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N Kadohama

Publications and source records attributed to N Kadohama.

At least 19 recordsLinked to original sources
PubMed1993

Tobacco alkaloid derivatives as inhibitors of breast cancer aromatase.

The inhibition of estrogen biosynthesis by the use of aromatase inhibitors is emerging as a valuable approach to breast cancer therapy. Because smoking has a profound effect on estrogen-related processes we examined the ability of tobacco constituents to suppress estrogen production by breast cancer aromatase. N-n-octanoylnornicotine and N-(4-hydroxyundecanoyl) anabasine suppressed aromatase activity in culture of two human breast cancer cell lines, MDA-MB-231 (IC50 of 310 and 20 microM, respectively) and SK-BR-3 (IC50 of 450 and approximately 2 microM, respectively). MDA-MB-231 cells induced by 250 nM dexamethasone or 1 mM (Bt)2cAMP were slightly more sensitive to both inhibitors. Kinetic analyses showed that inhibition by N-(4-hydroxyundecanoyl)anabasine is competitive with respect to androstenedione as substrate, with apparent Ki values of 0.2 microM against microsomal aromatase activity derived from both (Bt)2cAMP-induced MDA-MB-231 cells and human breast tumor tissue. The corresponding apparent Ki against human placental microsomal aromatase activity was 0.4 microM. These results indicate that acyl derivatives of nornicotine and anabasine block estrogen formation in breast tumor cells and tissue and could contribute to the decreased intra-tissue estrogen levels in women who smoke.

Adenocarcinoma↗
PubMed1993

Catalytic efficiency of expressed aromatase following site-directed mutagenesis.

Mutant aromatase cytochrome P-450s, expressed in CHO cells after transfection with cDNAs, have been characterized in terms of their catalytic efficiencies. After solubilization from microsomes, specific aromatase P-450 content of wild-type and mutants Pro308Phe, Asp309Asn, Asp309Ala and Phe406Arg was quantitated by a sandwich enzyme-linked immunosorbent assay (ELISA). Microsomal aromatase activity was determined by the 3H-water method using [1 beta-3H]androstenedione as substrate. Estimations of the actual turnover rate (catalytic efficiency) were derived from the combined data. The P-450 content in the mutants varied but was always less than that in the wild type. Hence, the decreases in the Vmax observed in the mutant enzymes did not correlate completely with reductions in catalytic effectiveness. In recent studies on the structure-function relationship of aromatase cytochrome P-450, the observed reduction of enzyme activity in terms of Vmax following site-directed mutagenesis led to the assumption that there was a corresponding loss of catalytic effectiveness. The present study reveals that a lower P-450 content can contribute significantly to decreasing catalytic activity in the mutants. In fact, in mutant Phe406Arg which exhibited virtually no catalytically active aromatase, the specific P-450 content was below the detectable level. Because of its location, the result of this latter mutation could be a major structural perturbation of the heme-binding property. Thus, interpretation of losses and reductions in aromatase activity resulting from single amino-acid replacement should take into account changes in the specific content of aromatase cytochrome P-450.

Androstenedione↗
PubMed1993

Structure-function studies of human aromatase.

Site-directed mutagenesis experiments have been carried out to determine the structure-function relationship of human aromatase. By sequence comparison, the region in aromatase that corresponds to the distal helix of cytochrome P-450cam has been identified to be Gln-298 to Val-313. Eight aromatase mutants with changes in this region, i.e. C299A, E302L, P308F, D309N, D309A, T310S, T310C, and S312C, have been generated using a mammalian cell stable-expression system. The results from site-directed mutagenesis studies indicate that the region containing Gln-298 to Val-313 is indeed a very important part of the active site of aromatase. The catalytic properties of P308F, D309N, and D309A have been examined in detail and are discussed. Active site-directed labeling is also an important approach to investigate the structure-function relationship of aromatase. HPLC-linked electrospray mass spectrometry is indicated as a useful technique for the characterization of active site-directed probe-modified enzyme. The mass spectral analysis of aromatase suggests that aromatase is glycosylated.

Amino Acid Sequence↗
PubMed1992

Kinetic properties of aromatase mutants Pro308Phe, Asp309Asn, and Asp309Ala and their interactions with aromatase inhibitors.

Mutant forms of aromatase cytochrome P-450 bearing modifications of amino acid residues Pro308 and Asp309 and expressed in transfected Chinese hamster ovary cells were subjected to kinetic analysis and inhibition studies. The Km for androstenedione for expressed wild type (11.0 +/- 0.3 nM SEM, n = 3) increased 4-, 25- and 31-fold for mutants Pro308Phe, Asp309Asn and Asp309Ala, respectively. There were significant differences in sensitivity among wild type and mutants to highly selective inhibitors of estrogen biosynthesis. 4-Hydroxyandrostenedione (4-OHA) a strong inhibitor of wild type aromatase activity (IC50 = 21 nM and Ki = 10 nM), was even more effective against mutant Pro308Phe (IC50 = 13 nM and Ki = 2.8 nM), but inhibition of mutants Asp309Asn and Asp309Ala was considerably less (IC50 = 345 and 330 nM and Ki = 55 and 79 nM, respectively). Expressed wild type aromatase and Pro308Phe aromatase were strongly inhibited by CGS 16949A (IC50 = 4.0 and 4.6 nM, respectively) whereas mutants Asp309Asn and Asp309Ala were markedly less sensitive (IC50 = 140 and 150 nM, respectively). CGS 18320B produced similar inhibition. Kinetic analyses produced Ki = 0.4 nM for CGS 16949A inhibition of wild type versus 1.1, 37 and 58 nM, respectively, against Pro308Phe, Asp309Asn and Asp309Ala. The results demonstrate significant changes in function resulting from single amino acid modifications of the aromatase enzyme. Our data indicate that mutation in Asp309 creates a major distortion in the substrate binding site, rendering the enzyme much less efficient for androstenedione aromatization. The substitution of Pro308 with Phe produces weaker affinity for androstenedione in the substrate pocket, but this alteration favors 4-OHA binding. Similarly, mutant Pro308Phe exhibits a slightly greater sensitivity to inhibition by CGS 18320B than does the wild type. These results indicate that residues Pro308 and Asp309 play critical roles in determining substrate specificity and catalytic capability in aromatase.

Animals↗
PubMed1991

Murine monoclonal antibodies reactive with a variety of androgen independent Dunning rat prostate adenocarcinoma sublines also reactive with human prostate adenocarcinoma.

Murine hybridoma-derived monoclonal antibody (MCA) to Dunning rat prostate adenocarcinoma R-3327HIS (androgen independent type) has been produced by fusing P3x63 Ag8-653 myeloma cells with splenocytes of BALB/c mice which were immunized with R-3327HIS tumor cell membranes. One monoclonal antibody designated MCA-R1 (IgG2a subtype) produced an intense immunostaining of various androgen independent Dunning rat prostate tumor sublines (HIS, HIM, HIF, AT-1, AT-2, AT-3, MAT-Lu and MAY-Ly-Lu), but did not stain other tumors of rat origin or normal rat tissues. Marginal immunostaining was detected in the androgen responsive R-3327H and R-3327G tumor subline. Although MCA-R1 antibody did not react with the regressed prostate tumor of R-3327H or R-3327G, it strongly reacted with the relapsed prostate tumor from either R-3327H or R-3327G tumor derived from rats were treated with diethyl stilbestrol (DES) or castration. MCA-R1 antibody also produced a strong cross-reaction with human prostate adenocarcinoma. Like the Dunning rat tumor, human adenocarcinoma exhibited distinct immunostaining patterns with respect to intracellular localization among well differentiated, moderately differentiated and poorly differentiated tumor. Benign prostatic hyperplasia or other normal tissues did not stain. Immunofluorescence, immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioautographic analysis revealed that the Dunning rat prostate tumor antigen recognized (m.wt. 50 and 120 Kd) by MCA-R1 antibody are localized on both cell surface and in the cytoplasm. This MCA may represent a potential reagent for the study of tumor biology and immunotherapy of prostate tumor.

Adenocarcinoma↗
PubMed1988

Growth inhibition by diethylstilbestrol and relapse of the Noble rat prostatic tumor.

The response of androgen-sensitive Noble (Nb) rat prostatic adenocarcinoma [2Pr-121D (1)] to varying doses (50 approximately 1,000 micrograms/kg body weight) of diethylstilbestrol (DES) was investigated and characterized with respect to cytosol and nuclear androgen binding profiles, histology and pattern of relapse. Inhibition of tumor growth was closely related to the dose of DES. Treatment at all but the lowest dose (50 micrograms/kg) initially caused tumor regression, whereas serum testosterone concentrations in all groups, including that receiving the lowest dose, were decreased to castrate levels. Histologically, while extensive cellular destruction was clearly evident at higher doses of DES, some active tumor cells appeared to survive. Tumors eventually relapsed when higher doses of DES were stopped or with the continued administration of low doses. The cytosol dihydrotestosterone (DHT) receptor in this tumor line, as determined by sucrose density gradient, dextran charcoal and Sephadex column methods, was negative. Nuclear binding, however, was positive. Salt-extractable and salt-resistant fractions of nuclei derived from the untreated primary tumor and relapsed tumor following DES treatment contained high affinity androgen receptor. Comparison of binding constants revealed no significant differences. Our findings, based on the Nb rat prostatic tumor model, indicate that DES acts not only by eliminating circulating testosterone, but also by a direct cytotoxic effect on malignant cells. The results also suggest the lack of an apparent relationship between the loss of hormone responsiveness associated with recurrence of prostatic tumor growth and nuclear androgen binding parameters.

Adenocarcinoma↗
PubMed1987

Immunotherapy in a spontaneously developed murine mammary carcinoma with syngeneic monoclonal antibody.

A new spontaneously arisen murine breast tumor, designated JC, has been established in immunocompetent BALB/c mice. Upon reestablishment of tumor in vitro and in vivo, the epithelial murine tumor cells retained their original papillary adenocarcinoma morphology. Various immunotherapy protocols have been performed in previously implanted and progressively growing JC tumor in syngeneic hosts with a murine monoclonal antibody (McAb), F36/22 (IgG3). Affinity of McAb binding to JC tumor cells was determined to be 6.1 x 10(7) L/M. Quantitatively 1.2 x 10(5) molecules of McAb bound to a JC tumor cell. Immunotherapeutic effectiveness in vivo on a tumor mass after its establishment is a major feature of this experimental tumor model. When four sequential administration of McAb, i.p., at a dose of 400 micrograms 4 days apart were used, McAb-treated animals showed statistically significant tumor regression and longer survival than those of control animals treated with an irrelevant McAb of the same isotype. Two temporal phases of tumoricidal activity were observed as measured by tumor volume reduction. The first phase of tumoricidal response (tumor regression) was detected within days upon the first administration of McAb. A distinct second phase followed within 3-5 weeks after the last McAb administration, which resulted in tumor necrosis even in large tumors. Histological examinations revealed heavy infiltration of inflammatory cells at the beginning of the second phase. Similar tumor regression was also obtained from animals treated with a single dose (400 micrograms) of McAb followed by injections of McAb with complete and incomplete adjuvant, respectively. These results demonstrate that this syngeneic murine mammary tumor can serve as a potential preclinical model for investigation of parameters and mechanisms associated with McAb immunotherapy.

Adenocarcinoma, Papillary↗
PubMed1987

Protein profiles of benign hypertrophic prostate: stroma-abundant distribution of BPH-associated nonhistone proteins.

Mechanically isolated epithelium and stroma from benign hypertrophic prostates were highly pure on the basis of histochemical and biochemical criteria. By electrophoretic analyses, whole cellular and nuclear proteins were compared among whole tissues, epithelium, and stroma. The characteristic protein profiles of benign hypertrophic prostates were reflected in the electrophoretic patterns of the stroma. Two-dimensional gel electrophoretic patterns of the epithelium were different from those of the stroma with exception of about 18 major spots that were common to both fractions. Of the protein species separated, 35K/6.7 (molecular weight/pI) and 36K, which was composed of two species with pI of 4.4 and 4.6, were abundant in the epithelium and stroma, respectively. Nuclei prepared from whole tissues of benign hypertrophic prostates contained three kinds of nonhistone proteins (NHP) closely associated with benign prostatic hypertrophy (BPH); 42 K-NHP, 55 K-NHP and 190 K-NHP. Electrophoretic analysis of the nuclear proteins revealed that all the BPH-associated nonhistone proteins were abundantly distributed in the nuclei of the stroma.

DNA↗
PubMed1985

Retardation of prostate tumor progression in the Noble rat by 4-methyl-4-aza-steroidal inhibitors of 5 alpha-reductase.

The effect of 4-methyl-4-aza-steroidal inhibitors of 5 alpha-reductase has been evaluated on tumor growth in the Noble rat model of prostatic adenocarcinoma. The growth characteristics of the tumor line 2Pr-121D(1) were consistent with heterogeneity of cell types, composed of androgen-sensitive and androgen-insensitive malignant cells. Both sodium 4-methyl-3-oxo-4-aza-5 alpha-pregnane-20 (s)-carboxylate and 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 and 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one significantly retarded tumor progression. Each agent increased tumor volume doubling time by approximately 62%. On the basis of their similarities to female rats and male castrate group, in terms of growth rate, tumor doubling time, and histologic characteristics, the treatments with the 4-methyl-4-aza-steroids appeared to produce effects common to both castration and estrogenization (chronic administration of pharmacologic doses of estrogen). The failure of 5 alpha-reductase inhibitors to be active as antiprostatic agents in vivo has hitherto detracted from their use of therapeutic agents. Present studies demonstrate that the 4-methyl-4-aza-steroidal inhibitors of 5 alpha-reductase may represent an alternative to orchiectomy and chronic estrogen therapy for the management of the hormone-dependent phase of prostate cancer.

5-alpha Reductase Inhibitors↗
PubMed1984

Selective inhibition of prostatic tumor 5 alpha-reductase by a 4-methyl-4-azasteroid.

The effect of sodium 4-methyl-3-oxo-4-aza-5 alpha-pregnane-20(s)-carboxylate (4-MAPC) on testosterone metabolism was investigated in rat and human prostates in organ culture. The general properties of the test system for androgen metabolism and response to inhibitors were in close agreement with in vivo observations. As an inhibitor of prostatic tumor 5 alpha-reductase, 4-MAPC was equally as effective as 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one, reported to be a potent 5 alpha-reductase inhibitor. Inhibition of 5 alpha-reductase activity by 4-MAPC, but not by 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one, was accompanied by concomitant stimulation of 17 beta-oxidation of testosterone. This differential effect was observed in explants of human prostatic carcinoma and benign prostatic hypertrophy containing a relatively high degree of glandular hyperplasia. It was also seen in explants of dorsolateral rat prostate but not in the ventral prostate. 4-MAPC exhibited low affinity for rat prostatic cytosol 8S androgen receptor. Steroid extraction of purified nuclei from inhibited rat tissues revealed substantial amounts of radioactivity derived from [3H]testosterone cochromatographed with other metabolites in addition to dihydrotestosterone. The endocrine changes produced by this inhibitor of 5 alpha-reductase are reconcilable with the responsiveness of androgen-sensitive malignant prostatic cells to hormonal therapy.

5-alpha Reductase Inhibitors↗
PubMed1983

Inhibitory effects of some steroidal 6-methylene derivatives on 5 alpha-reductase activity in human and rat prostate.

Using a short-term organ culture assay, some 6-methylene derivatives of progesterone and testosterone have been evaluated for their effects on testosterone metabolism in rat and human prostatic tissues, and on DNA synthesis in explants from 7-day castrated rats. Comparative studies showed that the ability to inhibit 5 alpha-reductase activity was fairly specific with respect to structural requirements. Methylene substitution at the C6 position of the progesterone molecule was associated with high inhibitory activity. In explants prepared from human prostates, 6-methylene progesterone (II) had 70-85% (mean of 79% for 4 BPH tissues) of the potency of unmodified progesterone (I). Its 17 alpha-acetoxy-6-methylene analog (III), however, had only 32-73% (mean of 53% for 5 BPH specimens) of the activity of (I). The degrees of inhibition in rat and human prostatic tissues were similar. Inhibition of 5 alpha-reductase activity in cultured explants by 6-methylene progesterone (II) could not be reversed by change in media. The 6-methylene derivatives had little or no effect on DNA synthesis. Histological examination confirmed a lack of effect on basal cell proliferation. However, morphological alterations affecting epithelial cell height and secretory activity were clearly evident. These results indicate that, under our experimental conditions, the main effect of inhibition of 5 alpha-reductase activity in prostatic tissues by 6-methylene derivatives of progesterone is related to suppression of differentiated function.

5-alpha Reductase Inhibitors↗
PubMed1983

Prostatic cancer--II. Inhibitors of rat prostatic 4-ene-3-ketosteroid 5 alpha-reductase derived from 6-methylene-4-androsten-3-ones.

The studied 6-methylene-4-androsten-3-ones proved to be significantly inferior to 6-methylene-4-pregnene-3,20-dione and its 17-acetoxy derivative described in Part 1 as inhibitors of 4-ene-3-ketosteroid 5 alpha-reductase [1] in vitro. Surprisingly, the 6-methylene derivative of testosterone was only weakly active until acetylated, when an effective inhibitor was obtained. Etherification of the hydroxyl-group, its replacement by a hydrocarbon chain, or introduction of a substituent at C17 or on the methylene group led to virtual loss of activity. 17 alpha-Chloro-6-methylene-4-androstene-3-one had ca 60-70% of the potency of progesterone, but was inactive as enzyme inhibitor in explants of rat prostate in tissue culture and in in vivo studies. 6-Methylenetestosterone acetate was weakly active as enzyme inhibitor in explants of human prostate in tissue culture and produced a histological picture closely resembling testosterone and differing from that of cyproterone acetate. In vivo in the rat it had 80% of the androgenic activity of testosterone propionate. The foregoing data have been used to define some structural characteristics necessary for enzyme inhibition and to draw some conclusions regarding the architecture of the androgen and progesterone receptors and of the enzyme active site.

5-alpha Reductase Inhibitors↗
PubMed1981

Fluid inhibition as a factor in estrogen-induced increase of prostatic weight in castrated rats.

The mechanism by which estrogens inhibit castration atrophy has been investigated morphologically and biochemically utilizing ventral prostate from Copenhagen rats. The suppression of weight loss and gross edematous appearance of the prostate associated with the in vivo effect of 17 beta-estradiol (E2) could not be accounted for by DNA and protein synthesis. Increase in the fluid content in the tissues was confirmed by demonstration of significant increase in the ratio of wet/dry tissue weights. Light microscopy demonstrated that the main effects were on the stroma, characterized by large interglandular areas almost totally devoid of collagen resulting in an edematous appearance. Electron microscope studies showed an abundance of fluid localized adjacent to the capillary endothelium and some red blood cells indicating disturbances in capillary permeability. The combination of a prolactin secretion inhibiting agent with E2 alone, indicating an involvement of prolactin the estrogen effect. Differences in blood prolactin concentration between the strains of rats may influence the sensitivity of the prostate to estrogens.

Animals↗
PubMed1978

Estramustine phosphate: metabolic aspects related to its action in prostatic cancer.

Estramustine phosphate, a drug effective in a substantial number of patients with cancer of the prostate who had failed on other forms of therapy, has been shown to be split into its constituents, that is estradiol-17beta and the carbamate nitrogen mustard by non-cancerous and cancerous human prostatic tissues. This fact may explain, in part, the mechanism of action and efficacy of the drug in patients with cancer of the prostate. In addition to presenting results on the hydrolysis and its products accomplished by human prostatic tissues we discuss the limitations of extrapolating animal results with estramustine phosphate to the human condition and possible parameters that bear upon the insignificant toxic and estrogenic effects observed in patients given estramustine phosphate.

Adenocarcinoma↗