Potentiation by caffeine of cytogenetic damage induced by steroidal derivatives in human lymphocytes in vitro.

We studied the effects of three newly synthesized steroidal derivatives of nitrogen mustards, alone or in combination with caffeine, on sister chromatid exchange (SCE) frequencies and on human lymphocyte proliferation kinetics. The agents have as alkylator functionalities either P-N,N-bis(2-chloroethyl)aminophenyl-buturate (CHL) or P-N,N-bis(2-chloroethyl)aminophenyl-acetate (PHE), esterified with a modified steroidal nucleus. An enhancement of SCE frequency was seen with compounds which contain either PHE or CHL as alkylators and are esterified with a steroidal nucleus having added a cholestene group in the 17-position of the D-ring. The exocyclic insertion of an -NHCO- group in the D-ring of the steroidal nucleus esterified with PHE (amide ester of PHE) gave a compound showing increased SCE frequency. Enhanced cytogenetic damage was observed when lymphocytes were exposed in vitro to caffeine. The compounds, alone or in combination with caffeine, caused a concentration-dependent increase in SCE frequencies and cell division delays, and caffeine was found to act synergistically with the steroidal alkylators.

Cytogenetic and antineoplastic effects by newly synthesised steroidal alkylators in lymphocytic leukaemia P388 cells in vivo.

New compounds with potential antitumour activity were synthesised by combining nitrogen mustard with the steroidal skeleton, in an effort to improve specificity and at the same time reduce systemic toxicity. The steroidal part is aimed to serve as a biological platform enabling the alkylating moiety to approach its site of action by altering its physicochemical properties. The purpose of the present investigation was to evaluate these compounds for anti-neoplastic activity. The compounds tested have as alkylators either para-NN-bis(2-chloroethyl)-aminophenyl-butyrate (CHL) or para-N,N-bis(2-chloroethyl)-aminophenyl-acetate (PHE) esterified with a differently modified steroidal nucleus. The eight newly synthesised compounds were compared on a molar basis with respect to their ability to induce sister chromatid exchanges (SCEs) and to modify proliferation rate indices (PRI) in lymphocytic leukaemia P388 cells in mice in vivo. The life span of BDF1 mice inoculated with P388 leukaemia cells was also estimated (anti-leukaemic activity). The compounds that were effective in inducing cytogenetic effects in lymphocytic leukaemia cells in vivo were also effective in inducing antineoplastic effects in BDF1 mice inoculated with P388 leukaemia cells. These results suggest that the in vivo cytogenetic effects in conjunction with the antineoplastic activity of modified steroidal alkylators depend on the configuration of the whole molecule and on the appropriate combination of the alkylator with the steroidal molecule: a pronounced cytogenetic and anti-neoplastic action was demonstrated by the compounds that contain either PHE or CHL as alkylators and are esterified with either a steroidal nucleus that carries a cholesten group in the 17 position of the D-ring, or with a steroidal nucleus having an exocyclic NHCO-group in the D-ring. In contrast, a ketone group or an NHCO-group in the D-ring inserted endocyclically in the steroidal nucleus esterified with either CHL or PHE failed to induce cytogenetic or anti-neoplastic effects.

Comparative study of genetic activity of chlorambucil's active metabolite steroidal esters: the role of steroidal skeleton on aneugenic potential.

p-N,N-bis(2-chloroethyl)aminophenylacetic acid (PHE), a nitrogen mustard analogue and chlorambucil's active metabolite used as chemotherapeutic agent, has been shown that, in addition to its clastogenic activity, induces chromosome delay. In the present study an efford has been made (a) to investigate if the steroidal analogues of PHE (EA-92, EA-97, AK-333, AK-409 and AK-433) exert the same genetic activity as the parent compound, (b) to further analyze the aneugenic activity of nitrogen mustard analogues, (c) to investigate the mechanism by which they exert aneugenic potential and (d) to correlate the genetic activity with chemical structure. For this purpose the Cytokinesis Block Micronucleus (CBMN) assay was conducted in human lymphocytes in vitro and the micronucleus (MN) frequency was determined to investigate their genetic activity. The mechanism of micronucleation was determined in combination with Fluorescence In Situ Hybridization (FISH) using pancentromeric DNA probe. Since one of the mechanisms that chemicals cause aneuploidy is through alterations in the mitotic spindle, we also investigated the effect of the above compounds on the integrity and morphology of the mitotic spindle using double immunofluorescence of beta- and gamma-tubulin in C(2)C(12) mouse cell line. We found that PHE and its steroidal analogues, EA-92, EA-97, AK-333, AK-409 and AK-433, affect cell proliferation in human lymphocytes and C(2)C(12) mouse cells. All studied compounds are capable of inducing chromosome breakage events, as indicated by the enhanced C(-)MN frequencies. The less lipophilic compounds are the most genetically active molecules. PHE and only two of the studied analogues, AK-409 and AK-433, the most hydrophilic ones, showed aneugenic potential, by increasing the frequencies of MN containing a whole chromosome. The aneugenic potential of the above referred analogues is associated with amplification of centrosome number, since they caused high multipolar metaphase frequencies.

Genotoxic, cytostatic, antineoplastic and apoptotic effects of newly synthesized antitumour steroidal esters.

In this study, we have investigated the genotoxic, cytostatic, antineoplastic and apoptotic effects of three newly synthesized modified steroidal esters, having as alkylating agent p-N,N-bis(2-chloroethyl) aminophenyl butyrate (CHL) or p-N,N-bis(2-chloroethyl) aminophenyl acetate (PHE) esterified with the steroidal nucleus modified in the B- and D-ring. The genotoxic and cytotoxic effects of the compounds were investigated both in vitro, in lymphocyte cultures obtained from blood samples of healthy donors and in vivo, in ascites cells of P388 leukemia obtained from the peritoneal cavity of DBA/2 mice. Preparations were scored for sister-chromatid exchange (SCE) and proliferation-rate indices (PRI). The newly synthesized compounds were also studied for antineoplastic activity against lymphocytic P388 and lymphoid L1210 leukemias in mice, by calculating the mean of the median survival of the drug-treated animals (T) versus the untreated control (C) (T/C%). The activity of caspase-2 and caspase-3, indicators of apoptosis, was assessed biochemically in primary cultures of human lymphocytes. Our results show that the newly synthesized compounds caused severe genotoxic effects by significantly increasing the frequency of SCE and decreasing the PRI values in cultures of peripheral lymphocytes in vitro and in ascites cells of lymphocytic P388 leukemia in vivo. A significant correlation was also observed in both the in vitro and in vivo experiments: the higher the SCE frequency the lower the PRI value (r=-0.65, P<0.001 and r=-0.99, P<0.01, respectively). The measured antileukemic potency was statistically increased by all test compounds in both types of tumours, while the activity of caspase-2 and caspase-3 showed a statistically significant increase after two periods of exposure. The genotoxic (increase of SCE), cytostatic/cytotoxic (decrease of PRI) and antileukemic effects (increase of T/C%) in combination with the induction of apoptosis (activation of caspase-2 and caspase-3) caused by the newly synthesized compounds, lead us to propose them as agents with potentially antineoplastic properties.

Aneugenic potential of the nitrogen mustard analogues melphalan, chlorambucil and p-N,N-bis(2-chloroethyl)aminophenylacetic acid in cell cultures in vitro.

Melphalan (MEL), chlorambucil (CAB) and p-N,N-bis(2-chloroethyl)aminophenylacetic acid (PHE) are nitrogen mustard analogues, which are clinically used as chemotherapeutic agents. They also exert carcinogenic activity. The aim of this study was to investigate the aneugenic potential of the above drugs and the possible mechanism responsible for this activity. The Cytokinesis Block Micronucleus (CBMN) assay in combination with fluorescence in situ hybridization (FISH) was used in human lymphocyte cultures to evaluate micronucleus (MN) frequency. Pancentromeric probe (alpha-satellite) was applied to identify chromosomes in micronuclei and an X-chromosome specific centromeric probe was used to asses micronucleation and non-disjunction of this chromosome in binucleated cells. The effect of the above compounds on the organization of mitotic apparatus, as a possible target of chemicals with aneugenic potential, was investigated in C(2)C(12) mouse cell line by double immunofluorescence of alpha- and gamma-tubulin. We found that the studied drugs increased MN frequency in a linear dose-dependent manner primarily by chromosome breakage and in a lesser extent by an aneugenic mechanism. Non-disjunction and micronucleation of X-chromosome were also induced. Abnormal metaphase cells were linearly increased with concentration and characterized by abnormal centrosome number. Interphase cells with micronuclei and abnormal centrosome number were also observed. Since nitrogen mustards are highly reactive agents, with low selectivity and form covalent bonds with different nucleophilic sites in proteins and nucleic acids, it is reasonable to consider that one possible pathway for nitrogen mustard analogues to exert their aneugenic activity is through reaction with nucleophilic moieties of proteins or genes that are involved in the duplication and/or separation of centrosomes, resulting in abnormal centrosome number. Based on our results the carcinogenicity of nitrogen mustard analogues studied may be attributed not only to their activity to trigger gene mutation and chromosome breakage, but also to their aneugenic potential. Further studies are warranted to clarify the above two hypotheses.

Antileukemic and cytogenetic activity by triple administration of three modified steroidal derivatives of nitrogen mustards.

Combination chemotherapy is widely and routinely used for most cancer patients. The main objective of this study is an effort to develop new anticancer drugs and procedures with enhanced antitumor activity and reduced toxicity. This study was designed to determine the antileukemic and cytogenetic activity of five mixtures of three specific steroidal esters of aromatic nitrogen mustards in different proportions. This is the next step of two previous studies where the combination of two such esteric analogues was investigated with promising results. All of the five mixtures used proved active against leukemia P388 and in the induction of sister chromatid exchanges, indicating that the combination of the same class of compounds can be successful, especially when a highly potent agent is combined with another less active but probably mechanistically supplementary one. These results can be used in future experiments in order to further scout the specific role of the steroidal part of these molecules in the antileukemic potency of them.

Rational design, synthesis and in vitro evaluation of three new alkylating steroidal esters.

Recent studies have indicated that minor functional changes on the steroidal part of complex molecules, comprising of an alkylating moiety and a steroidal congener, lead to compounds with enhanced biological activity. The observed induction of the genotoxic, cytotoxic and antileukemic effects suggest a determinative role of the steroidal congener on the mechanism of action. In order to further elucidate the structural requirements responsible for this, we designed and synthesized a new modified steroid, carrying a 17beta-acetamide substituent and a B lactamic ring, and studied the ability of its esters with three potent nitrogen mustards to induce sister chromatid exchange (SCEs) and to inhibit cell proliferation in normal human lymphocytes in vitro. The role of the steroidal skeleton was clearly stated by the results of the in vitro evaluation of the final compounds, as all three derivatives proved better inducers of SCE (58-102 SCE/cell) and cell division delays (1.18-1.25 PRI) than the simple nitrogen mustards (24-38 SCE/cell and 1.51-1.62 PRI). Obviously, the steroidal module enhances the formation of DNA adducts that cannot be repaired by excision repair enzymes probably through the induction of the interaction of these complex compounds with different base sequences or by disabling the repair mechanisms through the blockage of the enzymes responsible for excision repair. On the other hand, it seems that these compounds also act through a parallel site of action responsible for cell death when their primary binding site becomes saturated, as in higher concentrations two of the derivatives tested showed enhanced cytotoxicity while their ability to induce SCE stabilized.

Estrogen receptors alpha and beta (ERalpha and ERbeta) and androgen receptor (AR) in human sperm: localization of ERbeta and AR in mitochondria of the midpiece.

BACKGROUND: The central role of estrogens and androgens in the male reproductive system has focused attention on the presence and distribution of their cognate receptors [estrogen receptor (ER) alpha, ERbeta and androgen receptor (AR)] in male reproductive tissues and cells. Since the presence of steroid hormone receptors in mitochondria of mammalian cells has been well documented, we investigated the possibility of mitochondrial localization of sex steroid hormone receptors in sperm. METHODS AND RESULTS: Applying immunofluorescence labelling and confocal laser scanning microscopy we show that the estrogen receptor beta and the AR of human sperm are specifically enriched in the midpiece, at the site of the mitochondria, which were visualized by labelling with the vital dye CMX. Nuclear and mitochondrial localization of AR was also detected in LnCap human prostate cancer cells. Differentially, most of the ERalpha immunostaining is in the form of a compact zone at a region corresponding to the equatorial segment of the upper post-acrosomal region of the sperm head. Immunoblotting experiments using sperm extracts revealed the presence of a 66 and a 45 kDa protein reacting with the ERalpha antibody, one 64 kDa protein reacting with the ERbeta antibody and a 110 and a 90 kDa protein reacting with the antibody against AR. CONCLUSIONS: Our findings suggest that the differential localization of AR and ER isoforms in human sperm reveals distinct roles of these receptors in the physiology of sperm cells and, perhaps, also in the process of fertilization.

7-keto-delta(5)-steroids: key-molecules owning particular biological and chemical interest.

7-keto-Delta(5)-steroids have been suggested for the treatment of several diseases. Their significant biological profile resulted in the development of a great number of methods and reagents for the allylic oxidation of Delta(5)-steroids. These methods and the biological evaluation of the main oxidized Delta(5)-steroids are summarized.

Comparative study of sister chromatid exchange induction and antitumor effects by homo-aza-steroidal esters of [p-[bis(2-chloroethyl)amino]phenyl]butyric acid.

The present work was undertaken in order to test the hypothesis that the Sister Chromatid Exchange (SCE) assay in vitro can be used for the prediction of in vivo tumor response to newly synthesized potential chemotherapeutics. The effect of three homo-aza-steroidal esters containing the -CONH- in the steroidal nucleus, 1, 2, and 3 on SCE rates and on cell kinetics in cultured human lymphocytes was studied. The antitumor activity of these compounds was tested on leukemia P388- and leukemia L1210-bearing mice. The three substances induced statistically significant enhancement of SCEs and of cell division delays. Compounds 1 and 3 were identified, on a molar basis, as more effective inducers of SCEs and of cell division delays compared with compound 2. Compounds 1 and 3 had upon both experimental tumors better therapeutic effects compared with compound 2 at equitoxic doses. Therefore, the order of the antitumor effectiveness of the three compounds coincided with the order of the cytogenetic effects they induced.

Synergistic antineoplastic and cytogenetic effects by the combined action of two homo-aza-steroidal esters of nitrogen mustards on P388 and L1210 leukaemias in vivo and in vitro.

In order to increase the damaging effects on specific DNA sequences and decrease the subsequent toxicity, the use of homo-aza-steroidal esters of nitrogen mustards is already known. Two specific homo-aza-steroidal esters were mixed at different proportions and the resultant final mixtures were tested in vivo and in vitro. The effects of these on P388 and L1210 leukaemias, on SCE rates and on human lymphocyte proliferation kinetics were studied. The results demonstrate that the combined substances enhanced SCE induction (p < 0.05) and antitumour activity (p < 0.02) in a synergistic manner. A correlation was observed (p < 0.001) between the magnitude of the SCE response and the depression of the cell proliferation index.

Enhanced cytogenetic and antineoplastic effects by the combined action of two esteric steroidal derivatives of nitrogen mustards.

The authors studied the effect of two modified steroids containing different proportions (%) of alkylating agents alone or in combination on sister chromatid exchange (SCE) rates and on human lymphocyte proliferation kinetics. The antitumor activity of these compounds was tested on leukemia P388- and leukemia L1210-bearing mice. The two chemicals in mixtures enhance SCE induction and antitumor activity in a synergistic manner. The homo-aza-steroidal ester of p-bis(2-chloroethyl)aminophenyl acetic acid was found to be more effective than the homo-aza-steroidal ester of o-bis(2-chloroethyl)aminobenzoic acid in causing cytogenetic damage and antineoplastic activity. A correlation was observed between the magnitude of the SCE response and the depression of the cell proliferation index. The order of the antitumor effectiveness of the five different treatments tested coincided with the order of the cytogenetic effects they induced.

Antitumor and cytogenetic effects of esteric (ASE) and amidic (ASA) steroidal derivative of p-bis (2-chloroethyl) amino phenylacetic acid (CAPA). A comparative study.

The homo-aza-steroidal ester of p-bis (2-chloroethyl) amino phenylacetic acid (ASE) (I) the homo-aza-steroidal amide of p-bis (2-chloroethyl) amino phenylacetic acid (ASA) (II), and the parent compound p-bis (2-chloroethyl) amino phenylacetic acid (III) were tested in an effort to evaluate their ability to inhibit a transplanted leukemia (P388) in vivo, and the DNA synthesis of P388 cell cultures in vitro. The compounds' effects on Sister Chromatid Exchange (SCE) rate and on human cell proliferation kinetics in vitro were also studied. The above mentioned compounds were identified as displaying cytogenetic, cytostatic and antineoplastic effects, the ester compound being the more potent. The main conclusion from this study is that the existence of the esteric bond is necessary for the expression of the antitumor activity. The synthetic route for the preparation of the amidic derivative (II), as new product, is also reported.

Heparin-binding keratinocyte growth factor is a candidate stromal-to-epithelial-cell andromedin.

The growth of isolated epithelial and stromal cells from both androgen-dependent normal rat prostate and an androgen-responsive model rat prostate tumor is androgen-independent. When added to co-cultures of epithelial and stromal cells separated by a semipermeable membrane, androgen stimulated epithelial cell growth without an effect on stromal cell growth. Northern blot and nuclease protection analysis of mRNA revealed that stromal cells specifically expressed an androgen-sensitive secreted member of the heparin-binding fibroblast growth factor family [keratinocyte growth factor (KGF)/fibroblast growth factor-7]. KGF was mitogenic for epithelial cells, but not for stromal cells. Epithelial cells expressed specifically a splice variant of the bek receptor gene that specifically binds KGF. Expression of the bek receptor gene in stromal cells was undetectable by Northern blot and nuclease protection analyses. The results suggest that stromal cell-derived KGF has the properties of an andromedin, which mediates the indirect control of epithelial cell proliferation by androgen through a directional stromal-to-epithelial cell paracrine mechanism.

Potential role of HBGF (FGF) and TGF-beta on prostate growth.

We review in this paper the role of heparin-binding growth factor (HBGF*) or fibroblast growth factor (FGF*), rat prostate cancer cells produce TGF-beta, IGF-II* and OGF*. Of these growth factors, TGF-beta and unknown labile factor with 19 kDa are the most probable candidates responsible for osteoblastic bony metastasis of prostate cancer. In vitro experiments suggest that TGF-beta modulates cell detachment of prostate cancer cells together with nutritional factors. HBGF-dependent growth of the prostate tumor epithelial cells is free from inhibition by TGF-beta, whereas normal prostate epithelial cells are sensitive to TGF-beta inhibition. Transfection experiments suggest that HBGF-2 (basic FGF) might be closely related to the malignant growth of prostate cancer, in addition to tumor angiogenesis.

Interaction of phosphorylase kinase with polymyxins.

Nonactivated rabbit skeletal muscle phosphorylase kinase is inhibited by the polymyxins A, B, D and E when assayed at pH 8.6. Polymyxin B is the most effective inhibitor, causing 50% inhibition at 0.3 mM. Following the effect of polymyxin B on the kinase activity toward troponin, no inhibition was observed. In contrast, polymyxin B was found to greatly stimulate the autophosphorylation of phosphorylase kinase. About 10 mol of phosphate per tetramer (alpha beta nu delta) were incorporated in presence of polymyxin B (full autophosphorylation). This incorporation was about 6-fold higher than that observed without polymyxin. The stimulation of autophosphorylation by polymyxin B was accompanied with enhancement of the rate of autoactivation at pH 6.8.

Phosphorylase kinase from chicken gizzard. Partial purification and characterization.

Phosphorylase kinase was partially purified (530-970-fold) from chicken gizzard smooth muscle by a procedure involving ammonium sulfate fractionation, chromatography on 8-(6-aminohexyl)adenosine-5'-phosphate--Sepharose 4B and glycerol density gradient ultracentrifugation. The final and most efficient purification step takes advantage of the relatively high molecular mass of gizzard phosphorylase kinase, which was found to be similar to that of rabbit skeletal muscle enzyme. The gizzard kinase, further purified to near homogeneity by calmodulin-Sepharose 4 B affinity chromatography, showed one main protein band of 61 kDa, upon dodecyl sulfate acrylamide gel electrophoresis. Four minor protein bands of higher molecular mass were also present but no protein stain was seen at the position of the gamma subunit. The gizzard phosphorylase kinase showed a high pH 6.8/8.2 activity ratio of 0.53, it was stimulated by Ca2+, inhibited up to 80% by EGTA and it was activated about 1.9-fold by calmodulin. The km value for ATP was 0.45 mM, while the K0.5 for rabbit muscle phosphorylase b was extremely low, more than 200-fold lower than the Km of nonactivated skeletal muscle phosphorylase kinase for its protein substrate. High concentrations of phosphorylase b were found to be inhibitory. At 10 mg/ml phosphorylase b, the maximum activity of the kinase was inhibited fivefold. No evidence has been obtained indicating autophosphorylation or the existence of active and inactive forms of gizzard phosphorylase kinase. Limited proteolysis of the smooth muscle kinase with trypsin was accompanied by a twofold activation at pH 6.8.

Interaction of aliphatic amines with glycogen phosphorylase.

A number of aliphatic amines was shown to stimulate AMP-dependent activity of phosphorylase b. The extent of stimulation depends on the molecular structure of amines. For linear amines, the longer the linear chain, the greater the stimulation observed. High concentrations of amines were able to induce a small activation of phosphorylase b in the absence of AMP. Kinetic studies of phosphorylase b indicated that the presence of n-hexylamine (a) results in lowering Km values for AMP and glucose 1-phosphate, (b) increases maximal velocity of the enzyme, and (c) modifies the glucose 6-phosphate, ATP, caffeine, and glucose binding sites of the enzyme by increasing the inhibition constants for these inhibitors. In contrast, the activity of phosphorylase b' is not altered by n-hexylamine. This fact suggests the possibility that amines interact with the N-terminal tail of phosphorylase b chain.