Plasmids encoding various topoIIα mutations were generated from Flag-TopoIIα (GeneCopoeia, Rockville, MD) by site-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Primers used to generate topoIIα mutations were as follows:

S1361A, 5′-TGCTAGTCCAC CTAAGACCAAAACTGCCCCAAAACTTAG-3′/5′-C TAAGTTTTGGGGCAGTTTTGGTCTTAGGTGGA CTAGCA-3′; S1365A, 5′-GAC-CAAAACTTCCCCA AAACTTGCTAACAAAGAACTGAAACCACAG-3′/5′-CTGTG-GTTTCAGTTCTTTGTTAGCAAGTT TTGGGGAAGTTTTGGTC-3′; E1368A, 5′-CCC CAAAACTTAGTAACAAAGCACTGAAACCACAGA AAAGTGT-3′/5′-ACAC-TTTTCTGTGGTTTCAGT GCTTTGTTACTAAGTTT-TGGGG-3′; S1393A, 5′-GGGC-AGTGTACCACTGTCTTCAGCCCCTCCT GCTAC-3′/5′-GTAGCAGGAGGGGCTGA-AGACAG TGGTACACTGCCC-3′; T1397A, 5′-CTTCAAGCC CTCCTGCTGCACATT-TCCCAGATGAA-3′/5′-TTC Poziotinib chemical structure ATCTGGGAAATGTGCAGCAGGAGGGCTTGAAG-3′. Female athymic nude mice (5-6 weeks of age) were obtained from Harlan Laboratories (Indianapolis, IN). All experimental procedures were done according to protocols approved by the Ohio State

University Institutional Laboratory Animal Care and Use Committee. Each mouse was injected subcutaneously with 1 × 106 PLC5 cells in 0.1 mL serum-free medium containing 50% Matrigel. Mice with established tumors (mean starting tumor volume, 223 ± 75 mm3)

were randomized to two groups (n = 5) that MCE公司 received the following Idasanutlin treatments daily by gavage (10 μL/g body weight) for 3 or 6 days: (1) methylcellulose/Tween 80 vehicle, and (2) AR42 at 25 mg/kg. At the study endpoint, tumors were snap-frozen and stored at −80°C for subsequent coimmunoprecipitation analysis. Pursuant to our finding that AR42 exhibits high in vivo efficacy against PLC5 tumor growth,6 we examined the effects of AR42 on various biomarkers pertinent to the aggressive phenotype of HCC, among which the concentration- and time-dependent suppression of topoIIα expression was noteworthy (Fig. 1A). As AR42 inhibited topoIIα expression at concentrations well below its median inhibitory concentration (IC50) of 0.72 μM in inhibiting cell viability,6 this down-regulation was not consequent to drug-induced cell death. This topoIIα repression was also noted with MS-275 and, to a lesser extent, vorinostat; however, at an order-of-magnitude higher concentration. This drug-induced suppression was topoIIα-selective because these HDAC inhibitors did not cause dramatic changes in topoIIβ expression.