Role of glycogen synthase kinase 3 beta (GSK3beta) in mediating the cytotoxic effects of the histone deacetylase inhibitor trichostatin A (TSA) in MCF-7 breast cancer cells

Abstract

Histone deacetylase inhibitors (HDACIs) have been shown to induce apoptotic and autophagic cell death in vitro and in vivo. The molecular mechanisms that underlie these cytotoxic effects are not yet clearly understood. Recently, HDACIs were shown to induce Akt dephosphorylation by disrupting HDAC-protein phosphatase 1 (PP1) complexes. This disruption results in the increased association of PP1 with Akt, resulting in the dephosphorylation and consequent inactivation of the kinase. Akt enhances cellular survival through the phosphorylation-dependent inhibition of several pro-apoptotic proteins. Akt is an important negative regulator of GSK3beta, a kinase that has been shown to regulate apoptosis in response to various stimuli. In the present study, we investigated the role of GSK3beta in mediating the cytotoxic effects in MCF-7 breast cancer cells treated with trichostatin A (TSA), a prototype HDACI. We show that TSA induces Akt dephosphorylation in a PP1-dependent manner, resulting in activation of GSK3beta in MCF-7 cells. Similarly, knockdown of HDAC1 and-2 by small interfering RNA (siRNA) resulted in the dephosphorylation of Akt and GSK3beta. Selective inhibition of GSK3beta attenuated TSA induced cytotoxicity and resulted in enhanced proliferation following drug removal. Our findings identify GSK3beta as an important mediator of TSA-induced cytotoxicity in MCF-7 breast cancer cells.

Figure 1

(A). TSA induces Akt and GSK3β dephosphorylation in MCF-7 breast cancer cells. MCF-7 cells were incubated with 1 μM TSA for the indicated times. Following incubation, the cells were harvested and lysates were resolved by SDS-PAGE. Proteins were detected using the indicated antibodies. (B). The relative amounts of pAkt and pGSK3β in A were measured by densitometry and normalised to the amount of p38/SAPK. Result is representative of at least three separate experiments. (C). Knockdown of class I HDAC proteins induces Akt and GSK3β dephosphorylation. MCF-7 cells were transfected with oligo pools specifically targeting HDAC1, 2, 3 or a non-targeting siRNA pool (NSC). 72 h after transfection, cells were harvested and lysed. Lysates were treated as in A and probed with the indicated antibodies. (D). siRNA-mediated GSK3β knockdown attenuates the cytotoxic effect of TSA on MCF-7 cells. MCF-7 cells were transfected with oligo pools specifically targeting GSK3β or a non-targeting siRNA pool (NSC). 24 h after transfection cells were harvested and reseeded in 96-well plates and incubated for 24 h. Cells were then treated with 1 μM TSA for 48 h and relative cell survival was measured as described in materials and methods. Results represent the mean ± S.E. from at least three separate experiments. * P < 0.001 TSA treated vs. untreated NSC siRNA cells, ** P < 0.001 TSA treated NSC vs. TSA treated GSK3β siRNA cells. Inset: Lysates from cells transfected in parallel were probed with antibodies directed against GSK3 to monitor siRNA efficiency. (E). Effect of GSK3β siRNA on TSA induced cytotoxicity. Cells were treated as in D and examined by flow cytometry (see materials and methods section). Result is representative of at least three separate experiments. (F). Effect of GSK3β inhibition on TSA induced cytotoxicity. MCF-7 cells were cultured in 96-well plates with 10^-9 – 10^-5 M TSA alone or in combination with 10 mM LiCl. Relative cell survival was determined after 48 h as described in materials and methods section. Result is representative of three separate experiments.