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



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.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. 1.

    H.B. El-Serag, K.L. Rudolph, Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 132, 2557-2576 (2007).

    Article  PubMed  CAS  Google 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).

    Article  PubMed  CAS  Google 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)

    Article  Google 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).

    Article  PubMed  CAS  Google 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).

    Article  PubMed  Google 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).

    Article  PubMed  PubMed Central  CAS  Google 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).

    Article  CAS  Google 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).

    Article  CAS  Google 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).

    Article  CAS  Google 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).

    Article  PubMed  CAS  Google 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)

    Article  PubMed  CAS  Google 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).

    Article  PubMed  CAS  Google 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).

    Article  PubMed  CAS  Google Scholar 

  15. 15.

    M. Bailly, G.E. Jones, Polarised migration: Cofilin holds the front. Curr. Biol. 13, R128-R130 (2003).

    Article  PubMed  CAS  Google Scholar 

  16. 16.

    T.D. Pollard, G.G. Borisy, Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453-465 (2003).

    Article  PubMed  CAS  Google 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).

    Article  PubMed  CAS  Google 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).

    PubMed  PubMed Central  CAS  Article  Google 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).

    Article  PubMed  CAS  Google 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).

    Article  PubMed  CAS  Google 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).

    Article  PubMed  CAS  Google 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)

    PubMed  CAS  Google 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)

    PubMed  CAS  Google 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)

    Article  PubMed  CAS  Google 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).

    Article  PubMed  CAS  Google 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).

    Article  PubMed  PubMed Central  CAS  Google 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)

    Article  CAS  Google 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).

    Article  PubMed  PubMed Central  CAS  Google 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).

    Article  PubMed  CAS  Google 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).

    Article  PubMed  PubMed Central  CAS  Google 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).

    Article  PubMed  CAS  Google 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).

    Article  PubMed  CAS  Google 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).

    Article  PubMed  PubMed Central  CAS  Google 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. 61–102

    Google 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).

    Article  CAS  Google Scholar 

  36. 36.

    M.C. Berenbaum, Synergy, additivism and antagonism in immunosuppression. A critical review. Clin. Exp. Immunol. 28, 1-18 (1977)

    PubMed  PubMed Central  CAS  Google 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).

    Article  PubMed  CAS  Google 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).

    Article  PubMed  CAS  Google 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).

    Article  PubMed  CAS  Google 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).

    Article  PubMed  PubMed Central  CAS  Google 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).

    Article  PubMed  CAS  Google 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).

    Article  PubMed  CAS  Google 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).

    Article  PubMed  CAS  Google 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).

    Article  PubMed  CAS  Google Scholar 

  45. 45.

    C. Huang, K. Jacobson, M.D. Schaller, MAP kinases and cell migration. J. Cell Sci. 117, 4619-4628 (2004).

    Article  PubMed  CAS  Google 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).

    Article  PubMed  CAS  Google 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).

    Article  PubMed  CAS  Google Scholar 

Download references


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).

Author information



Corresponding author

Correspondence to Brian Irving Carr.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material


(PDF 749 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

D’Alessandro, R., Refolo, M.G., Lippolis, C. et al. Strong enhancement by IGF1-R antagonists of hepatocellular carcinoma cell migration inhibition by Sorafenib and/or vitamin K1. Cell Oncol. 41, 283–296 (2018).

Download citation


  • Hepatocellular carcinoma
  • Combination therapy
  • Sorafenib
  • Vitamin K1
  • IGF1
  • Microenvironment
  • Cytoskeleton