| Vitamin B6, one of the B vitamers, is an essential water soluble vitamin required for normal growth and development. Pyridoxine (PN), pyridoxal (PL) , pyridoxamine (PM) as well as their phosphated forms are structural analogues in which pyridoxal-5'-phosphate(PLP) and pyridoxamine-5'- phosphate (PMP) are the biological active forms in vivo which act as the cofactors of aminotransferases, amino acid decarboxylases, cysteine desufbrase and cystathionine , etc by constant transconversions. Recently, it was discovered that B6 played a novel role in tumor growth suppression. Severe deficiency of the vitamin may impair cell-mediated-immunity and stimulate tumor growth by activating gene transcription, while megadose of B6 could improve the celluar-imunity against development of neoplasms. It was elucidated that large doses of vitamin B6 may kill a number of neoplasm cell lines in vivo or in vitro. All those above suggest that there is a close relationship between tumor metabolizing and the B6 vitamer, which has now been regarded as an effective bioregulator. In order to discuss the potential mechanism by which B6 arrested tumor growth, we performed a series of experiments both from cell level and molecular level using H22 murinehepatoma and human SMMC-7721 hepatoma cell lines as the in vitro models. All our work was to provide some sound experimental proves for farther therapeutic use of vitamin B6. Methods and results are as follows:1. Cytotoxic effects of 14 kinds of chemotherapeutic drugs and PN on H22 hepatoma and SMMC-7721 human hepatoma cell lines: Tumor cells grown in DMEM supplemented with 15% inactivated PCS, 100U.ml-1 penicillin and lOOug/ml streptomycin were plated in 96 well culture plates with a density of 2X 104.0.2ml-1. After incubation in situ with different drugs with diversed doses in the medium at 37, 5%CO2 for different periods, cytotoxicities were assessed using MTT assay, while optical densities were read at 570nm. As a result, there were 6 among the 14 drugs inhibited cell proliferation and so did 11 of them to SMMC-7721 hepatoma. Only eADM, Ara-c and ICTX were effective to both of the cell lines. The H22 hepatoma subjected to 2,4and 6mmol-L-1 of pyridoxine exerted inhibition rates of 26.5%, 45.1% and 55.6% respectively, while the SMMC-7721 hepatoma expressed its growth inhibition by 29.7%, 31.1% and 55.3% separately. The results implied that tumors with similar histological classification react differently to certain drugs, let alone the diversed genera. However, the similar effects of PN indicated that the drug might have broad spectrum of anti-neoplasm potence.2. Dose and time effects of PN on H22 hepatoma cell line:Cytotoxic effects were tested after 24, 48 and 72 hours' incubation with 2, 3, 4 and 6mmol.L-1 PN in the culture medium mentioned before. The results suggested that the drug was sensitive to H22 cells from the dose of 3mmol/L and the IC50 proved to be 5mmol.L-1 ,which led to about 50% retardance of cell growth. A regression analyse indicating a positive correlation between drug doses and cytotoxicities according to a 48 hours' incubation, which implied that the drug effects were dose and time dependent. 3. Changes in the intracellular GST activities and MDA contents as well as activities of certain aminotransferases :Cells were collected after 24 hours' incubation with or without 5mmol/L PN in the culture medium, washed twice with pH7.4 PB supplemented with 20mmol.L-12-ME. Then the cell pellets were subjected to an ultrasonic comminution followed by a 10000rpm centrifugation at 4 for 30min. The supertanent was used separately for the analyses of GST activities and MDA contents using a Nanjing JianCheng kit and of aminotransferase activities using an autobiochemical analytic system. The results suggested that GST activities were higher in trial groups than in control ones(P < 0.002), while the control groups had higher MDA contents than the trial groups(P < 0.05). The results implied that PN treatment could elevate intrace... |
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