formazans and Kidney-Neoplasms

Metastatic renal cell carcinoma (RCC) is highly resistant to systemic chemotherapy. Unfortunately, nearly all patients die of the metastatic and chemoresistant RCC. Recent studies have shown the atypical PKCζ is an important regulator of tumorigenesis. However, the correlation between PKCζ expression and the clinical outcome in RCC patients is unclear. We examined the level of PKCζ expression in human RCC.. PKCζ mRNA and protein expressions were examined by real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC) respectively in RCC tissues of 144 patients. Cellular cytotoxicity and proliferation were assessed by MTT.. PKCζ expression was significantly higher in normal than in cancerous tissues (P<0.0001) by real-time PCR and IHC. Similarly, PKCζ expression was down-regulated in four renal cancer cell lines compared to immortalized benign renal tubular cells. Interestingly, an increase of PKCζ expression was associated with the elevated tumor grade (P=0.04), but no such association was found in TNM stage (P=0.13). Tumors with higher PKCζ expression were associated with tumor size (P=0.048). Expression of higher PKCζ found a poor survival in patients with high tumor grade. Down-regulation of PKCζ showed the significant chemoresistance in RCC cell lines. Inactivation of PKCζ expression enhanced cellular resistance to cisplatin and paclitaxel, and proliferation in HK-2 cells by specific PKCζ siRNA and inhibitor.. PKCζ expression was associated with tumorigenesis and chemoresistance in RCC.

Topics: Aged; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Cisplatin; Down-Regulation; Drug Resistance, Neoplasm; Female; Formazans; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Immunohistochemistry; Kidney Neoplasms; Male; Middle Aged; Neoplasm Staging; Paclitaxel; Protein Kinase C; Real-Time Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Tetrazolium Salts

The hydrogen acceptor 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) is commonly utilized to estimate cellular viability in drug screening protocols. The present investigation was prompted, in part, by observations that reduction of MTT to its colored reaction product, MTT formazan, varied between cell lines and with culture age. A correlation was established between the D-glucose concentration of the culture medium at the time of assay and the production of MTT formazan for cell lines representing seven tumor histologies. A decrease in the concentration of D-glucose from culture medium was accompanied by a decrease in MTT specific activity (MTT formazan/microgram cell protein) for a number of cell lines. Cells which extensively metabolized D-glucose exhibited the greatest reduction in MTT specific activity. Further evidence that the D-glucose concentration of the culture medium played an important role in MTT reduction was provided by experiments which demonstrated that transfer of cells to a glucose-free medium (L-15) was accompanied by an immediate decrease in MTT reduction which was pH independent. These studies suggested that cellular transport and constant metabolism of glucose were required for maximum MTT reduction. Decreases in the cellular concentration of the reduced pyridine nucleotides NADH and NADPH were accompanied by concomitant decreases in MTT formazan production. MTT formazan varied significantly among cell lines in both the kinetics of its formation and the degree of saturability exhibited. Apparent IC50 values for Adriamycin varied, in a cell line-specific manner, with MTT exposure time. These results indicate that MTT specific activity is significantly influenced by a number of parameters and suggest that assay conditions should be established which minimize their effects.

Topics: Carcinoma, Renal Cell; Cell Division; Cell Line; Cell Survival; Coloring Agents; Drug Screening Assays, Antitumor; Formazans; Glucose; Humans; Kidney Neoplasms; Kinetics; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; NAD; NADP; Tetrazolium Salts; Thiazoles