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Yoshiyuki Mizushina, Norihisa Kato, Hiromi Yoshida et al.
HUVEC on reconstituted basement membrane migrated, attached to each other and formed tube structures. PLP and PL did not affect HUVEC tube formation on the reconstituted basement membrane at the concentration at which they strongly inhibited HUVEC proliferation (Figure 4); thus, vitamin B6 would have no effect on such HUVEC functions. Table 4. LD50 values of vitamin B6 compounds on HUVEC proliferation Vitamin B6 compound PL PN PM PLP
LD50 values (μM) 53.9 >1000 >1000 112
HUVEC was purchased from Kurabo Industries (Osaka, Japan). HUVEC was dispersed with trypsin and suspended in HuMedia EG2 medium. A cell suspension (15,000 cell/ml) was plated onto 6-well culture plates (2 ml/well), and incubated at 37 °C in a humidified 5 % CO2 for 24 h. The medium was replaced with fresh HuMedia EG2 containing vitamin B6 compounds. After 72 h, cells were dispersed with trypsin, suspended in the medium, and counted.
Figure 4. Effect of PL and PLP on HUVEC tube formation on reconstituted basement membrane gel. (A) control, (B) 250 μM PLP, and (C) 250 μM PL. Tube formation assay was performed using an In Vitro Angiogenesis Assay Kit (Chemicon International, Inc., Temecula, CA, U.S.A.). Cells were plated on reconstituted gel and observed 12 h later. Tube formation was observed under an inverted light microscope at 40 X magnification.
6. EFFECTS OF PL AND PLP ON CULTURED HUMAN CANCER CELLS To investigate the anti-cancer effects of vitamin B6 compounds, human epitheloid carcinoma of the cervix cell line HeLa was tested. Cell viability was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay [20]. Cell growth inhibition dose-curves are shown in Figure 5. These results indicated that PL had potent cell proliferation inhibitory effects on this cancer cell line with an LD50 value of 224 μM. Surprisingly, none of the PLP tested showed such an inhibitory effect. PLP was more