BioProject

HTI-286, an analogue of hemiasterlin, interferes with microtubule dynamics and circumvents transport-based resistance to taxanes. In this study we evaluate the inhibitory effects of HTI-286 on human prostate cancer growth in vitro and in different in vivo models. The results show that HTI-286 is a potent inhibitor of proliferation and induced marked increases in apoptotic rates in all cell lines tested. Despite similar transcriptomic changes regarding cell death and cell cycle-regulating genes after exposure to HTI-286 or docetaxel, the gene array revealed distinct molecular signatures for both compounds. Keywords: Drug comparison Overall design: LNCaP cells of the same passage were treated in 14 cm dishes for 72 h with 0.75 nM HTI-286 or 3 nM docetaxel and their corresponding vehicle controls (<0.0001% in solution). We chose a three day drug exposure because alterations of gene expression have been reported more evident after prolonged treatment. Total RNA of three biological replicates for 12 samples in total was isolated from cultured cell lines using the Trizol method (Invitrogen). The quality and quantity of RNA was assessed with an Agilent 2100 bioanalyzer (Agilent Technologies, Mississauga, ON, Canada). Total RNA from each treatment sample (HTI-286 or docetaxel) was compared to its vehicle control sample on the same microarray. A dye swap for each pair was performed to account for dye bias. Microarrays of 21,000 (70-mer) human oligonucleotides representing 21,000 genes (Operon Technologies, Alameda, CA) printed in duplicate in 3xSSC onto aminosilane-coated slides (ERIE C28) were supplied by the Array Facility of The Prostate Centre at Vancouver General Hospital. Arrays were hybridized with 3DNA DNA Dendrimer Probes generated from 10 µg total RNA from LNCaP cells (n=3 for HTI-286 and vehicle control or n=3 for docetaxel and vehicle control) according to the manufacturer’s directions (Genisphere, Hatfield, PA). Briefly, reverse transcription incorporated a specific sequence present on the 5’ end of the RT primer supplied with the kit. The cDNA was hybridized to the array overnight at 42ºC. After stringent washes, the fluorescent 3DNA reagent which includes a ‘capture sequence’ complementary to the sequence at the 5’ end of the RT primer, was hybridized to the cDNA (47ºC for 2-3 h). Following further washes the arrays were immediately scanned on a ScanArrayExpress Microarray Scanner (PerkinElmer, Waltham, MA). Signal quality and quantity were assessed using Imagene 5.6 (BioDiscovery, San Diego, CA). Data from Imagene were analyzed in GeneSpring 6.1 (Silicon Genetics, Redwood City, CA) in order to profile changes in gene expression. Analyses performed in GeneSpring included background correction, LOWESS normalization and hierarchical clustering using standard correlation. Genes demonstrating normalized fold-changes >1.5 or <-1.5 in 3 of 3 experiments were then categorized by function using Ingenuity Pathways Analysis 5.0 (Ingenuity Systems, Redwood City, CA).