Selective inhibition of the effects of phorbol ester on doxorubicin resistance and P-glycoprotein by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) in multidrug-resistant MCF-7/Dox human breast carcinoma cells

Abstract

The possible regulation of the multidrug-resistant (MDR) phenotype and P-glycoprotein by protein kinase C (PKC) was investigated in the doxorubicin (Dox)-resistant MCF-7 cell line (MCF-7/Dox). In a clonogenic assay, cells exposed to 100 nM phorbol 12-myristate 13-acetate (PMA) for 1 hr were about 3-fold more resistant to Dox than were cells exposed to Dox alone. The PKC inhibitor l-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7, 30 μM) completely blocked the PMA-induced effect, but did not reverse the MDR phenotype. Complete down-regulation of PKC from MCF-7/Dox cells by 24-hr preincubation with PMA did not alter the degree of Dox resistance. Intracellular accumulation of [ 14C]Dox decreased from a baseline of 28 pmol/10 6 cells to 15 pmol/10 6 cells in the presence of 100 nM PMA. The reduced Dox accumulation in the presence of PMA. was not blocked by pretreatment of cells with H7. Following a 24-hr pretreatment with PMA, the cells accumulated almost equal amounts of [ 14C]Dox in the absence or presence of PMA. Cells from PMA-treated colonies showed significantly higher levels of expression of P-glycoprotein when compared with those from control colonies. H7 did not affect the basal level of P-glycoprotein in cells from control colonies or PMA-induced overexpression of P-glycoprotein in cells from PMA-treated colonies. Upon stimulation with PMA (100 nM), PKC α and β translocated to the cell membrane and nucleus and PKC δ and ϵ to the perinuclear membrane and the nucleus, respectively. H7 (30 μM) completely inhibited PMA-induced translocations of PKC δ and ϵ, whereas it only partially blocked the translocations of PKC α and β. These results suggest that PMA appears to alter Dox resistance and intracellular Dox accumulation in a PKC-dependent manner and to induce increased expression of P-glycoprotein in MCF-7/Dox cells. Differential effects of H7 on the PMA-induced changes suggest that different isoforms of PKC may be involved in cell growth and drug accumulation processes as well as P-glycoprotein expression.