Tumor Biology

, Volume 37, Issue 8, pp 11039–11048 | Cite as

C6 ceramide sensitizes the anti-hepatocellular carcinoma (HCC) activity by AZD-8055, a novel mTORC1/2 dual inhibitor

Original Article


Aberrant activation of mammalian target of rapamycin (mTOR) plays pivotal roles in promoting hepatocellular carcinoma (HCC) tumorigenesis and chemoresistance. Here, we tested the potential anti-HCC activity by a novel mTOR complex 1/2 (mTORC1/2) dual inhibitor AZD-8055 and, more importantly, the potential AZD-8055 sensitization effect by a cell-permeable short-chain ceramide (C6). We showed that AZD-8055 mainly exerted moderate cytotoxic effect against a panel of HCC cell lines (HepG2, Hep3B, and SMMC-7721). Co-treatment of C6 ceramide remarkably augmented AZD-8055-induced HCC cytotoxicity. Meanwhile, C6 ceramide dramatically potentiated AZD-8055-induced HCC cell apoptotic death. Further studies demonstrated that AZD-8055 and C6 ceramide synergistically induced anti-survival and pro-apoptotic activity in primary cultured human HCC cells, but not in the non-cancerous human hepatocytes. Signaling studies showed that AZD-8055 and C6 ceramide synergistically suppressed Akt-mTOR complex 1/2 cascade activation. In vivo, AZD-8055 oral administration suppressed HepG2 hepatoma xenograft growth in nude mice, while moderately improving mice survival. Its anti-tumor activity was dramatically potentiated with co-administration of a liposome-packed C6 ceramide. Together, these results demonstrate that concurrent targeting mTORC1/2 by AZD-8055 exerts anti-tumor ability in preclinical HCC models, and its activity is further sensitized with co-administration of C6 ceramide.


Hepatocellular carcinoma (HCC) Mammalian target of rapamycin (mTOR) AZD-8055 C6 ceramide Chemo-sensitization 


Compliance with ethical standards

Human HCC tissue specimens were collected from three patients undergoing hepatectomy, with informed consents. All studies using human samples were conducted according to the principles expressed in the Declaration of Helsinki and national and international guidelines. The study was approved by the Ethics Review Board (ERB) of Xinjiang Medical University (No. 2014035).

Conflicts of interest



  1. 1.
    Farazi PA, DePinho RA. Hepatocellular carcinoma pathogenesis: from genes to environment. Nat Rev Cancer. 2006;6:674–87.CrossRefPubMedGoogle Scholar
  2. 2.
    Yang JD, Roberts LR. Hepatocellular carcinoma: a global view. Nat Rev Gastroenterol Hepatol. 2010;7:448–58.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin. 2014;64:9–29.CrossRefPubMedGoogle Scholar
  4. 4.
    Llovet JM, Bruix J. Systematic review of randomized trials for unresectable hepatocellular carcinoma: chemoembolization improves survival. Hepatology. 2003;37:429–42.CrossRefPubMedGoogle Scholar
  5. 5.
    Singh S, Singh PP, Roberts LR, Sanchez W. Chemopreventive strategies in hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol. 2014;11:45–54.CrossRefPubMedGoogle Scholar
  6. 6.
    Tanaka S, Arii S. Molecular targeted therapies in hepatocellular carcinoma. Semin Oncol. 2012;39:486–92.CrossRefPubMedGoogle Scholar
  7. 7.
    Chen JS, Wang Q, Fu XH, Huang XH, Chen XL, Cao LQ, et al. Involvement of PI3K/PTEN/AKT/mTOR pathway in invasion and metastasis in hepatocellular carcinoma: association with MMP-9. Hepatol Res. 2009;39:177–86.CrossRefPubMedGoogle Scholar
  8. 8.
    Villanueva A, Chiang DY, Newell P, Peix J, Thung S, Alsinet C, et al. Pivotal role of mTOR signaling in hepatocellular carcinoma. Gastroenterology. 2008;135:1972–83. 1983 e1971-1911.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Chen BW, Chen W, Liang H, Liu H, Liang C, Zhi X, et al. Inhibition of mTORC2 induces cell-cycle arrest and enhances the cytotoxicity of doxorubicin by suppressing MDR1 expression in HCC cells. Mol Cancer Ther. 2015;14:1805–15.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Zaytseva YY, Valentino JD, Gulhati P, Evers BM. mTOR inhibitors in cancer therapy. Cancer Lett. 2012;319:1–7.CrossRefPubMedGoogle Scholar
  11. 11.
    Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012;149:274–93.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Garcia-Echeverria C. Allosteric and ATP-competitive kinase inhibitors of mTOR for cancer treatment. Bioorg Med Chem Lett. 2010;20:4308–12.CrossRefPubMedGoogle Scholar
  13. 13.
    Konings IR, Verweij J, Wiemer EA, Sleijfer S. The applicability of mTOR inhibition in solid tumors. Curr Cancer Drug Targets. 2009;9:439–50.CrossRefPubMedGoogle Scholar
  14. 14.
    Vilar E, Perez-Garcia J, Tabernero J. Pushing the envelope in the mTOR pathway: the second generation of inhibitors. Mol Cancer Ther. 2011;10:395–403.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Li Q, Song XM, Ji YY, Jiang H, Xu LG. The dual MTORC1 and MTORC2 inhibitor AZD8055 inhibits head and neck squamous cell carcinoma cell growth in vivo and in vitro. Biochem Biophys Res Commun. 2013;440:701–6.CrossRefPubMedGoogle Scholar
  16. 16.
    Pike KG, Malagu K, Hummersone MG, Menear KA, Duggan HM, Gomez S, et al. Optimization of potent and selective dual MTORC1 and MTORC2 inhibitors: the discovery of AZD8055 and AZD2014. Bioorg Med Chem Lett. 2013;23:1212–6.CrossRefPubMedGoogle Scholar
  17. 17.
    Morad SA, Cabot MC. Ceramide-orchestrated signalling in cancer cells. Nat Rev Cancer. 2013;13:51–65.CrossRefPubMedGoogle Scholar
  18. 18.
    Henry B, Moller C, Dimanche-Boitrel MT, Gulbins E, Becker KA. Targeting the ceramide system in cancer. Cancer Lett. 2011;332(2):286–94.CrossRefPubMedGoogle Scholar
  19. 19.
    Dimanche-Boitrel MT, Rebillard A, Gulbins E. Ceramide in chemotherapy of tumors. Recent Pat Anticancer Drug Discov. 2011;6:284–93.CrossRefPubMedGoogle Scholar
  20. 20.
    Lin CF, Chen CL, Lin YS. Ceramide in apoptotic signaling and anticancer therapy. Curr Med Chem. 2006;13:1609–16.CrossRefPubMedGoogle Scholar
  21. 21.
    Chen MB, Jiang Q, Liu YY, Zhang Y, He BS, Wei MX, et al. C6 ceramide dramatically increases vincristine sensitivity both in vivo and in vitro, involving AMP-activated protein kinase-p53 signaling. Carcinogenesis. 2015;36:1061–70.CrossRefPubMedGoogle Scholar
  22. 22.
    Ji C, Yang B, Yang YL, He SH, Miao DS, He L, et al. Exogenous cell-permeable C6 ceramide sensitizes multiple cancer cell lines to doxorubicin-induced apoptosis by promoting AMPK activation and MTORC1 inhibition. Oncogene. 2010;29:6557–68.CrossRefPubMedGoogle Scholar
  23. 23.
    Yang L, Zheng LY, Tian Y, Zhang ZQ, Dong WL, Wang XF, et al. C6 ceramide dramatically enhances docetaxel-induced growth inhibition and apoptosis in cultured breast cancer cells: a mechanism study. Exp Cell Res. 2015;332:47–59.CrossRefPubMedGoogle Scholar
  24. 24.
    Yu T, Li J, Sun H. C6 ceramide potentiates curcumin-induced cell death and apoptosis in melanoma cell lines in vitro. Cancer Chemother Pharmacol. 2010;66:999–1003.CrossRefPubMedGoogle Scholar
  25. 25.
    Rahmani M, Reese E, Dai Y, Bauer C, Payne SG, Dent P, et al. Coadministration of histone deacetylase inhibitors and perifosine synergistically induces apoptosis in human leukemia cells through Akt and ERK1/2 inactivation and the generation of ceramide and reactive oxygen species. Cancer Res. 2005;65:2422–32.CrossRefPubMedGoogle Scholar
  26. 26.
    Zhu QY, Wang Z, Ji C, Cheng L, Yang YL, Ren J, et al. C6-ceramide synergistically potentiates the anti-tumor effects of histone deacetylase inhibitors via AKT dephosphorylation and alpha-tubulin hyperacetylation both in vitro and in vivo. Cell Death Dis. 2011;2:e117.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Schulze-Bergkamen H, Untergasser A, Dax A, Vogel H, Buchler P, Klar E, et al. Primary human hepatocytes—a valuable tool for investigation of apoptosis and hepatitis B virus infection. J Hepatol. 2003;38:736–44.CrossRefPubMedGoogle Scholar
  28. 28.
    Zhen YF, Wang GD, Zhu LQ, Tan SP, Zhang FY, Zhou XZ, et al. P53 dependent mitochondrial permeability transition pore opening is required for dexamethasone-induced death of osteoblasts. J Cell Physiol. 2014;229:1475–83.CrossRefPubMedGoogle Scholar
  29. 29.
    Xu L, Tu Z, Xu G, Wang Y, Pan W, Zhan X, et al. Epirubicin directly promotes hepatitis B virus (HBV) replication in stable HBV-expressing cell lines: a novel mechanism of HBV reactivation following anticancer chemotherapy. Mol Med Rep. 2014;9:1345–50.PubMedGoogle Scholar
  30. 30.
    Renaud J, Bournival J, Zottig X, Martinoli MG. Resveratrol protects DAergic pc12 cells from high glucose-induced oxidative stress and apoptosis: effect on p53 and GRP75 localization. Neurotox Res. 2014;25:110–23.CrossRefPubMedGoogle Scholar
  31. 31.
    Cao C, Huang X, Han Y, Wan Y, Birnbaumer L, Feng GS, et al. Galpha(i1) and Galpha(i3) are required for epidermal growth factor-mediated activation of the Akt-mTORC1 pathway. Sci Signal. 2009;2:ra17.PubMedPubMedCentralGoogle Scholar
  32. 32.
    Zolnik BS, Stern ST, Kaiser JM, Heakal Y, Clogston JD, Kester M, et al. Rapid distribution of liposomal short-chain ceramide in vitro and in vivo. Drug Metab Dispos. 2008;36:1709–15.CrossRefPubMedGoogle Scholar
  33. 33.
    Fruman DA, Rommel C. PI3K and cancer: lessons, challenges and opportunities. Nat Rev Drug Discov. 2014;13:140–56.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Dienstmann R, Rodon J, Serra V, Tabernero J. Picking the point of inhibition: a comparative review of PI3K/AKT/mTOR pathway inhibitors. Mol Cancer Ther. 2014;13:1021–31.CrossRefPubMedGoogle Scholar
  35. 35.
    Sun SY. mTOR kinase inhibitors as potential cancer therapeutic drugs. Cancer Lett. 2013;340:1–8.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    O'Reilly KE, Rojo F, She QB, Solit D, Mills GB, Smith D, et al. mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res. 2006;66:1500–8.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Carracedo A, Ma L, Teruya-Feldstein J, Rojo F, Salmena L, Alimonti A, et al. Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer. J Clin Invest. 2008;118:3065–74.PubMedPubMedCentralGoogle Scholar
  38. 38.
    Zhou HY, Huang SL. Current development of the second generation of mTOR inhibitors as anticancer agents. Chin J Cancer. 2012;31:8–18.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Altomare DA, Testa JR. Perturbations of the AKT signaling pathway in human cancer. Oncogene. 2005;24:7455–64.CrossRefPubMedGoogle Scholar
  40. 40.
    Song G, Ouyang G, Bao S. The activation of Akt/PKB signaling pathway and cell survival. J Cell Mol Med. 2005;9:59–71.CrossRefPubMedGoogle Scholar
  41. 41.
    Tran MA, Smith CD, Kester M, Robertson GP. Combining nanoliposomal ceramide with sorafenib synergistically inhibits melanoma and breast cancer cell survival to decrease tumor development. Clin Cancer Res. 2008;14:3571–81.CrossRefPubMedGoogle Scholar
  42. 42.
    Stover TC, Sharma A, Robertson GP, Kester M. Systemic delivery of liposomal short-chain ceramide limits solid tumor growth in murine models of breast adenocarcinoma. Clin Cancer Res. 2005;11:3465–74.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  1. 1.Department of Intervention RadiologyThe Affiliated Tumor Hospital of Xinjiang Medical UniversityUrumqiChina
  2. 2.Department of Cancer Research InstituteThe Affiliated Tumor Hospital of Xinjiang Medical UniversityUrumqiChina

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