Proteins were visualized by Western blotting. Real time PCR Total RNA was extracted from cells selleck chem inhibitor 24 hours post transfection with HDAC1 siRNA using TrizolW and following the manufacturers instructions. P21 and HDAC1 expression was quantified by TaqManW real time PCR using specific primers. SPRR2A was done with SYBR Green using previously described primers. Gene expression was normalized to GAPDH using the comparative 2 CT method, with expression levels in the untreated control set to a value of 1. 0. Statistics All statistical analyses were performed using SigmaStat software. A P value of 0. 05 was considered statistically significant, and all tests were two tailed. All interval values are expressed as mean SD. Group comparisons were analyzed with Kruskal Wallis ANOVA or one way ANOVA.
Background Multidrug resistance constitutes a major obstacle for success of cancer treatment. The MDR phenotype is responsible for resistance to a wide variety of anticancer drugs, such as anthracyclines, vinca alkaloids and others. Although several mechanisms could be involved in the acquisition of this phenotype, the role of two different membrane proteins, P glycoprotein and multidrug resistance associated protein, has been well established. Both proteins are members of the same ATP binding cassette superfamily of trans port proteins. Pgp was first identified as a consequence of its overexpression in multidrug resistant tumour cells, where it mediates the ATP dependent efflux of a variety of chemotherapeutic agents.
In addition to its role during the acquisition of the MDR phenotype, Pgp is expressed in normal tissues, both as a consequence of differentiation and also in response to environmental challenges, and it has been proposed to play a role as a cell protector against cellular toxins. In addition, a general antiapoptotic role for Pgp has been proposed. It is clear that Pgp has several functions in different cells and tissues. Pgp is encoded by a multigene family in higher eukaryotes. The ABCB1 gene encodes the human Pgp. In cultured cells, constitutive overexpression of Pgp is mediated by changes in gene dosage or transcription. Pgp can also be transiently induced in cultured cells by a variety of stimuli, such as heat shock, UV radiation, and che motherapeutic agents. The regulation of Pgp expres sion has been mostly related to transcriptional control of the ABCB1 gene expression.
The proximal promoter of ABCB1 contains several regulatory regions, such as an inverted CCAAT box and a GC element, both of which are required for constitutive promoter activity in several cell lines. It has been reported that in the colon carcin oma cell line SW620, the histone deacetylase inhibitor trichostatin A, induces an Cilengitide increase in ABCB1 transcription through the inverted CCAAT box element, with the requirement of the NF Y transcription factor.