Cellular Oncology

, Volume 41, Issue 3, pp 283–296 | Cite as

Strong enhancement by IGF1-R antagonists of hepatocellular carcinoma cell migration inhibition by Sorafenib and/or vitamin K1

  • Rosalba D’Alessandro
  • Maria Grazia Refolo
  • Catia Lippolis
  • Nicola Carella
  • Caterina Messa
  • Aldo Cavallini
  • Brian Irving CarrEmail author
Original Paper



Emerging evidence indicates that combining Sorafenib with vitamin K1 (VK1) may result in a synergistic inhibition of hepatocellular carcinoma (HCC) cell migration and proliferation. Despite this synergy, its benefits may be limited due to drug resistance resulting from cross-talk with the tumor microenvironment. Insulin-like growth factor-1 (IGF1) signaling acts as an important modulator of HCC cell growth, motility and drug resistance. Therefore, we aimed to explore the effects of Sorafenib in combination with VK1 and/or IGF1-R antagonists on HCC cells.


Scratch wound migration assays were performed to assess the motility of HCC-derived PLC/PRF/5, HLF and Hep3B cells. The synergistic, additive or antagonistic effects of Sorafenib, VK1 and IGF1-R antagonists on HCC cell motility were assessed using CompuSyn software. The effects mediated by these various compounds on HCC cytoskeleton organization were evaluated using DyLight 554 Phalloidin staining. Proliferation and migration-associated signaling pathways were analyzed in PLC/PRF/5 cells using Erk1/2 and Akt activation kits and Western blotting (Mek, JNK, Akt, Paxillin and p38), respectively.


The effects of the IGF1-R antagonists GSK1838705A and OSI-906 on HCC cell migration inhibition after Sorafenib and/or VK1 administration, individually or in combination, were evaluated. We found a synergistic effect in PLC/PRF/5, HLF and Hep3B cells for combinations of fixed doses of GSK1838705A or OSI-906 together with different doses of Sorafenib and/or VK1. The levels of synergy were found to be stronger at higher Sorafenib and/or VK1 concentrations and lower or absent at lower concentrations, with some variation among the different cell lines tested. In addition, we found that in PLC/PRF/5 and HLF cells IGF1-R blockage strongly enhanced the reduction and redistribution of F-actin induced by Sorafenib and/or VK1 through alterations in the phosphorylation levels of some of the principal proteins involved in the MAPK signaling cascade, which is essential for cell migration.


Our results indicate that modulation of the efficacy of Sorafenib through combinations with VK1 and/or IGF1-R antagonists results in synergistic inhibition of HCC cell migration.


Hepatocellular carcinoma Combination therapy Sorafenib Vitamin K1 IGF1 Microenvironment Cytoskeleton 



This research was supported by the Italian Ministry of Public Health (n.11/2016).


This research was supported by the Italian Ministry of Public Health (n.11/2016).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

13402_2018_370_MOESM1_ESM.pdf (749 kb)
ESM 1 (PDF 749 kb)


  1. 1.
    H.B. El-Serag, K.L. Rudolph, Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 132, 2557-2576 (2007). CrossRefPubMedGoogle Scholar
  2. 2.
    M. Shimada, K. Takenaka, T. Gion, Y. Fujiwara, K. Kajiyama, T. Maeda, K. Shirabe, T. Nishizaki, K. Yanaga, K. Sugimachi, Prognosis of recurrent hepatocellular carcinoma: A 10-year surgical experience in Japan. Gastroenterology 111, 720-726 (1996). CrossRefPubMedGoogle Scholar
  3. 3.
    E. Adachi, S. Maehara, E. Tsujita, K. Taguchi, S. Aishima, T. Rikimaru, Y. Yamashita, S. Tanaka, Clinicopathologic risk factors for recurrence after a curative hepatic resection for hepatocellular carcinoma. Surgery 131, 148–152 (2002)CrossRefGoogle Scholar
  4. 4.
    S. Aishima, Y. Basaki, Y. Oda, Y. Kuroda, Y. Nishihara, K. Taguchi, A. Taketomi, Y. Maehara, F. Hosoi, Y. Maruyama, A. Fotovati, S. Oie, M. Ono, T. Ueno, M. Sata, H. Yano, M. Kojiro, M. Kuwano, M. Tsuneyoshi, High expression of insulin-like growth factor binding protein-3 is correlated with lower portal invasion and better prognosis in human hepatocellular carcinoma. Cancer Sci. 97, 1182-1190 (2006). CrossRefPubMedGoogle Scholar
  5. 5.
    G. Giannelli, F. Pierri, P. Trerotoli, F. Marinosci, G. Serio, O. Schiraldi, S. Antonaci, Occurrence of portal vein tumor thrombus in epatocellular carcinoma affects prognosis and survival. A retrospettive clinical study of 150 cases. Hepatol. Res. 24, 50-59 (2002). CrossRefPubMedGoogle Scholar
  6. 6.
    G. Wei, M. Wang, T. Hyslop, Z. Wang, B.I. Carr, Vitamin K enhancement of Sorafenib-mediated HCC cell growth inhibition in vitro and in vivo. Int. J. Cancer 127, 2949-2958 (2010). CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    R. Gopal, K. Selvarasu, P.P. Pandian, K. Ganesan, Integrative transcriptome analysis of liver cancer profiles identifies upstream regulators and clinical significance of ACSM3 gene expression. Cell. Oncol. 40, 219-233 (2017). CrossRefGoogle Scholar
  8. 8.
    V. Ramesh, K. Selvarasu, J. Pandian, S. Myilsamy, C. Shanmugasundaram, K. Ganesan, NFκB activation demarcates a subset of hepatocellular carcinoma patients for targeted therapy. Cell. Oncol. 39, 523-536 (2016). CrossRefGoogle Scholar
  9. 9.
    J. Liu, X. Wei, Y. Wu, Y. Wang, Y. Qiu, J. Shi, H. Zhou, Z. Lu, M. Shao, L. Yu, L. Tong, Giganteaside D induces ROS-mediated apoptosis in human hepatocellular carcinoma cells through the MAPK pathway. Cell. Oncol. 39, 333-342 (2016). CrossRefGoogle Scholar
  10. 10.
    S.M. Wilhelm, C. Carter, L. Tang, D. Wilkie, A. McNabola, H. Rong, C. Chen, X. Zhang, P. Vincent, M. McHugh, Y. Cao, J. Shujath, S. Gawlak, D. Eveleigh, B. Rowley, L. Liu, L. Adnane, M. Lynch, D. Auclair, I. Taylor, R. Gedrich, A. Voznesensky, B. Riedl, L.E. Post, G. Bollag, P.A. Trail, BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res. 64, 7099-7109 (2004). CrossRefPubMedGoogle Scholar
  11. 11.
    J.M. Llovet, S. Ricci, V. Mazzaferro, P. Hilgard, E. Gane, J.F. Blanc, A.C. de Oliveira, A. Santoro, J.L. Raoul, A. Forner, M. Schwartz, C. Porta, S. Zeuzem, L. Bolondi, T.F. Greten, P.R. Galle, J.F. Seitz, I. Borbath, D. Häussinger, T. Giannaris, M. Shan, M. Moscovici, D. Voliotis, J. Bruix, SHARP Investigators Study Group. Sorafenib in advanced hepatocellular carcinoma. N. Engl. J. Med. 359, 378–390 (2008)CrossRefPubMedGoogle Scholar
  12. 12.
    S.M. Wilhelm, L. Adnane, P. Newell, A. Villanueva, J.M. Llovet, M. Lynch, Preclinical overview of sorafenib, a multikinase inhibitor that targets both Raf and VEGF and PDGF receptor tyrosine kinase signaling. Mol. Cancer Ther. 7, 3129-3140 (2008). CrossRefPubMedGoogle Scholar
  13. 13.
    L. Gao, C. Shay, F. Lv, X. Wang, Y. Teng. Implications of FGF19 on sorafenib-mediated nitric oxide production in hepatocellular carcinoma cells - a short report. Cell. Oncol. 41, 85-91 (2018).
  14. 14.
    Z. Wang, M. Wang, B.I. Carr, Involvement of receptor tyrosine phosphatase DEP-1 mediated PI3K-cofilin signaling pathway in sorafenib-induced cytoskeletal rearrangement in hepatoma cells. J. Cell. Physiol. 224, 559-565 (2010). CrossRefPubMedGoogle Scholar
  15. 15.
    M. Bailly, G.E. Jones, Polarised migration: Cofilin holds the front. Curr. Biol. 13, R128-R130 (2003). CrossRefPubMedGoogle Scholar
  16. 16.
    T.D. Pollard, G.G. Borisy, Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453-465 (2003). CrossRefPubMedGoogle Scholar
  17. 17.
    B.I. Carr, Z. Wang, M. Wang, G. Wei, Differential effects of vitamin K1 on AFP and DCP levels in patients with unresectable HCC and in HCC cell lines. Dig. Dis. Sci. 56, 1876-1883 (2011). CrossRefPubMedGoogle Scholar
  18. 18.
    G. Wei, M. Wang, B.I. Carr, Sorafenib combined vitamin k induces apoptosis in human pancreatic cancer cell lines through RAF/MEK/ERK and c-Jun NH2-terminal kinase pathways. J. Cell. Physiol. 224, 112-119 (2010). PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    T. Shibayama-Imazu, S. Sakairi, A. Watanabe, T. Aiuchi, S. Nakajo, K. Nakaya, Vitamin K(2) selectively induced apoptosis in ovarian TYK-nu and pancreatic MIA PaCa-2 cells out of eight solid tumor cell lines through a mechanism different from geranylgeraniol. J. Cancer Res. Clin. Oncol. 129, 1-11 (2003). CrossRefPubMedGoogle Scholar
  20. 20.
    T. Yokoyama, K. Miyazawa, M. Naito, J. Toyotake, T. Tauchi, M. Itoh, A. You, Y. Hayashi, M.M. Georgescu, Y. Kondo, S. Kondo, K. Ohyashiki, Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells. Autophagy 4, 629-640 (2008). CrossRefPubMedGoogle Scholar
  21. 21.
    M. Ma, X.J. Qu, G.Y. Mu, M.H. Chen, Y.N. Cheng, N. Kokudo, W. Tang, S.X. Cui, Vitamin K2 inhibits the growth of hepatocellular carcinoma via decrease of des-gamma-carboxy prothrombin. Chemotherapy 55, 28-35 (2009). CrossRefPubMedGoogle Scholar
  22. 22.
    S. Kuriyama, M. Hitomi, H. Yoshiji, T. Nonomura, T. Tsujimoto, A. Mitoro, T. Akahane, M. Ogawa, S. Nakai, T. Masaki, N. Uchida, Vitamins K2, K3 and K5 exert in vivo antitumor effects on hepatocellular carcinoma by regulating the expression of G1 phase-related cell cycle molecules. Int. J. Oncol. 27, 505-511 (2005)PubMedGoogle Scholar
  23. 23.
    T.Y. Ha, S. Hwang, H.N. Hong, Y.I. Choi, S.Y. Yoon, Y.J. Won, G.W. Song, N. Kim, E. Tak, B.Y. Ryoo, Synergistic effect of sorafenib and vitamin K on suppression of hepatocellular carcinoma cell migration and metastasis. Anticancer Res. 35, 1985–1995 (2015)PubMedGoogle Scholar
  24. 24.
    B.I. Carr, Z. Wang, M. Wang, A. Cavallini, R. D’Alessandro, M.G. Refolo, c-Met-Akt pathway-mediated enhancement of inhibitory c-Raf phosphorylation is involved in vitamin K1 and sorafenib synergy on HCC growth inhibition. Cancer Biol Ther 12, 531–538 (2011)CrossRefPubMedGoogle Scholar
  25. 25.
    R. D'Alessandro, C. Messa, M.G. Refolo, B.I. Carr, Modulation of sensitivity and resistance to multikinase inhibitors by microenvironmental platelet factors in HCC. Expert. Opin. Pharmacother. 16, 2773-2780 (2015). CrossRefPubMedGoogle Scholar
  26. 26.
    R. D'Alessandro, M.G. Refolo, C. Lippolis, G. Giannuzzi, N. Carella, C. Messa, A. Cavallini, B.I. Carr, Antagonism of sorafenib and regorafenib actions by platelet factors in hepatocellular carcinoma cell lines. BMC Cancer 14, 351 (2014). CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    M.G. Refolo, R. D'Alessandro, C. Lippolis, C. Messa, N. Carella, A. Cavallini, B.I. Carr, Modulation of Doxorubicin mediated growth inhibition of hepatocellular carcinoma cells by platelet lysates. Anti Cancer Agents Med. Chem. 14, 1154–1160 (2014)CrossRefGoogle Scholar
  28. 28.
    C. Lippolis, M.G. Refolo, R. D'Alessandro, N. Carella, C.A. Messa, A. Cavallini, B.I. Carr, Resistance to multikinase inhibitor actions mediated by insulin like growth factor-1. J. Exp. Clin. Cancer Res. 34, 90 (2015). CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    P. Sabbatini, S. Korenchuk, J.L. Rowand, A. Groy, Q. Liu, D. Leperi, C. Atkins, M. Dumble, J. Yang, K. Anderson, R.G. Kruger, R.R. Gontarek, K.R. Maksimchuk, S. Suravajjala, R.R. Lapierre, J.B. Shotwell, J.W. Wilson, S.D. Chamberlain, S.K. Rabindran, R. Kumar, GSK1838705A inhibits the insulin-like growth factor-1 receptor and anaplastic lymphoma kinase and shows antitumor activity in experimental models of human cancers. Mol. Cancer Ther. 8, 2811-2820 (2009). CrossRefPubMedGoogle Scholar
  30. 30.
    H.X. Chen, E. Sharon, IGF-1R as an anti-cancer target--trials and tribulations. Chin J Cancer 32, 242-252 (2013). CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    J.G. Scharf, W. Schmidt-Sandte, S.A. Pahernik, G. Ramadori, T. Braulke, H. Hartmann, Characterization of the insulin-like growth factor axis in a human hepatoma cell line (PLC/PRF/5). Carcinogenesis 19, 2121-2128 (1998). CrossRefPubMedGoogle Scholar
  32. 32.
    C.C. Liang, A.Y. Park, J.L. Guan, In vitro scratch assay: A convenient and inexpensive method for analysis of cell migration in vitro. Nat. Protoc. 2, 329-333 (2007). CrossRefPubMedGoogle Scholar
  33. 33.
    B.I. Carr, R. D'Alessandro, M.G. Refolo, P.A. Iacovazzi, C. Lippolis, C. Messa, A. Cavallini, M. Correale, A. Di Carlo, Effects of low concentrations of Regorafenib and Sorafenib on human HCC cell AFP, migration, invasion, and growth in vitro. J. Cell. Physiol. 228, 1344-1350 (2013). CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    T.C. Chou, in Synergism and Antagonism in Chemotherapy, ed. by T. C. Chou, D. C. Rideout. The median-effect principle and the combination index for quantitation of synergism and antagonism (Academic Press, San Diego, 1991), pp. 61–102Google Scholar
  35. 35.
    T.C. Chou, P. Talalay, Quantitative analysis of dose-effect relationships: The combined effects of multiple drugs or enzyme inhibitors. Adv. Enzym. Regul. 22, 27–55 (1984). CrossRefGoogle Scholar
  36. 36.
    M.C. Berenbaum, Synergy, additivism and antagonism in immunosuppression. A critical review. Clin. Exp. Immunol. 28, 1-18 (1977)PubMedPubMedCentralGoogle Scholar
  37. 37.
    R. D'Alessandro, A. Klajn, L. Stucchi, P. Podini, M.L. Malosio, J. Meldolesi, Expression of the neurosecretory process in PC12 cells is governed by REST. J. Neurochem. 105, 1369-1383 (2008). CrossRefPubMedGoogle Scholar
  38. 38.
    F. Gao, B. Liang, S.T. Reddy, R. Farias-Eisner, X. Su, Role of inflammation-associated microenvironment in tumorigenesis and metastasis. Curr. Cancer Drug Targets 14, 30-45 (2014). CrossRefPubMedGoogle Scholar
  39. 39.
    A.H. Rosendahl, C. Gundewar, K. Said Hilmersson, L. Ni, M.A. Saleem, R. Andersson, Conditionally immortalized human pancreatic stellate cell lines demonstrate enhanced proliferation and migration in response to IGF-I. Exp. Cell Res. 330, 300-310 (2015). CrossRefPubMedGoogle Scholar
  40. 40.
    S. Sarkissyan, M. Sarkissyan, Y. Wu, J. Cardenas, H.P. Koeffler, J.V. Vadgama, IGF-1 regulates Cyr61 induced breast cancer cell proliferation and invasion. PLoS One 9, e103534 (2014). CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    H.Z. Sun, S.F. Wu, Z.H. Tu, Blockage of IGF-1R signaling sensitizes urinary bladder cancer cells to mitomycin-mediated cytotoxicity. Cell Res. 11, 107-115 (2001). CrossRefPubMedGoogle Scholar
  42. 42.
    G.L. Johnson, R. Lapadat, Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 298, 1911-1192 (2002). CrossRefPubMedGoogle Scholar
  43. 43.
    C. Huang, Z. Rajfur, C. Borchers, M.D. Schaller, K. Jacobson, JNK phosphorylates paxillin and regulates cell migration. Nature 424, 219-223 (2003). CrossRefPubMedGoogle Scholar
  44. 44.
    Z. Huang, D.P. Yan, B.X. Ge, JNK regulates cell migration through promotion of tyrosine phosphorylation of paxillin. Cell. Signal. 20, 2002-2012 (2008). CrossRefPubMedGoogle Scholar
  45. 45.
    C. Huang, K. Jacobson, M.D. Schaller, MAP kinases and cell migration. J. Cell Sci. 117, 4619-4628 (2004). CrossRefPubMedGoogle Scholar
  46. 46.
    Y.P. Ching, V.Y. Leong, M.F. Lee, H.T. Xu, D.Y. Jin, I.O. Ng, P21-activated protein kinase is overexpressed in hepatocellular carcinoma and enhances cancer metastasis involving c-Jun NH2-terminal kinase activation and paxillin phosphorylation. Cancer Res. 67, 3601-3608 (2007). CrossRefPubMedGoogle Scholar
  47. 47.
    C.T. Hu, C.C. Cheng, J.R. Wu, S.M. Pan, W.S. Wu, PKCε-mediated c-met endosomal processing directs fluctuant c-met-JNK-paxillin signaling for tumor progression of HepG2. Cell. Signal. 27, 1544-1555 (2015). CrossRefPubMedGoogle Scholar

Copyright information

© International Society for Cellular Oncology 2018

Authors and Affiliations

  • Rosalba D’Alessandro
    • 1
  • Maria Grazia Refolo
    • 1
  • Catia Lippolis
    • 1
  • Nicola Carella
    • 1
  • Caterina Messa
    • 1
  • Aldo Cavallini
    • 1
  • Brian Irving Carr
    • 2
    Email author
  1. 1.Laboratory of Cellular and Molecular Biology, Department of Clinical Pathology, National Institute of Gastroenterology“S. De Bellis” Research HospitalCastellana GrotteItaly
  2. 2.Program for Targeted Experimental Therapeutics, Izmir Biomedicine and Genome CenterDokuz Eylul UniversityIzmirTurkey

Personalised recommendations