The Effect Of Melatonin On The Expression Of C-Myc And P53in The Murine Gastric Cancer

ObjectiveTo investigate the effect of melatonin on proliferation and apoptosis of murineforegastric carcinoma (MFC) cells, and further more to explore some probablemechanisms of melatonin-mediated of tumor cell’s apoptosis by analyzing theexpression change of c-Myc and p53in MFC cells and tumor tissues.Methods1. Mode of murine foregastric carcinoma cell MFC treated with melatonin wasestablished: murine foregastric carcinoma cell MFC was subcultured; The cells in theexponential phase were divided into different groups according to the differentconcentration of melatonin,2mM，4mM，6mM，8mM groups(24hours);2mM，4mM，6mM groups(48hours);2mM，4mM groups(72hours). The control group withoutmelatonin intervention was established at the same time. All the index of each groupwere detected after melatonin intervention of different hours.2. Animal models of gastric cancer was established by inoculatingsubcutaneously with5×105MFC cells under the right axilla of the615-(H-2Kk)mouse strain. One week after inoculation, the tumor-bearing mice models weresuccessfully established and the experimental animal groups were set up as follows:group A: tumor-bearing control mice with daily intraperitoneal (i.p.) injection of100mg/kg saline water; group B: tumor-bearing mice injected with25mg/kg melatonin;group C: tumor-bearing mice injected with50mg/kg melatonin; and group D:tumor-bearing mice injected with100mg/kg melatonin. Melatonin was given to theassigned groups at17:00hr every day for1week via i.p. injection3. Immunocytochemistry was used to detect the location of c-Myc and p53inmurine foregastric carcinoma cell MFC.4. Real-time fluorescence quantitative PCR was used to detect the expressionchange of c-Myc and p53mRNA in each group. 5. Western blot was used to detect the expression change of c-Myc and p53protein in each group.6. Immunohistochemical was used to detect the expression change of c-Myc andp53protein in each group.Results1. Morphological changes, growth inhibition and elevated apoptosis of MFCwere detected after intervention of different concentrations and times of melatonin.2. The results of Immunocytochemistry showed the expression of c-Myc and p53protein in murine foregastric carcinoma cell MFC.3. Real-time fluorescence quantitative PCR analyses demonstrated that c-MycmRNA expression in each melatonin treatmented group of MFC and tumor tissuesdecreased compared with the control group. Each group showed significantdifferences compared with the control group except the2mM(48h)group. While thep53mRNA expression of each melatonin treated group significantly increasedcompared with the control group.4. Western bolt results showed that c-Myc protein expression of each melatonintreatmented group of MFC and tumor tissues decreased significantly compared withthe control group except the low dose group. The p53protein of each melatonintreatmented group significantly decreased compared with the control group except2mM（24、48h）group、4mM（48h）group in MFC cells, while the p53protein of eachmelatonin treatmented group significantly increased compared with the control groupexcept the low dose group in tumer tissues.5. The results of immunohistochemistry showed that the average optical densityof c-Myc positive-protein in tumor tissues reduced in each melatonin treatmentedgroup compared with the control group, and the result of each group showedsignificant differences.The average optical density of p53positive-protein in tumortissues significantly increased in each melatonin treatmented group compared with thecontrol group, which consisted with the western bolt results. ConclusionAfter melatonin intervention of24、48、72hours, growth inhibition and elevatedapoptosis of MFC cells were detected, which showed in a dose-dependent manner. Atthe same time, the transcription, translation and synthesis levels of c-Myc and p53gene changed in varying degrees, which also showed in a probable dose-dependentmanner. The changes in tumor tissues were similar with the MFC cells. This indictedthat melatonin could down-regulate the expression of c-Myc and up-regulate theexpression of p53, which might be one of the mechanisms of melatonin anti-cancereffect.