Figure S1 - Effectiveness of the Histone Deacetylase Inhibitor (S)-2 against LNCaP and PC3 Human Prostate Cancer Cells

Preliminary in vivo experiments with a murine xenograft model. Male nude (nu/nu) athymic mice (Harlan Laboratories, Srl, San Pietro al Natisone, UD, Italy) were cared for and maintained in accordance with applicable European Animal Welfare regulations under an approved Institutional Animal Care and Use Protocol in an animal facility at University of Florence accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Aliquots of LNCaP cell suspension (3×106 cell/100 µl containing an equal volume of RPMI and matrigel) were implanted subcutaneously on the left flank of 12 mice [50]. A week later the tumor mass was present only in eight out of the originally injected mice (66% of tumor incidence) which were then randomized into two equal groups and drug treatment was started. (S)-2 was formulated as a DMSO solution and injected intraperitoneally. One group was treated with the drug (50 µl final volume, at 65 mg/kg, corresponding to approximately 2 mg/mouse) three times a week for two weeks, while the control group was treated with 50 µl DMSO alone as the vehicle. Mice were killed after 6 treatments and 24 h post-dose by cervical dislocation. (A) – Tumors were measured by calipers after the sacrifice (bottom); the tumor mass was weighed and the volume was calculated according to the formula length × width2 × π/6 (top). In all cases, tumor volumes in untreated mice were significantly larger relative to those of drug-treated mice to suggest that (S)-2 was capable of reaching the cancer cells and decreasing their growth rates. Photographs are representative of a tumor mass from a mouse treated with either the vehicle or (S)-2, respectively. (B) – For immunohistochemistry, slides with 2.5–5 µm sections of paraffin embedded tumor mass were first deparaffinized, boiled in 1 mM EDTA pH 9 for 15 min and after cooling aspecific peroxidases were blocked with 3% H2O2 for 10 min. Then, slides were according to standard procedures and incubated with a primary antibody against γ-H2AX (see Materials and Methods) followed by a peroxidase-conjugated IgG preparation; 3,3′-diaminobenzidine was used as the chromogen for development. Slides were counterstained with aqueous Meyer hematoxylin and mounted with glycerol for visual inspection and photography; pictures are representative of four randomly chosen microscopic fields (magnification: x400). γ-H2AX-positive cells were frequently observed within the tumor mass of drug-treated mice but not of mice injected with the vehicle only as depicted by the pictures (bottom) and clearly indicated by the histograms (top). Statistical analysis was carried out by Student’s t-test and significant differences between the two groups were indicated by the asterisks (*P[27], no specific drug-induced histologic alteration in May-Grünwald Giemsa-stained liver parenchymal cells was observed (data not shown). (EPS)