Effect of PNB-028 on Global gene expression in MAC-16 xenograft

Microarray studies were conducted with RNA isolated from vehicle- or PNB-028- treated MAC-16 colon cancer xenograft tumors (n=3/group). PNB-028 significantly regulated over 1400 genes compared to vehicle-treated samples with approximately 450 genes up-regulated and 1100 genes down-regulated by 1.5 fold or greater. All xenograft animal protocols were approved by The University of Tennessee Animal Care and Use Research Committee. MAC-16 cells were harvested, counted under microscope using trypan blue exclusion, and a suspension in growth medium was prepared. The cell suspension (5 million cells/mouse) was implanted subcutaneously in NSG mice. Once the tumors reached 100-200 mm3, the animals were randomized within the respective cell line and treated orally at 50 mg/kg/day. PNB-028 was dissolved in 20% DMSO+80% PEG-300 and administered by oral gavage. Body weight and tumor volumes were measured thrice weekly. Animals were sacrificed (6 hrs after the last dose) when tumors reached 1500 mm3 or when the animals lost over 20% body weight. Tumors were excised, weighed, and stored for further analysis (two parts snap frozen and stored at -80°C and one part stored in formalin for histology). RNA was isolated from MAC-16 tumors (n=3/group) using Qiagen RNAeasy kit. RNA was quantified using nanodrop and diluted to equal concentrations of 100 ng/µl. RNA quality was determined using Agilent Bioanalyzer and RNA samples with RNA Integrity Number (RIN) greater than 7 were used for microarray experiments (ST 2.0 mouse array from Affymetrix). Results from microarray studies were analyzed using Affymetrix software (Transcriptome Analysis Console (TAC)). Pathways differentially regulated by PNB-028 were identified using Ingenuity Pathway Analysis software.