Recently, a frequent gene fusion between the androgenregulated prostatespecific protease TMPRSS and the ERG transcription factor was discovered. In addition to TMPRSSERG,otherdr iver genes, as well as oncogen ic ETS fac to rs have been identified as gene fusions in prostate tumors.The costs of publication of this article were defrayed in part by the payment of page charges.Histone deacetylase, and monensin were identified as the only agents that had antitumor efficacy at nanomolar concentrations without affecting nonmalignant control cells.Because DSF has a favorab le sa fety profi le, it was se lec ted for fur ther stud ies. Gene express ion ana lyses ind icated that DSF did not influence androgen receptor expression, but induced metallothionein genes and downregulated DNA replication.Copper potentiated the antiproliferative effects of DSF in cultured prostate cancer cells, suggesting potential for increasing efficacy by combinatorial approaches.The AR and ETS status of the Isoborneol available prostate cancer model cell lines are relatively well known.The ARTA mutation has also been found from prostatic tissues derived from patients with a metastatic prostate cancer, indicating potential biological relevance. Due to this selection of compounds analyzed, any interesting drugs that show efficacy would be possible to rapidly test in vivo in preclinical models and also in clinical trials.We focused our analysis toward identifying antiproliferative compounds that act in prostate cancer cells but lack impact on normal or transformed prostate epithelial cells.Plates containing nL of compound stock solutions or DMSO as controls were diluted with L of cell culture media, and appropriate amount of cells were plated in L of media.The statistical outliers were downweighted when a polynomial surface was fitted to the intensities within each assay plate using local regression.This ensured a robust fit, although some plates had a much higher hitrate than others. The fit, representing a systematic background signal, was then subtracted from the raw signal values.The data were then logtransformed and centered on the plate median.The following formula was used to calculate the absolute percentagewise response: loesslogvalue.EC assays were done on well plates with, cells per well plated in their respective growth media and left to attach ove rn ight.A fo lddi lution series of selected compounds were added to the cells, and the plates were incubated for h.The cell extracts were separated by SDSPAGE and transferred to a nitrocellulose membrane.D, multidimensional scaling with principal coordinates analysis visualizes the distribution of prostate cell lines based on their compound responses.Because the growth rate of these different cell lines varied, cell amounts were carefully titrated to be on a linear range before HTS.Compound libraries compr is ing, small mo lecu le compounds, inc lud ing most currently marketed drugs, were screened with at least two different concentrations.The cell viability was determined after day incubation with compounds using a fluorescent assay.Multidimensional scaling with principal coordinates analysis was used to visualize the distributionofprostatecelllines based on theircompoundre sponses, with normal epitheliumderived cell lines forming one group and the different prostate cancer cell lines forming separate clusters.Tumor volumes were calculated by caliper measurements done week ly to mon itor tumor growth.

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