Biomedical subjects

J Lankelma

Publications and source records attributed to J Lankelma.

At least 55 records · Page 3Linked to original sources

PubMed1993

Prolonged retention of high concentrations of 5-fluorouracil in human and murine tumors as compared with plasma.

Concentrations of 5-fluorouracil (5-FU) and its active metabolite 5-fluoro-2'-deoxy-5'-monophosphate (FdUMP) were measured in biopsy specimens of tumor tissue, normal mucosa, metastatic liver nodules, and normal liver tissue obtained from 39 patients and in two murine colon tumors (colon 26 and colon 38) after a single injection of 5FU at a therapeutic dose (500 mg/m2 and 100 mg/kg, respectively). These data were compared with plasma concentrations. Peak plasma concentrations (300-500 microM) of 5FU were comparable in human and murine plasma. The half-life of plasma elimination (during the period from 15 to 120 min) in both mouse and man ranged from 10 to 20 min, whereas at between 2 and 8 h, plasma concentrations varied from 0.1 to 1 microM, the half-life being about 100 min. In both species, 5FU could be measured in plasma at concentrations ranging from 0.01 to 1 microM for several days after 5FU treatment. 5FU concentrations in tissue samples obtained from 14 patients were measured during the time range of 1-6 h, those in samples taken from 7 patients, during the interval of 19-27 h; and those in samples obtained from 18 patients, within the interval of 40-48 h after injection. 5FU tumor concentrations varied between 0.78-21.6, 0.44-6.1, and 0.17-10.8 mumol/kg wet wt., respectively. Some of the 48-h samples were obtained from patients who had received leucovorin plus 5FU; coadministration of leucovorin did not alter 5FU tissue concentrations. At between 4 and 48 h, the tissue concentration/plasma concentration ratio was at least 10. 5FU concentrations in murine tumors were measured for up to 10 days after 5FU administration, with plateau 5FU tumor concentrations being about 50 mumol/kg wet wt. in colon 38 and about 200 mumol/kg wet wt. in colon 26 at 2 h after treatment; after 4 days, values of 0.5 and 4.8 mumol/kg, respectively, were obtained and after 10 days, respective concentrations of 0.1 and 0.07 mumol/kg were detected. The FdUMP concentrations measured in colon 26 and colon 38 tumors were 214 and 46 pmol/g, respectively, at 2 h after 5FU administration, and these values subsequently decreased to about 15 pmol/g in both tumors. In human tumors the initial FdUMP concentration ranged from 10 to 1000 pmol/g; at later time points the level of FdUMP was just above the detection limit of the assay. In liver metastases, high 5FU concentrations seemed to be related to high levels of FdUMP, which was likely of importance for the antitumor effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗

PubMed1993

Changes in subcellular doxorubicin distribution and cellular accumulation alone can largely account for doxorubicin resistance in SW-1573 lung cancer and MCF-7 breast cancer multidrug resistant tumour cells.

Doxorubicin accumulation defects in multidrug resistant tumour cells are generally small in comparison to the resistance factors. Therefore additional mechanisms must be operative. In this paper we show by a quantitative approach that doxorubicin resistance in several P-glycoprotein-positive non-small cell lung cancer and breast cancer multidrug resistant cell lines can be explained by a summation of accumulation defect and alterations in the efficacy of the drug once present in the cell. This alteration of efficacy was partly due to changes in intracellular drug localisation, characterised by decreased nuclear/cytoplasmic doxorubicin fluorescence ratios (N/C-ratios). N/C-ratios were 2.8-3.6 in sensitive cells, 0.1-0.4 in cells with high (> 70-fold) levels of doxorubicin resistance and 1.2 and 1.9 in cells with low or intermediate (7.5 and 24-fold, respectively) levels of doxorubicin resistance. The change of drug efficacy was reflected by an increase in the total amount of doxorubicin present in the cell at equitoxic (IC50) concentrations. N/C ratios in highly resistant P-glycoprotein-containing cells could be increased with the resistance modifier verapamil to values of 1.3-2.7, a process that was paralleled by a decrease of the cellular doxorubicin amounts present at IC50. At the low to moderate residual levels of resistance, obtained with different concentrations of verapamil, a linear relationship between IC50 and cellular doxorubicin amounts determined at IC50 was found. This shows that at this stage of residual resistance, extra reversal by verapamil should be explained by further increase of drug efficacy rather than by increase of cellular drug accumulation. A similar relationship was found for P-glycoprotein-negative MDR cells with low levels of resistance. Since in these cells N/C ratios could not be altered, verapamil-induced decrease of IC50 must be due to increased drug efficacy by action on as yet unidentified targets. Although the IC50 of sensitive human cells cannot be reached with resistance modifiers, when using these relationships it can be shown by extrapolation that cellular and nuclear doxorubicin amounts at IC50 at complete reversal of resistance were the same as in sensitive cells. It is concluded that doxorubicin resistance factors for multidrug resistant cells can for a large part, and in the case of P-glycoprotein-containing cells probably fully, be accounted for by decreased amounts of drug at nuclear targets, which in turn is characterised by two processes only: decreased cellular accumulation and a shift in the ratio nuclear drug/cytoplasmic drug.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Genistein modulates the decreased drug accumulation in non-P-glycoprotein mediated multidrug resistant tumour cells.

In tumour cells the pharmacological basis for multidrug resistance (MDR) often appears to be a reduced cellular cytostatic drug accumulation caused by the drug efflux protein, P-glycoprotein (Pgp MDR), or by other drug transporters (non-Pgp MDR). Here we report the reversal of the decreased daunorubicin (DNR) accumulation in five non-Pgp MDR cell lines (GLC4/ADR, SW-1573/2R120, HT1080/DR4, MCF7/Mitox and HL60/ADR) by genistein. Genistein inhibited the enhanced DNR efflux in the GLC4/ADR cells. In these cells the decreased VP-16 accumulation was also reversed by genistein. Three other (iso)flavonoids biochanin A, apigenin and quercetin also increased the DNR accumulation in the GLC4/ADR cells. In contrast to the effects on non-Pgp MDR cells, 200 microM genistein did not increase the reduced DNR accumulation in three Pgp MDR cell lines (SW-1573/2R160, MCF7/DOX40 and KB8-5) or in the parental cell lines. In conclusion the use of genistein provides a means to probe non-Pgp related drug accumulation defects.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Cortisol is transported by the multidrug resistance gene product P-glycoprotein.

The physiology of the multidrug transporter P-glycoprotein (Pgp) is still poorly understood. We now show evidence that cell lines with a high expression of Pgp display a reduced accumulation of cortisol and an ATP-dependent outward transport of the hormone. Cortisol efflux from Pgp negative cells does not have such an active component. Further we show that the steroid hormones cortisol, testosterone, and progesterone cause an immediate, dose-dependent increase of daunorubicin accumulation in Pgp overexpressing cells. These effects are particularly apparent for the more lipophilic steroids. These results demonstrate that Pgp may function as a transporter for cortisol and suggest a physiological role of the protein in steroid handling by organs such as the adrenal.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Kinetics of daunorubicin transport by P-glycoprotein of intact cancer cells.

Drug permeation across the plasma membrane of multidrug-resistant cells depends on the kinetics of the P-glycoprotein-mediated pump activity as well as on the passive permeation of the drug. We here demonstrate a method to characterize kinetically the pump in intact cells. To this purpose, we examined the membrane-transport properties of daunorubicin in various sensitive cancer cell lines and in their multidrug resistant (MDR) counterparts. First, we determined the passive permeability coefficient for daunorubicin. Then, using a flow-through system, the drug flux into the cell was measured after inhibition of the P-glycoprotein-mediated efflux pump. Combining the two results allowed us to calculate the intracellular free concentration of the drug. In the steady-state, the pump rate must equal the net rate of passive diffusion of the drug and, therefore, the same experiments gave us the pumping rate of daunorubicin. These experiments were then repeated at various extracellular drug concentrations. By plotting the pumping rate versus the intracellular drug concentration, we then characterized the P-glycoprotein kinetically. Four independent methods were used to measure the passive permeability coefficient for the cell line A2780. Similar values were obtained. Maximal pump rates (Vmax) showed a good correlation with the amount of P-glycoprotein in the cell lines used. We obtained saturation curves for the variation of the pump rates with the intracellular daunorubicin concentrations. These curves were typical for positive cooperativity, which provides evidence that at least two binding sites for daunorubicin are present on the active transport system of daunorubicin. The apparent Km values for P-glycoprotein-mediated transport, the intracellular free cytosolic daunorubicin concentrations at half-maximal velocity for the cell lines used, were approximately 1.5 microM. Except for the cell lines with the highest amount of P-glycoprotein, the passive efflux rate of daunorubicin proved to be a substantial part of the total daunorubicin efflux rate for the cell lines used. In cell lines with relatively low levels of P-glycoprotein, passive daunorubicin efflux was even the main route of daunorubicin transport from the cells, determining the intracellular steady-state concentrations of daunorubicin.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Reactions of glutathione with the catechol, the ortho-quinone and the semi-quinone free radical of etoposide. Consequences for DNA inactivation.

Etoposide [4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta- D-glucopyranoside)] can be metabolized to DNA-inactivating catechol, ortho-quinone and semi-quinone free radical derivatives which may contribute to its cytotoxicity. In this paper, we examined in vitro whether glutathione (GSH), which is known to react easily with quinoid compounds, could interact with the active etoposide intermediates and in this way influence the cytotoxicity of the parent compound. To this end, reactions of GSH with the etoposide intermediates were studied, using HPLC and ESR measurements, together with the effects of GSH on the biological inactivation of single-stranded (ss) and double-stranded (RF) phi X174 DNA by these compounds. From the results it could be determined that: (a) GSH does not react with the catechol and, as a consequence, has no effect on the reaction of this intermediate of etoposide with ss and RF phi X174 DNA; (b) GSH reacts with the ortho-quinone most likely by formation of a conjugate and by two-electron reduction to the catechol, resulting in a partial protection of ss and RF phi X174 DNA against inactivation by this species; and (c) GSH protects ss phi X174 DNA against inactivation by the semi-quinone free radical of etoposide probably by conjugation with this species.

Bacteriophage phi X 174↗

PubMed1992

Probing daunorubicin accumulation defects in non-P-glycoprotein expressing multidrug-resistant cell lines using digitonin.

Multidrug resistance (MDR) in tumor cells is frequently associated with reduced cellular cytostatic drug accumulation, caused by the drug efflux protein, P-glycoprotein (Pgp). The action of Pgp in tumor cells can be detected by measuring the increase of daunorubicin accumulation upon blocking Pgp with drugs such as verapamil. A number of MDR cell lines have been described, characterized by decreased drug accumulation without Pgp being present. For such non-Pgp MDR cells no gene probes or functional assays are available to study this phenotype in clinical tumor specimens. We have worked out a method which enables the detection of drug-transport-related decreases in cellular daunorubicin accumulations without the need for the use of specific Pgp blockers. The cells used were SW-1573-, GLC4- and HT1080-sensitive cell lines, which accumulated (corrected for DNA content) 272%, 1,288% and 203% more daunorubicin than the non-Pgp MDR sublines SW-1573/2R120, GLC4/ADR and HT1080/DR4. When the plasma membranes of these MDR lines were permeabilized with 20 microM digitonin an increase to 282%, 1,260% and 239% of 14C-daunorubicin control accumulation was measured (at pH = 7.35). The intracellular pH measured with BCECF was the same in parent and corresponding MDR cells, excluding the role of pH differences in the measured effects. This method provides a tool allowing the detection of cellular mechanisms (including Pgp) which are related to active outward transport of daunorubicin.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Energy-dependent processes involved in reduced drug accumulation in multidrug-resistant human lung cancer cell lines without P-glycoprotein expression.

Mechanisms contributing to reduced cytotoxic drug accumulation were studied in two multidrug-resistant (MDR) human lung cancer cell lines without P-glycoprotein expression. In these (non-small cell) SW-1573/2R120 and (small cell) GLC4/ADR MDR cells, the steady-state accumulation of [14C]daunorubicin was 30 and 12%, respectively, of that in the parent cells. When cells, at steady state, were permeabilized with digitonin, the amount of daunorubicin binding increased only in the resistant cells. The reduced accumulation of daunorubicin in the SW-1573/2R120 and GLC4/ADR cells was accompanied by a lower initial (2 min) uptake rate of this drug. No difference in initial efflux rate of daunorubicin from preloaded cells could be detected between sensitive and resistant SW-1573 cells. However, daunorubicin was extruded 5-fold faster from GLC4/ADR cells than from the parental cells. In the presence of the energy metabolism inhibitors sodium azide and deoxyglucose, the reduced daunorubicin accumulations in the SW-1573/2R120 and GLC4/ADR MDR cells were (almost) completely reversed. The effects of these inhibitors on drug uptake were already apparent during the earliest measured time points (less than 15 s). Also, the enhanced efflux of daunorubicin from GLC4/ADR cells was inhibited. In ATP-depleted cells, the intracellular pH was lowered by approximately 0.3 units in resistant as well as in sensitive cells. The lower intracellular pH, however, could not account for the increase in daunorubicin accumulation in the resistant cells. Also, for vincristine and etoposide, the increases in drug accumulation under energy-deprived conditions were more pronounced in the resistant SW-1573/2R120 cells than in the parent SW-1573 cells. These results suggest that accumulation of drugs in the non-P-glycoprotein MDR human lung carcinoma cell lines SW-1573/2R120 and GLC4/ADR is reduced by an energy-dependent drug export mechanism which prevents efficient transport of drug to the target. Since P-glycoprotein expression in lung tumors is generally low, these MDR lung cancer cell lines can be used as a model to study alternative mechanisms leading to multidrug resistance in this tumor type.

Adenosine Triphosphate↗

PubMed1992

Quantification by laser scan microscopy of intracellular doxorubicin distribution.

Changes in intracellular drug localization accompany doxorubicin resistance in multidrug resistant tumor cells. The purpose of this study was to develop a method to quantify these changes and so detect different levels of resistance. Tumor cells were incubated with the fluorescent anthracycline doxorubicin (excitation at 480 nm; emission maximum at 560-590 nm) and were quantified using laser scanning microscopy. The fluorescent mode was used to record the intracellular drug distribution, whereas the absorption mode was used to define the nuclear and cytoplasmic boundaries. The cell compartments were delineated interactively on an image processing system and the ratio nuclear fluorescence/cytoplasmic fluorescence (N/C ratio) was determined. N/C ratios were: 1.8 in the Chinese hamster ovarian cell line AUXB1 and 0.1 in its MDR subline CHRC5; 3.8 in the human squamous lung cancer cell line SW-1573 and 1.8 and 0.4 in its MDR sublines SW-1573/2R120 and SW-1573/2R160, respectively; and 3.6 in the human myeloma cell line 8226/S and 2.1 and 1.0 in its MDR sublines 8226/Dox4 and 8226/Dox40, respectively. The doxorubicin distribution was independent of the doxorubicin concentration within a range from 1-32 microM. Furthermore, the progressive mean of the nuclear/cytoplasmic doxorubicin fluorescence ratio showed that a minimal sample size of 30 cells is necessary for reliable results. The results of two independent assessments showed a high reproducibility (r = 0.97). Thus, with the method described in this paper, it is possible to detect relatively low levels of doxorubicin resistance (factor 8).

Animals↗

PubMed1992

Modulation by D,L-buthionine-S,R-sulphoximine of etoposide cytotoxicity on human non-small cell lung, ovarian and breast carcinoma cell lines.

Treatment with 25 mumol/l D,L-buthionine-S,R-sulphoximine (BSO) for at least 24 h depleted glutathione (GSH) in human non-small cell lung (SW-1573), ovarian (A2780) and breast carcinoma (MCF-7) cell lines to about 20% of control, and was accompanied by a 2-fold potentiation of the cytotoxicity of etoposide, doxorubicin and cisplatin. Cellular etoposide, but not doxorubicin or cisplatin, concentrations were increased 2-fold due to decreased efflux. This occurred independently of the presence of BSO during 1 h of etoposide exposure, but required prolonged exposure to BSO (at least 24 h). Energy depletion as well as cotreatment, but not pretreatment, of the cells with daunomycin, doxorubicin, vinblastine or vincristine increased cellular etoposide accumulation. Treatment of control cells with verapamil caused similar changes in etoposide cytotoxicity and cellular pharmacokinetics as GSH depletion, but did not further increase etoposide cytotoxicity and accumulation in GSH-depleted cells. Etoposide efflux may have been inhibited, not because of (competitive) inhibition by BSO or disturbance of the energy required for this process, but probably because of plasma membrane alterations.

Antimetabolites, Antineoplastic↗

PubMed1992

Electron spin resonance spectroscopy of oxygen radicals generated by synthetic fecapentaene-12 and reduction of fecapentaene mutagenicity to Salmonella typhimurium by hydroxyl radical scavenging.

Fecapentaenes form a class of potent fecal mutagens and have been suggested to play an initiating role in colon carcinogenesis. Although several indications have been found that fecapentaenes may induce oxidative DNA damage as well as DNA alkylation, the mechanism of genotoxicity remains unknown. In this study, electron spin resonance spectroscopy with several spin traps has been used in order to determine whether reactive oxygen species can be formed by fecapentaene-12 (FP-12). No specific conditions could be defined that resulted in the direct formation of oxygen radicals from FP-12. However, peroxidation of FP-12 by various peroxidative enzymes has been shown to result in the formation of superoxide adducts of the spin traps alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Addition of superoxide dismutase resulted in a decreased spectrum intensity, whereas the hydroxyl radical scavenger t-butyl alcohol (tBA) appeared of no influence on the signal, both confirming the formation of superoxide. The formation of hydroxyl radical spin adducts has been demonstrated after peroxidation of FP-12 in incubations with the spin-trapping agent 2,2,6,6-tetramethyl-piperidine (TMP). Further, the effects of scavenging hydroxyl radicals with respect to the genotoxic potential of FP-12 in the Salmonella mutagenicity assay has been investigated. It was clearly shown that radical scavenging reduced the number of revertants in Salmonella strains TA100, TA102 and TA104. This mutagenicity-reducing effect was more convincing using both spin traps DMPO and TMP as compared to the effect of hydroxyl radical scavengers tBA and DMSO. Based on these findings, a reaction scheme is proposed that suggests the formation of superoxide after peroxidation of FP-12, which is subsequently converted to hydroxyl radicals by the iron-catalysed Haber-Weiss reaction.

Benzene Derivatives↗

PubMed1991

Formation of different reaction products with single- and double-stranded DNA by the ortho-quinone and the semi-quinone free radical of etoposide (VP-16-213).

In this report, the types of DNA damage introduced by the ortho-quinone and the semiquinone free radical of 4'-demethylepipodophyllotoxin-9-(4-6-O-ethylidene-beta-D- glucopyranoside) (etoposide) and their relevance for the inactivation of single-stranded (ss) and double-stranded (ds) replicative form (RF) phi X174 DNA have been examined in vitro. The ortho-quinone yielded in both ss and ds DNA only chemical adducts, of which on the average about 1 out of 3 and 1 out of 12 per DNA molecule led to inactivation of ss or RF phi X174 DNA, respectively. The semi-quinone free radical, on the other hand, generated both frank and alkali-labile strand-breaks in ss and in ds DNA which, however, did not contribute significantly to DNA inactivation. The radical introduced, in addition, chemical DNA adducts. Unlike the ortho-quinone adducts, however, each of the semi-quinone adducts was lethal in ss phi X174 DNA, while more than 40 were required for the inactivation of RF DNA. The excision repair system of Escherichia coli did not operate on semi-quinone-modified RF DNA but removed about half of the ortho-quinone adducts [van Maanen JMS, Lafleur MVM, Mans DRA, van den Akker E, de Ruiter C, Koostra PR, Pappie D, de Vries J, Retèl J and Pinedo HM, Biochem Pharmacol 37: 3579-3589, 1988]. When ortho-quinone-modified ss or ds DNA was subjected to a post-alkaline treatment, the adducts remained stably bound to the DNA and the degree of biological inactivation was not influenced. In contrast, post-alkaline treatment removed about 70 and 60% of the semi-quinone adducts from ss and ds DNA, respectively, which, in the case of ss phi X174 DNA, resulted in a partial restoration of the biological activity. It is concluded that the ortho-quinone and the semi-quinone free radical of etoposide produce different types of damage in DNA which have different effects on the biological activity.

DNA↗

PubMed1991

Cellular daunomycin fluorescence in multidrug resistant 2780AD cells and its relation to cellular drug localisation.

Multidrug resistant (MDR) 2780AD human ovarian carcinoma cells were loaded with the fluorescent anticancer agent daunomycin (DN). Fluorescence anisotropy was lower than for corresponding A2780 wild-type cells, indicating that DN was less rigidly bound than in the wild-type cells. Average fluorescence quenching of DN was lower for 2780AD cells. Data were fitted into a model with a highly quenched fraction (fraction A), corresponding to DN intercalated in DNA, and an unquenched fraction (fraction B). The ratio A/B was one order of magnitude lower for the MDR cells than for the wild-type cells. Two other MDR cell lines were investigated and low A/B ratios were found in both cases. Thus, evidence has been provided that in MDR cells the DNA-bound fraction is relatively low and that more free DN is present, for example in acidic vesicles.

DNA, Neoplasm↗

PubMed1991

P-glycoprotein drug efflux pump involved in the mechanisms of intrinsic drug resistance in various colon cancer cell lines. Evidence for a saturation of active daunorubicin transport.

We studied the resistance of colon tumors to anticancer agents in vitro. Using daunorubicin (DN), a number of cellular parameters which normally indicate acquired or multidrug resistance (MDR), were compared for several human wild-type colon cell lines, i.e. HT29, SW1116 and COLO 320, and the murine colon cell line C-26. The sensitive/MDR human ovarian cancer cell line couple A2780/2780AD was used as a reference. The amount of P-glycoprotein (P-gp) was in the order HT29, A2780 less than or equal to SW1116 less than C26 less than or equal to COLO 320 less than 2780AD. The MDR modifiers verapamil, Cremophor EL, cyclosporin A and Ro 11-2933/001 had significant effects on DN cytotoxicity, total DN accumulation and efflux, only if P-gp was present. A flow-through system was used to study the mechanism of DN transport. For the first time, evidence for saturation of an active transport of DN from the cells is reported. We discussed the possible presence of cooperative activity between at least two binding sites on the protein responsible for DN efflux, likely to be P-gp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1991

Measurement of in vitro cellular pharmacokinetics of 5-fluorouracil in human and rat cancer cell lines and rat hepatocytes using a flow-through system.

A flow-through system was used to study the cellular pharmacokinetics of 5-fluorouracil (5-FU) in four human cell lines (squamous-cell carcinoma HEp-2, colon carcinoma WiDr, hepatoma Hep G2, and breast carcinoma MCF-7) as well as in the rat hepatoma H35 cell line and in freshly isolated rat hepatocytes. The system made it possible to restrict the decrease in the concentration of 5-FU in the medium, to keep the volume in which the metabolites accumulated relatively small, and to study the dynamics of a response during and after a change in the composition of the eluent. Clearance of 5-FU from the eluent was achieved predominantly (greater than 95%) by its catabolism to dihydrofluorouracil in the tumor cell lines and to 2-fluoro-beta-alanine in the hepatocytes. Not only rat hepatocytes but also HEp-2 cells showed relatively high clearance values. A concentration-dependent 5-FU elimination was observed, indicating saturation of 5-FU elimination according to Michaelis-Menten kinetics (Km 14-22 microM). The maximal velocity (Vmax) values ranged from 0.025 to 0.13 nmol 5-FU/10(6) cells per minute. For HEp-2 cells, high-concentration pulse injections of 5-FU, thymine, uridine, or uracil immediately led to a reduction in 5-FU conversion, followed by recovery within 5 min. The flow-through system proved to be adequate for the study of the non-linear pharmacokinetics of 5-FU in different intact cells and for the comparison of various manipulations of these pharmacokinetics.

Animals↗

PubMed1990

Evidence for daunomycin efflux from multidrug-resistant 2780AD human ovarian carcinoma cells against a concentration gradient.

Using a flow-through system human multidrug-resistant 2780AD ovarian carcinoma cells were exposed to flowing culture medium containing the anticancer agent daunomycin (5 microM). A pulse of medium containing verapamil caused increased cellular daunomycin accumulation, resulting in a dip in the fluorescence signal from daunomycin in the effluent. After passage of this pulse we observed an efflux of more than 90% of this extra accumulated daunomycin within 10 min. This daunomycin efflux against a concentration gradient provides evidence for drug efflux from these cells being an active process. After the addition of methylamine to the medium to increase intravesicular pH, the dip in the fluorescence signal was not decreased, indicating that vesicular transport was not an important component of this efflux.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1990

Differential sensitivity of multi-drug-resistant and -sensitive cells to resistance-modifying agents and the relation with reversal of anthracycline resistance.

Calcium channel blockers, calmodulin inhibitors, and some other classes of non-related compounds reverse multi-drug resistance. In the present study, we found that several resistance modifiers are more toxic for MDR cells than for the corresponding sensitive parent cells, whereas others show the opposite effect. Several calcium channel blockers including bepridil, diltiazem, nifedipine and verapamil, as well as the calmodulin inhibitor trifluoperazine, were more toxic for several MDR cell lines than for the parent cell lines. In contrast, cross-resistance for cyclosporin A and the verapamil analogue Ro II-2933/001 was observed in the MDR/sensitive cell couple CHRC5/AUXB1, probably due to a concentration-dependent stimulation of cell growth in the range of 0-4 microM cyclosporin A and of 1-4 microM Ro II-2933/001. In partially revertant CHRC5 cells, growth inhibition by Ro II-2933/001 at concentrations below I microM, as seen in CHRC5 cells, changed into growth stimulation, and the collateral sensitivity to verapamil and bepridil disappeared almost completely. In the MDR cells CHRC5, 2780AD and DC3F/DMXX, cross-resistance to another calcium channel blocking agent, Ro II-1781/001 (tiapamil), was observed as well. This compound showed exceptional behavior: it induced marked potentiation of Dx cytotoxicity as well as stimulation of Dx accumulation in AUXB1 cells, even at low tiapamil concentrations, but not in the CHRC5 cells, even at high concentrations. It is concluded that resistance modifiers can selectively influence growth of MDR cells via more than one process, and resulting in either strong growth inhibition in MDR cells relative to the effect on sensitive cells or in growth stimulation.

Animals↗

PubMed1990

Role of the semi-quinone free radical of the anti-tumour agent etoposide (VP-16-213) in the inactivation of single- and double-stranded phi X174 DNA.

The mechanism of action of the anti-tumour agent etoposide (VP-16-213) could involve its bioactivation to metabolites which can damage DNA. Active metabolites of etoposide, generated in vitro, are the 3',4'-dihydroxy-derivative (catechol) and its oxidation product, the ortho-quinone. The conversion of the catechol into the ortho-quinone (and vice versa) proceeds via formation of a semi-quinone free radical. We investigated the role of this radical species in the inactivation of biologically active single- (ss) and double-stranded (RF) phi X174 DNA. Since the formation of semi-quinone free radicals from the ortho-quinone of etoposide is pH dependent, experiments were performed, in which the ortho-quinone was incubated at pH 4, 7.4 and greater than or equal to 9. ESR measurements showed no formation of radical species from the ortho-quinone at pH 4, but an increased rate of generation of the primary semi-quinone free radical at pH values 7.4 to 10; at still higher pH values a secondary semi-quinone free radical was produced. HPLC analyses demonstrated chemical stability of the ortho-quinone at pH 4, but an accelerated decay was observed when the pH was elevated from 7.4 to 9, with its concomitant conversion into more polar components and into the catechol of etoposide. Ss phi X174 DNA, exposed to the ortho-quinone, was inactivated at an increasing rate at pH values increasing from 4 to 7.4 and subsequently to 9. RF phi X174 DNA was only significantly inactivated in incubations with the ortho-quinone at pH 4, not at pH values 7.4 and 9. From these data it is concluded that the primary semi-quinone free radical of etoposide may to a great extent be responsible for the ortho-quinone-induced ss phi X174 DNA inactivation, but that this radical species is not lethal towards RF phi X174 DNA.

Bacteriophage phi X 174↗