Phase IIa Evaluation of Early Bactericidal Activity

The enzymatic activities of Lenalidomide MMP 2 and MMP 9 were determined by gelatin zymography. In brief, conditioned media were prepared with standard SDS gel loading buffer containing 0. 01% SDS without mercaptoethanol or DTT and not boiled before loading. An amount of 50 g conditioned media underwent SDS PAGE with 0. 1% gel. After electrophoresis, gels were washed twice with 1 TBST then incubated with 2% Triton 100 for 30 min at room temperature to remove SDS, then in 30mL reaction buffer for 24hr at 37 C. Before scanning, gels were stained with Coomassie Brilliant Blue R 250 for 30min and destained with destaining solution. An amount of 2 106 of each RCC cell line was treated with DMSO or ovatodiolide for 24 hr, and cytoplasmic and nuclear extracts were separated with the use ofNE PERNuclear andCytoplasmicExtractionReagents. In all, 20 g nuclear and cytoplasmic extracts were used for SDS PAGE and Western blot analysis to compare catenin nuclear and cytoplasmic distribution. Aftermeasuring tumor weight, a small part of each Gene expression tumor was flash frozen in liquid nitrogen for western blot analysis and other parts were fixed with formalin for immunohistochemistry. Real time PCR data and cell numbers from transwell assay were recorded as continuous data and analyzed by Students t test. To confirm the specificity of ovatodiolide in suppressing catenin signaling, we compared the ovatodiolide effects with NF AT, CRE, and NF B luciferase reporter assays, with their agonistic compounds used as inductive controls. Ovatodiolide specifically inhibited the luciferase activity of TOP flash but had no effect in NF AT, CRE, and NF B reporters. Thesuppressive effects Cediranib of ovatodiolide were further evaluated with catenin/TCF/LEF downstream genes by immunocytochemistry. The staining for nuclear catenin and its downstream genes cyclin D1 and survivin was less in ovatodiolide treated RCC cells than in DMSO vehicle controls. In 4 RCC cell lines, ovatodiolide reduced levels of active catenin and its downstream genes but not other WNT molecules and S1D. However, ovatodiolide had no inhibitory effects in HEK293T, a low constitutive WNT signaling cell, or in normal kidney epithelial HK 2 cells. Ovatodiolide treatment at 40 M reduced mRNA levels of catenin signaling target genes Axin2, Sp5, and Nkd1 by 60% to 80% in both RCC cells. To evaluate the cytotoxicity of ovatodiolide in RCC and normal kidney cell lines, we analyzed cell viability. Ovatodiolide had a significantly higher cytotoxic effect in 4 RCC cell lines but less effect in HK 2 cells, S2A, and S2B. The IC50 with 48 hr treatment for HK 2 cells was 88. 20 M, which is much higher than that for RCC cells.