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Molecular and Cellular Biochemistry

, Volume 408, Issue 1–2, pp 123–137 | Cite as

Synergistic anticancer effects of combined γ-tocotrienol and oridonin treatment is associated with the induction of autophagy

  • Roshan V. Tiwari
  • Parash Parajuli
  • Paul W. Sylvester
Article

Abstract

γ-Tocotrienol and oridonin are natural phytochemicals that display potent anticancer activity. Studies showed that combined treatment with subeffective doses of γ-tocotrienol with oridonin resulted in synergistic autophagic and apoptotic effects in malignant +SA, but not normal CL-S1 mouse mammary epithelial cells in vitro. Specifically, combined treatment with low doses of γ-tocotrienol (8 µM) and oridonin (2 µM) for 24 h resulted in synergistic inhibition of +SA mammary cancer cells viability. This combination significantly enhanced the expression of autophagy cellular markers including the conversion of LC3B-I to LC3B-II, beclin-1, Atg3, Atg7, Atg5–Atg12, LAMP-1 and cathepsin-D, and pretreatment with the autophagy inhibitors 3-methyladenine (3-MA) or bafilomycin A1 (Baf1) blocked these effects. Furthermore, blockade of γ-tocotrienol and oridonin-induced autophagy with 3-MA or Baf1 induced a modest, but significant reduction in cytotoxicity resulting from the combined treatment of these phytochemicals. The anticancer effects of combination treatment was also associated with a large suppression in Akt/mTOR mitogenic signaling and corresponding increase in the levels of apoptotic cellular marker including cleaved caspase-3 and PARP, and Bax/Bcl-2 ratio in these tumor cells. These effects were also found to be selective against cancer cells, since similar combined treatment with γ-tocotrienol and oridonin did not induce autophagy or reduce viability of normal mouse CL-S1 mammary epithelial cells. These findings indicate that combined γ-tocotrienol and oridonin-induced autophagy plays a role in mediating the synergistic anticancer effects of these phytochemicals.

Keywords

γ-Tocotrienol Autophagy Breast cancer Beclin-1 LAMP-1 Cathepsin-D 

Notes

Acknowledgments

The authors would like to thank the First Tech International Ltd. for generously providing γ-tocotrienol for use in these studies. This work was performed at the School of Pharmacy, University of Louisiana at Monroe, Monroe, LA USA, and supported in part by grants from First Tec International Ltd. (Hong Kong), the Louisiana Cancer Foundation and the Louisiana Campuses Research Initiative (LACRI). The authors would also like to thank Dr. Karen P Briski for her assistance in studies with the confocal microscope.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no personal, financial or competing interests. First Tech International Ltd. provided a grant that partially paid for the funding of these experiments and the purified γ-tocotrienol that was used in these experiments.

References

  1. 1.
    Polyak K (2011) Heterogeneity in breast cancer. J Clin Invest 121:3786–3788PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Fischgrabe J, Wulfing P (2008) Targeted therapies in breast cancer: established drugs and recent developments. Curr Clin Pharmacol 3:85–98CrossRefPubMedGoogle Scholar
  3. 3.
    Amin A, Gali-Muhtasib H, Ocker M, Schneider-Stock R (2009) Overview of major classes of plant-derived anticancer drugs. Int J Biomed Sci 5:1–11PubMedCentralPubMedGoogle Scholar
  4. 4.
    Akl MR, Ayoub NM, Abuasal BS, Kaddoumi A, Sylvester PW (2013) Sesamin synergistically potentiates the anticancer effects of gamma-tocotrienol in mammary cancer cell lines. Fitoterapia 84:347–359CrossRefPubMedGoogle Scholar
  5. 5.
    McIntyre BS, Briski KP, Gapor A, Sylvester PW (2000) Antiproliferative and apoptotic effects of tocopherols and tocotrienols on preneoplastic and neoplastic mouse mammary epithelial cells. Proc Soc Exp Biol Med 224:292–301CrossRefPubMedGoogle Scholar
  6. 6.
    Sylvester PW, Akl MR, Malaviya A, Parajuli P, Ananthula S, Tiwari RV, Ayoub NM (2014) Potential role of tocotrienols in the treatment and prevention of breast cancer. BioFactors 40:49–58CrossRefPubMedGoogle Scholar
  7. 7.
    Tiwari RV, Parajuli P, Sylvester PW (2014) gamma-Tocotrienol-induced autophagy in malignant mammary cancer cells. Exp Biol Med (Maywood) 239:33–44CrossRefGoogle Scholar
  8. 8.
    Zhang W, Huang Q, Hua Z-C (2010) Oridonin: a promising anticancer drug from China. Front Biol 5:540–545CrossRefGoogle Scholar
  9. 9.
    Li C-Y, Wang E-Q, Cheng Y, Bao J-K (2011) Oridonin: an active diterpenoid targeting cell cycle arrest, apoptotic and autophagic pathways for cancer therapeutics. Int J Biochem Cell Biol 43:701–704CrossRefPubMedGoogle Scholar
  10. 10.
    Kang N, Zhang JH, Qiu F, Chen S, Tashiro S, Onodera S, Ikejima T (2010) Induction of G(2)/M phase arrest and apoptosis by oridonin in human laryngeal carcinoma cells. J Nat Prod 73:1058–1063CrossRefPubMedGoogle Scholar
  11. 11.
    Cui Q, Tashiro S, Onodera S, Minami M, Ikejima T (2007) Autophagy preceded apoptosis in oridonin-treated human breast cancer MCF-7 cells. Biol Pharm Bull 30:859–864CrossRefPubMedGoogle Scholar
  12. 12.
    Chen Y, Azad MB, Gibson SB (2010) Methods for detecting autophagy and determining autophagy-induced cell death. Can J Physiol Pharmacol 88:285–295CrossRefPubMedGoogle Scholar
  13. 13.
    Klionsky DJ, Emr SD (2000) Autophagy as a regulated pathway of cellular degradation. Science 290:1717–1721PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Choi CH, Jung YK, Oh SH (2010) Autophagy induction by capsaicin in malignant human breast cells is modulated by p38 and extracellular signal-regulated mitogen-activated protein kinases and retards cell death by suppressing endoplasmic reticulum stress-mediated apoptosis. Mol Pharmacol 78:114–125CrossRefPubMedGoogle Scholar
  15. 15.
    Klionsky DJ (2005) The molecular machinery of autophagy: unanswered questions. J Cell Sci 118:7–18PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Rabinowitz JD, White E (2010) Autophagy and metabolism. Science 330:1344–1348PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Tassa A, Roux MP, Attaix D, Bechet DM (2003) Class III phosphoinositide 3-kinase-Beclin1 complex mediates the amino acid-dependent regulation of autophagy in C2C12 myotubes. Biochem J 376:577–586PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Baehrecke EH (2005) Autophagy: dual roles in life and death? Nat Rev Mol Cell Biol 6:505–510CrossRefPubMedGoogle Scholar
  19. 19.
    Ghavami S, Yeganeh B, Stelmack GL, Kashani HH, Sharma P, Cunnington R, Rattan S, Bathe K, Klonisch T, Dixon IM, Freed DH, Halayko AJ (2012) Apoptosis, autophagy and ER stress in mevalonate cascade inhibition-induced cell death of human atrial fibroblasts. Cell Death Dis 3:e330PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19:5720–5728PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Tanida I, Ueno T, Kominami E (2004) LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol 36:2503–2518CrossRefPubMedGoogle Scholar
  22. 22.
    Sylvester PW (2012) Synergistic anticancer effects of combined gamma-tocotrienol with statin or receptor tyrosine kinase inhibitor treatment. Genes Nutr 7:63–74PubMedCentralCrossRefPubMedGoogle Scholar
  23. 23.
    Anderson LW, Danielson KG, Hosick HL (1979) New cell line. Epithelial cell line and subline established from premalignant mouse mammary tissue. In Vitro 15:841–843CrossRefPubMedGoogle Scholar
  24. 24.
    Danielson KG, Anderson LW, Hosick HL (1980) Selection and characterization in culture of mammary tumor cells with distinctive growth properties in vivo. Cancer Res 40:1812–1819PubMedGoogle Scholar
  25. 25.
    Anderson LW, Danielson KG, Hosick HL (1981) Metastatic potential of hyperplastic alveolar nodule derived mouse mammary tumor cells following intravenous inoculation. Eur J Cancer Clin Oncol 17:1001–1008CrossRefPubMedGoogle Scholar
  26. 26.
    Shah SJ, Sylvester PW (2005) Tocotrienol-induced cytotoxicity is unrelated to mitochondrial stress apoptotic signaling in neoplastic mammary epithelial cells. Biochem Cell Biol 83:86–95CrossRefPubMedGoogle Scholar
  27. 27.
    Biederbick A, Kern HF, Elsasser HP (1995) Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur J Cell Biol 66:3–14PubMedGoogle Scholar
  28. 28.
    Munafo DB, Colombo MI (2001) A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation. J Cell Sci 114:3619–3629PubMedGoogle Scholar
  29. 29.
    Kanzawa T, Germano IM, Komata T, Ito H, Kondo Y, Kondo S (2004) Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells. Cell Death Differ 11:448–457CrossRefPubMedGoogle Scholar
  30. 30.
    Wali VB, Sylvester PW (2007) Synergistic antiproliferative effects of gamma-tocotrienol and statin treatment on mammary tumor cells. Lipids 42:1113–1123CrossRefPubMedGoogle Scholar
  31. 31.
    Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354PubMedCentralCrossRefPubMedGoogle Scholar
  32. 32.
    Tallarida RJ (2001) Drug synergism: its detection and applications. J Pharmacol Exp Ther 298:865–872PubMedGoogle Scholar
  33. 33.
    Glunde K, Guggino SE, Solaiyappan M, Pathak AP, Ichikawa Y, Bhujwalla ZM (2003) Extracellular acidification alters lysosomal trafficking in human breast cancer cells. Neoplasia 5:533–545PubMedCentralCrossRefPubMedGoogle Scholar
  34. 34.
    Glick D, Barth S, Macleod KF (2010) Autophagy: cellular and molecular mechanisms. J Pathol 221:3–12PubMedCentralCrossRefPubMedGoogle Scholar
  35. 35.
    Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140:313–326PubMedCentralCrossRefPubMedGoogle Scholar
  36. 36.
    Petiot A, Ogier-Denis E, Blommaart EF, Meijer AJ, Codogno P (2000) Distinct classes of phosphatidylinositol 3′-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J Biol Chem 275:992–998CrossRefPubMedGoogle Scholar
  37. 37.
    Yamamoto A, Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y (1998) Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct Funct 23:33–42CrossRefPubMedGoogle Scholar
  38. 38.
    Eskelinen EL, Saftig P (2009) Autophagy: a lysosomal degradation pathway with a central role in health and disease. Biochim Biophys Acta 1793:664–673CrossRefPubMedGoogle Scholar
  39. 39.
    White E, DiPaola RS (2009) The double-edged sword of autophagy modulation in cancer. Clin Cancer Res 15:5308–5316PubMedCentralCrossRefPubMedGoogle Scholar
  40. 40.
    Jiang Q, Rao X, Kim CY, Freiser H, Zhang Q, Jiang Z, Li G (2012) Gamma-tocotrienol induces apoptosis and autophagy in prostate cancer cells by increasing intracellular dihydrosphingosine and dihydroceramide. Int J Cancer 130:685–693PubMedCentralCrossRefPubMedGoogle Scholar
  41. 41.
    Tiwari RV, Parajuli P, Sylvester PW (2015) gamma-Tocotrienol-induced endoplasmic reticulum stress and autophagy act concurrently to promote breast cancer cell death. Biochem Cell Biol 12:1–15Google Scholar
  42. 42.
    Kihara A, Kabeya Y, Ohsumi Y, Yoshimori T (2001) Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network. EMBO Rep 2:330–335PubMedCentralCrossRefPubMedGoogle Scholar
  43. 43.
    Eskelinen EL, Tanaka Y, Saftig P (2003) At the acidic edge: emerging functions for lysosomal membrane proteins. Trends Cell Biol 13:137–145CrossRefPubMedGoogle Scholar
  44. 44.
    Rochefort H, Capony F, Garcia M (1990) Cathepsin D: a protease involved in breast cancer metastasis. Cancer Metastasis Rev 9:321–331CrossRefPubMedGoogle Scholar
  45. 45.
    Sylvester PW, Ayoub NM (2013) Tocotrienols target PI3 K/Akt signaling in anti-breast cancer therapy. Anticancer Agents Med Chem 13:1039–1047CrossRefPubMedGoogle Scholar
  46. 46.
    Singh BN, Kumar D, Shankar S, Srivastava RK (2012) Rottlerin induces autophagy which leads to apoptotic cell death through inhibition of PI3 K/Akt/mTOR pathway in human pancreatic cancer stem cells. Biochem Pharmacol 84:1154–1163CrossRefPubMedGoogle Scholar
  47. 47.
    Guo Y, Shan Q, Gong Y, Lin J, Yang X, Zhou R (2012) Oridonin in combination with imatinib exerts synergetic anti-leukemia effect in Ph+ acute lymphoblastic leukemia cells in vitro by inhibiting activation of LYN/mTOR signaling pathway. Cancer Biol Ther 13:1244–1254PubMedCentralCrossRefPubMedGoogle Scholar
  48. 48.
    Wang YY, Lv YF, Lu L, Cai L (2014) Oridonin inhibits mTOR signaling and the growth of lung cancer tumors. Anticancer Drugs 25:1192–1200CrossRefPubMedGoogle Scholar
  49. 49.
    Scopa CD, Vagianos C, Kardamakis D, Kourelis TG, Kalofonos HP, Tsamandas AC (2001) bcl-2/bax ratio as a predictive marker for therapeutic response to radiotherapy in patients with rectal cancer. Appl Immunohistochem Mol Morphol 9:329–334PubMedGoogle Scholar
  50. 50.
    Reed JC (1994) Bcl-2 and the regulation of programmed cell death. J Cell Biol 124:1–6CrossRefPubMedGoogle Scholar
  51. 51.
    Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, Packer M, Schneider MD, Levine B (2005) Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 122:927–939CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Roshan V. Tiwari
    • 1
  • Parash Parajuli
    • 1
  • Paul W. Sylvester
    • 1
  1. 1.School of PharmacyUniversity of Louisiana at MonroeMonroeUSA

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