FIGURES AND TABLES

Figure 1.

TR inhibits DNA synthesis in various cell types. DNA synthesis was measured by the incorporation of [³H]-thymidine into the cells, as described in Materials and Methods. TR at various concentrations was incubated with BCE, BSMC, BALB/MK, C6 rat glioma cells and with D122 cells as described in Materials and Methods. TR inhibited in a dose-dependent manner DNA synthesis in various cell types. Experiments were done in triplicates and inhibition was calculated as percentage of DNA synthesis of non-treated control.

Figure 2.

TR inhibits SOD-1 activity in vitro. Various concentrations of TR were incubated with 20 ng ml^-1 of human recombinant SOD-1. SOD-1 activity was measured as described in Materials and Methods and calculated as per cent from the activity of control non-treated 20 ng ml^-1 SOD-1. TR had markedly inhibited human recombinant SOD-1 enzymatic activity (85%). Experiments were repeated twice.

Figure 3.

Antioxidants prevent the inhibitory effect of TR on endothelial cells. N-acetylcysteine: NAC (3 mM), glutathione: Glut. (3 mM) and Tiron (5 mM) were co-incubated with 0.1 M of TR for 24 h and DNA synthesis was measured as described in Materials and Methods. The effect of antioxidants was calculated as per cent of DNA synthesis compared with that in control cells treated with antioxidants alone. Inhibition of DNA synthesis in BCE by TR was fully restored by co-incubation with antioxidants. DNA synthesis in control cells was about 50 000 c.p.m.

Figure 4.

Over-expression of SOD-1 in PC12 cells renders them more resistant to the inhibitory effect of TR. Parental non-transfected (open circle) and SOD-1-transfected (solid circle) PC12 cells were incubated in the presence of various concentrations of TR, and DNA synthesis was measured as described in Materials and Methods. The effect of TR was calculated as per cent of DNA synthesis compared with that in control cells not treated with TR. PC12 cells transfected with human SOD-1 were more resistant to the inhibitory effect of TR than parental non-transfected PC12 cells. DNA synthesis in PC12 non-treated cells was about 30 000 c.p.m.

Figure 5.

TR induces apoptosis in endothelial cells. BCE, BSMC, BALB/MK keratinocytes, C6 glioma cells and 3T3 fibroblasts following incubation with the indicated concentrations of TR for 6 h or for 20 h with BALB/MK, were analysed by the TUNEL method as described in Materials and Methods. Only the nuclei of treated BCE were labelled using the TUNEL staining method. The nuclei of treated BSMC, BALB/MK C6 glioma cells and 3T3 fibroblasts were not labelled. Experiments were repeated twice with triplicates.

Figure 6.

TR inhibits neovascularisation in CD1 mice. Agarose beads containing bFGF (10 g bead^-1) were implanted subcutaneously as described in Materials and Methods. (A) The angiogenic potential of bFGF in vivo is demonstrated 4 days after implantation in skin specimens. TR (13–30 g mouse^-1 day^-1) was introduced p.o. every day starting with bead implantation. TR inhibited almost completely in a dose dependent manner neovascularisation inside and around the bead. Bar is 1 mm. (B) Quantitative analysis of blood vessel density around and inside the beads. Quantitative analysis was done by use of NIH Image 1.61 software. TR at 13–30 g mouse^-1 day^-1 inhibited angiogenesis by 80–95% respectively (n=4).

Figure 7.

Histological sections of C6 tumours stained with Haematoxylin-Eosin and light green. C6 tumours from TR-treated animals are different from C6 tumours from control non-treated animals. Cells in tumours from control non-treated animals have a more pleomorphic appearance, with a bizarre form. Cell size and shape in tumours from TR-treated (TR 25 g) or (TR 120 g) animals are more uniform and are more differentiated (nuclei stain and shape). Magnification ( 120).

Figure 8.

TR reduces blood vessel number in C6 tumours. Histological sections of C6 glioma tumours were stained for Haematoxylin-Eosin and light green followed by staining with the endothelial-specific GSL lectin (dark blue staining) as described in Materials and Methods. The number of blood vessels per field in C6 tumours derived from mice treated with TR (25 g) was reduced by 33% compared with non-treated control (10.80.41 s.e. and 16.10.47 s.e., accordingly (P<0.0003).

Figure 9.

TR inhibits Lewis lung metastatic growth in the lungs. D122 tumour cells were injected into C57/BL mice and 3 days later TR (13–40 g mouse^-1) was introduced p.o. three times a week. Twenty-four days following tumour injection, the lungs were weighed. Metastatic growth in the lungs of TR-treated animals, compared with that in water-fed control was significantly smaller (P=0.019–0.05) (n=6). At 13–40 g mouse^-1 TR decreased metastatic growth in the lungs 5–3-fold accordingly. Experiments were repeated twice. The weight of lungs derived from normal mice was subtracted from that of the metastasised lungs. P by student's t-test.

Table 1 - Copper impedes the inhibitory effect of TR on BCE cells^a .