Western Blot AnalysisUntreated cells and cells treated with BOU and MHCU at concentrations of 1 1 and 50 M, were lysed after 24 and 72 h in lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.2 mM EGTA, 10% glycerol, 1% Triton X-100) and protease inhibitor cocktail (Roche, Basel, Switzerland). the inhibitor results pointed to HDACs of class I as potential molecular targets for compound BOU. The significant decrease of activity of HDAC of classes I and/or II, in the entire SW620 cell lysate treated with Hexacosanoic acid BOU at 50 M concentration (Physique 2D), supported this obtaining. The HDAC enzymes of class I are overexpressed in CRC  and it has been reported that HDAC inhibitors might induce cell cycle arrest in SW620 cells in dependence on the inhibitor concentration . We found that BOU induced cell cycle arrest in SW620 cells as well, suggesting that its inhibition of cancer cell growth might be mediated, at least in part, by arrest of the cell cycle progression caused by inhibition of HDAC of class I and/or II. According to the docking analysis, BOU probably inhibits class I HDACs 1C3 due to favorable occupancy of Hexacosanoic acid an available foot pocket near the zinc binding place by its docking analysis Hexacosanoic acid showed that conversation with the foot pocket near the zinc binding place of HDACs was not possible for MHCU. This result was substantiated by the HDAC colorimetric assay kit results as well (Physique 2D). HDAC assay exhibited a stimulating activity of MHCU on the activity of HDAC enzymes. Induction of HDACs activity is in agreement with the altered regulation of several inflammatory proteins (Table S3 in Supplementary Information). It was already reported that anti-inflammatory effects of some drugs might be attributed to the activation of HDACs and specific acetylation/deacetylation patterns in cells [39,40] (ultimately leading to suppression of the inflammatory response). The obtained information around the envisaged molecular conversation with cellular targets may provide a good basis for further optimization for improved amino acid hydroxyurea derivatives binding to HDACs and development of lead compounds. 2.5. Activity of BOU BOU exerted stronger antiproliferative effect compared to MHCU and was detected as a potential HDAC inhibitor. Therefore, its effect was evaluated on Balb/C mice inoculated with the colon carcinoma cell line CT26.WT. Rather high cytotoxicity observed and in the pilot experiment (data not shown) prompted us to diminish BOU dosages compared to the standard hydroxyurea doses used for studies in mice [41,42]. The mean survival time in the control group of Balb/C mice inoculated with the colon carcinoma cell line CT26.WT was 40 days, while it increased to 45.5 days in BOU; ILS % was 13.757% (data not shown). The overall survival period and tumor size after 45 days was not significantly different for mice treated with BOU (Physique 3). However, the treatment of animals showed a death reduction between 30 and 35 days upon treatment with BOU even though the tumor mass remained the same. Open in a separate window Physique 3. (A) Kaplan-Meyer survival graph for Balb/C mice inoculated intramuscularly with CT26WT tumor cells (1 106 cells/mice) and treated with BOU at 1 mM/kg given intraperitoneally on days 1, 5, 10, 15 and 20. No statistical differences in overall survival growth of treated mice was observed in comparison with control mice (= 0.1915; log-Rank test); (B) Tumor size in Balb/C mice inoculated intramuscularly with CT26WT cells (1 106 cells/mice) and treated with BOU at 1 mM/kg given intraperitoneally on days 1, 5, 10, 15 and 20. The absence of an overall effect on animal survival might be partially attributed to the low doses used for the experiments. This raises the question of toxicity and substantiates the need for further chemical optimization of BOU in relation to toxicity. Nevertheless, the therapeutic potential for BOU might be seen in Hexacosanoic acid combination with other small molecules with a complementary mechanism of action  or in chronic or autoimmune inflammatory disorders . 3.?Experimental Section 3.1. Tested Compounds Synthesis and antiproliferative effect of Analyses 3.2.1. Cell CulturingThe SW620 cells (colon carcinoma, metastasis) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), cultured as monolayers and maintained in Dulbeccos altered Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 U/mL penicillin and 100 g/mL streptomycin in a humidified atmosphere with 5% CO2 at 37 C. 3.2.2. Cell Viability AssayViability Rabbit Polyclonal to PRKAG1/2/3 of cells was assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay..