| OBJECTIVE1. Via establish solid tumor model of S180 tumor-bearing mice, to observe the anti-tumor effects of melatonin and selenium yeast treatment, the expressions of cyclin-dependent kinase 2(CDK2), p53, p21 and apoptosis in S180 sarcoma cells. To find whether the anti-tumor effect can be improved or not when the melatonin combined with selenium.2. Via establish ascites type tumor model of S180 tumor-bearing mice, to observe the effects of melatonin and selenium yeast treatment on the survival time.METHODS 1. The tumor cells were inoculated subcutaneously on the right axilla of male mice. And the mice were randomized into seven groups: Model group; Se 100μg·kg-1 group; Se 200μg·kg-1 group; MT 40 mg·kg-1 group; MT 80 mg·kg-1 group; MT 40 mg·kg-1 + Se 100μg·kg-1 group; Cyclophosphamide (CTX) group. Then, each group will give the corresponding drugs. After ten days, the mice were euthanized. The expressions of CDK2, p53, p21 in the tumor tissues were examined by Immunohistochemistry and Western blotting. The tumor cells apoptosis was observed by TUNEL and DNA Ladder.2. The tumor cells were inoculated in the abdominal cavity of male mice. And the mice were randomized into seven groups: Model group; Se 100μg·kg-1 group; Se 200μg·kg-1 group; MT 40 mg·kg-1 group; MT 80 mg·kg-1 group; MT + Se group; CTX group. Then, each group will give the corresponding drugs until the mice dead and recorded the survival time.RESULTS1.1 Effects of melatonin and selenium yeast treatment on the tumor inhibiting rate and the weight of tumorsSe (100, 200μg·kg-1), MT (40, 80 mg·kg-1), MT + Se, CTX can inhibit tumor growth. The tumor inhibiting rate of every group exceeded 30%: 43.51%, 66.91%, 38.40%, 61.30%, 60.42%, 81.05%, and compared to the model group, the average weight of tumors were significantly reduced in intervening groups (P<0.01). Melatonin in combination with selenium treatment significantly reduced the average weight of tumors in comparison with Se 100μg·kg-1 group (P<0.05) and MT 40 mg·kg-1 group (P<0.01).1.2 Effects of melatonin and selenium yeast treatment on the expressions of CDK2, p53 and p21 in S180 sarcoma cells by immunohistochemistryCompared to the model group, the expression of CDK2 in Se (100, 200μg·kg-1), MT (40, 80 mg·kg-1), MT + Se, CTX groups significantly decreased (P<0.01). The expression of CDK2 in the MT + Se group is lower than the Se 100μg·kg-1 group (P<0.05) or MT 40 mg·kg-1 group (P<0.01).Compared to the model group, the expression of p53 in Se 200μg·kg-1, MT 80 mg·kg-1, MT + Se groups significantly increased (P<0.01). The expression of p53 in the MT + Se group is higher than the Se 100μg·kg-1 group (P<0.05) or MT 40 mg·kg-1 group (P<0.05).Compared to the model group, the expression of p21 in Se 200μg·kg-1, MT 80 mg·kg-1, MT + Se groups significantly increased (P<0.01). The expression of p21 in the MT + Se group is higher than the Se 100μg·kg-1 group (P<0.01) or MT 40 mg·kg-1 group (P<0.01).1.3 Effects of melatonin and selenium yeast treatment on the expressions of CDK2 and p53 in S180 sarcoma cells by Western blottingCompared to the model group, the expression of CDK2 in Se 200μg·kg-1, MT 80 mg·kg-1, MT + Se groups significantly decreased (P<0.01), the expression of p53 significantly increased (P<0.01); Compared to Se 100μg·kg-1 group or MT 40 mg·kg-1 group, the expression of CDK2 significantly decreased (P<0.01), the expression of p53 significantly increased (P<0.01);1.4 Effects of melatonin and selenium yeast treatment on DNA Ladder in S180 sarcoma cellsDNA fragmentations were observed in Se 200μg·kg-1 group, MT 80 mg·kg-1 group and MT + Se group. But DNA fragmentations were not observed in model group, Se 100μg·kg-1 group and MT 40 mg·kg-1 group. 1.5 Effects of melatonin and selenium yeast treatment on apoptosis in S180 sarcoma cells by TUNELThere was no apoptosis in model group. But apoptotic cells appeared in Se (100, 200μg·kg-1), MT (40, 80 mg·kg-1), MT + Se, CTX groups. And the apoptotic cells in Se 200μg·kg-1 group, MT 80 mg·kg-1 group and MT + Se group significantly more than Se 100μg·kg-1 group or MT 40 mg·kg-1 group.1.6 Effects of melatonin in combination with selenium yeast treatment on pathology of tumor.The necrosis did not observed in the edge of tumor tissue in model group. But in intervening groups, the different degrees of necrosis were observed in the edge of tumor tissue. Compared to the Se 100μg·kg-1 group and MT 40 mg·kg-1 group, the necrosis range of MT + Se group was greater.2 Effects of melatonin in combination with selenium yeast treatment on the survival time.The rates of prolonged life in every intervening group were 10.42%, 11.58%, 15.44%, 20.85%, 10.04%, 32.43%. But all the rates of prolonged life in intervening groups were not more than 30% except the CTX group.CONCLUSIONS1. The S180 sarcoma cells were inoculated into the right axilla of the mice by sc. Injection. Then, it was treated with MT and Se. The tumor inhibiting rate of every group exceeded 30%. The tumor weight of every group reduced, and the effect increased with dose. Both MT and Se have antitumor effect. Compare to the same dose of single agent, the antitumor effect of MT combined with Se was more evident. Although MT and Se can prolong the survival time of ascites type S180 tumor-bearing mice, but there was not statistically significant.2. Both MT and Se can significantly reduce the expression level of CDK2 and increase the expression level of wild-type p53 and p21. The effect of reducing the the expression level of CDK2, increasing the expression level of wild-type p53 and p21 was more obvious when MT combined with Se.These induced that MT and Se exerted antitumor effect via arresting cell cycle.3. The number of apoptotic cells of each treatment group significantly increased with the dose. DNA fragmentations were observed in Se 200μg·kg-1 group, MT 80 mg·kg-1 group and MT + Se group. Compare to the same dose of single agent, the number of apoptotic cells significantly increased and DNA fragmentation was more evident when MT combined with Se. Pathology also showed that compare to the same dose of single agent, the degrees of necrosis which was observed in the edge of tumor tissue significantly increased. All these suggest that inducing apoptosis of tumor cells is one of antitumor mechanisms of MT and Se.
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