra, Panobinostat induces apoptosis via production of reactive oxygen species and synergizes with topoisomerase inhibitors in cervical cancer cells. Biomed Pharmacother 84, 1393–1405 (2016).  https://doi.org/10.1016/j.biopha.2016.10.057 CrossRefPubMedGoogle Scholar
  10. 10.
    R.S. Hotchkiss, A. Strasser, J.E. McDunn, P.E. Swanson, Cell Death. N Engl J Med 361, 1570–1583 (2009).  https://doi.org/10.1056/NEJMra0901217
  11. 11.
    J.F. Kerr, C.M. Winterford, B.V. Harmon, Apoptosis. Its significance in cancer and cancer therapy. Cancer 73, 2013–2026 (1994).  https://doi.org/10.1002/1097-0142(19940415)73:8<2013::AID-CNCR2820730802>3.0.CO;2-J CrossRefPubMedGoogle Scholar
  12. 12.
    K.T. Thurn, S. Thomas, A. Moore, P.N. Munster, Rational therapeutic combinations with histone deacetylase inhibitors for the treatment of cancer. Future Oncol 7, 263–283 (2011).  https://doi.org/10.2217/fon.11.2 CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    O. Martinez-Iglesias, L. Ruiz-Llorente, R. Sanchez-Martinez, L. Garcia, A. Zambrano, A. Aranda, Histone deacetylase inhibitors: Mechanism of action and therapeutic use in cancer. Clin Transl Oncol 10, 395–398 (2008).  https://doi.org/10.1007/s12094-008-0221-x CrossRefPubMedGoogle Scholar
  14. 14.
    D.R. Budman, A. Calabro, L. Rosen, M. Lesser, Identification of unique synergistic drug combinations associated with downexpression of survivin in a preclinical breast cancer model system. Anti-Cancer Drugs 23, 272–279 (2012).  https://doi.org/10.1097/CAD.0b013e32834ebda4 CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Y. Cai, X. Yan, G. Zhang, W. Zhao, S. Jiao, The predictive value of ERCC1 and p53 for the effect of panobinostat and cisplatin combination treatment in NSCLC. Oncotarget 6, 18997–19005 (2015).  https://doi.org/10.18632/oncotarget.3620 PubMedCentralPubMedGoogle Scholar
  16. 16.
    S. Venkannagari, W. Fiskus, K. Peth, P. Atadja, M. Hidalgo, A. Maitra, K.N. Bhalla, Superior efficacy of co-treatment with dual PI3K/mTOR inhibitor NVP-BEZ235 and pan-histone deacetylase inhibitor against human pancreatic cancer. Oncotarget 3, 1416–1427 (2012).  https://doi.org/10.18632/oncotarget.724 CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    G. Wang, H. Edwards, J.T. Caldwell, S.A. Buck, W.Y. Qing, J.W. Taub, Y. Ge, Z. Wang, Panobinostat synergistically enhances the cytotoxic effects of cisplatin, doxorubicin or etoposide on high-risk neuroblastoma cells. PLoS One 8, e76662 (2013).  https://doi.org/10.1371/journal.pone.0076662 CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    F. Bruzzese, M. Rocco, S. Castelli, E. Di Gennaro, A. Desideri, A. Budillon, Synergistic antitumor effect between vorinostat and topotecan in small cell lung cancer cells is mediated by generation of reactive oxygen species and DNA damage-induced apoptosis. Mol Cancer Ther 8, 3075–3087 (2009).  https://doi.org/10.1158/1535-7163.MCT-09-0254 CrossRefPubMedGoogle Scholar
  19. 19.
    D. Chen, J. Cao, L. Tian, F. Liu, X. Sheng, Induction of apoptosis by casticin in cervical cancer cells through reactive oxygen species-mediated mitochondrial signaling pathways. Oncol Rep 26, 1287–1294 (2011).  https://doi.org/10.3892/or.2011.1367 PubMedGoogle Scholar
  20. 20.
    L. Gao, M. Gao, G. Yang, Y. Tao, Y. Kong, R. Yang, X. Meng, G. Ai, R. Wei, H. Wu, X. Wu, J. Shi, Synergistic activity of carfilzomib and Panobinostat in multiple myeloma cells via modulation of ROS generation and ERK1/2. Biomed Res Int 2015, 459052 (2015)PubMedCentralPubMedGoogle Scholar
  21. 21.
    O. Sordet, Q.A. Khan, I. Plo, P. Pourquier, Y. Urasaki, A. Yoshida, S. Antony, G. Kohlhagen, E. Solary, M. Saparbaev, J. Laval, Y. Pommier, Apoptotic topoisomerase I-DNA complexes induced by staurosporine-mediated oxygen radicals. J Biol Chem 279, 50499–50504 (2004).  https://doi.org/10.1074/jbc.M410277200 CrossRefPubMedGoogle Scholar
  22. 22.
    M.D. Brand, C. Affourtit, T.C. Esteves, K. Green, A.J. Lambert, S. Miwa, J.L. Pakay, N. Parker, Mitochondrial superoxide: Production, biological effects, and activation of uncoupling proteins. Free Radic Biol Med 37(6), 755–767 (2004).  https://doi.org/10.1016/j.freeradbiomed.2004.05.034 CrossRefPubMedGoogle Scholar
  23. 23.
    M. Ott, V. Gogvadze, S. Orrenius, B. Zhivotovsky, Mitochondria, oxidative stress and cell death. Apoptosis 12, 913–922 (2007).  https://doi.org/10.1007/s10495-007-0756-2 CrossRefPubMedGoogle Scholar
  24. 24.
    J.K. Brunelle, A. Letai, Control of mitochondrial apoptosis by the Bcl-2 family. J Cell Sci 122, 437–441 (2009).  https://doi.org/10.1242/jcs.031682 CrossRefPubMedCentralPubMedGoogle Scholar
  25. 25.
    B. Leibowitz, J. Yu, Mitochondrial signaling in cell death via the Bcl-2 family. Cancer Biol Ther 9, 417–422 (2010).  https://doi.org/10.4161/cbt.9.6.11392 CrossRefPubMedCentralPubMedGoogle Scholar
  26. 26.
    V.J. Bouchard, M. Rouleau, G.G. Poirier, PARP-1, a determinant of cell survival in response to DNA damage. Exp Hematol 31, 446–454 (2003).  https://doi.org/10.1016/S0301-472X(03)00083-3 CrossRefPubMedGoogle Scholar
  27. 27.
    D.A. Altomare, J.R. Testa, Perturbations of the AKT signaling pathway in human cancer. Oncogene 24, 7455–7464 (2005).  https://doi.org/10.1038/sj.onc.1209085 CrossRefPubMedGoogle Scholar
  28. 28.
    G. Song, G. Ouyang, S. Bao, The activation of Akt/PKB signaling pathway and cell survival. J Cell Mol Med 9, 59–71 (2005).  https://doi.org/10.1111/j.1582-4934.2005.tb00337.x CrossRefPubMedGoogle Scholar
  29. 29.
    K. Vazquez-Santillan, J. Melendez-Zajgla, L. Jimenez-Hernandez, G. Martinez-Ruiz, V. Maldonado, NF-kappaB signaling in cancer stem cells: A promising therapeutic target? Cell Oncol 38, 327–339 (2015).  https://doi.org/10.1007/s13402-015-0236-6 CrossRefGoogle Scholar
  30. 30.
    F. Chang, J.T. Lee, P.M. Navolanic, L.S. Steelman, J.G. Shelton, W.L. Blalock, R.A. Franklin, J.A. McCubrey, Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: A target for cancer chemotherapy. Leukemia 17, 590–603 (2003).  https://doi.org/10.1038/sj.leu.2402824 CrossRefPubMedGoogle Scholar
  31. 31.
    M. Barkett, T.D. Gilmore, Control of apoptosis by Rel/NF-kappaB transcription factors. Oncogene 18, 6910–6924 (1999).  https://doi.org/10.1038/sj.onc.1203238 CrossRefPubMedGoogle Scholar
  32. 32.
    C. Bubici, S. Papa, C.G. Pham, F. Zazzeroni, G. Franzoso, The NF-kappaB-mediated control of ROS and JNK signaling. Histol Histopathol 21, 69–80 (2006).  https://doi.org/10.14670/HH-21.69 PubMedGoogle Scholar
  33. 33.
    C. Yu, B.B. Friday, J.P. Lai, A. McCollum, P. Atadja, L.R. Roberts, A.A. Adjei, Abrogation of MAPK and Akt signaling by AEE788 synergistically potentiates histone deacetylase inhibitor-induced apoptosis through reactive oxygen species generation. Clin Cancer Res 13, 1140–1148 (2007).  https://doi.org/10.1158/1078-0432.CCR-06-1751 CrossRefPubMedGoogle Scholar
  34. 34.
    S. Cagnol, J.C. Chambard, ERK and cell death: Mechanisms of ERK-induced cell death--apoptosis, autophagy and senescence. FEBS J 277, 2–21 (2010).  https://doi.org/10.1111/j.1742-4658.2009.07366.x CrossRefPubMedGoogle Scholar
  35. 35.
    Z. Lu, S. Xu, ERK1/2 MAP kinases in cell survival and apoptosis. IUBMB Life 58, 621–631 (2006).  https://doi.org/10.1080/15216540600957438 CrossRefPubMedGoogle Scholar
  36. 36.
    J.A. McCubrey, L.S. Steelman, W.H. Chappell, S.L. Abrams, E.W. Wong, F. Chang, B. Lehmann, D.M. Terrian, M. Milella, A. Tafuri, F. Stivala, M. Libra, J. Basecke, C. Evangelisti, A.M. Martelli, R.A. Franklin, Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochim Biophys Acta 1773, 1263–1284 (2007).  https://doi.org/10.1016/j.bbamcr.2006.10.001 CrossRefPubMedGoogle Scholar
  37. 37.
    M.J. Chuang, S.T. Wu, S.H. Tang, X.M. Lai, H.C. Lai, K.H. Hsu, K.H. Sun, G.H. Sun, S.Y. Chang, D.S. Yu, P.W. Hsiao, S.M. Huang, T.L. Cha, The HDAC inhibitor LBH589 induces ERK-dependent prometaphase arrest in prostate cancer via HDAC6 inactivation and down-regulation. PLoS One 8, e73401 (2013).  https://doi.org/10.1371/journal.pone.0073401 CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© International Society for Cellular Oncology 2017

Authors and Affiliations

  1. 1.Laboratory of Molecular Modeling and Anticancer Drug Development, Dr. B. R. Ambedkar Center for Biomedical ResearchUniversity of DelhiDelhiIndia

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