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Biomedical subjects

U Regenass

Publications and source records attributed to U Regenass.

33 records · Page 2Linked to original sources

New S-adenosylmethionine decarboxylase inhibitors with potent antitumor activity.

Methylglyoxal bis(guanylhydrazone) (MGBG) has been studied clinically as an antitumor and antileukemic agent and is recognized as a potent but nonspecific inhibitor of the key polyamine biosynthetic enzyme, S-adenosylmethionine decarboxylase (SAMDC). A series of four SAMDC inhibitors with structural features similar to MGBG have been found to have improved potency and specificity toward the target enzyme, SAMDC. Relative to MGBG, the new derivatives were much more effective in inhibiting partially purified preparations of SAMDC (50% inhibitory concentration, 10 to 100 nM), much less effective at inhibiting diamine oxidase, and inactive toward ornithine decarboxylase. The inhibitors varied relative to MGBG in their ability to compete with spermidine for uptake, with two being similar and two being less effective. Against L1210 leukemic cells and T24 bladder carcinoma cells, the compounds were slightly less effective than MGBG at inhibiting cell growth, with 50% inhibitory concentration values of 1 to 10 microM as compared with 0.5 and 1.1 microM, respectively, for MGBG. Under 50% growth-inhibitory conditions, the inhibitors decreased SAMDC activity, increased ornithine decarboxylase activity and putrescine pools, and markedly depleted spermidine and spermine pools of L1210 cells. At the same time, mitochondrial integrity as assessed by whole-cell pyruvate oxidation and mitochondrial DNA content was not affected as it was with MGBG. At doses less than one tenth that of the maximally tolerated dose, all of the new inhibitors strongly suppressed the growth of B16 melanoma in vivo with minimal weight loss or toxicity. At doses less than one sixth the maximally tolerated dose, they effectively inhibited the growth of T24 human bladder carcinoma xenografts. In these same systems, MGBG showed only marginal antitumor activity. These studies identify two potent and efficacious inhibitors of SAMDC as potential antitumor agents and reaffirm the importance of SAMDC as a target in anticancer drug discovery.

Adenosylmethionine Decarboxylase↗

2-substituted 3-(aminooxy)propanamines as inhibitors of ornithine decarboxylase: synthesis and biological activity.

1-Amino-3-(aminooxy)-2-propanol (6a) has been synthesized and found to inhibit rat liver ornithine decarboxylase (ODC) with an IC50 in the nanomolar range. Compound 6a served as a basis for the design of new enzyme inhibitors, which led to the identification of 3-(aminooxy)-2-fluoropropanamine (15) as a new powerful enzyme blocker. Compound 15 inhibited ODC at 3 times lower concentrations than 6a and 3-(aminooxy)propanamine (APA), and it was superior to APA as an antiproliferative agent in inhibiting the growth of human T24 bladder carcinoma cells in vitro.

Animals↗

Indole alkaloids with in vitro antiproliferative activity from the ammoniacal extract of Nauclea orientalis.

Nine angustine-type alkaloids were isolated from ammoniacal extracts of Nauclea orientalis L. (Rubiaceae). Three of them, 10-hydroxyangustine and the two diastereoisomeric 3,14-dihydroangustolines, have not been described in the literature thus far. The structures of the isolates were determined with spectroscopic methods, mainly 1D- and 2D-NMR spectroscopy. The compounds were found to exhibit in vitro anti-proliferative activity against the human bladder carcinoma T-24 cell line and against EGF (epidermal growth factor)-dependent mouse epidermal keratinocytes. By using overpressure layer chromatography, it was shown that minor quantities of these alkaloids occur in dried Nauclea orientalis leaves. The use of ammonia in the extraction process results in a significant increase in the formation of angustine-type alkaloids from strictosamide-type precursors.

Alkaloids↗

Sulfonylbenzoyl-nitrostyrenes: potential bisubstrate type inhibitors of the EGF-receptor tyrosine protein kinase.

The synthesis and biological activities of a series of sulfonylbenzoyl-nitrostyrene derivatives, a novel class of selective bisubstrate type inhibitors of the EGF-receptor tyrosine protein kinase, are described. The most potent derivatives inhibited the EGF-R tyrosine kinase, using angiotensin II as exogenous substrate, with IC50 values of less than or equal to 1 microM. No inhibition of the v-abl tyrosine kinase or the serine/threonine kinases PKC and PK-A was observed. In addition, active derivatives (compounds 5 and 12) effectively blocked the autophosphorylation of the EGF-R in vitro. Starting from the acids 5, 7, and 9, a series of esters, amides, and peptides was synthesized with the aim of increasing cellular penetration. Amides 14-18 showed potent antiproliferative effects using the EGF-dependent Balb/MK mouse epidermal keratinocyte cell line. Additionally, with the amide 14 inhibition of EGF-R autophosphorylation was demonstrated in the A431 cell line. CAMM studies using a computer-generated model for the transition state of the gamma-phosphoryl transfer from ATP to a tyrosine moiety and fitting experiments using the highly potent derivative 7 (IC50 value = 54 nM) support the hypothesis that the sulfonylbenzoyl group mimics a diphosphate moiety in the transition state. These results demonstrate that the rational design of tyrosine kinase inhibitors, using the inhibitory nitrostyrene moiety as a tyrosine mimic together with the sulfonylbenzoyl moiety as a diphosphate mimic, leads to highly potent and selective multisubstrate type inhibitors.

Angiotensin II↗

Thiazolidine-diones. Biochemical and biological activity of a novel class of tyrosine protein kinase inhibitors.

Various derivatives of thiazolidine-diones have been identified as tyrosine protein kinase inhibitors. The epidermal growth factor (EGF) receptor kinase and c-src kinase were inhibited in vitro with IC50 values in the range of 1-7 microM. The v-abl tyrosine protein kinase was not inhibited by thiazolidine-diones. Inhibition was found to be specific for tyrosine protein kinases. Inhibition of serine/threonine protein kinases was not observed. The active derivatives were shown to inhibit EGF-induced receptor autophosphorylation, either in vitro or in intact cells, and were also found to inhibit growth of the EGF-dependent BALB/MK and A431 cell lines (IC50 1-3 microM). Growth of the interleukin-3-dependent myeloid cell line FDC-P1 was inhibited with equal efficiency. Thus, in these cell lines, members of the c-src kinase family are also potential targets for inhibition by the compounds.

Animals↗

Cytogenetics of breast cancer.

Chromosome counts were performed on 1,100 cells from 17 malignant breast carcinomas and on 168 cells of four normal tissue samples after amethopterin treatment and G-banding. Karyotypes were established from 216 cells of 11 tumor-derived cultures and from 47 cells of four nonmalignant tissue-derived cultures. Karyotypes of cells from nonmalignant samples showed a normal diploid chromosomal constitution with no consistent loss or gain of a specific chromosome. Structural chromosomal abnormalities were not observed. Tumor-derived cultures could be distinguished from normal cultures on the basis of a significantly increased incidence of numerical changes and structural chromosomal aberrations. In nine of 11 tumor-derived cultures, numerically normal cells were shown to be pseudodiploid, with frequencies ranging to 43% (mean, 13.2%) of the diploid cells. In agreement with previous reports, cytogenetic analyses showed predominantly diploid cells. Clonal numerical changes of chromosomes 17, 18, 20, and 21 could be detected in three tumor samples. Clonal structural abnormalities could be observed in two of 11 analyzed tumours. A t(6;12)(p21;p13) and an enlarged chromosome 7 (7q+) were found in a patient with invasive ductal carcinoma. An inversion of chromosome 7 [inv(7)(q11.2q32)] was observed in one case, also diagnosed as invasive ductal carcinoma. The significance of these findings in relation to clinical data is discussed.

Adult↗

A derivative of staurosporine (CGP 41 251) shows selectivity for protein kinase C inhibition and in vitro anti-proliferative as well as in vivo anti-tumor activity.

Analogues of staurosporine were synthesized and their ability to inhibit protein kinases was examined. Staurosporine is a potent but non-selective inhibitor of in vitro protein kinase C(PKC) activity (IC50 6.0 nM). The derivative CGP 41 251 had reduced PKC activity with an IC50 of 50 nM but showed a high degree of selectivity when assayed for inhibition of cyclic AMP-dependent protein kinase (IC50 2.4 microM), S6 kinase (IC50 5.0 microM) and tyrosine-kinase-specific activity of epidermal growth factor receptor (IC50 3.0 microM). Staurosporine and CGP 41 251 exerted growth inhibition in the human bladder carcinoma line T-24, human promyelocytic leukemia line HL-60 and bovine corneal endothelial cells at concentrations which correlated well with in vitro PKC inhibition. In addition, both compounds inhibited the release of H2O2 from human monocytes pre-treated with 12-O-tetradecanoyl-phorbol-13-acetate at non-toxic concentrations. In vivo anti-tumor activity was examined in T-24 human bladder carcinoma xenografts in athymic nude mice. Tumor growth inhibition tests revealed significant anti-tumor activity (2p less than 0.001) at 1/10 of the maximum tolerated doses for both compounds. By contrast, a closely related derivative of staurosporine (CGP 42 700) was inactive at concentrations of over 100 microM in all in vitro enzyme and anti-proliferative assays as well as in animal tumor models. Our data suggest an association between PKC inhibition and anti-proliferative and anti-tumor activity.

Alkaloids↗

Anti-tumor effects of tumor necrosis factor in combination with chemotherapeutic agents.

The anti-tumor activity of partially purified tumor necrosis factor (TNF) was analysed in combination with chemotherapeutic drugs against intradermally transplanted Meth A sarcoma. Tumors were allowed to grow until they reached an average diameter of about 8 mm. TNF was given once i.v. (16,000 units as determined on L-M cells) and the chemotherapeutic agents adriamycin (1-10 mg/kg), 5-FU (3-100 mg/kg), cyclophosphamide (50-200 mg/kg) were applied once i.p. 4 hr after TNF injection. The strongest anti-tumor effects in normal BALB/c mice were observed when TNF was combined with the following doses of chemotherapeutic agents: cyclophosphamide 100 mg/kg, adriamycin 5 mg/kg, 5-FU 30-100 mg/kg. The most effective combinations induced complete regressions. When TNF was combined with varying doses of adriamycin and cyclophosphamide, bell-shaped dose-response curves were obtained. Experiments were repeated in Meth A sarcoma-bearing BALB/c nu/nu mice. In this case TNF combinations with cyclophosphamide and 5-FU did not induce tumor regressions. The highest dose of the chemotherapeutic agent was the most effective in drug combinations. Histological analysis revealed a potentiation of the TNF-induced necrosis by cyclophosphamide. Increased hyperemia and extravasation of erythrocytes could be found in tumors of animals treated with the drug combination.

Animals↗

Immunolocalization of cytoskeletal elements in human mammary epithelial cells.

The expression of cytokeratins, vimentin and actin was analysed by immunohistochemical methods in primary cultures derived from breast carcinomas and non-malignant breast tissues, in tissue sections and in permanent breast cancer cell lines. A polyclonal antibody specific for cytokeratins bound to the majority of cells in all primary cultures. Cells positive for simple-epithelial cytokeratins were less frequent in primary cultures derived from malignant tissues than in cultures derived from non-malignant tissues. Established cell lines MCF-7 and BT-20 expressed high amounts of simple-epithelial cytokeratins. All outgrowing cells in primary cultures expressed high amounts of vimentin whereas vimentin expression in established carcinoma lines was variable but generally low. When paraffin sections of the original mammary tissues were analysed, epithelial cells were devoid of vimentin as expected. Anti-actin antibody decorated fine fibres in all cells of primary cultures in contrast to the diffuse staining found in established mammary carcinoma cell lines.

Actins↗

An efficient method for the purification of tumor necrosis factor from rabbit serum.

Tumor necrosis factor (TNF) has been purified to homogeneity from rabbit serum. TNF was isolated by a purification scheme consisting of chromatography on DEAE-Trisacryl M, concentration on an Amicon YM10 membrane filter, molecular exclusion on TSK-3000 SWG (HPLC), and then either by Reversed phase HPLC on a Vydac C 4 column or by hydrophobic interaction chromatography. This procedure gives a recovery of 50% of pure TNF. The purified material has a specific activity of 2 X 10(8) U/mg in vitro and is active in vivo causing necrosis in Meth A sarcoma bearing mice. It gives a single band at a molecular weight of 17.000 on 15% polyacrylamide gel electrophoresis and 40.000 on gel exclusion chromatography at pH 7.0. The protein has a pI of 5.0 and is stable when heated to 70 degrees C for 10 minutes.

Animals↗

Mouse genital ridges in organ culture: the effects of temperature on maturation and experimental induction of teratocarcinogenesis.

Testicular teratomas can be induced experimentally by grafting genital ridges from male mouse fetuses to the testes of adults. A high incidence of teratomas occurs in genital ridges grafted to scrotal testes, but not in genital ridges grafted to testes maintained at body temperature. Genital ridges were cultured at 32 degrees C or 37 degrees C prior to grafting to the testes to determine the effect of temperature on the incidence of teratomas. Genital ridges cultured at 32 degrees C for two or three days produced a high incidence of teratomas when grafted to the testes, in contrast to genital ridges cultured at 37 degrees C for two or three days which produced a low incidence. Histologically, genital ridges cultured at 32 degrees C contained disorganized testicular tubules and were retarded in development. Genital ridges continued to develop in vitro at 37 degrees C, but were histologically different from genital ridges maturing in the fetus. Genital ridges cultured at 32 degrees C for 10 to 12 days did not develop teratomas in vitro or after grafting to the testes. Further characterization of temperature effects in vitro may lead to a better understanding of teratocarcinogenesis in vivo.

Animals↗

Experimental induction of testicular teratomas in dissociated-reaggregated chimaeric gonads.

Testicular teratomas can be experimentally induced in some strains of mice by grafting 12.5-day male genital ridges to the testes of adults. The grafts develop into testes and most of them have teratomas. The cells of 12.5-day foetal gonads were dissociated and the germ cells and somatic cells were separated. When germ cells were reaggregated with somatic cells and implanted in adult testes, they formed seminiferous tubules with teratomas. The somatic cell populations were contaminated with about 1% germ cells, and when they were implanted in adult testes, they formed testes with a comparatively low incidence of teratomas. When germ cells of a highly susceptible strain were combined with somatic cells from a resistant strain, they formed chimaeric testes with a high incidence of teratomas. When germ cells from a resistant strain were combined with somatic cells from a susceptible strain they formed chimaeric gonads and the incidence of teratomas was low. This indicates that at 12.5 days the genotype of the germ cells is responsible for susceptibility. When germ cells from older foetal gonads were combined with somatic cells of 12.5-day gonads, the incidence of teratomas was low. This showed that 12.5-day somatic cells cannot 'rejuvenate' older germ cells in a way to regain their susceptibility. When 12.5-day germ cells of highly susceptible strains were combined with older somatic cells the incidence of tumours was low indicating that the age of the somatic cells influences susceptibility to teratocarcinogenesis.

Animals↗

Sequence specific antisense oligonucleotide analog interference with spermidine/spermine N1-acetyltransferase gene expression.

Induction of the polyamine acetylating enzyme, spermidine/spermine N1-acetyltransferase (SSAT), is one of several biochemical effects associated with the antiproliferative action of polyamine analogs such as N1, N11 diethylnorspermine (DENSPM). Findings to date indicate that this complex and extremely potent gene response involves increased gene transcription, stabilization of mRNA, enhanced translation and protein stabilization. In this study, SSAT-directed antisense oligonucleotide analogs (AOs) were studied for their ability to prevent enzyme induction by DENSPM. Nine 18-mer fully phosphorothioate modified AOs targeting the start codon, exon 6, stop codon and polyadenylation regions of the human SSAT mRNA were synthesized and evaluated in MALME-3M human melanoma cells prior to and during a 6 hr treatment with 10 microM DENSPM. The most effective AOs were those targeting sequences in the stop codon region. Of these, AO-82 suppressed DENSPM induction of SSAT activity, enzyme protein and mRNA by 70-80%. The quantitative similarity of these effects suggests AO interference with mRNA stabilization, a property apparently mediated by sequences located in the stop codon region. Growth inhibition by DENSPM in the presence of the terminally phophorothioated analogs of AO-82 remained similar to that produced by DENSPM alone. While it is possible that SSAT induction may not be involved in analog-mediated antiproliferative activity, a more likely interpretation is that the approximately 50% suppression of the enzyme response achieved in growth studies is not sufficient to abrogate growth inhibition.

Acetyltransferases↗