Kazimierz Studzian

Cytotoxicity, cellular uptake and DNA damage by daunorubicin and its new analogues with modified daunosamine moiety

Daunorubicin (DRB) and its two analogues containing a trisubstituted amidino group at the C-3′ position of the daunosamine moiety have been compared regarding their cytotoxic activity, cellular uptake, subcellular localization and DNA damaging properties. An analogue containing in the amidino group a morpholine moiety (DRBM) as well as an analogue with a hexamethyleneimine moiety (DRBH), tested against cultured L1210 cells, exhibited lower cytotoxicity then DRB. The decrease of cytotoxic activity was not related to cellular uptake and subcellular localization of drugs. Although all tested drugs were active in the induction of DNA breaks and DNA–protein crosslinks, they differed in the mechanism of induction of DNA lesions. DRB produced DNA breaks mediated solely by topoisomerase II, whereas DRBM and DRBH induced two types of DNA breaks by two separate processes. The first is related to the inhibition of topoisomerase II and the second presumably reflects a covalent binding of drug metabolites to DNA. It is hypothesized that the replacement of the primary amino group (–NH2) at the C-3′ position of the daunosamine moiety by a trisubstituted amidino group (–N=CH–NRR) may be a route to the synthesis of anthracycline derivatives with enhanced ability to form covalent adducts to DNA.

H2O2 as a DNA fragmenting agent in the alkaline elution interstrand crosslinking and DNA-protein crosslinking assays

A method for DNA fragmentation by H2O2 in the DNA alkaline elution procedure is described. Treatment of cell suspensions for 1 h with 100 microM H2O2 or 5 mM H2O2 at 0-1 degree C resulted in DNA breakage equivalent to doses of 300 and 3000 rad of gamma-rays, respectively. The elution profiles were reproducible and H2O2 was used for measurements of interstrand crosslinks and DNA-protein crosslinks induced in HeLa cells by mitomycin C, cis-diamminedichloroplatinum(II), and trans-diamminedichloroplatinum(II). The comparison of data obtained with the use of H2O2 and gamma-rays has shown that both methods have similar sensitivity and reproducibility.

Functional comparison of two evolutionary conserved insect neurokinin-like receptors

Tachykinins are multifunctional neuropeptides that have been identified in vertebrates as well as invertebrates. The C-terminal FXGXRa-motif constitutes the consensus active core region of invertebrate tachykinins. In Drosophila, two putative G protein-coupled tachykinin receptors have been cloned: DTKR and NKD. This study focuses on the functional characterization of DTKR, the Drosophila ortholog of the stable flys tachykinin receptor (STKR). Tachykinins containing an alanine residue instead of the highly conserved glycine (FXAXRa) display partial agonism on STKR-mediated Ca(2+)-responses, but not on cAMP-responses. STKR therefore seems to differentiate between a number of tachykinins. Gly- and Ala-containing tachykinins are both encoded in the Drosophila tachykinin precursor, thus raising the question of whether DTKR can also distinguish between these two tachykinin types. DTKR was activated by all Drosophila tachykinins and inhibited by tachykinin antagonists. Ala-containing analogs did not produce the remarkable activation behavior previously observed with STKR, suggesting different mechanisms of discerning ligands and/or activating effector pathways for STKR and DTKR.

A convenient synthesis and cytotoxic evaluation of β-aryl-α-methylidene-γ-lactones and β-aryl-α-methylidene-γ-lactams

3-Aryl-2-diethoxyphosphoryl-4-nitrohexanoates 8, obtained by Michael addition of ethyl diethoxyphosphorylacetate 6 to 1-aryl-2-nitro-1-butenes 7, were utilized as convenient common intermediates in the synthesis of β-aryl-γ-ethyl-α-methylidene-γ-lactones 17 and β-aryl-γ-ethyl-α-methylidene-γ-lactams 21. Transformation of the nitro functionality in 8 into a hydroxyl or amino group and cyclization yielded lactones 16 or lactams 19, which were used in Horner–Wadsworth–Emmons olefination of formaldehyde to give target compounds in good yields. Cytotoxicity of these compounds was evaluated in vitro against mouse leukemia cell line L-1210 and two human leukemia cell lines, HL-60 and NALM-6. Two of the obtained compounds 17b,c with 4-bromophenyl and 4-methylphenyl substituents in the β position proved to be very potent against all three cell lines with IC50 values lower than 6 μM.

Synthesis and cytotoxic evaluation of β-alkyl or β-aryl-δ-methyl-α-methylene-δ-lactones. Comparison with the corresponding γ-lactones

We present a simple and general strategy for the synthesis of beta,delta-disubstituted-alpha-methylene-delta-lactones starting from easily available tert-butyl 2-(diethoxyphosphoryl)alk-2-enoates. The elaborated synthetic protocol includes pyrrolidine-catalyzed Michael addition of acetone, diastereoselective reduction of the carbonyl group, lactonization and finally the Horner-Wadsworth-Emmons reaction with formaldehyde. All alpha-methylene-delta-lactones were evaluated in vitro against mouse leukemia cell line L-1210 and two human leukemia cell lines HL-60 and NALM-6. Comparison of cytotoxic activity with corresponding alpha-methylene-gamma-lactones is also discussed.

Comparison of antagonist activity of spantide family at human neurokinin receptors measured by aequorin luminescence-based functional calcium assay.

Neurokinin receptors (NK1, NK2, NK3) are G-protein-coupled receptors, which upon activation by a peptide agonist induce a transient increase in the concentration of intracellular calcium. The functional assay based on aequorin-derived luminescence triggered by receptor-mediated changes in Ca2+ levels was used to compare the effect of spantides I-III on SP-, NKA- and NKB-stimulated NK1, NK2 and NK3 receptors, respectively. Recombinant cell lines expressing neurokinin receptors and apoaequorin were used in the study. The obtained results indicate that all three spantides acted as competitive antagonists at the NK1 and NK2 receptors and inhibited agonist-induced calcium responses. The rank order of antagonism at the NK1 receptor was spantide II>spantide III>spantide I and at the NK2 receptor was spantide III>spantide II>spantide I. All three spantides failed to antagonize NKB-induced calcium responses at the NK3 receptor.

Cytotoxic activity and chemical reactivity of cis-platinum(II) and trans-palladium(II) complexes with diethyl (pyridinylmethyl)phosphates.

A series of square-planar platinum(II) and palladium(II) complexes of the formula cis-[PtCl2L2] and trans-[PdCl2L2] [L stands for diethyl (pyridin-2-ylmethyl)phosphate (2-pmOpe) or diethyl (pyridin-3-ylmethyl)phosphate (3-pmOpe) or diethyl (pyridin-4-ylmethyl)phosphate (4-pmOpe)] have been synthesized and tested in vitro for their cytotoxicity against mouse leukemia L1210 cells. The results indicated that the cis-platinum complexes showed superior activity than trans-palladium complexes, but lower in comparison to cisplatin. The chemical reactivity of the tested complexes has been determined in an in vitro NBP test. The platinum complexes exhibited very high chemical reactivity in NBP test, higher than cisplatin. The results showed no correlation between cytotoxicity and chemical reactivity for platinum complexes. Two platinum(II) complexes {cis-[PtCl2(2-pmOpe)2], cis-[PtCl2(3-pmOpe)2]} have been synthesized and characterized by IR, 1H NMR, 31P NMR, and elemental analysis.

Effects of anticancer drugs on transcription in vitro.

Abstract The effects of DNA interacting drugs on: (1) total RNA synthesis catalyzed by E.coli and T7 RNA polymerase; (2) synthesis of the initiating dinucleotide (pppApU) by E .coli RNA polymerase (“abortive initiation“); (3) elongation of RNA chains synthesized by T7 RNA polymerase on pT7-7 plasmid DNA bearing T7 RNA polymerase promoter ϕ 10 with human Cu/Zn superoxide dismutase coding sequence, (4) interaction of transcription factor Sp1 and its binding site were studied. Intercalating ligands which form quickly dissociating complexes with DNA (anthracyclines, proflavine, ethidium bromide) are compared with the slowly dissociating drug of d(G · C ) specificity (actinomycin D), the non-intercalating, d(A · T ) specific pyrrole antibiotics (netropsin and distamycin A) and covalently binding to DNA 1-nitroacridine derivative (nitracrine). The obtained results indicate that rapidly dissociating ligands, proflavine and ethidium bromide, inhibit total RNA synthesis in vitro and the abortive initiation to a similar extent while they do not induce discrete elongation stops of RNA polymerase. Actinomycin D and nitracrine exhibit a high inhibitory effect on total RNA synthesis and induce stops of RNA polymerase while not affecting abortive initiation. Pyrrole antibiotics primarily inhibit the initiation, while no elongation stops are induced. Actinomycin D inhibits complex formation between nuclear proteins and the Sp1 binding site. Netropsin, ethidium bromide, proflavine and other intercalating acridines do not affect Sp1 binding. The results indicate that the effects primarily depend on sequence specificity and secondarily on the dissociation rate of ligands from their complexes with DNA.

DNA damage and cytotoxicity of nitracrine in cultured HeLa cells

The effects of nitracrine (1-nitro-9-(3,3-N,N-dimethylaminopropylamino)acridine on DNA of cultured HeLa cells were studied. DNA strand breakage and interstrand cross-linking as well as DNA-protein cross-linking were measured by means of an alkaline elution technique and were compared with the cytotoxic effect of the drug. Interstrand cross-links were not detectable in the concentration range that inhibited cell growth up to 99%. DNA single-strand breaks were found when cells were treated with highly cytotoxic doses of the drug. DNA breakage was not reparable and exhibited a tendency to increase during incubation after drug removal. The only chromatin lesion induced by sublethal doses of nitracrine were DNA-protein cross-links which persisted for 24 h after drug treatment. It is concluded that DNA breaks represent degraded DNA from dying cells, whereas DNA-protein cross-links are specific cellular lesions, which may be responsible for the cell-killing effect of nitracrine.

Interactions of Novel Morpholine and Hexamethylene Derivatives of Anthracycline Antibiotics with DNA

Abstract Doxorubicin (DOX), daunorubicin (DRB), epidoxorubicin (EDOX) and their analogues with a 3′-NH2 group in daunosamine form a covalent bond with a 2-NH2 group of guanine via a methylene group from formaldehyde (CH2O). It is assumed that a Schiff base type intermediate is formed between CH2O and the 3′-NH2 group in the reaction. This reaction is supposed to occur in the cell. New analogues of anthracyclines with formamidine functionality bound to C-3′ of daunosamine and containing the bulky morpholine (DRBM, DOXM and EDOXM) or hexamethyleneimine rings attached are studied in our laboratory. These substituents decrease the association of the drugs to DNA and potentially hinder the formation of Schiff base-intermediates. Our experiments indicate that the formation of the covalent complexes by DRB, DOX and EDOX under these conditions is confirmed by a high enhancement (17-40x) of the inhibition of overall RNA synthesis by E. coli RNA polymerase on T7 DNA. DRBM and DOXM exhibit a lower enhancement of the inhibition by CH2O (7-13x). The other analogues show a 1.6-3x increase of inhibition. Hence, their covalent binding is lower than that of the parent compounds. These conclusions are confirmed by spectrophotometric estimations following removal of non-covalently associated drugs. Electrophoretic analysis of drug-DNA complexes formed in the presence of CH2O indicates that DRBM and DOXM as their parent compounds induce labile cross-links in DNA. Comparison of the results obtained at the subcellular level with cytotoxicity estimations indicates that there is a correlation between cytotoxicity of the anthracyclines on L1210 cells and transcriptional template activity of drug-DNA complexes formed in the presence of CH2O (r = 0.64; n = 9). These data confirm a notion that covalent attachment of anthracyclines to DNA is an essential event leading to cytotoxicity.