t. for 7 consecutive days. For i.p. injection protocols, BSc2118 was given at dose of 15, 30, or 60 mg per kg body weight for seven consecutive days. Bortezomib was given at 1 mg/kg i.p. 7 times every second day. Each group contained 7 animals. Appropriate volumes of the solvents were given as control. During the experiments melanoma bearing mice were

observed daily for survival and adverse effects. Tumor size of melanoma-bearing mice was measured every 2 days. Tumor volume was determined according to the formula: tumor volume = (shorter diameter2 × longer diameter)/2. Differences in tumor volume were analyzed for significance by the Student’s t test. A P value of < 0.05 was considered to be statistically significant. Log-rank test was used to analyze survival. Mice were anesthetized and received sterile abdominal injections of 250 μl of Matrigel (Becton Dickinson, Germany) subcutaneously

containing 50 nM βFGF (Sigma selleck chemical Aldrich, Germany). Thereafter, Bleomycin mice were i.p. treated with BSc2118 at 30 mg/kg for 7 consecutive days. At day 8, vascularization of the Matrigel was quantified by intravenous injecting of 0.1 ml (0.25 mg/ml stock solution) of FITC-dextran (125,000 molecular weight, Sigma Aldrich, Germany) into mice, which allowed blood vessels within plugs to be visualized. Animals were sacrificed 20 minutes after injection, when Matrigel plugs were removed and digested in Dispase reagent (Becton

Dickinson, Germany). The fluorescence of the solution obtained was measured using a fluorimeter (POLARstar, BMG Labtech, Germany) with an excitation at 480 nm and an emission wavelength at 520 nm. Differences between groups were calculated by Student’s t test. A P value of < 0.05 was considered to be statistically significant. Experimental lung metastases were performed as described by Feleszko et al. [32]. Briefly, experimental lung metastases were induced by injection of 2 × 105 of B16F10 cells/100 μl PBS into the tail vein of anesthetized female C57BL/6 mice. Mice (5 to 7 per group) were i.p. injected for 7 consecutive days with either DMSO or BSc2118 (15 mg/kg body weight/day). The animals were then sacrificed on day 21 after inoculation of tumor cells. An average number of metastases Fossariinae were calculated for every mouse by two independent observers blinded to the experimental groups. Differences between experimental groups were analyzed using the Mann–Whitney U test. A P value of < 0.05 was considered to be statistically significant. An In Vitro cytotoxicity screening was performed to characterize the anti-tumor potential of BSc2118. For this purpose, a panel of solid tumor cell lines, most of them originating from malignant melanoma, was incubated either with BSc2118 or with bortezomib as a reference inhibitor (Figure 1). The average GI50 value was estimated for each cell line and across the entire tumor cell panel.