I. Núñez de Castro

Determination of 27 dansyl amino acid derivatives in biological fluids by reversed-phase high-performance liquid chromatography

The concentrations of free amino acids in plasma and in ascitic liquid of mice with Ehrlich ascitic tumours were determined by reversed-phase high-performance liquid chromatography using pre-column derivatization with Dns chloride and UV detection at 254 nm. Sample preparation is simple, and the Dns derivatives are stable. Complete separation of 27 amino acids, including proline and cysteine, was achieved in 70 min with detection limits of less than 25 pmol. There was no interference from Dns-Cl, Dns-OH and Dns-NH2. Retention time reproducibility was better than 1%. The described method enables a rapid, economical and reproducible quantification of free amino acids in biological fluids.

Increased histidine decarboxylase (HDC) activity in human colorectal cancer: Results of a study on ten patients

Following numerous reports of high histidine decarboxylase (HDC) activity in tumour-bearing animals, the present work was designed to determine the activity of this enzyme in normal and tumour tissues in a series of ten surgical patients with colorectal carcinoma.Significantly increased HDC activity, almost double that of normal tissues, was found in specimens from extirpated human tumours. These results, obtained under reliable sampling and measurement conditions indicated that changes in the enzymic activity of HDC may have a significant role in the development of colorectal tumour cells. Inhibition of the enzyme activity in human cancer subjects may retard or impede tumour growth and perhaps limit the spread of metastases.

Chlorpheniramine inhibits the ornithine decarboxylase induction of Ehrlich carcinoma growing in vivo

The antihistaminic (±)‐chlorpheniramine significantly reduced the progression of Ehrlich carcinoma when it was administered at 0.5 mg/mouse/day from the third day on, after tumour inoculation. The ODC activity of tumour cells was diminished by 70% on day 7 after tumour transplantation, when maximum ODC activity is detected in non‐treated tumour growing ‘in vivo’. Northern blot analyses indicated that the inhibitory effect of this 1,4‐diamine takes place at a post‐transcriptional level. Results obtained from serum‐free cultured cells indicated that chlorpheniramine inhibits the ODC synthesis rate.

A comparative study of the effects of genistein and 2-methoxyestradiol on the proteolytic balance and tumour cell proliferation.

The cytotoxicity of two compounds described as anti-angiogenic, the isoflavone genistein and the oestrogen metabolite 2-methoxyestradiol, has been studied in different human tumour cell lines. Since the degradation of the extracellular matrix is one of the essential steps in angiogenesis, the potential modulatory effects of both compounds on the proteolytic balance in media conditioned by different human tumour cells have been also investigated. The IC50 values for 2-methoxyestradiol were lower than those for genistein on all the cell lines tested. In all the cell lines expressing measurable amounts of active enzymes, genistein induced a shift towards antiproteolysis in both matrix metalloproteinase/tissue inhibitor of metalloproteinase and urokinase/plasminogen activator inhibitor proteolytic balances. On the other hand, 2-methoxyestradiol did not produce any clear net shift of the proteolytic balance, with the significant exception of the matrix metalloproteinase/tissue inhibitor of metalloproteinase balance in WAC-2 cells, a neuroblastoma cell line with enhanced expression of the N-myc oncogene.

Simultaneous fluoremetric determination of intracellular polyamines separated by reversed-phase high-performance liquid chromatography

A reversed-phase HPLC technique in combination with fluorescent detection is described for simulataneous quantification of the precolumn Dansyl derivatives of intracellular amines. The derivatives were stable for at least one week, kept protected from the light at −20 °C. The detection limit was between 1 and 5 pmol for all tested polyamines. Serotonin coeluted with tryptamine. The method has a very good reproducibility for both, retention times and chromatographic peak areas. The average recovery of standard amine solutions added to cellular extracts was estimated to be higher than 90%. The described method enables a rapid, reliable and reproducible quantification of biogenic and related polyamines in biological fluids and tissues.

Changes in histamine synthesis, tissue content and catabolism in human breast cancer

The present study in 10 breast cancer patients supports the concept that newly synthetized, nascent histamine is involved in tumour growth. Histidine decarboxylase (HDC) activity is increased in mammary tumour tissue compared to healthy mammary gland-, skin- and muscle tissue in all but one patient studied. The newly formed histamine is probably not stored in the tumor tissue. Significantly decreased histamine concentrations were measured in parallel samples in the tumour tissue. Moreover, the preliminary results from urinary analysis of histamine and Nτ-methylhistamine in 3 of the 10 patients studied showed a significant decline after tumour extirpation compared to preoperative values.

Polyamine contents of human breast cancer cells treated with the cytotoxic agents chlorpheniramine and dehydrodidemnin B

The cytotoxic agents chlorpheniramine and dehydrodidemnin B decreased the cell growth of estrogen receptor-negative human breast cancer cells MDA-MB231 and estrogen receptor-positive MCF-7, after 48 h treatment. Both agents reduced ornithine decarboxylase activity, but polyamine levels were increased in MDA-MB231 cells treated with dehydrodidemnin B. MCF-7 cells when treated with dehydrodidemnin B showed significant increases in spermidine and spermine contents. The results suggest that besides other effects, the cytotoxicity of DDB could be explained in part by the over-accumulation of spermidine and spermine.

Mouse liver free amino acids during the development of Ehrlich ascites tumour

Sequential amino acid concentrations were determined in the liver of mice infested with a highly malignant strain of Ehrlich ascites tumour cells. The liver concentrations of a certain group of amino acids showed changes consistent with those previously reported for plasma, ascitic liquid and tumour cells during tumour growth. Shortly after tumour transplantation a significant decrease of the essential amino acids methionine, threonine, valine, isoleucine + phenylalanine, leucine, lysine and histidine, was detected. Some non-essential amino acids, mainly the gluconeogenic substrates alanine and serine, showed a strong reduction in hepatic concentrations during the first days; these amino acids remained significantly lower than controls until animal death. Interestingly, hepatic glutamine increased at days 1 and 2 after inoculation, and proline showed a sustained increase from the seventh day onwards, reaching a value double the control at the end of animal life.

Histamine and serotonin inhibit induction of ornithine decarboxylase by ornithine in perifused Ehrlich ascites tumour cells

Ornithine induced more than 36‐fold the ornithine decarboxylase activity in confined Ehrlich ascites tumour cells after 3.5 h of continuous perifusion with 0.5 mM ornithine; arginine and glutamine also induced the activity 3‐ and 4‐fold, respectively. The addition of cycloheximide or actinomycin D antibiotics to the perifusion medium confirmed that the regulation of the enzyme synthesis takes place at the level of translation. Perifusion in the presence of 0.5. mM ornithine and 55, 25, and 10 μM histamine suppressed the induction by 91, 53, and 35%, respectively. Similar results were obtained in the presence of serotonin. Histidine also showed inhibitory effect but 5 mM histidine was required to produce 21% inhibition; other basic amino acids were ineffective.

Plasma membrane redox system activity in tumour cell lines of mammary origins with different proliferation rates

SummaryPlasma membrane redox systems seem to play a role in the control of cell growth. In fact, we have found that in mammary tumour cell lines the increase in the proliferation rate is accompanied by a decrease in the plasma membrane redox activity. The oxygen consumption rates, the glycolytic fluxes and other bioenergetic parameters have been studied in two cell strains of Ehrlich ascites tumour with different proliferation rates. In the more proliferative Ehrlich cell strain, the decrease in plasma membrane redox system activity is accompanied by decreased oxygen consumption and glycolytic flux and to a generally less energised status.