Renáta Minorics

Antiproliferative effects of some novel synthetic solanidine analogs on HL-60 human leukemia cells in vitro.

There is increasing evidence of the direct antiproliferative effects of various steroidal structures, including cardenolides, steroidal alkaloids and sexual hormones. The aim of the present study was to characterize the antiproliferative effects of three synthetic solanidine analogs (1-3) on HL-60 human leukemia cells. The three compounds exerted similar cytostatic effects (IC(50) values: 1.27-2.94 μM after a 72-h exposure) and the most effective (2) was selected for further investigations. Incubation with compound 2 resulted in a marked chromatin condensation followed by a gradual increase in cell membrane permeability detected by Hoechst dye 33258-propidium iodide double staining. A flow cytometric analysis revealed a marked decrease in the G1 phase and substantial increases in the S and G2/M phases after 24-h incubation, while after 48 h the proportion of cells in the subG1 phase was increased significantly with a concomitant decrease in cells in the G1 and G2/M phases. Compound 2 at 6.0 μM significantly decreased the activity of ribonucleotide reductase and proved to be a potent antioxidant in the lipid peroxidation and DPPH assays (IC(50) values: 2.0 and 13.1 μM, respectively). The antiproliferative effect of the test compound on the non-cancerous human lung fibroblast cell line (MRC-5) was significantly weaker than that on the leukemia cells. These results lead to the conclusion that compound 2 induces a marked disturbance in the cell cycle, which is, at least partially, a consequence of the inhibition of DNA synthesis.

Synthesis and receptor-binding examinations of the normal and 13-epi-D-homoestrones and their 3-methyl ethers.

An effective epimerization of the normal estrone 3-methyl and 3-benzyl ethers by using o-phenylenediamine and AcOH made the possibility for facile entry into the 13alpha-estrone series. Combination of this synthetic methodology with an isolation step carried out by means of the Girard-P reagent, the corresponding ethers of 13-epi-estrone were obtained in excellent yields. The 3-hydroxy and 3-methoxy D-homoestrone derivatives in both the normal and the 13alpha-estrone series were then synthesized and tested in vitro in a radioligand-binding assay. The estrogen receptor recognizes these compounds, but their relative binding affinities (RBAs) are lower than that of the reference compound 3,17beta-estradiol. The progesterone receptor-binding affinities of the four D-homo derivatives were also tested showing low values for 13alpha-D-homoestrone and its 3-methyl ether. Pharmacologically, these 13alpha-D-homoestrone derivatives are estrogen receptor-selective molecules.

Analysis of the role of central and peripheral α2-adrenoceptor subtypes in gastric mucosal defense in the rat

The present study confirmed our previous assumption on the crucial role of central alpha2B-like adrenoceptor subtype in gastric mucosal defense. It was found that beside clonidine, rilmenidine, an alpha2/imidazoline receptor agonist and ST-91, an alpha2B-adrenoceptor preferring agonist inhibited the mucosal lesions induced by ethanol given intracerebroventricularly (i.c.v.). The ED50 values for clonidine, rilmenidine and ST-91 are 0.2, 0.01 and 16 nmol/rat i.c.v., respectively. The effect was reversed by the intracerebroventricularly injected alpha2B/2C-adrenoceptor antagonists prazosin and ARC-239, indicating the potential involvement of central alpha2B/2C-adrenoceptor subtype in the protective action. The gastroprotective effect of adrenoceptor stimulants was reversed by bilateral cervical vagotomy, suggesting that vagal nerve is likely to convey the central action to the periphery. In gastric mucosa both nitric oxide and prostaglandins may mediate the centrally-induced effect, since both indomethacin and N(G)-nitro-L-arginine reversed the protective effect of alpha2-adrenergic stimulants. Though expression of mRNA of alpha2B-, as well as alpha2A- and alpha2C-adrenoceptor subtypes was demonstrated in gastric mucosa of the rat, the hydrophilic ST-91, given peripherally (orally, subcutaneously), failed to exert mucosal protection, in contrast with clonidine and rilmenidine which were also effective. Consequently, while peripheral alpha2B-adrenoceptors are not likely to be involved in gastric mucosal protection, activation of central alpha2B-like adrenoceptor subtype may initiate a chain of events, which result in a vagal dependent gastroprotective action.

Antiproliferative effect of normal and 13-epi-d-homoestrone and their 3-methyl ethers on human reproductive cancer cell lines

The possibility of the therapeutic use of estrogens emerged following the recognition that certain estradiol analogs, and particularly metabolites (e.g. the A-ring metabolite 2-hydroxyestrone, etc.) inhibit the differentiation of diverse tumor cell lines. Until recently, despite the investigation of numerous synthetic d-ring-substituted estrone derivatives, no analysis had been published on the effects of D-ring expansion of estrone on its tumor-suppressing activity. The aim of the present study was to characterize the antiproliferative effects of normal and 13-epi-D-homoestrone and their 3-methyl ethers (1-4) on human reproductive cancer cell lines. The antitumor activities of the two epimer pairs on HeLa, MCF-7 and Ishikawa cells were determined. Normal D-homoestrone exerted the greatest cytostatic effect on HeLa cells (IC(50)=5.5 μM) and was subjected to further investigations to elucidate its mechanism of action on apoptosis induction. Morphological changes detected by Hoechst 33258-propidium iodide double staining, the cell cycle arrest at phase G2/M and the subsequent increase in the proportion of the subG1 fraction determined by flow cytometric analysis and the significant increase in the activity of caspase-3 confirmed the induction of apoptosis in HeLa cells treated with D-homoestrone. D-Homoestrone was also tested on a non-cancerous human lung fibroblast cell line (MRC-5) to determine its selective toxicity. The concentration in which it inhibited cell proliferation by 50% was at least six times higher for the fibroblast cells than for cervical cancer cells. No significant in vivo estrogenic activity was observed as concerns the uterus weight of gonadectomized rats after a 7-day treatment with normal D-homoestrone. These results led to the conclusion that normal D-homoestrone is a novel antitumor compound with a similar activity on HeLa cells as that of the reference agent cisplatin, but its selectivity toward non-cancerous cells is significantly higher than that of cisplatin. It may be considered to be a basic lead molecule for the preclinical development of potential anticancer agents.

Different roles of α2-adrenoceptor subtypes in non-pregnant and late-pregnant uterine contractility in vitro in the rat

The roles of the alpha2-adrenoceptor (alpha2-AR) subtypes (alpha2A-, alpha2B- and alpha2C-AR) in uterine contractility have not been investigated. The aims of this study were to identify these receptors in the non-pregnant and the late-pregnant rat myometrium and to determine their roles in contractions. We found that the myometrial alpha2-AR subtypes are involved differently in the control of late-pregnant contractions, while they have no influence on the contractions of the non-pregnant myometrium. The myometrial expressions of the alpha2-AR subtypes were determined by RT-PCR and Western blotting techniques. In vitro contractions were stimulated with noradrenaline, and its effect was modified with the selective antagonists BRL 44408 (alpha2A), ARC 239 (alpha2B/C) and spiroxatrine (alpha2C). cAMP production was followed by noradrenaline stimulation in the presence of isobutylmethylxanthine and forskolin, and alterations induced in it by the antagonists were determined with an Enzyme Immunoassay Kit. The most effective antagonist was tested on labour-induced uteri in vitro. All the alpha2-AR subtypes were identified in both non-pregnant and pregnant uteri. Noradrenaline was not able to contract the non-pregnant tissue in the presence of propranolol and doxazosin, while its contracting effect in the pregnant uteri was enhanced by BRL 44408, spiroxatrine and the combination BRL 44408+spiroxatrine. ARC 239 exerted a strong inhibitory effect on noradrenaline-stimulated contractions. The increasing and the decreasing effects of the compounds were confirmed by the changes in the intracellular cAMP levels. The effect of ARC 239 on the labour-induced myometrium was similar to that on the 22-day-pregnant myometrium. The stimulation of alpha2-ARs does not evoke contractions in the non-pregnant uterus. The alpha2A- and alpha2C-ARs mediate decreases, while the alpha2B-AR mediates an increase in the contractions in the 22-day-pregnant myometrium. These differences may offer new targets for drugs against premature contractions in pregnancy.

Synthesis and receptor-binding examination of 16-hydroxymethyl-3,17-estradiol stereoisomers

The four 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers were synthesized and tested in a radioligand-binding assay. The estrogen receptor recognizes these compounds, but their relative binding affinities are lower than 2.0% relative to that of the reference molecule estra-1,3,5(10)-triene-3,17beta-diol. The affinities of the tested compounds for the androgen and progesterone receptors are very low (K(i)> 100 microm and 1 microM, respectively). The prepared 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers are therefore estrogen receptor-selective molecules.

Anticancer and Multidrug Resistance-Reversal Effects of Solanidine Analogs Synthetized from Pregnadienolone Acetate

A set of solanidine analogs with antiproliferative properties were recently synthetized from pregnadienolone acetate, which occurs in Nature. The aim of the present study was an in vitro characterization of their antiproliferative action and an investigation of their multidrug resistance-reversal activity on cancer cells. Six of the compounds elicited the accumulation of a hypodiploid population of HeLa cells, indicating their apoptosis-inducing character, and another one caused cell cycle arrest at the G2/M phase. The most effective agents inhibited the activity of topoisomerase I, as evidenced by plasmid supercoil relaxation assays. One of the most potent analogs down-regulated the expression of cell-cycle related genes at the mRNA level, including tumor necrosis factor alpha and S-phase kinase-associated protein 2, and induced growth arrest and DNA damage protein 45 alpha. Some of the investigated compounds inhibited the ABCB1 transporter and caused rhodamine-123 accumulation in murine lymphoma cells transfected by human MDR1 gene, expressing the efflux pump (L5178). One of the most active agents in this aspect potentiated the antiproliferative action of doxorubicin without substantial intrinsic cytostatic capacity. The current results indicate that the modified solanidine skeleton is a suitable substrate for the rational design and synthesis of further innovative drug candidates with anticancer activities.

Synthesis of trans-16-triazolyl-13α-methyl-17-estradiol diastereomers and the effects of structural modifications on their in vitro antiproliferative activities.

Novel 16-triazoles in the 13α-estrone series were synthesized via Cu(I)-catalyzed azide-alkyne cycloaddition of the two diastereomeric (on C-16 and on C-17) 16-azido-13α-estra-1,3,5(10)-trien-17-ol 3-benzyl ethers with substituted phenylacetylenes. The new heterocyclic derivatives were evaluated in vitro by means of MTT assays for antiproliferative activity against a panel of human adherent cancer cell lines (HeLa, MCF-7, A431, A2780, T47D, MDA-MB-231 and MDA-MB-361). The inversion of the configurations at C-16 and C-17 selectively affected the growth-inhibitory properties of the tested compounds. The 16β,17α isomers generally proved to be potent on all cell lines, with IC50 values comparable to those of the reference agent cisplatin. Change of the substitution pattern of the phenyl group of the acetylene led to great differences in antiproliferative properties. Exclusively the p-phenyl-substituted triazoles exerted high cytostatic effects. One of the most potent compounds activated caspase-3 and caspase-9 without influencing caspase-8, confirming the induction of apoptosis via the intrinsic pathway.

Synthesis and biological evaluation of 13α-estrone derivatives as potential antiproliferative agents.

13α-Estrone derivatives containing various substituents on C-3 and C-17 were synthesized, and evaluated by means of MTT assays for in vitro antiproliferative activity against a panel of human adherent cancer cell lines (HeLa, MCF-7, A2780 and A431). Compounds with N-benzyltriazolylmethoxy moieties on C-3 proved to be more potent than their 3-hydroxy or 3-ether counterparts. Some triazoles exerted substantial cytostatic effects against particular tumor cell lines, with submicromolar IC50 values.

Syntheses and antiproliferative effects of d-homo- and d-secoestrones

Substituted and/or heterocyclic d-homoestrone derivatives were synthetized via the intramolecular cyclization of a δ-alkenyl-d-secoaldehyde, -d-secoalcohol or -d-secocarboxylic acid of estrone 3-benzyl ether. The d-secoalcohol was modified at three sites in the molecule. The in vitro antiproliferative activities of the new d-homo- and d-secoestrone derivatives were determined on HeLa, MCF-7, A431 and A2780 cells through use of MTT assay. d-Homoalcohols 3 and 5 displayed cell line-selective cytostatic effects against ovarian and cervical cell lines, respectively. Two d-secoestrones (6 and 12c) proved to be effective, with IC50 values comparable with those of the reference agent cisplatin. A selected compound (6) was tested by tubulin polymerization assay and its cancer specificity was additionally determined by using noncancerous human fibroblast cells.