Alterations in F-actin distribution in cells treated with melatonin.

One of the possible pathways of action of melatonin is its effect on the cytoskeleton. In this work we looked for alterations in the cytoskeleton of cells treated with melatonin at physiological concentrations. T-47D, Hs-578T (human breast carcinoma cell lines), and MDCK (normal dog kidney) cells were maintained in MCDB 153 supplemented with 1% fetal bovine serum (FBS), or in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% FBS and treated with melatonin (10(-9) M or 10(-10) M) for 2 and 5 days, with or without 10(-8) M estradiol. F-actin was stained with phalloidin-fluorescein isothiocyanate (FITC). Cytokeratin 19 and beta-tubulin filaments were detected with specific monoclonal antibodies and secondary antibodies bound to FITC. Melatonin-treated T-47D cells observed in a transmission electronic microscope (TEM) showed an irregular nuclear shape and intermediate filaments disposed around the nucleus, which was not observed in control cells. Immunofluorescence analysis of cytokeratin filaments did not show significant differences between their distribution in control and treated cells. Melatonin did not induce significant alterations in cytokeratin filaments of T-47D, Hs578T or MDCK cells in DMEM and MCDB 153, or T-47D cells in DMEM. Melatonin induced the derangement of F-actin both in T-47D and MDCK cells kept in MCDB 153. The same was not observed when estradiol was also present. We did not observe significant alterations in the distribution of F-actin in T-47D or Hs-578T cells grown in DMEM. In DMEM, melatonin-treated MDCK cells were more elongated, with a slight concentration of F-actin on the cell boundary. Melatonin induced very slight alterations in microtubule organization of all cell lines studied.

Cytotoxic activity of a methanol extract of Phallusia nigra (Tunicata, Ascidiacea).

Tunicates have been reported to be a rich source of biologically active compounds. In this study, we demonstrate the presence of cytotoxic substances in Phallusia nigra, a common tunicate from Brazilian coastal waters. An extract of tunicate tissue was obtained by homogenizing the visceral organs from 50 specimens in methanol, followed by filtration and concentration in a rotary vacuum evaporator. Finally, the concentrate was partitioned with chloroform to remove lipids. The resulting extract possessed antimitotic and hemolytic activity. The former was demonstrated as a delay in the development of sea urchin eggs by partially inhibiting the process of cleavage (first cleavage, EC50 +/- SEM = 3.44 +/- 0.84 mg/ml). The < 500 molecular fraction of the extract obtained by ultrafiltration also inhibited cell proliferation (the number of viable cells was decreased by 68% with 500 micrograms/ml) and DNA synthesis of T47D cells derived from human breast carcinoma as measured by [3H]-thymidine incorporation (66% of the control value after 24-h incubation with 100 micrograms/ml). Dose-dependent hemolysis obtained with P. nigra extract on mouse erythrocytes had an EC50 +/- SEM = 1.12 +/- 0.02 mg/ml for a 0.5% erythrocyte suspension. Hemolysis could be reduced by pre-incubating the cells with choline-containing phospholipid. Sphingomyelin (40 micrograms/ml) increased the EC50 by two-fold to 2.86 +/- 0.04 mg/ml, but phosphatidylcholine (80 micrograms/ml) did not modify hemolysis.

Cytotoxic activity of a methanol extract of Phallusia nigra (Tunicata, Ascidiacea)

Tunicates have been reported to be a rich source of biologically active compounds. In this study, we demonstrate the presence of cytotoxic substances in Phallusia nigra, a common tunicate from Brazilian coastal waters. An extract of tunicate tissue was obtained by homogenizing the visceral organs from 50 specimens in methanol, followed by filtration and concentration in a rotary vacuum evaporator. Finally, the concentrate was partitioned with chloroform to remove lipids. The resulting extract possessed antimitotic and hemolytic activity. The former was demonstrated as a delay in the development of sea urchin eggs by partially inhibiting the process of cleavage (first cleavage, EC50 ñ SEM = 3.44 ñ 0.84 mg/ml). The <500 molecular fraction of the extract obtained by ultrafiltration also inhibited cell proliferation (the number of viable cells was decreased by 68 per cent with 500 mug/ml) and DNA synthesis of T47D cells derived from human breast carcinoma as measured by [3H]-thymidine incorporation (66 per cent of the control value after 24-h incubation with 100 mug/ml). Dose-dependent hemolysis obtained with P. nigra extract on mouse erythrocytes had an EC50 ñ SEM = 1.12 ñ 0.02 mglml for a 0.5 per cent erythrocyte suspension. Hemolysis could be reduced by pre-incubating the cells with choline-containing phospholipid. Sphingomyelin (40 mug/ml) increased the EC50 by twofold to 2.86 ñ 0.04 mg/ml, but phosphatidylcholine (80 mug/ml) did not modify hemolysis.