Renal cell carcinoma (RCC) is relatively resistant to chemotherapy and radiotherapy. Recent advances in drug development are providing novel agents for the treatment of RCC, but the effects are still minimal. In addition, there is an urgent need to identify diagnostic markers for RCC. In this report, to discover potential diagnostic markers and therapeutic targets, we subjected RCC samples to a quantitative proteomic analysis utilizing 2-nitrobenzenesulfenyl (NBS) reagent. Proteins were extracted from RCC and adjacent normal tissue, obtained surgically from patients, and labeled with NBS reagent containing six 12 C or 13 C. This was followed by trypsin digestion and the enrichment of labeled peptides. Samples were then subjected to analysis by MALDI-TOF MS. NBS-labeled peptides with a 6 Da difference were identified by MS/MS. Thirtyfour proteins were upregulated in more than 60% of the patients of which some were previously known, and some were novel. The identity of a few proteins was confirmed by Western blotting and quantitative real time RT-PCR. The results suggest that NBS-based quantitative proteomic analysis is useful for discovering diagnostic markers and therapeutic targets for RCC.

In our previous study, the MDR1/Pglycoprotein-overexpressing multidrug resistant subline, Hvr100-6, was established from the human cervical carcinoma cell line HeLa-Ohio (HeLa) by stepwise exposure to an anti-microtubule agent, vinblastine sulfate, a typical substrate of MDR1. Their gene and protein expression profiles were analyzed herein, and 148 genes were identified to be differentially expressed by cDNA microarray analysis. The up-regulation of sorcin, a soluble resistance-related calcium-binding protein of 22 kDa, was confirmed in Hvr100-6 cells by the proteome analysis. To clarify the relationship between MDR1 and sorcin, HeLa cells were treated with small interfering RNAs (siRNAs) targeted for theirs mRNAs. The siRNA for MDR1 mRNA resulted in its decrease by 86% and 61% on the days 1 and 2 after the treatment, whereas the expression level of sorcin mRNA was not changed. On the other hand, the siRNA for sorcin mRNA suppressed its expression by 80-90% on days 1-3 after the treatment. Interestingly; suppression of sorcin induced a more than 3-fold increase in the expression level for MDR1 mRNA. An efflux function of MDR1 evaluated with using rhodamine 123 as a probe showed a tendency to be increased in HeLa cells treated with siRNA for sorcin, compared with that in the cells treated with scramble siRNA. The activity and the expression of caspase-3 in the sorcin knock-down HeLa cells were relatively higher than those in the cells treated with scramble siRNA. Thus, we demonstrated that sorcin might be a partial suppressor of MDR1 expression. Furthermore, the present study suggested that sorcin repressed apoptosis via dysfunction of caspase-3.