View More View Less
  • 1 Alanya Alaaddin Keykubat University, Turkey
  • 2 Akdeniz University, Turkey
Restricted access

Background and aims

Targeted chemotherapeutics such as cetuximab can cause many side effects such as skin toxicity when used in high concentrations. In addition, cancer cells can develop resistance to some of the anticancer agents during treatment. The lack of the desired success in chemotherapy and the development of resistance to chemotherapeutics, such as epirubicin HCl, suggest that there is a need for combined therapies. The combination of targeted chemotherapeutics and conventional chemotherapy drugs may lead to the emergence of new strategies in the treatment of cancer. In this study, cytotoxic, antiproliferative, cell cycle inhibitive, oxidative stress generation, and apoptotic effects and effect mechanisms of cetuximab alone and together with epirubicin HCl on parental liver cancer cells (P-Hep G2) and epirubicin HCl-resistant liver cancer cells (R-Hep G2) were investigated.

Materials

Cytotoxic effects of cetuximab alone and with epirubicin-HCl on cells were determined by Cell Titer-Blue® Cell Viability and Lactate Dehydrogenase Activity tests. Cell cycle distributions and apoptosis were detected by reverse transcription polymerase chain reaction (RT-PCR).

Results

Cetuximab with epirubicin HCl treatment increased the cytotoxic effect on both cells. Caspase-3/7 activity increased 3 and 1.5 times in comparison with control group in P-Hep G2 and R-Hep G2 cells, respectively, after treating with cetuximab alone, whereas the increase was found to be approximately 4.7 and 2.5 times when cetuximab was treated with epirubicin HCl in P-Hep G2 and R-Hep G2 cells, respectively. Both cetuximab alone and together with epirubicin HCl treatments caused increases in Bax/Bcl-2 ratio in both cells.

Discussion

Treatment of cetuximab with epirubicin HCl to P-Hep G2 and R-Hep G2 cells was found to be more effective in cytotoxic effect and inducing apoptosis comparison to cetuximab alone treatment. In addition, combination treatment showed different effects on pro-apoptotic/anti-apoptotic genes expression according to cells drug resistance properties.

  • Ashworth, R. E., Wu, J. (2014) Mammalian target of rapamycin inhibition in hepatocellular carcinoma. World J. Hepatol. 6, 776782.

  • Berthiaume, J. M., Wallace, K. B. (2007) Adriamycin-induced oxidative mitochondrial cardiotoxicity. Cell. Biol. Toxicol. 23, 1525.

  • Bijwaard, K. E., Aguilera, N. S. I., Monczak, Y. T. M., Taubenberger, J. K., Jack, H., Lichy, J. H. (2001) Quantitative real-time reverse transcription-PCR assay for cyclin D1 expression: utility in the diagnosis of mantle cell lymphoma. Clin. Chem. 47, 195201.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Bosetti, C., Turati, F., La Vecchia, C. (2014) Hepatocellular carcinoma epidemiology. Best. Pract. Res. Clin. Gastroenterol. 28, 753770.

  • Bradford, M. M. (1976) A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248254.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Buckley, A. F., Burgart, L. J., Sahai, V., Kakar, S. (2008) Epidermal growth factor receptor expression and gene copy number in conventional hepatocellular carcinoma. Am. J. Clin. Pathol. 129, 245251.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Carvalho, A. C., Franklin, G., Dias, A. C. P., Lima, C. F. (2014) Methanolic extract of Hypericum perforatum cells elicited with Agrobacterium tumefaciens provides protection against oxidative stress induced in human HepG2 cells. Ind. Crop. Prod. 59, 177183.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Chen, W., Hu, Q. D., Xia, X. F., Liang, C., Liu, H., Zhang, Q., Ma, T., Liang, F., Liang, T. B. (2014) Rapamycin enhances cetuximab cytotoxicity by inhibiting mTOR-mediated drug resistance in mesenchymal hepatoma cells. Cancer. Biol. Ther. 15, 992999.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Chen, W., Shen, X., Xia, X., Xu, G., Ma, T., Bai, X., Liang, T. (2012) NSC 74859-mediated inhibition of STAT3 enhances the anti-proliferative activity of cetuximab in hepatocellular carcinoma. Liver. Int. 32, 7077.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Chuma, M., Terashita, K., Sakamoto, N. (2015) New molecularly targeted therapies against advanced hepatocellular carcinoma: from molecular pathogenesis to clinical trials and future directions. Hepatol. Res. 45, E1E11.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • D’angelo, D., Mussnich, P., Rosa, R., Bianco, R., Tortora, G., Fusco, A. (2014) High mobility group A1 protein expression reduces the sensitivity of colon and thyroid cancer cells to antineoplastic drugs. BMC Cancer 14, 851.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Daveau, M., Scotte, M., François, A., Coulouarn, C., Ros, G., Tallet, Y., Hiron, M., Hellot, M. F., Salier, J. P. (2003) Hepatocyte growth factor, transforming growth factor alpha, and their receptors as combined markers of prognosis in hepatocellular carcinoma. Mol. Carcinog. 36, 130141.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Ellence. (1999) Epirubicin Hydrochloride Injection, Revised Package Insert. Pharmacia & Upjohn, Perth, Australia.

  • Escudier, B., Eisen, T., Stadler, W. M., Szczylik, C., Oudard, S., Siebels, M., Negrier, S., Chevreau, C., Solska, E., Desai, A. A., Rolland, F., Demkow, T., Hutson, T. E., Gore, M., Freeman, S., Schwartz, B., Shan, M., Simantov, R., Bukowski, R. M. (2007) Sorafenib in advanced clear-cell renal-cell carcinoma. N. Engl. J. Med. 356, 125134.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Flohe, L., Gunzler, W. A. (1984) Glutathione peroxidase. Methods. Enzymol. 105, 115121.

  • Forner, A., Llovet, J. M., Bruix, J. (2012) Hepatocellular carcinoma. Lancet 379, 12451255.

  • Gao, A. M., Ke, Z. P., Shi, F., Sun, G. C., Chen, H. (2013) Chrysin enhances sensitivity of BEL-7402/ADM cells to doxorubicin by suppressing PI3K/Akt/Nrf2 and ERK/Nrf2 pathway. Chem. Biol. Interact. 206, 100108.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Gruenwald, V., Wilkens, L., Gebel, M., Greten, T. F., Kubicka, S., Ganser, A., Manns, M. P., Malek, N. P. (2007) A phase II open-label study of cetuximab in unresectable hepatocellular carcinoma: final results [Abstract]. J. Clin. Oncol. 25, 4598.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Han, Y., Zhang, J. P., Qian, J. Q., Hu, C. Q. (2015) Cardiotoxicity evaluation of anthracyclines in zebrafish (Danio rerio). J. Appl. Toxicol. 35, 241252.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Harari, P. M. (2004) Epidermal growth factor receptor inhibition strategies in oncology. Endocr. Relat. Cancer 11, 689708.

  • Harding, J., Burtness, B. (2005) Cetuximab: an epidermal growth factor receptor chemeric human-murine monoclonal antibody. Drugs Today (Barc) 41, 107127.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Huang, S. T., Yang, R. C., Yang, L. J., Lee, P. N., Pang, J. H. S. (2003) Phyllanthus urinaria triggers the apoptosis and Bcl-2 down regulation in Lewis lung carcinoma cells. Life Sci. 72, 17051716.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Huether, A., Hopfner, M., Baradari, V., Schuppan, D., Scherubl, H. (2005) EGFR blockade by cetuximab alone or as combination therapy for growth control of hepatocellular cancer. Biochem. Pharmacol. 70, 15681578.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Jansson, O., Motlagh, P. B., Persson, M., Henrikson, R., Grankvist, K. (1999) Increase in doxorubicin cytotoxicity by carvedilol inhibition of P-glycoprotein activity. Biochem. Pharmacol. 58, 18011806.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Javitt, N. B. (1990) HepG2 cells as a resource for metabolic studies: lipoprotein, cholesterol, and bile acids. FASEB J. 4, 161168.

  • Kirkman, T. W. (1996) Statistics to Use [Online]. Retrieved August 17, 2008, from http://www.physics.csbsju.edu/stats/1996

  • Llovet, J. M., Bruix, J. (2008) Molecular targeted therapies in hepatocellular carcinoma. Hepatology 48, 13121327.

  • Lupberger, J., Zeisel, M. B., Xiao, F., Thumann, C., Fofana, I., Zona, L., Davis, C., Mee, C. J., Turek, M., Gorke, S., Royer, C., Fischer, B., Zahid, M. N., Lavillette, D., Fresquet, J., Cosset, F. L., Rothenberg, S. M., Pietschmann, T., Patel, A. H., Pessaux, P., Doffoël, M., Raffelsberger, W., Poch, O., Mckeating, J. A., Brino, L., Baumert, T. F. (2011) EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy. Nat. Med. 17, 589595.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Mao, C., Liao, R. Y., Chen, Q. (2010) BRAF mutation predicts resistance to anti-EGFR monoclonal antibodies in wild-type KRAS metastatic colorectal cancer. J. Cancer. Res. Clin. Oncol. 136, 12931294.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Nasr, M., Nafee, N., Saad, H., Kazem, A. (2014) Improved antitumor activity and reduced cardiotoxicity of epirubicin using hepatocyte-targeted nanoparticles combined with tocotrienols against hepatocellular carcinoma in mice. Eur. J. Pharm. Biopharm. 88, 216225.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Nishiwaki-Matsushima, R., Ohta, T., Nishiwaki, S., Suganuma, M., Kohyama, K., Ishikawa, T., Carmichael, W. W., Fujiki, H. (1992) Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR. J. Cancer. Res. Clin. Oncol. 118, 420424.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Ozkan, A. (2007) Lymphokine-activated killer cell susceptibility in epirubicin resistant and parental human non-small cell lung cancer (NSCLC). Biologia 62, 232237.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Parfitt, K., Sweetman, S. C., Blake, P. S., Parsons, A. V. (1999) The Complete Drug Reference (32nd ed.). Pharmaceutical Press, London, p. 543.

    • Search Google Scholar
    • Export Citation
  • Radford, J. A., Margison, J. M., Swindell, R., Mad, M. J., Wilkinson, P. M., Thatcher, N. (1991) The stability of ifosfamide in aqueous solution and its suitability for continuous 7-day infusion by ambulatory pump. J. Cancer. Res. Clin. Oncol. 117, 151156.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Ramalingam, M., Kim, S. J. (2014) Insulin on hydrogen peroxide-induced oxidative stress involves ROS/Ca2+ and Akt/Bcl-2 signaling pathways. Free. Radic. Res. 48, 347356.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Rampias, T., Giagini, A., Siolos, S., Matsuzaki, H., Sasaki, C., Scorilas, A., Psyrri, A. (2014) RAS/PI3K crosstalk and cetuximab resistance in head and neck squamous cell carcinoma. Clin. Cancer. Res. 20, 29332946.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Rihova, B., Strohalm, J., Kobackova, K. (2002) Acquired and specific immunological mechanisms co-responsile for efficacy of polymer-bound drugs. J. Control. Release 78, 97114.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Rothenberg, M. L., Blanke, C. D. (1999) Topoisomerase I inhibitors in the treatment of colorectal cancer. Colorectal cancer: past accomplishments and future directions II. Semin. Oncol. 26, 632639.

    • Search Google Scholar
    • Export Citation
  • Spinzi, G., Paggi, S. (2008) Sorafenib in advanced hepatocellular carcinoma. N. Engl. J. Med. 359, 24972498.

  • Stotz, M., Gerger, A., Haybaeck, J., Kiesslich, T., Bullock, M. D., Pichler, M. (2015) Molecular targeted therapies in hepatocellular carcinoma: past, present and future. Anticancer Res. 35, 57375744.

    • Search Google Scholar
    • Export Citation
  • Trissel, L. A., Parks, N. P. T., Santiago, N. M. (1991) Visual compatibility of fludarabine phosphate with antineoplastic drugs, antiinfectives, and other selected drugs during simulated Y-site injection. Am. J. Hosp. Pharm. 48, 21862189.

    • Search Google Scholar
    • Export Citation
  • Van, I. S. C., Zegers, M. M., Kok, J. W., Hoekstra, D. (1997) Segregation of glucosylceramide and sphingomyelin occurs in the apical to basolateral transcytotic route in HepG2 cells. J. Cell. Biol. 137, 347357.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Williams, D. A., Lokich, J. (1992) A review of the stability and compatibility of antineoplastic drugs for multiple-drug infusions. Cancer. Chemother. Pharmacol. 31, 171181.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Yang, J. D., Roberts, L. R. (2010) Hepatocellular carcinoma: a global view. Nat. Rev. Gastroenterol. Hepatol. 7, 448458.

  • Yang, Y., Yan, J., Huang, Y., Xu, H., Zhang, Y., Hu, R., Jiang, J., Chen, Z., Jiang, H. (2015) The cancer pain related factors affected by celecoxib together with cetuximab in head and neck squamous cell carcinoma. Biomed. Pharmacother. 70, 181189.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Zhao, Y. N., Cao, J., Wu, F. X., Ou, C., Yuan, W. P., Mo, Q. G., Wei, W., Li, Y., Su, J. J., Liang, A. M. (2004) Expression and significance of EGF mRNA and EGFR mRNA in hepatocellular carcinoma. Ai. Zheng. 23, 762766.

    • Search Google Scholar
    • Export Citation
  • Zhu, A. X., Stuart, K., Blaszkowsky, L. S., Muzikansky, A., Reitberg, D. P., Clark, J. W., Enzinger, P. C., Bhargava, P., Meyerhardt, J. A., Horgan, K., Fuchs, C. S., Ryan, D. P. (2007) Cetuximab Phase 2 study of cetuximab in patients with advanced hepatocellular carcinoma. Cancer 110, 581589.

    • Crossref
    • Search Google Scholar
    • Export Citation