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/

The possible regulation of the multidrug-resistant (MDR) phenotype and P-glycoprotein by protein kinase C (PKC) was investigated in the doxo rubicin (Dox)-resistant MCF-7 cell line (MCF-7/Dox). In a clonogenic a ssay, cells exposed to 100 nM phorbol 12-myristate 13-acetate (PMA) fo r 1 hr were about 3-fold more resistant to Dox than were cells exposed to Dox alone. The PKC inhibitor 1-(5-isoquinolinesulionyl)-2-methylpi perazine (H7, 30 mu M) completely blocked the PMA-induced effect, but did nut reverse the MDR phenotype. Complete down-regulation of PKC fro m MCF-7/Dox cells by 24-hr preincubation with PMA did not alter the de gree of Dox resistance. Intracellular accumulation of [C-14]Dox decrea sed from a baseline of 28 pmol/10(6) cells to 15 pmol/10(6) cells in t he presence of 100 nM PMA. The reduced Dox accumulation in the presenc e of PMA was not blocked by pretreatment of cells with H7. Following a 24-hr pretreatment with PMA, the cells accumulated almost equal amoun ts of [C-14]Dox in the absence or presence of PMA. Cells from PMA-trea ted colonies showed significantly higher levels of expression of P-gly coprotein when compared with those from control colonies. H7 did not a ffect the basal level of P-glycoprotein in cells from control colonies or PMA induced overexpression of P-glycoprotein in cells from PMA-tre ated colonies. Upon stimulation with PMA (100 nM), PKC alpha and beta translocated to the cell membrane and nucleus and PKC delta and epsilo n to the perinuclear membrane and the nucleus, respectively. H7 (30 mu M) completely inhibited PMA-induced translocations of PKC delta and e psilon, whereas it only partially blocked the translocations of PKC al pha and beta. These results suggest that PMA appears to alter Dox resi stance and intracellular Dox accumulation in a PKC-dependent manner an d to induce increased expression of P-glycoprotein in MCF-7/Dox cells. Differential effects of H7 on the PMA-induced changes suggest that di fferent isoforms of PKC may be involved in cell growth and drug accumu lation processes as well as P-glycoprotein expression.