ABSTRACT
The potent kinase inhibitor staurosporine and its protein kinase C (PKC)-selective analogue CGP 41251 are known to sensitise cells with the multidrug resistance (MDR) phenotype mediated by P-glycoprotein (P-gp) to cytotoxic agents. Here four PKC-selective staurosporine cogeners, CGP 41251, UCN-01, RO 31 8220 and GF 109203X, were compared with staurosporine in terms of their MDR-reversing properties and their susceptibility towards P-gp-mediated drug efflux from MCF-7/Adr cells. Staurosporine was the most potent and the bisindolylmaleimides RO 31 8220 and GF 109203X the least potent cytostatic agents. When compared with MCF-7 wild-type cells, MCF-7/Adr cells were resistant towards the growth-arresting properties of RO 31 8220 and UCN-01, with resistance ratios of 12.6 and 7.0 respectively. This resistance could be substantially reduced by inclusion of the P-gp inhibitor reserpine. The ratios for GF 109203X, staurosporine and CGP 41251 were 1.2, 2.0 and 2.9 respectively, and they were hardly affected by reserpine. These results suggest that RO 31 8220 and UCN-01 are avidly transported by P-gp but that the other compounds are not. Staurosporine and CGP 41251 at 10 and 20 nM, respectively, decreased efflux of the P-gp probe rhodamine 123 (R123) from MCF-7/Adr cells, whereas RO 31 8220 and GF 109203X at 640 nM were inactive. CGP 41251 was the most effective and GF 109203X the least effective inhibitor of equilibrium binding of [3H]vinblastine to its specific binding sites, probably P-gp, in MCF-7/Adr cells. Overall, the results imply that for this class of compound the structural properties that determine susceptibility towards P-gp-mediated substrate transport are complex. Comparison with ability to inhibit PKC suggests that the kinase inhibitors affect P-gp directly and not via inhibition of PKC. Among these compounds CGP 41251 was a very potent MDR-reversing agent with high affinity for P-gp and least affected by P-gp-mediated resistance, rendering it an attractive drug candidate for clinical development.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Alkaloids/toxicity , Antineoplastic Agents/toxicity , Cell Division/drug effects , Drug Resistance, Multiple , Alkaloids/metabolism , Antineoplastic Agents/metabolism , Breast Neoplasms , Cell Line , Doxorubicin/metabolism , Female , Flow Cytometry , Fluorescent Dyes , Humans , Indoles/metabolism , Indoles/toxicity , Maleimides/toxicity , Protein Kinase C/antagonists & inhibitors , Rhodamine 123 , Rhodamines , Staurosporine , Structure-Activity Relationship , Tumor Cells, Cultured , Vinblastine/metabolismABSTRACT
We have performed a Phase I clinical trial with the naturally occurring flavonoid quercetin (3,3',4',5,7-pentahydroxyflavone). Quercetin has antiproliferative activity in vitro and is known to inhibit signal transduction targets including tyrosine kinases, protein kinase C, and phosphatidyl inositol-3 kinase. Quercetin was administered by short i.v. infusion at escalating doses initially at 3-week intervals. The first dose level was 60 mg/m2; at the 10th dose level of 1700 mg/m2, dose-limiting nephrotoxicity was encountered, but no myelosuppression. At the preceding dose level of 1400 mg/m2, five patients were treated at 3-week intervals, and another eight patients were treated on a once-weekly schedule; overall, 2 of 10 evaluable patients had renal toxicity, 1 at grade 2 and 1 at grade 4. We therefore treated other patients at 945 mg/m2 (eight at 3-week intervals and six at weekly intervals); 3 of 14 patients had clinically significant renal toxicity, 2 patients with grade 2 and 1 patient with grade 3. Patients treated on the weekly schedule did not have cumulative renal impairment but did have a fall in the glomerular filtration rate of 19 +/- 8% in the 24 h after drug administration. We recommend 1400 mg/m2 as the bolus dose, which may be given either in 3-week or weekly intervals, for Phase II trials. Quercetin pharmacokinetics were described by a first-order two-compartment model with a median t(1/2)alpha of 6 min and median t(1/2)beta of 43 min. The median estimated clearance was 0.28 liter/min/m2, and median volume of distribution at steady state was 3.7 liter/m2. In 9 of 11 patients, lymphocyte protein tyrosine phosphorylation was inhibited following administration of quercetin at 1 h, which persisted to 16 h. In one patient with ovarian cancer refractory to cisplatin, following two courses of quercetin (420 mg/m2), the CA 125 had fallen from 295 to 55 units/ml, and in another patient with hepatoma, the serum alpha-fetoprotein fell. In conclusion, quercetin can be safely administered by i.v. bolus at a dose injection. The plasma levels achieved inhibited lymphocyte tyrosine kinase activity, and evidence of antitumor activity was seen.
Subject(s)
Enzyme Inhibitors/adverse effects , Neoplasms/drug therapy , Protein-Tyrosine Kinases/antagonists & inhibitors , Quercetin/adverse effects , Adult , Aged , Female , Humans , Kidney/drug effects , Male , Middle Aged , Potassium/blood , Quercetin/pharmacokineticsABSTRACT
Cell membranes were prepared from the multidrug resistant, P-glycoprotein expressing human lymphoblastoid cell line CCRF-ADR 5000. The P-glycoprotein of these membranes possessed high affinity binding sites for [3H]vinblastine, with a Kd of 8 +/- 2 nM and Bmax of 17 +/- 8 pmol/mg of protein. The binding of [3H]vinblastine to P-glycoprotein was not ATP-dependent, and was inhibited by cytotoxic drugs with the following potency order; vincristine > doxorubicin > etoposide. The 1,4-dihydropyridine and multidrug resistance reversing agent, dexniguldipine-HCl, inhibited binding with a Ki value of 37 nM. The multidrug resistance reversing agent cyclosporin A, and the cytotoxics doxorubicin and etoposide did not alter the kinetics of [3H]vinblastine dissociation from P-glycoprotein; however, the 1,4-dihydropyridines dexniguldipine-HCl and nicardipine accelerated dissociation of [3H]vinblastine. These data suggest that P-glycoprotein possesses at least two allosterically coupled drug acceptor sites; receptor site 1 which binds vinblastine, doxorubucin, etoposide and cyclosporin A, and receptor site 2 which binds dexniguldipine-HCl and other 1,4-dihydropyridines.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Calcium Channel Blockers/pharmacology , Dihydropyridines/pharmacology , Vinblastine/metabolism , Allosteric Regulation , Binding, Competitive/drug effects , Cell Division/drug effects , Cyclosporine/pharmacology , Doxorubicin/pharmacology , Drug Resistance, Multiple , Etoposide/pharmacology , Humans , Kinetics , Mathematics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Tritium/metabolism , Tumor Cells, Cultured , Vincristine/pharmacologyABSTRACT
Pretreatment of NG108-15 cells for 1 h with 1 microM phorbol 12-myristate,13-acetate produced no significant effect on the subsequent stimulation of adenylate cyclase activity by the IP receptor agonist, iloprost, the adenosine A2 receptor agonist, N-ethylcarboxamidoadenosine (NECA), or sodium fluoride, suggesting that protein kinase C activation does not produce desensitization in this system. Pretreatment of cells with 10 microM iloprost or forskolin for 17 h produced a decrease in the specific binding of [3H]iloprost, consistent with a decrease in IP receptor number. Iloprost pretreatment produced a decrease in responses to iloprost, NECA and sodium fluoride, whereas forskolin pretreatment produced a decrease in subsequent responsiveness to iloprost and NECA, but the response to sodium fluoride remained unaffected. The desensitization produced by forskolin could be completely inhibited by the inhibitor of protein kinase A and protein kinase C, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), but H7 had no effect on the desensitization produced by iloprost.