Cancer Chemotherapy and Pharmacology

, Volume 64, Issue 3, pp 497–507 | Cite as

Synergistic anti-tumor efficacy of lovastatin and protein kinase C-beta inhibitor in hepatocellular carcinoma

  • Won Kim
  • Jung-Hwan YoonEmail author
  • Jung-Ryul Kim
  • In-Jin Jang
  • Yung-Jue Bang
  • Yoon-Jun Kim
  • Hyo-Suk Lee
Original Article



Hepatocellular carcinoma (HCC) is characterized by hypervascularity and chemoresistance. Protein kinase C (PKC) participates in cancer progression by enhancing anti-apoptotic signals, angiogenesis, and chemoresistance. Statins have a selective anti-cancer effect due to over-expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoAR) in cancer cells, but statins may also activate PKC. Thus, we hypothesized that simultaneous treatment with statin and PKC inhibitor might synergistically enhance their anti-tumor efficacies against HCCs.


Hepatocellular carcinoma cell growth was assessed using MTS assays, apoptotic cell death by DAPI staining, and apoptotic signaling cascades were explored by immunoblotting. An in vivo model of HCC was established in C3H mice intradermally implanted with MH134 cells. Lovastatin and/or a PKC inhibitor (enzastaurin) was subsequently administered. Anti-tumor efficacies were evaluated by measuring tumor volumes and quantifying apoptotic cells and microvessel densities (MVD).


Co-treatment with lovastatin and enzastaurin was found to synergistically suppress HCC cell growth in vitro. Lovastatin induced HCC cellular apoptosis by activating mitochondrial apoptotic signals, and although enzastaurin alone did not induce apoptosis, its addition significantly enhanced lovastatin-induced apoptosis. This enhanced apoptosis was attributed to increased caspase-9 activation in these cells. Moreover, tumor growth was significantly suppressed in mice co-treated with lovastatin and enzastaurin, and percentages of TUNEL-positive cells were significantly increased and MVDs were significantly decreased in those mice.


Combinatorial treatment with statin and PKC inhibitor was found to enhance anti-tumor efficacy in vivo and in vitro. Further studies are warranted to prove anti-tumor efficacy of this potential therapy in human HCCs.


Hepatocellular carcinoma Statin Protein kinase C Apoptosis 



Hepatocellular carcinoma


Protein kinase C


3-Hydroxy-3-methylglutaryl-coenzyme A reductase


Microvessel densities


Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling


X-linked inhibitor of apoptosis protein



This study was supported by the Seoul National University Hospital Research Fund (#03-2006-007), and by the Korean Foundation of Liver Research (2006) and the Korea Health 21 R&D Project (#0412-CR01-0704-0001).


  1. 1.
    Llovet JM, Burroughs A, Bruix J (2003) Hepatocellular carcinoma. Lancet 362:1907–1917PubMedCrossRefGoogle Scholar
  2. 2.
    El-Serag HB, Mason AC (1999) Rising incidence of hepatocellular carcinoma in the United States. N Engl J Med 340:745–750PubMedCrossRefGoogle Scholar
  3. 3.
    Parkin DM, Bray F, Ferlay J, Pisani P (2001) Estimating the world cancer burden: Globocan 2000. Int J cancer 94:153–156PubMedCrossRefGoogle Scholar
  4. 4.
    Yuen MF, Cheng CC, Lauder IJ, Lam SK, Ooi CG, Lai CL (2000) Early detection of hepatocellular carcinoma increases the chance of treatment: Hong Kong experience. Hepatology 31:330–335PubMedCrossRefGoogle Scholar
  5. 5.
    Myung SJ, Yoon JH, Kim KM, Gwak GY, Kim YJ, Yu JW, Chung JW, Lee HS (2006) Diffuse infiltrative hepatocellular carcinomas in a hepatitis B-endemic area: diagnostic and therapeutic impediments. Hepatogastroenterology 53:266–270PubMedGoogle Scholar
  6. 6.
    Lee HS, Kim KM, Yoon JH, Lee TR, Suh KS, Lee KU, Chung JW, Park JH, Kim CY (2002) Therapeutic efficacy of transcatheter arterial chemoembolization as compared with hepatic resection in hepatocellular carcinoma patients with compensated liver function in a hepatitis B virus-endemic area: a prospective cohort study. J Clin Oncol 20:4459–4465PubMedCrossRefGoogle Scholar
  7. 7.
    Louafi S, Boige V, Ducreux M, Bonyhay L, Mansourbakht T, de Baere T, Asnacios A, Hannoun L, Poynard T, Taïeb J (2007) Gemcitabine plus oxaliplatin (GEMOX) in patients with advanced hepatocellular carcinoma (HCC): results of a phase II study. Cancer 109:1384–1390PubMedCrossRefGoogle Scholar
  8. 8.
    Matsunaga T, Shirasawa H, Hishiki T, Enomoto H, Kouchi K, Ohtsuka Y, Iwai J, Yoshida H, Tanabe M, Kobayashi S, Asano T, Etoh T, Nishi Y, Ohnuma N (1998) Expression of MRP and cMOAT in childhood neuroblastomas and malignant liver tumors and its relevance to clinical behavior. Jpn J Cancer Res 89:1276–1283PubMedGoogle Scholar
  9. 9.
    Chou YY, Cheng AL, Hsu HC (1997) Expression of P-glycoprotein and p53 in advanced hepatocellular carcinoma treated by single agent chemotherapy: clinical correlation. J Gastroenterol Hepatol 12:569–575PubMedCrossRefGoogle Scholar
  10. 10.
    Lai CL, Wu PC, Chan GC, Lok AS, Lin HJ (1988) Doxorubicin versus no antitumor therapy in inoperable hepatocellular carcinoma. A prospective randomized trial. Cancer 62:479–483PubMedCrossRefGoogle Scholar
  11. 11.
    Thorgeirsson SS, Grisham JW (2002) Molecular pathogenesis of human hepatocellular carcinoma. Nat Genet 31:339–346PubMedCrossRefGoogle Scholar
  12. 12.
    Hentosh P, Yuh SH, Elson CE, Peffley DM (2001) Sterol-independent regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in tumor cells. Mol Carcinog 32:154–166PubMedCrossRefGoogle Scholar
  13. 13.
    Wong WW, Tan MM, Xia Z, Dimitroulakos J, Minden MD, Penn LZ (2001) Cerivastatin triggers tumor-specific apoptosis with higher efficacy than lovastatin. Clin Cancer Res 7:2067–2075PubMedGoogle Scholar
  14. 14.
    Kubota T, Fujisaki K, Itoh Y, Yano T, Sendo T, Oishi R (2004) Apoptotic injury in cultured human hepatocytes induced by HMG-CoA reductase inhibitors. Biochem Pharmacol 67:2175–2186PubMedCrossRefGoogle Scholar
  15. 15.
    Shibata MA, Ito Y, Morimoto J, Otsuki Y (2004) Lovastatin inhibits tumor growth and lung metastasis in mouse mammary carcinoma model: a p53-independent mitochondrial-mediated apoptotic mechanism. Carcinogenesis 25:1887–1898PubMedCrossRefGoogle Scholar
  16. 16.
    Clemens MJ, Trayner I, Menaya J (1992) The role of protein kinase C isoenzymes in the regulation of cell proliferation and differentiation. J Cell Sci 103(Pt 4):881–887PubMedGoogle Scholar
  17. 17.
    Teicher BA, Alvarez E, Menon K, Esterman MA, Considine E, Shih C, Faul MM (2002) Antiangiogenic effects of a protein kinase Cbeta-selective small molecule. Cancer Chemother Pharmacol 49:69–77PubMedCrossRefGoogle Scholar
  18. 18.
    Suzuki S, Hayashi H, Takagi K, Kondo T, Takagi K, Ueyama J, Wakusawa S (2006) Protein kinase Cbeta isoform down-regulates the expression of MDR3 P-glycoprotein in human Chang liver cells. Biochim Biophys Acta 1760:1552–1557PubMedGoogle Scholar
  19. 19.
    Tanaka K, Honda M, Takabatake T (2004) Anti-apoptotic effect of atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitor, on cardiac myocytes through protein kinase C activation. Clin Exp Pharmacol Physiol 31:360–364PubMedCrossRefGoogle Scholar
  20. 20.
    Welch PA, Sinha VP, Cleverly AL, Darstein C, Flanagan SD, Musib LC (2007) Safety, tolerability, QTc evaluation, and pharmacokinetics of single and multiple doses of enzastaurin HCl (LY317615), a protein kinase C-beta inhibitor, in healthy subjects. J Clin Pharmacol 47:1138–1151PubMedCrossRefGoogle Scholar
  21. 21.
    Carducci MA, Musib L, Kies MS, Pili R, Truong M, Brahmer JR, Cole P, Sullivan R, Riddle J, Schmidt J, Enas N, Sinha V, Thornton DE, Herbst RS (2006) Phase I dose escalation and pharmacokinetic study of enzastaurin, an oral protein kinase C beta inhibitor, in patients with advanced cancer. J Clin Oncol 24:4092–4099PubMedCrossRefGoogle Scholar
  22. 22.
    Feleszko W, Balkowiec EZ, Sieberth E, Marczak M, Dabrowska A, Giermasz A, Czajka A, Jakóbisiak M (1999) Lovastatin and tumor necrosis factor-alpha exhibit potentiated antitumor effects against Ha-ras-transformed murine tumor via inhibition of tumor-induced angiogenesis. Int J Cancer 81:560–567PubMedCrossRefGoogle Scholar
  23. 23.
    Higuchi H, Bronk SF, Takikawa Y, Werneburg N, Takimoto R, El-Deiry W, Gores GJ (2001) The bile acid glycochenodeoxycholate induces trail-receptor 2/DR5 expression and apoptosis. J Biol Chem 276:38610–38618PubMedCrossRefGoogle Scholar
  24. 24.
    Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J (1982) Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res 42:3858–3863PubMedGoogle Scholar
  25. 25.
    Yamashita YI, Shimada M, Hasegawa H, Minagawa R, Rikimaru T, Hamatsu T, Tanaka S, Shirabe K, Miyazaki JI, Sugimachi K (2001) Electroporation-mediated interleukin-12 gene therapy for hepatocellular carcinoma in the mice model. Cancer Res 61:1005–1012PubMedGoogle Scholar
  26. 26.
    Beal SL (1984) Population pharmacokinetic data and parameter estimation based on their first two statistical moments. Drug Metab Rev 15:173–193PubMedCrossRefGoogle Scholar
  27. 27.
    Wong WW, Dimitroulakos J, Minden MD, Penn LZ (2002) HMG-CoA reductase inhibitors and the malignant cell: the statin family of drugs as triggers of tumor-specific apoptosis. Leukemia 16:508–519PubMedCrossRefGoogle Scholar
  28. 28.
    Wu J, Wong WW, Khosravi F, Minden MD, Penn LZ (2004) Blocking the Raf/MEK/ERK pathway sensitizes acute myelogenous leukemia cells to lovastatin-induced apoptosis. Cancer Res 64:6461–6468PubMedCrossRefGoogle Scholar
  29. 29.
    Siperstein MD, Fagan VM (1964) Deletion of the cholesterol-negative feedback system in liver tumors. Cancer Res 24:1108–1115PubMedGoogle Scholar
  30. 30.
    Buchwald H (1992) Cholesterol inhibition, cancer, and chemotherapy. Lancet 339:1154–1156Google Scholar
  31. 31.
    Cafforio P, Dammacco F, Gernone A, Silvestris F (2005) Statins activate the mitochondrial pathway of apoptosis in human lymphoblasts and myeloma cells. Carcinogenesis 26:883–891PubMedCrossRefGoogle Scholar
  32. 32.
    Paragh G, Kertai P, Kovacs P, Paragh G Jr, Fulop P, Foris G (2003) HMG CoA reductase inhibitor fluvastatin arrests the development of implanted hepatocarcinoma in rats. Anticancer Res 23:3949–3954PubMedGoogle Scholar
  33. 33.
    Littman ML, Taguchi T, Mosbach EH (1966) Effect of cholesterol-free, fat-free diet and hypocholesteremic agents on growth of transplantable animal tumors. Cancer Chemother Rep 50:25–45PubMedGoogle Scholar
  34. 34.
    Kawata S, Yamasaki E, Nagase T, Inui Y, Ito N, Matsuda Y, Inada M, Tamura S, Noda S, Imai Y, Matsuzawa Y (2001) Effect of pravastatin on survival in patients with advanced hepatocellular carcinoma. A randomized controlled trial. Br J Cancer 84:886–891PubMedCrossRefGoogle Scholar
  35. 35.
    Blais L, Desgagne A, LeLorier J (2000) 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors and the risk of cancer: a nested case-control study. Arch Intern Med 160:2363–2368Google Scholar
  36. 36.
    Graaf MR, Beiderbeck AB, Egberts AC, Richel DJ, Guchelaar HJ (2004) The risk of cancer in users of statins. J Clin Oncol 22:2388–2394PubMedCrossRefGoogle Scholar
  37. 37.
    Marais R, Light Y, Mason C, Paterson H, Olson MF, Marshall CJ (1998) Requirement of Ras-GTP-Raf complexes for activation of Raf-1 by protein kinase C. Science 280:109–112PubMedCrossRefGoogle Scholar
  38. 38.
    O’Brian C, Vogel VG, Singletary SE, Ward NE (1989) Elevated protein kinase C expression in human breast tumor biopsies relative to normal breast tissue. Cancer Res 49:3215–3217PubMedGoogle Scholar
  39. 39.
    Selzer E, Okamoto I, Lucas T, Kodym R, Pehamberger H, Jansen B (2002) Protein kinase C isoforms in normal and transformed cells of the melanocytic lineage. Melanoma Res 12:201–209PubMedCrossRefGoogle Scholar
  40. 40.
    Wu TT, Hsieh YH, Wu CC, Hsieh YS, Huang CY, Liu JY (2007) Overexpression of protein kinase Calpha mRNA in human hepatocellular carcinoma: a potential marker of disease prognosis. Clin Chim Acta 382:54–58PubMedCrossRefGoogle Scholar
  41. 41.
    Keyes KA, Mann L, Sherman M, Galbreath E, Schirtzinger L, Ballard D, Chen YF, Iversen P, Teicher BA (2004) LY317615 decreases plasma VEGF levels in human tumor xenograft-bearing mice. Cancer Chemother Pharmacol 53:133–140PubMedCrossRefGoogle Scholar
  42. 42.
    Yoshiji H, Kuriyama S, Ways DK, Yoshii J, Miyamoto Y, Kawata M, Ikenaka Y, Tsujinoue H, Nakatani T, Shibuya M, Fukui H (1999) Protein kinase C lies on the signaling pathway for vascular endothelial growth factor-mediated tumor development and angiogenesis. Cancer Res 59:4413–4418PubMedGoogle Scholar
  43. 43.
    Goekjian PG, Jirousek MR (2001) Protein kinase C inhibitors as novel anticancer drugs. Expert Opin Investig Drugs 10:2117–2140PubMedCrossRefGoogle Scholar
  44. 44.
    Lee JY, Hannun YA, Obeid LM (1996) Ceramide inactivates cellular protein kinase Calpha. J Biol Chem 271:13169–13174PubMedCrossRefGoogle Scholar
  45. 45.
    Goss VL, Hocevar BA, Thompson LJ, Stratton CA, Burns DJ, Fields AP (1994) Identification of nuclear beta II protein kinase C as a mitotic lamin kinase. J Biol Chem 269:19074–19080PubMedGoogle Scholar
  46. 46.
    Shi RX, Ong CN, Shen HM (2005) Protein kinase C inhibition and x-linked inhibitor of apoptosis protein degradation contribute to the sensitization effect of luteolin on tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in cancer cells. Cancer Res 65:7815–7823PubMedCrossRefGoogle Scholar
  47. 47.
    Brady SC, Allan LA, Clarke PR (2005) Regulation of caspase-9 through phosphorylation by protein kinase C zeta in response to hyperosmotic stress. Mol Cell Biol 25:10543–10555PubMedCrossRefGoogle Scholar
  48. 48.
    Skaletz-Rorowski A, Kureishi Y, Shiojima I, Walsh K (2004) The pro- and antiangiogenic effects of statins. Semin Vasc Med 4:395–400PubMedCrossRefGoogle Scholar
  49. 49.
    Kureishi Y, Luo Z, Shiojima I, Bialik A, Fulton D, Lefer DJ, Sessa WC, Walsh (2000) The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals. Nat Med 6:1004–1010PubMedCrossRefGoogle Scholar
  50. 50.
    Thibault A, Samid D, Tompkins AC, Figg WD, Cooper MR, Hohl RJ, Trepel J, Liang B, Patronas N, Venzon DJ, Reed E, Myers CE (1996) Phase I study of lovastatin, an inhibitor of the mevalonate pathway, in patients with cancer. Clin Cancer Res 2:483–491PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Won Kim
    • 1
  • Jung-Hwan Yoon
    • 2
    Email author
  • Jung-Ryul Kim
    • 3
  • In-Jin Jang
    • 3
  • Yung-Jue Bang
    • 4
  • Yoon-Jun Kim
    • 2
  • Hyo-Suk Lee
    • 2
  1. 1.Department of Internal MedicineSeoul National University Boramae HospitalSeoulSouth Korea
  2. 2.Department of Internal Medicine and Liver Research InstituteSeoul National University College of MedicineSeoulSouth Korea
  3. 3.Department of Pharmacology and Clinical Pharmacology UnitSeoul National University College of MedicineSeoulSouth Korea
  4. 4.Department of Internal Medicine and Cancer Research InstituteSeoul National University College of MedicineSeoulSouth Korea

Personalised recommendations