Abstract
The ability of 1,9-dideoxyforskolin (DDF), 1-deoxyforskolin (DF) and forskolin to modulate cellular sensitivity to vinblastine (VBL) was examined in drug-sensitive parental KB-3-1 cells and a multidrug-resistant subline, KB-GRC1, derived by transfection of mdr1. Fifty microM DF and forskolin enhanced the 1 h uptake of VBL by 8.0 +/- 0.7 (s.d.) and 4.7 +/- 2.5-fold, respectively, with 50 microM DDF producing a 13.6 +/- 1.9-fold increase. The greater effect of DDF relative to forskolin indicated that the effect was independent of activation of cAMP, and this was supported by a lack of effect of dibutyryl cAMP on the uptake. The effect of these agents on uptake were < or = 1.4-fold in KB-3-1 cells. DDF selectively inhibited initial efflux in cells expressing a functional P-glycoprotein (PGP), but both forskolin and DDF inhibited the terminal phase of efflux irrespective of PGP expression. Neither agent affected membrane permeability of polarisation and forskolin did not enhance the uptake of VBL in protein-free liposomes. At a non-toxic concentration of 20 microM, DDF and forskolin decreased the IC50 of VBL from 18.9 to 2.7 and 13 nM in KB-GRC1 cells, respectively, and DDF acted synergistically with VBL as shown by median effect analysis [combination index = 0.20 +/- 0.05 (s.d.)]. In contrast, these diterpenes did not affect VBL sensitivity in KB-3-1 cells. These results indicate that the diterpenes modulate VBL sensitivity predominantly by inhibiting PGP-mediated efflux activity.
Highlights
We report here that the primary mechanism by which DDF and forskolin modulate VBL sensitivity is by inhibition of a rapidly-acting PGP efflux transporter
Comparison of diterpenes as modulators whFyhidgirucrohexydl1iffcegorrmopfuarprosemsaettahcehthseottrhuoecnrteurbeyanovdfirftonuriesnkeooflipenoi,stihDteirFonosan.nedHoyDrdDrtFow,o philicity increases with the number of hydroxyl groups with forskolin being most hydrophilic, least hydrophilic
Forskolin was chosen for investigation because it activates protein kinase A indirectly (Laurenza et al, 1989) and alteration of protein kinase A has been shown to alter drug sensitivity (Abraham et al, 1990) and phosphorylation of PGP (Meyers, 1989; Mellado & Horwitz, 1987)
Summary
Drugs and chemicalsDDF, DF, and forskolin were purchased from Sigma Chemical Co. DDF, DF, and forskolin were purchased from Sigma Chemical Co. Stock solutions of these drugs were made by dissolving them in DMSO. TPP+ (97% pure) was purchased from Aldrich Chemical Co. Working solutions were prepared by further dilution in tissue culture medium. ['4C]-doxorubicin HCl (55 mCi mmol -) and [3H]-TPP+ (23 Ci mmol-') were purchased from Amersham Radiopharmaceuticals Inc. [3H]-VBL (10-20Cimmol'1) in methanol was purchased under a special quality-control contract to ensure high purity from Moravek Biochemicals (Brea, CA), stored in the dark at - 80'C and protected from light during experiments. The purity of [3H]-VBL was confirmed by HPLC analysis according to the method of Thimmaiah and Sethi (1985). [3H]-VBL was stable under the experimental conditions of these studies The purity of [3H]-VBL was confirmed by HPLC analysis according to the method of Thimmaiah and Sethi (1985). [3H]-VBL was stable under the experimental conditions of these studies
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