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. novel strategy to increase sorafenib efficacy in HCC management, and point to target the mitochondria as the subcellular location where sorafenib therapy could be potentiated. synthesis, respectively [7, 8], can be limited by the concurrent activation of ceramide-degrading enzymes, which reduce the efficacy of drug therapy on tumor cells [8, 9]. For instance, glucosylceramide synthase (GCS) catalyzes the generation of glucosylceramide from ceramide while ceramidases (CDases) deacylate ceramide to sphingosine, which is then phosphorylated to sphingosine-1-phosphate by sphingosine kinases. Both pathways have been characterized in drug-resistance as protective mechanisms triggered by tumor cells after cancer treatment [8, 10, 11]. In liver cancer, increasing intratumoral ceramide levels with nanoliposomal administration has been used as a strategy in the treatment of HCC [12], while targeting acid CDase (ACDase) potentiated the cytotoxic effect of daunorubicin in hepatoma cells [13]. Regarding sorafenib action, recent data has shown the efficacy of combining sorafenib with recombinant acid sphingomyelinase, a ceramide-generating enzyme, in experimental liver cancer [14], or with nanoliposomal ceramide in melanoma or breast cancer [15]. These findings have proposed a role for sphingolipids in sorafenib toxicity [16], but a detailed analysis of ceramide metabolism and HCC models after sorafenib treatment has not been previously reported. Our data indicate that, although sorafenib alters the sphingolipidic metabolism in hepatoma cells via ASMase activation, ceramide toxicity is partially reduced by the simultaneous induction of ceramide-eliminating enzymes, in particular GCS. Moreover, pharmacological or genetic GCS antagonism sensitized hepatoma cells to sorafenib by a caspase-independent mitochondrial-dependent mechanism. Moreover, GCS is upregulated in resistant hepatoma cells after long-term exposure to sorafenib, pointing to GCS targeting as an effective approach to re-sensitize tumor cells to sorafenib. Therefore, our results validate the interest of ceramide-focused strategies to increase sorafenib effectiveness in HCC and confirm mitochondria as the subcellular site responsible for these effects. RESULTS Sorafenib increases ceramide levels and the expression of enzymes involved in ceramide metabolism in Hep3B cells Despite several evidences showing the influence of ceramide-related compounds in sorafenib efficacy [14, 15], the effect of sorafenib on ceramide metabolism has not been evaluated. Among critical sphingolipidic genes (Suppl. Fig. 1), we found that overnight sorafenib exposure increased expression of genes responsible for ceramide production (Table ?(Table1)1) by sphingomyelin hydrolysis (acid sphingomyelinase, ASMase) or synthesis (serine palmitoyl transferase, SPT, ceramide synthase 2, CerS2). In parallel, genes involved in ceramide modification via ceramidase degradation (acid Edivoxetine HCl ceramidase, ACDase, and sphingosine kinase 1, SK1) or glycosylation (glucosylceramide synthase, GCS) were also increased. Moreover, in another hepatoma cell line, HepG2, sorafenib also increased ceramide formation through ASMase and glycosylation via GCS (Suppl. Table 1). Table 1 mRNA levels of main sphingolipidic enzymes in Hep3B cells after sorafenib exposure ceramide synthesis (SPT and CerS2). Moreover, sorafenib induced the expression of GCS and ACDase, which metabolize ceramide, as well as SK1. These effects were accompanied by changes in ceramide levels upon sorafenib treatment. Ceramide increased dose-dependently, being significant for all doses (from 2.5 to 20 M) after 4 h of sorafenib exposure (Figure ?(Figure1B1B). Open in a separate window Figure 1 Sorafenib administration to hepatoma cells Edivoxetine HCl induces changes in ceramide metabolismA. Time-course analysis of mRNA levels of main sphingolipidic enzymes in Hep3B cells exposed to sorafenib (5M). B. Ceramide levels were quantified Edivoxetine HCl in Hep3B cells treated with increased doses of SOR (2.5, 5, 10 and 20 M) Mouse monoclonal to CD18.4A118 reacts with CD18, the 95 kDa beta chain component of leukocyte function associated antigen-1 (LFA-1). CD18 is expressed by all peripheral blood leukocytes. CD18 is a leukocyte adhesion receptor that is essential for cell-to-cell contact in many immune responses such as lymphocyte adhesion, NK and T cell cytolysis, and T cell proliferation during different times of incubation, after lipid extraction, TLC running and PhosphoImager quantification. C. and D. Cell viability of Hep3B cells, preincubated (30 min) with imipramine (IMIP, 15 M) or myriocin (MYR, 5 M), and treated with sorafenib for 16 h. (n=3). *, p<0.05 vs. control. Pharmacologic inhibition of sphingolipid enzymes modulates sorafenib-induced toxicity in hepatoma cells To examine the role of the ceramide production/degradation pathways in sorafenib cytotoxicity, we administered sphingolipid inhibitors combined with sorafenib in.