Is stearate induced p21^cip1/waf1 and p27^kip1 protein expression dependent on inhibition of RhoC activity? Free

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There is in vitro and in vivo evidence that stearate (C18:0), a long chain dietary saturated fatty acid, inhibits solid tumor growth, as well as promotes breast cancer cell morphological changes; however the mechanism(s) underlying these effects are unclear. We have demonstrated that treatment of Hs578t human breast cancer cells with 50 μM stearate for 6 hours results in a reduction of actin stressfibers and a decrease in focal adhesion contact sites. We have also shown that at physiological concentrations, stearate inhibits both epidermal growth factor and complete media induced cell cycle progression of Hs578t human breast cancer cells in both G1 and G2 phases; ( n=3; p<0.001, control vs. all other treated groups). Stearate inhibition of the cell cycle was associated with increased accumulation of cell cycle inhibitors p21^cip1/waf1 and p27^kip1. This increase of p21^cip1/waf1 (n=3, p<0.005 control vs. all other treated groups) is due to an increase in synthesis, whereas the p27^kip1 increase was most likely due to inhibition of degradation. Members of Ras superfamily, Rho are known to affect both cell proliferation and the organization of the actin cytoskeleton. We hypothesized that stearate decreases Rho activity. After detecting Rho expression in Hs578T human breast cancer cells, we examined whether stearate affects Rho activity in these cells treated with or without stearate for 6 hrs and EGF post-treatment for 2-24 hrs. We found that stearate inhibited Rho activation and RhoC protein expression. In order to determine if stearate inhibition of Rho activity was dependent on p190 Rho-GAP, we used constitutively active Rho contructs which were resistant to inhibition by p190 Rho-GAP. Using constitutively active RhoA, RhoB and RhoC proteins, we demonstrated that RhoC but not RhoA was able to reverse the stimulatory effect of stearate on p21^cip1/waf1 and p27^kip1. RhoB had an intermediate effect on reversing stearate induced p21^cip1/waf1 and p27^kip1. In Hs578T human breast cancer cells transfected with constitutively active RhoA, RhoB, or RhoC, stearate had no effect on Rho activity based on the Rhotekin assay. These data are consistent with stearate increasing p21^cip1/waf1 and p27^kip1 by inhibition of RhoC via a p190 Rho-GAP mediated mechanism. Ongoing studies include using RhoC siRNA to further confirm the role of RhoC in mediating stearate induced p21^cip1/waf1 and p27^kip1 expression and investigating the effects of stearate on p190 Rho-GAP.