A urine sample preparation workflow for the iTRAQ (isobaric tag for relative and absolute quantitation) technique was established. The reproducibility of this platform was evaluated and applied to discover proteins with differential levels between pooled urine samples from nontumor controls and three bladder cancer patient subgroups with different grades/stages (a total of 14 controls and 23 cancer cases in two multiplex iTRAQ runs). Combining the results of two independent clinical sample sets, a total of 638 urine proteins were identified. Among them, 55 proteins consistently showed >2-fold differences in both sample sets. Western blot analyses of individual urine samples confirmed that the levels of apolipoprotein A-I (APOA1), apolipoprotein A-II, heparin cofactor 2 precursor and peroxiredoxin-2 were significantly elevated in bladder cancer urine specimens (n = 25-74). Finally, we quantified APOA1 in a number of urine samples using a commercial ELISA and confirmed again its potential value for diagnosis (n = 126, 94.6% sensitivity and 92.0% specificity at a cutoff value of 11.16 ng/mL) and early detection (n = 71, 83.8% sensitivity and 94.0% specificity). Collectively, our results provide the first iTRAQ-based quantitative profile of bladder cancer urine proteins and represent a valuable resource for the discovery of bladder cancer markers.

These results suggested that the T3 upregulates proliferation of LNCaP cells by downregulating BTG2 gene expression through the consensus TRE pathway.

Luteolin is a polyphenolic flavone and has antitumor activity for many cancers. The prostate-derived Ets factor (PDEF), a novel epithelium-specific Ets transcription factor, acts as an androgen-independent transcriptional activator of the prostate-specific antigen (PSA) promoter. We determined the antitumor function of luteolin via upregulation of PDEF gene expression in human prostate carcinoma LNCaP cells. Results from flow cytometry and Prostate derived Ets transcription factor (PDEF), a novel epithelium-specific Ets transcription factor, not only acts as an androgen-independent transcriptional activator of the prostate-specific antigen (PSA) promoter but also directly interacts with the DNA binding domain of androgen receptors (ARs) and enhances androgen-mediated activation of the PSA promoter.1 Studies indicate that silibinin and tectorigenin treatments decrease PSA secretion in human LNCaP cells through the downregulation of PDEF gene expression.2,3 Furthermore, transient overexpression of PDEF enhances PSA gene expression at the transcriptional and translational levels under androgen-free condition; however, curcumin treatments block PSA gene expression by a mechanism other than inhibition of the PDEF gene expression. 4,5 A recent study using proteomic analysis identified 286 proteins in the PDEF-associated protein complex involved in the cell cycle, DNA repair, cytoskeleton organization, mRNA processing and cell signaling in MDA-MB-231 human breast cancer cells. 6 A global gene expression analysis identified several genes, which are associated with PDEF expression, regulating cell migration during cancer progression. 7 In vitro and xenograft studies revealed that PDEF knock-down in prostate LNCaP and PC-3 cancer cells increased migration and invasiveness suggesting that PDEF is involved in the tumor biology of the human prostate. 8 Expression of B-cell translocation gene 2 (BTG2) in cycling cells induced accumulation of growth-inhibitory forms of pRb and led to G1 cell-cycle arrest.9 BTG2 is more abundantly expressed in prostate tissue with high epithelial