05) ( Fig. 3A–F). Cell invasion is one of the steps involved in metastasis. To determine whether biflorin was involved in this process, the authors first ensured that the inhibition of invasion was not due to cell death. Thus, the viability of MDA-MB-435 melanoma cancer cells was assessed after 8 and 12 h of treatment with 1, 2.5 and 5 μM biflorin. As shown in Fig. 3C, cell death was not observed in any of the concentrations

of biflorin and durations of incubation tested. However, a strong and dose-dependent reduction in the invasion of MDA-MB-435 cells through the Matrigel matrix was observed after the treatment with 1, 2.5 and 5 μM biflorin (38.25 ± 9.53; 16.5 ± 3.31 and 2.25 ± 0.95, respectively).In comparison, this was not observed in the negative Protease Inhibitor Library supplier control (55.00 ± 3.9) (Fig. 4A and B). Additionally, biflorin did not inhibit the adhesion of MDA-MB-435 cells to any of the ECM substrates tested (data not shown). The cadherins are a family of a cell to cell adhesion molecules that have been implicated in the invasive process (Hanahan and Weinberg, 2011). To determine whether the inhibition of invasion by biflorin was related to N-cadherin protein levels, a western blot

was performed. After 12 h of biflorin treatment, the protein levels of N-cadherin were down-regulated in a dose dependent manner Birinapant in vivo (Fig. 4C and D). To further understand the signaling pathways involved in the inhibition of invasion, 4��8C the expression levels of AKT-1 was assessed. 36B4, acidic ribosomal phosphoprotein P0, was used as a reference gene. AKT-1 mRNA levels were down-regulated in a dose-dependent manner by 94.65, 76.25 and 21.35%, by 1, 2.5 and 5 μM biflorin, respectively ( Fig. 4E). After 12 h of treatment with 5 μM biflorin, the AKT-1 (p < 0.05) mRNA level was decreased by 5-fold (p < 0.05). Melanoma is one of the most invasive and deadly forms of skin cancer, and only a few agents are available for treating advanced disease to enable long-term patient survival. However, these agents are relatively ineffective, with overall response rates of 5–20%. This finding supports

the need for identifying new compounds that inhibit the pathways that are deregulated in melanoma (Eggermont and Robert, 2012 and Sharma et al., 2009). Anticancer drug development strategies are usually aimed at directly inhibiting the growth of the primary tumor or reducing the existing tumor burden. Therapeutic agents that can inhibit metastasis could be an option for preventing colonization, thereby enabling the containment of the primary tumors in a chemically manageable form (Pérez and Danishefsky, 2007 and Hedley et al., 2004). In this study, using melanoma cell lines as a model for invasion studies, we investigated the ability of biflorin, an ortho-naphthoquinone, to treat solid tumors. We also investigated the EMC substrates, Fibronectin and types I and IV collagen, and the expression of N-cadherin and AKT1.