Leszek Szmigiero

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.

Anthracyclines potentiate activity against murine leukemias L1210 and P388 in vivo and in vitro

Abstract:  The interactions of 2‐chlorodeoxyadenosine (2‐CdA, cladribine) and three anthracyclines: doxorubicin (DOX), idarubicin (IDA) and mitoxantrone (MIT) were evaluated on murine leukemias P388 and L1210. Prolongation of survival time of animals receiving drugs in combination compared to mice treated with drugs in monotherapy was tested. We have also evaluated interactions of the cytostatics on murine leukemias in vitro by measuring their inhibitory effects on P388 and L1210 cell proliferation. We have observed a synergistic effect of MIT and IDA in combination with 2‐CdA on P388 leukemia resulting in an increase of life span (ILS)=226% in case of MIT+2‐CdA and ILS=126% in the case of IDA+2‐CdA, whereas 2‐CdA used as a sole drug resulted in an ILS=47%. The survival time of animals inoculated with P388 leukemic cells and treated with DOX+ 2‐CdA was similar to ILS gained by DOX monotherapy (178% and 200% respectively). The mice bearing L1210 leukemia receiving combined chemotherapy lived significantly longer than the animals on single agent regimens. The animals treated with schedule 2‐CdA+MIT lived significantly longer (P=0.004) as compared to the groups receiving drugs in monotherapy (ILS of 2‐CdA+MIT group=60%, ILS of MIT group 33%, and 2‐CdA group 33%). Finally, combination of DOX or IDA with 2‐CdA resulted in ILS =73% (2‐CdA+DOX regimen), and ILS=60% in case of 2‐CdA+IDA regimen, which is significantly higher than ILS gained on monotherapy schedules. In vitro tests revealed that all tested anthracyclines enhance the antiproliferative activity of 2‐CdA against L1210 and P388 leukemic cells (P<0.05).

Effect of moderate-intensity exercise on oxidative stress indices in metabolically healthy obese and metabolically unhealthy obese phenotypes in postmenopausal women: a pilot study.

Objective:The aim of this work was to determine whether the level of oxidative stress induced by moderate-intensity exercise depends on obesity phenotypes: metabolically healthy but obese (MHO) and non-metabolically healthy obese (at-risk obesity; non-MHO). Methods:We performed the study on 161 postmenopausal women aged 50 to 60 years. A metabolically healthy nonobese (MH-NO) group (n = 73), an MHO group (n = 27), and a non-MHO group (n = 61) exercised on a cycloergometer for 30 minutes at 50% of their peak oxygen consumption and were evaluated for oxidative status by determination of thiobarbituric acid-reactive substances (TBARS) and serum antioxidant activity (AS). Results:No difference was found in AS between the MH-NO group and the MHO group. The AS of the non-MHO group was significantly lower than that of the MH-NO group (P < 0.05) and that of the MHO group (P = 0.011). The insulin resistance index homeostasis model assessment was the only biochemical parameter that correlated with AS. After exercise, a significant increase in the TBARS concentration in all tested groups of women was observed, but differences in the increment of TBARS level between groups were not found. Conclusions:Antioxidant status in obese postmenopausal women depends on obesity phenotypes and is higher for women with the MHO than those with the non-MHO phenotype. Independently of obesity phenotype, obese postmenopausal women exposed to moderate-intensity exercise seem to be at similar risk for oxidative stress compared with their nonobese counterparts. We suggest that homeostasis model assessment be taken into account when planning physical exercise for obese people.

Synthesis, single-crystal and solution structure analysis and in vitro cytotoxic activity of two novel complexes of ruthenium(II) with in situ formed flavanone-based ligands.

Synthesis, structure and properties of two new flavanone complexes of Ru(ii) are described. The new complexes form during the reaction of ruthenium(iii) chloride with 3-aminoflavone (3-af) dissolved in an aliphatic alcohol. The formed products depend on the alcohol used and were found to be: cis-dichloridobis(3-imino-2-methoxyflavanone)ruthenium(ii)·3H(2)O (1) from a methanolic solution and cis-dichloridobis(3-imino-2-ethoxyflavanone)ruthenium(ii)·2H(2)O (2) from an ethanolic solution, in which the original ligand 3-af had been converted by dehydrogenative alcoholysis to an entirely new ligand. This paper presents the X-ray structure and detailed (1)H-NMR analysis of both new compounds, as well as the study of their antiproliferative activity. The coordination of Ru(ii) is octahedral with [RuCl(2)N(2)O(2)] chromophores, having trans chlorides and common Ru-L distances. Both 1 and 2 are highly cytotoxic towards the cisplatin resistant EJ and L1210 cell lines, and both complexes are as active as cisplatin in the sensitive cell lines. They display the ability to overcome cisplatin resistance in the drug resistant sub-lines EJcisR and L1210R. The present evidence suggests that the mechanism of biological activity may be different for these ruthenium compounds compared to cisplatin.

Some properties of the irreversible complexes of nitracrine (Ledakrin, C-283) with polynucleotides

In the presence of sulfhydryl compounds an anticancer drug nitracrine (NA), 1-nitro-9-aminoalkylacridine derivative forms strong, probably covalent complexes with DNA. It has been found that it binds with similar efficiency to RNA and DNA exhibiting a certain preference for single-stranded structure. At NA/polynucleotide ratio of 0.15 and nucleic acids concentration 100 microgram/ml the numbers of drug molecules bound per 10(3) nucleotides were about 10--13 for native calf thymus DNA, 19--28 for denatured DNA and 23--36 for RNA. Some base specificity to guanine is observed both in polydeoxyribo- and polyribonucleotides. The complexes of NA with DNA and double-stranded synthetic polynucleotides exhibit decreased transcriptional template activity in bacterial RNA synthesis in vitro system except poly(A) synthesis on poly(dA) x poly(dT) which is insensitive to the drug. The drug binding in vitro leads to cross-link formation in DNA as shown by means of ultraviolet spectrophotometry and hydroxylapatite chromatography of heat-denatured NA-DNA complexes. The amount of the double bonds introduced by the drug is however relatively low as compared with cross-linking of irradiated 8-methoxy-psoralen-DNA (MOP-DNA) complexes.

Proapoptotic activity in vitro of two novel ruthenium(II) complexes with flavanone-based ligands that overcome cisplatin resistance in human bladder carcinoma cells

In this study we examined their proapoptotic activity of cis-dichloridobis(3-imino-2-methoxyflavanone)ruthenium(II)3H(2)O (1) and cis-dichloridobis(3-imino-2-ethoxyflavanone)ruthenium(II)2H(2)O (2) towards human bladder carcinoma cell line EJ and its cisplatin resistant subline EJcisR. On the basis of the experiments we carried out, it may be concluded, that: CDDP (cis-diamminedichloridoplatinum) resistance of EJcisR cells is probably based on partial loss of apoptotic pathway activating caspase-8 and increased resistance to DNA strand breaks and/or alkali-labile sites. Increased glutathione levels, as well as activity of P-gp transporter seems to be not relevant in this case. The proapoptotic activity of the ruthenium compounds is higher than that of cisplatin. Higher proapoptotic activity of 1 and 2 when compared to CDDP may be due to the presence of large, lipophilic flavanone-based ligands that may facilitate their trans-membrane transport and their redox activity. 1 and 2 induce apoptosis apparently in more than one way. Although caspase-8 activation and DNA strand breaks and/or alkali-labile sites are caused by the compounds, their ability to cause the oxidative stress in the cells may also participate in apoptosis induction.

Post-exercise oxidative stress and obesity in postmenopausal women: The role of beta3-adrenergic receptor polymorphism

Aim. Some studies indicate that the Trp64Arg polymorphism in the gene encoding the β3-adrenergic receptor (ADRB3) is associated with obesity, insulin resistance and earlier onset of type 2 diabetes mellitus. The aim of the present study was to evaluate the frequency of this polymorphism and its relationship with obesity and oxidative stress in postmenopausal women. Material and methods. We performed the study on 200 women, aged 50–60 years. Estimation of anthropometric parameters and total body fat, android and gynoid fat deposits was carried out using dual-energy X-ray absorptiometry. Oxidative stress was estimated by measurement of thiobarbituric acid-reactive substances (TBARS) in serum. Blood for analysis was collected before, directly after and 6 h after a 30-min physical test on a cycle ergometer. ADRB3 genotyping was performed by polymerase chain reaction. Results. The frequency of Trp64/Arg64 genotype in the investigated population was 12%, and of Trp64/Trp64 was 87%. The Arg64/Arg64 genotype was present in only 1% of women. Women bearing the Trp64/Arg64 genotype did not differ in any measured anthropometric parameters from women bearing the Trp64/Trp64 genotype. Moreover, genotype had no influence on oxidative stress parameters. Likewise, in both groups, mean plasma level of TBARS was increased significantly (p < 0.05) directly after the endurance test and remained elevated 6 h after the test. Conclusions. The Trp64Arg polymorphism of ADRB3 seems to not be related to obesity in postmenopausal women. Moreover, the Trp64Arg polymorphism has no influence on oxidative stress intensification after standardized physical effort in postmenopausal women.

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.

ADRB3 and PPARγ2 gene polymorphisms and their association with cardiovascular disease risk in postmenopausal women

Abstract Objectives The contribution of heritability to the development of cardiovascular disease (CVD) is of interest as the identification of genes enhancing the susceptibility of individuals to CVD may help the design of clinical interventions optimized for the individuals genome. Methods We studied the associations of polymorphism of ADRB3 and PPARγ2 genes with obesity indices, unfavorable lipid profile parameters and insulin resistance index HOMA in 343 postmenopausal women. Results No association was found between tested polymorphisms and CVD risk factors such as total cholesterol ≥ 5.0 mmol/l, high density lipoprotein cholesterol < 1.2 mmol/l, low density lipoprotein cholesterol > 3.0 mmol/l and triacylglycerols > 1.7 mmol/l. The presence of arterial hypertension and HOMA value ≥ 1.95 were also not related to these polymorphisms. A significant association between PPARγ2 gene polymorphism and total body fat mass (odds ratio = 1.90 at p = 0.037) as well as android fat deposit mass (odds ratio = 1.82 at p = 0.048) was found. Conclusions CVD risk factors in postmenopausal women are not directly associated with the polymorphisms of PPARγ2 and ADRB3 genes. We suggest that some indirect link between PPARγ2 gene polymorphism and susceptibility of postmenopausal women to CVD may exist. This suggestion is based on our finding that high total body fat mass and high android fat deposits are associated with the presence of the Pro12Ala allele of the PPARγ2 gene.