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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.

P-glycoprotein (PGP) is an energy-dependent efflux pump that serves to protect cells against the cytotoxicity of many natural product drugs including vinblastine (VBL). In this study we investigated the role of PGP in regulating initial VBL influx. The apparent influx of VBL, measured over the first 20 s, was 2-fold lower in KB-GRC1 cells expressing a transfected mdr1 gene at high level than in non-expressing parental KB-3-1 cells. Inhibition of PGP efflux function with dipyridamole increased the influx rate constant by 4.0-fold in the KB-GRC1 cells but only 2.1-fold in the KB-3-1 cells. Verapamil, another inhibitor of PGP-mediated efflux, increased the initial influx rate constant by 2.7-fold in the KB-GRC1 cells but only 1.4-fold in the KB-3-1 cells. Inhibition of PGP function by depletion of ATP increased influx by 6.8-fold and 2.2-fold in the two cell types, respectively. Mutation of PGP at both ATP binding sites abolished its ability to limit initial influx. Thus, VBL is serving as an efficient substrate for the efflux pump even within the first few seconds of drug exposure, consistent with the hypothesis that PGP may directly efflux drug from the cell membrane.

Gene amplification is one mechanism whereby tumor cells can become resistant to antineoplastic agents. Unstably amplified genes occur either on submicroscopic circular pieces of extrachromosomal DNA called episomes or on small acentric chromosomes called double minutes. Double minutes are frequently associated with cells containing amplified drug-resistance genes. Human epidermoid carcinoma KBV1 cells contain unstably amplified mdr1 genes and overexpress P glycoprotein, resulting in decreased intracellular drug accumulation. In this cell line, a nonlethal concentration of hydroxyurea accelerated the rate of loss of vinblastine resistance once vinblastine had been removed from the culture medium. After removal of vinblastine, KBV1 cells exposed to hydroxyurea for the time required to complete 12 cell doublings accumulated more vinblastine than control cells grown in the absence of hydroxyurea. In contrast, hydroxyurea had no effect on vinblastine sensitivity and accumulation in parental drug-sensitive KB-3-1 cells. Hydroxyurea also had no effect on sensitivity to cisplatin in cisplatin-resistant human ovarian carcinoma 2008/C13* cells, indicating that hydroxyurea's effect on drug sensitivity was specific for a drug-resistance phenotype associated with unstably amplified drug-resistance genes. These results indicate that, after removing selective pressure, hydroxyurea accelerates loss of resistance to vinblastine and increases accumulation of vinblastine in KBV1 cells, presumably by accelerating loss of amplified mdr1 genes and thus P glycoprotein.

We examined the ability of dipyridamole (DPM) to act synergistically with vinblastine (VBL) in HT1080 fibrosarcoma cells and a drug-resistant variant, HT1080/DR4, which lacks mdr1 expression, in order to determine whether DPM requires P-glycoprotein to modulate drug sensitivity. Median effect analysis of clonogenic assay was used to produce the combination index (CI50, values less than 1 indicate synergy). DPM was mildly synergistic with VBL producing a CI50 of 0.83 +/- 0.13 for HT1080 cells and 0.80 +/- 0.16 for HT1080/DR4 cells. HT1080 and HT1080/DR4 cells accumulated 6.7 +/- 0.7 and 5.6 +/- 0.9 pmol 3H-VBL mg cells-1 at steady state (Css) and 20 microM DPM elevated the Css by 1.8 and 2.9-fold, respectively. In comparison, the CI50 was 1.1 +/- 0.2 in parental KB-3-1 cells and 0.1 +/- 0.1 in mdr1-expressing KB-GRC1 cells. The KB-3-1 and KB-GRC1 cells had a Css of 3.8 +/- 0.8 and 0.7 +/- 0.2 pmol 3H-VBL mg cells-1, respectively, and DPM elevated the Css by 9.2-fold in KB-GRC1 cells. These studies demonstrate that DPM can produce synergy independently of mdr1 expression but that much greater levels of synergy are achievable in mdr1-expressing tumour cells.