Research Article Synergistic Antitumor Effect of Doxorubicin and Tacrolimus (FK506) on Hepatocellular Carcinoma Cell Lines Francesca Capone, 1 Eliana Guerriero, 1 Angela Sorice, 1 Giovanni Colonna, 2 Gabriella Storti, 3 Jessica Pagliuca, 1 Giuseppe Castello, 1 and Susan Costantini 1 1 Istituto Nazionale per lo Studio E la Cura dei Tumori “Fondazione Giovanni Pascale”, IRCCS, 80131 Naples, Italy 2 Biochemistry, Biophysics and General Pathology Department, Second University of Naples, 80138 Naples, Italy 3 Department of Onco-Hematology, S.G. Moscati Hospital, 83100 Avellino, Italy Correspondence should be addressed to Susan Costantini; s.costantini@istitutotumori.na.it Received 5 November 2013; Accepted 24 December 2013; Published 20 February 2014 Academic Editors: C.-H. Chen and D. Morris Copyright © 2014 Francesca Capone et al. his is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Hepatocellular carcinoma is the ith most common cancer worldwide and shows a complex clinical course, poor response to pharmacological treatment, and a severe prognosis. hus, the aim of this study was to investigate whether tacrolimus (FK506) has synergistic antitumor efects with doxorubicin on two human hepatocellular carcinoma cell lines, Huh7 and HepG2. Cell viability was analyzed by Sulforhodamine B assay and synergic efect was evaluated by the sotware CalcuSyn. Cell apoptosis was evaluated using Annexin V and Dead Cell assay. Apoptosis-related protein PARP-1 cleaved and autophagy-related protein expressions (Beclin- 1 and LC3B) were measured by western blotting analysis. Cytokines concentration in cellular supernatants ater treatments was studied by Bio-Plex assay. Interestingly the formulation with doxorubicin and tacrolimus induced higher cytotoxicity level on tumor cells than single treatment. Moreover, our results showed that the mechanisms involved were (i) a strong cell apoptosis induction, (ii) contemporaneous decrease of autophagy activation, understood as prosurvival process, and (iii) downregulation of proinlammatory cytokines. In conclusion, future studies could relate to the doxorubicin/tacrolimus combination efects in mice models bearing HCC in order to see if this formulation could be useful in HCC treatment. 1. Introduction Hepatocellular carcinoma (HCC) is the ith most common cancer worldwide, and more than half a million new cases occur annually. HCC generally develops from chronic liver injury, which leads to inlammation, hepatocyte regeneration, liver matrix remodeling, ibrosis, and, inally, cirrhosis [1]. he main risk factors for HCC are hepatitis B (HBV) or C virus (HCV) infection, alcohol-induced liver disease (ALD), nonalcoholic fatty liver disease (NAFLD), primary biliary cirrhosis, exposure to environmental carcinogens (particu- larly alatoxin), and then type 2 diabetes and obesity [2]. Less common causes include hereditary hemochromatosis, alpha1-antitrypsin deiciency, autoimmune hepatitis, some porphyrias, and Wilson’s disease. he distribution of these risk factors among patients with HCC is highly variable, depending on geographic region and race or ethnic group [3]. Although the clinical diagnosis and management of early- stage HCC have improved signiicantly, its prognosis is still extremely poor [4]. However, this cancer is oten diagnosed at an advanced stage when most potentially curative thera- pies such as resection, transplantation or percutaneous, and transarterial interventions are of limited eicacy and no response is obtained to common therapies. herefore, new efective and well-tolerated therapy strategies are urgently needed [5, 6]. Chemotherapy is one of the common strategies in HCC treatment, especially for unresectable tumors. Dox- orubicin (adriamycin, DOX), antineoplastic chemotherapy drug that is a standard component in treating advanced HCC for its antitumor action, has shown insuicient eicacy, with a response rate of about 15–20% [7]. Other chemotherapeutics, such as epirubicin, cisplatin, 5-luorouracil, and etoposide and their combinations, demonstrate even lower eicacy [8]. Combined therapy with multiple drugs or modalities Hindawi Publishing Corporation e Scientific World Journal Volume 2014, Article ID 450390, 9 pages http://dx.doi.org/10.1155/2014/450390