Experimental Investigation On Melatonin's Actions Of Antimutation And Antitumor In Vitro

Objective: Melatonin, chemically named N-acetyl-5-methoxy-tryptamine, is a kind of indolamine hormone mainly secreted by pineal gland.lt has extensive roles to induce natural sleep, postpone senescence and increase immunity.Recent years,people have been more and more paying close attention to its actions of tumor prevention,tumor treatment and postponing cancer progression.In this article we not only observe melatonin's actions of antimutation and antitumor respectively by Ames test and tumor cells culture but also try to discuss those principles tentatively.Method: (1) Mutagenic tests of Mel: The Ames test was used(plate incorporation method).Select two tester strains:TA98 and TA100,use 0. Iml/dish DMSO as negative control, add 5000 y g, 500 vg^ 50 u g% 5 n g, 0. 5 u g Mel respectively in every dish and lay three parallel samples each dosage group. Observed backward mutated-colony numbers of different dosage groups after cultured for 48 to 72 hours at 37 C.A11 tests were repeatedly made twice regardless of adding S9 or not adding S9. (2)Antimutagenic tests of Mel: The Ames test was used(plate incorporation method).When not adding S9, we use 12. 5y g/dish Dexon and 0.25 u g/dish sodium agide as positive control of TA98 and TA100 respectively.Both TA98 and TA100 use 0. Iml/dish DMSO as negative control. Add 5000u g, 500 ug, 50pg, 5ug, 0. 5u g Mel respectively per dish and lay three parallel samples each dosage group. Observe colony numbers of different dosage groups after cultured for 48 to 72 hours at 37C.A11 tests were repeatedly done two times. When adding S9, bothTA98 and TA100 use 2-AF of 10 y g/dish as their positive concrol with the rest procedures identical with the ones when not adding S9. (3) Employ in vitro cultured human breast cancer cell line MCF-7 cells to do cytotoxicity test,observe in different dosage groups the growth status of tumor cells (which involve cell mitotic index,cell anchoring rate,cell growth curve,dye exclusion test,plate clone forming test,and microscopic evaluation of apoptotic cells) and detect the expressions of tumor related genes:bcl-2 and p53 immunohistochemically.Result: (1) Mutagenic tests of Mel: Adding or not adding S9, there is no significant differences of the backward mutated colony numbers between different dosage groups and negative contro(P0. 05).(2) Antirautagenic tests of Mel: Adding or not adding S9, the colony numbers of different dosage groups are lower than positive concrol (KO. 05) . (3) Cytotoxicity test:The IC?of Mel is 0. 89mmol/L and the I CM 2. 38mmol/L. (4) Cell mitotic index:When cultured for 48 hours,the MI of negative control group and ICM group have no significant differences (P>0. 05) . Both MI of these two groups are higher than that of ICso group (KO. 05) . When cultured for 96 hours,the MI of negative control group, IC30 group and ICso group are all mutually significantly different (/KO. 05) .And there is a tendency that the MI of different dosage groups decrease with the increase of melatonin's dosage.(5) Cell anchoring rate: When cultured for 4 hours,the cell anchoring rates in IC30 group and ICso group are identical (?0.05) .Both are lower than 5%DMSO group (K0.05) . When cultured for 8 hours, significant differences of cell anchoring rates in different groups are not shown (P>Q. 05) . When cultured for 16 hours, various groups' cell anchoring rates are significantly different (7^0.05) .The 5%DMSO group' rate is less than the ICso group' and the ICy, group' less than the ICso group' (KO. 05) .It seems as if there is a trend of dose-effect relationship between Mel'dosage and cell anchoring rate.(6) Cell growth curve:Mel of ICso elongate the doubling time(TD) of MCF-7 cells from 3. 51 days in control group to 5. 75 days in ICso group. (7) dye exclusion test: There is significant differences of cell survival rates among all Mel dosage groups (P<0. 05) . The rate in higher dosage group is less than that in lower one (P<0. 05) . (8) Plate clone forming test:When inoculating 50,100 and 200 cells in every plate respectively,the numbers of clo...