Frederika Mandelbaum-Shavit

Antitumor steroidal-cis-platinum(II)-o-catecholato conjugates: preliminary evaluation on breast cancer MCF-7 cells

Abstract Six new steroidal-cis-platinum(II)-o-catecholato complexes (5–9 and 15) were prepared by treatment of either [4-(2-aminoethyl)1,2-benzenediolato(2-)-O, O′]-bis(triphenylphosphine)platinum(II) or [3,4-dihydroxybenzenepropionic acid (2-)-O3, O4]-bis-(triphenylphosphine)platinum(II) with appropriate functionalized steroids. The biological effect of the air-stable conjugates on a human breast tumor cell line, MCF-7, was compared with that of cis-dichloro-diaminoplatinum(II) (cis-DDP). The activity of the new compounds proved to be of the same order of magnitude as cis-DDP.

Acetylation of p-aminobenzoic acid by human blood

Abstract Acetylation of p -aminobenzoic acid was studied in human blood cell lysates. The rate of acetylation with acetyl-coenzyme A was 3.4 nmoles per min per 0.5 ml lysate (corresponding to 0.5 ml blood with a 50% hematocrit). In the presence of a coenzyme A generating system, the rate was only 0.1 nmole per min per 0.5 ml lysate. N -Acetyltransferase (acetyl-CoA : arylamine N -acetyltransferase, EC 2.3.1.5) activity exhibited a temperature optimum within the range of 34–37° with a Q 10 of 2 between 24–34°. Heating for 3 min at 50° caused 50 per cent inactivation of enzymatic activity. The pH activity profile showed an optimum at pH 6.0–6.5. p -Chloromercuribenzoate was a potent inhibitor causing 50 per cent inhibition at 9 μM. The apparent K m value for p -aminobenzoic acid was 0.4 mM and for acetylcoenzyme A, 0.3 mM. The enzyme activity with p -aminosalicyclic acid was about 50 per cent that obtained with p -aminobenzoic acid. Acetylation of sulfamethazine was either very low or in several individuals undetectable. Isonicotinic acid hydrazide at a concentration 10 times that of p -aminobenzoic acid did not interfere with acetylation of the latter, nor did trimethoprim, another compound with an amine moiety. Folic acid and amethopterin competitively inhibited the acetylation of p -aminobenzoic acid, with respective K i values of 0.01 mM and 0.06 mM. It is concluded that the activity of N -acetyltransferase in human blood may have important clinical implications.

Preparation and anticancer activity of two tryptamine derived platinum complexes

Abstract In search for new antitumor drugs with target specificity [4-[10-(1H-indol-3-yl)-4,7-dioxo-3,8-diazadecyl]- 1,2-benzenediolato(2−)-O,O′]bis(triphenylphosphine)platinum (3) and L -[4-(9-carboxy-10-(1H-indol-3- yl)-4,7-dioxo-3,8-diazadecyl]-1,2-benzenediolato(2−)-O,O′]bis(triphenylphosphine)platinum methyl ester (4) were prepared by condensation of [4-(6-carboxy-4-oxo-3-azahexyl)-1,2-benzenediolato(2−)-O,O′]-bis(triphenylphosphine)platinum (2) with tryptamine and tryptophane methyl ester, respectively. Compounds 3 and 4 were found to have cytotoxic activity against MDA-MB 231, a human breast cancer cell line, albeit in a somewhat lower effectiveness than that of the cis-diamminedichloroplatinum(II) drug.

Dihydrofolate reductase activity in adriamycin and methotrexate sensitive and resistant P388 leukemia cells

P388 murine leukemia cells 18.4-fold more resistant to methotrexate (MTX) than the parent, drug susceptible line, were shown to possess a 1.5-fold higher dihydrofolate reductase (EC1.5.1.3) (DHFR) activity. This is in contrast to a MTX-resistant line, obtained from adriamycin-resistant cells, which is 27.9-fold more resistant to MTX and exhibits a 22.4-fold higher DHFR activity than that of the parent. The susceptibility of the enzyme to inhibition by MTX does not markedly change with the acquired drug resistance of the cell lines studied. Thus MTX-resistant cells obtained from an adriamycin-resistant line acquired resistance due to increased activity of the target enzyme, whereas other mechanisms are responsible for the resistance of cells derived from the adriamycin-sensitive parent.

Resistance of Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability I. Dihydrofolate reductase, thymidylate synthethase and formyltetrahydrofolate synthetase in amethopterin-resistant and -sensitive strains of Pediococcus cerevisiae

Pediococcus cerevisiae/AMr, resistant to amethopterin, possesses a higher dihydrofolate reductase (5, 6, 7, 8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) activity than the parent, a folate-permeable and thus amethopterin-susceptible strain and than the wild-type. The properties of dihydrofolate reductase from the three strains have been compared. Temperature, pH optima, heat stability, as well amethopterin binding did not reveal significant differences between the enzymes from the susceptible and resistant strains. The enzyme from the wild-type was 10 times more sensitive to inhibition by amethopterin and more susceptible to heat denaturation. The apparent Km values for dihydrofolate in enzymes from the three strains were in the range of 4.8--7.2 muM and for NADPH 6.5--8.0 muM. The amethopterin-resistant strain exhibited cross-resistance to trimethoprim and was about 40-fold more resistant to the latter than the sensitive parent and the wild-type. The resistance to trimethoprim appears to be a direct result of the increased dihydrofolate reductase activity. Inhibition of dihydrofolate reductase activity by this drug was similar in the three strains. 10--20 nmol caused 50% inhibition of 0.02 enzyme unit. Trimethoprim was about 10 000 times less effective inhibitor of dihydrofolate reductase than amethopterin. The cell extract of the AMr strain possessed a folate reductase activity three times higher than that of the sensitive strain. The activities of other folate-related enzymes like thymidylate synthetase and 10-formyltetrahydrofolate synthetase (formate: tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) were similar in the three strains studied.

Activity of dihydrofolate reductase in the mollicutesAcholeplasma laidlawii andMycoplasma gallisepticum and their susceptibility to antifolates

Mycoplasma gallisepticum andAcholeplasma laidlawii were found to possess dihydrofolate reductases exhibiting similar specific activities and kinetics, with values in the range of those reported for other microorganisms. The apparent Km values for dihydrofolate in enzymes ofM. gallisepticum andA. laidlawii are 7.95±0.13 and 7.50±0.11 μM, and for NADPNH 8.46±0.25 and 9.32±0.18 μM, respectively.M. gallisepticum is 3300-fold more resistant to methotrexate than isA. laidlawii; concentrations causing 50% inhibition were 200.00 and 0.06 μM, respectively. This is in contrast to almost the same sensitivity to that drug exhibited by the dihydrofolate reductases of both microorganisms.M. gallisepticum is also 3600-fold more resistant to trimethoprim than isA. laidlawii, and the concentrations for 50% inhibition of growth were 1800.0 and 0.5 μM, respectively. The high resistance was found to be due partially to a 130-fold lower affinity of the target enzyme for this antifolate, but another mechanism, presumably impaired transport, must also be involved. This is the first report of dihydrofolate reductase activity in Mollicutes.

Dihydrofolate Reductase in Pediococcus cerevisiae Strains Susceptible and Resistant to Amethopterin

A mutant strain of Pediococcus cerevisiae was obtained which had 1,000-fold greater resistance to amethopterin than the P. cerevisiae/PteGlu strain. The dihydrofolate reductase level of the mutant was about 60-fold higher than that of the parent. The mutant accumulated folate, though less efficiently than its parent.

Resistance of Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability: II. Permeability changes to amethopterin and other folates in the drug-resistant mutant

the accumulation of amethopterin in a Pediococcus cerevisiae strain resistant to this analogue was about 30% of that in P. cerevisiae/PteGlu, the sensitive parent. The uptake in the resistant strain was strictly glucose dependent, whereas in the sensitive parent about 16% accumulation occurred in absence of glucose. The transport in both strains was inhibited by iodoacetate and KF. Amethopterin uptake exhibited saturation kinetics with an apparent Km of 5 muM in P. cerevisiae/AMr and 0.5 muM in P. cerevisiae/PteGlu. The apparent V was 0.2 nmol per min per mg cells (dry weight); the same for both strains. The optimum pH for the uptake of amethopterin by P. cerevisiae/AMr and P. cerevisiae/PteGlu was pH 6.0. Folate and methyltetrahydrofolate competitivity inhibited amethopterin uptake with apparent Ki values of 8 and 0.7 muM, respectively. The uptake of folate exhibited a slightly increased Km value as compared to that of the sensitive strain, whereas the uptake activity velocity was in the same range. Methyltetrahydrofolate accumulated up to about 60-fold higher intracellular concentration than that of the medium, which is a markedly lower accumulation from that in the sensitive strain. The uptake was glucose dependent and inhibited by iodoacetate and KF. The pH optimum for methyltetrahydrofolate uptake in the resistant strain was the same as that in the sensitive parent (pH 5.7--6). In contrast to the increase in the apparent Km value for amethopterin in the resistant strain, the affinity of the carrier for methyltetrahydrofolate was apparently unchanged, whereas the V value was about 16 times lower than that in the sensitive strain. The Ki for amethopterin when added to increasing concentrations of methyltetrahydrofolate was 5.2 muM, a value about the same as that of the Km.

Up-Regulated Transport of Methotrexate and 5-Methyltetrahydrofolate in a Human Breast Cancer Cell Line

In a majority of tumor cells, a high affinity/low capacity transport system mediates the uptake of 5-substituted reduced folates and the analog methotrexate (MTX),1–3 an important chemotherapeutic agent. The relatively low levels of the MTX carrier prompted studies on selecting variants able to up-regulate this system. Such strategy was first introduced by Sirotriak4, who by cultivating L1210 cells in progressively decreasing and growth limiting concentrations of (6R,S)-5-formyltetrahydrofolate (5-HCO-H4PteGlu), selected cells with increased transport capacities for this compound. A similar approach was used to obtain a variant of human erythroleukemia line that exhibited elevated influx.Vmax for MTX and 5-HCO-H4PteGlu relative to the parental cells5. Up-regulation of the reduced folate carrier was also found in human CCRF-CEM leukemia cells adapted to grow on < 1 nM 5-HCO-H4PteGIu,t6 whereas cultivation in the presence of low concentrations of folate induced an increased level of a membrane-associated folate-binding protein7,8.