The inhibition of N-acetyltransferase activity and gene expression in human bladder cancer cells (T24) by shikonin.

Shikonin has the potential to prevent, or be used in the treatment of, bladder transitional cell carcinoma induced by arylamines. We evaluated its effectiveness by measuring the amount of acetylated 2-aminofluorene (AF), AF-DNA adducts, changes of NAT mRNA and the amount of NAT enzyme. T24 human bladder cancer cells were incubated with 30 microM AF with different concentrations of shikonin for various times. T24 cells treated with shikonin (16 microM) were then harvested and used in 2 experiments: 1). T24 cells were incubated with 22.5 microM AF and shikonin (0, 16 microM) (co-treatment) for 6, 12, 18, 24 and 48 h. 2). T24 cells were incubated with various concentrations of AF and shikonin (0, 16 microM) for 24 h. AF and AAF were measured by HPLC. Then in the prepared human T24 cell cytosols different concentrations of AF and shikonin were added to measure the kinetic constants of NAT. Next, AF-DNA adducts in human T24 cells with or without treatment with shikonin were detected and measured. The final two steps included measuring the NAT Ag-Ab complex after treatment with and without shikonin and evaluating the effect of shikonin on the NAT genes. Higher concentrations of shikonin induced decreasing AF acetylation. We found that the longer the culture period, the greater the difference in AF acetylation in the same shikonin concentrations. It was also noted that increase in AAF was proportional to incubation time. In the presence of 16 microM of shikonin, N-acetylation of AF decreased by up to 72-84%. Shikonin decreased the amount of AAF production in human T24 cells in all examined AF doses. Both Km and Vmax values in the cytosolic NAT decreased after the addition of shikonin to the cytosol. Finally, shikonin decreased the amount of AAF production and AF-DNA adducts formation in human 724 cells in all examined AF doses. The percentage of cells stained by antibody was significantly different after treatment with shikonin, especially with the higher shikonin concentrations. The NAT1 mRNA level and the NAT1/beta-actin ratio decreased significantly with higher concentrations (16-24 microM) of shikonin. Shikonin affected NAT activity, gene expression (NAT1 mRNA), AF-DNA adducts formation and formation of NAT Ag-Ab in human bladder tumor T24 cells. Therefore, shikonin should be considered as a candidate agent for the prevention or treatment of transitional cell carcinoma.