Figure 6

Suppression of T24 cell growth with protein kinase inhibitors and genistein. (A) When 50 μ M genistein and either PD98059 or U0126 were applied to T24 cells simultaneously, the cell growth rates were reduced in a dose-dependent manner. The difference reached a significance level at 20 μ M PD98059 (marked by ^*) or 10 μ M U0126 (marked by ^**), as compared to that of the cells treated only with the respective inhibitor without genistein (both P<0.05 by t-test). (B) Other protein kinase inhibitors such as H7 (PKC inhibitor) and LY294002 (PI3 K inhibitor) did not reverse the T24 drug resistance phenotype but exhibited only cytotoxicity, as H7 or LY294002 alone could also reduce cell growth rates to the levels similar to the cell cultures simultaneously treated with the inhibitors and the drug at the high concentrations. (C) Adding JNK inhibitor SP600125 to T24 cells rendered the dose-dependent growth suppression in the presence of genistein. Statistical analysis (t-test) revealed that the reduction of the cell growth rate reached significance (P<0.05) only when genistein was added to T24 cells that were previously treated with 30 μ M SP600125 (*) as compared to that of the cells treated only with SP600125. The dose-dependent suppression of cell growth was even more substantial when inhibitors for both JNK, at the concentrations of 10, 20, or 30 μ M, and MEK/ERK (U0126) were simultaneously added to T24 cell cultures, as compared to those cells treated only with 10, 20, or 30 μ M SP600125, respectively (all P<0.05 and indicated by ^**). Furthermore, comparing the growth rates of T24 cells pretreated only with 10 μ M U0126 and the indicated amounts of SP600125, the rates were again significantly reduced when the anticancer drug was simultaneously applied (all P<0.05 and indicated by ^***). In this experiment, the growth rate reached levels comparable to those of the genistein-sensitive cell lines. The symbols used in all panels are: cells without any treatment, □; and cells treated with 0.5% DMSO, ▪; 50 μ M genistein only, ; cells only treated with protein kinase inhibitor(s), ▨; cells treated with both protein kinase inhibitor(s) and genistein, ⍁.