Proteomic analysis of drug metabolizing networks in renal cell carcinomas with differential drug sensitivity and VHL expression.

Abstract

e13137 Background: Kidney tumors with mutated VHL constitute 75% of renal cancers. The VHL activity dependent therapeutic outcome is the current focus of translational research in renal oncology. We observed that CAKI-2 kidney cancer cells with wt VHL are more sensitive to sorafenib than 786-O cells with mt VHL, which are more sensitive to temsirolimus (Singhal SS et al 2009). Hence, we performed proteomic analysis to investigate the molecular basis of differential drug sensitivity. Methods: The cell cultures were performed in RPMI medium at 37°C in 5% CO2. The cells were lysed in buffer containing 20mM Tris, 50mM Nacl, 6M urea, 10mM NaPP, 1mM NaF, and 1mM Na3VO4. The lysate (200μg of protein) was subjected to reduction and alkylation of cysteins using 2.5mM DTT and 7mM idoacetamide followed by trypsin digestion and solid phase extraction using a C-18 cartridge (Waters, USA). Liquid chromatography–tandem mass spectrometry (LC-MS/MS) was performed on a linear ion trap–Fourier transform ion cyclotron resonance tandem mass spectrometer (LTQ-FT, Thermo) coupled to an Eksigent nano-LC system. MS/MS spectra were searched against a human protein database by the Mascot software (Matrix Science) and Label-free quantification was done by using spectral counts from the Scaffold program (Proteome Software) with previously validated method (Prokai et al 2009). Results: We detected significant differences in the levels of phase I and II drug metabolizing enzymes in two cell types (Table). Carbonyl reduction catalyzed by AKR1C1 and UGDH involved glucorunidation are vital both in the activation and inactivation of drugs. NQO1 mediates the anticancer effects of beta lapachone (Bey et al. 2006). Conclusions: Our proteomic identification of vital differences in the principal enzymes of drug metabolizing networks identify key molecular determinants of drug sensitivity, which may be further investigated and developed as drug sensitivity biomarkers for clinical use. Proteins identified by LC-MS/MS Fold increase in spectra in CAKI-2 cells comparedto 786-O cells Aldo-keto reductase (family 1 member C1, AKRC1) 6.1 NAD(P)H quinone dehydrogenase 1 (NQO1) 2.8 UDP-glucose 6- dehydrogenase (UGHD) 5.2 No significant financial relationships to disclose.