Study On The Effect Of Melatonin On Adriamycin In Treating Breast Cancer In Vitro And In Vivo

Breast cancer is one of the most common malignant diseases.Now the morbidity is first and the mortality is second in female malignant tumor in world.According to statistics in 2007,there are 79,000 new cases per year, which is 31%of total number of female malignant tumor.Every year,the average morbidity of breast cancer is 30.30/100,000,and the population standardized rate is 32.97/100,000 in world.60%of new cases are in developed country.Even though the diversity of morbidity is great,the tendency is climbing,the age of onset is promoting younger staff.As one of the developing country,we are not exception.Breast cancer has become the most common malignant diseases in woman,and the number one of killer to female.Thus it is the immense problem to increase therapeutic effect and cure rate.The combined therapy mode including operation,chemotherapy, radiotherapy and endocrine therapy has become the standard one.With the establishment of concept that breast cancer is a kind of systemic disease, people pay more and more attention to the chemotherapy.In NCCN guide of 2008 edition,most of the content involve in chemotherapy.Adriamycin,as one member of anthracene nucleus cytotoxic drug,plays the indispensable part in treating breast cancer.which exerts important role in ensuring effect,improving prognosis and decreasing recurrence especially for high risk ones.however we have to face two questions:(1) As one of the first line drugs,the drug-resistance of adriamycin,regardless of natural or acquiered one,has a direct influence on its application.In spite of many study on the mechanism of drug-resistance,there are no drugs to reverse it and be used in clinical generally.(2) cardiotoxicity is the dose-restricted toxicity of adrimycin,which has a great effect on the general application:many patients suffering from heart disease have to give up it,and greater dose intensity become impossible.Also the inreversible damage of heart caused by adriamycin has a bad influence on patients' quality of life,which seems more urgent today.Melatonin(MLT) has been proven to counteract chemotherapy toxicity such as:thrombocytopenia,immunosuppression,insomnia and anxiety,to significantly improve the quality of life with advanced solid tumor patients in poor clinical status.The organ protection is also one of the hot topic,especially that melatonin can abate the cardiac damage caused by adriamycin,which will make for expanding application of them.Besides there are reports that melatonin maybe improve the curative effect of cytotoxic drugs such as cisplatin and flurouracil,and clinical research has proved that the therapic effect in chemotherapy plus melatonin group is better than that of common chemotherapy group.In view of above,we do the experiment which investigate the effect of melatonin on adriamycin from molecule,cell and in vivo level.Partâ… :Study on effect and mechanism of melatonin on antitumor effect of adriamyeinObjective:To study the inhibition of melatonin on MCF-7(ER+) and MDA-MB-231(ER-),as well as the effect and mechanism of melatonin on antitumor role of adriamycin.Methods:(1) MTT was applied to check the inhibition of different concentrations of melatonin and adriamycin,and observed the effect of physiological and pharmacological concentrations of melatonin on antitumous effect of adriamycin.(2) FCM was used to detect the cell cycle distribution and apoptosis of different groups.(3) Western blotting was utilized to detect the expression of P53 and bcl-2 protein of groups.Results:(1) MTTmethod showed that melatonin have antiproliferation effect on MCF-7 and MDA-MB-231 by dose and time dependence.The IC50 value of MCF-7 and MDA-MB-231 for adrimycin were 0.62ug/ml and 1.00ug/ml,and there were significant difference between them.The IC50 value dropped to 0.59ug/ml and 0.42ug/ml while these cells were incubated respectively with physiological concentrations(10^-9mol/l) and pharmacological(10^-5mol/l) concentrations of melatonin,resulted in no significant difference existing between before and after cultured with Physiological concentrations(10^-9mol/l) of melatonin,but significant difference be observed with pharmacological(10-⁵mol/1) concentrations of melatonin.The IC50 value of MDA-MB-231 for adriamycin was 1.00ug/ml, They dropped to 0.85ug/ml and 0.47ug/ml when the cells were incubated respectively with Physiological concentrations(10^-9mol/l) and pharmacological(10^-5mol/l) concentrations of melatonin,the difference of the former was not significant and that of the latter was significant compared with the IC50 before being cultured.(2) The results of FCM:Physiological concentrations of melatonin has significant G0/G1 cell cycle block and apoptosis effect on cell line MCF-7 by dose dependence(P＜0.05).The effect of same concentrations was not so clearly for MDA-MB-231(P＞0.05),that of pharmacological concentration was significant.Adriamycin exerts its antiproliferative and apoptosis promotion effects on two cell lines.The proliferation index(PI) kept dropping with the melatonin concentrations(P＜0.05),but the apoptosis rate changed little.Compared to the same concentrations of adriamycine,the PI of MCF-7 was decreased(P＜0.05) and the apoptosis rate was not increased (P＞0.05) by physiological concentrations of melatonin plus adriamycin,on the other hand,the PI was decreased and the apoptosis rate was increased by pharmacological concentrations of melatonin plus adriamycin.For MDA-MB-231 cell line,the same results were observed only at high concentrations adriamycin plus physiological concentrations of melatonin,and the PI was not changed(P＞0.05) and apoptosis index was increased(P＜0.05) by pharmacological concentrations of melatonin plus low concentrations of adriamycin,the PI was significantly decreased with the adriamycin(P＜0.05),and the apoptosis index was not(P＞0.05).(3) The two cell lines showed p53 low expression and bcl-2 high expression.For MCF-7,physiological concentrations of melatonin could increase p53 and decrease bcl-2 expression by dose dependence(P＜0.05) For MDA-MB-231,physiological concentrations of melatonin has no effect on two protein,Pharmacological concentrations of melatonin upregulated p53 and downregulated bcl-2 of two cell lines,but there were no significant difference between melatonin plus adriamycin and melatonin groups(P＞0.05). Adramycin has no effect on the expression of two protein(P＞0.05).Conclusion:(1) melatonin inhibit proliferation of ER+ and ER-breast cancer cell lines by dose dependent in.The diffrence of sensitiveness degree shows that there is other mechanism except for estrogen passageway.(2) physiological concentrations of melatonin has no significantly effect on the anticancer action of adriamycin,pharmacological concentrations of melatonin or higher concentrations showed sensitization for adriamycin.(3) Apoptosis was one part of the sensitization of melatonin at low concentrations of adriamycin,the cytotoxic sensitization become more important at high concentration level.(4) Pharmacological concentrations of melatonin increased p53 and decrease bcl-2 protein expression of breast cancer cells. p53 and bcl-2 passageway are at least one part of apoptosis mechanism.The sensitization of melatonin for high concentrations of adriamycin may not involve in p53 and bcl-2 expression.Partâ…¡:the reverse effect and mechanism of melatonin on breast carcinoma cell line MCF-7/ADM resistant to adriamycinObjective:To investigate the reverse effect and the mechanism of physiological(10^-9mol/1) and pharmacological(≥10^-5mol/l) concentrations of melatonin on cell line MCF-7/ADM or adriamycin before resistant to sdriamycin.Method:MTT was applied to test the changes in IC50 value of cell line MCF-7/ADM or adriamycin before and after incubated with melatonin. Electron microscope was used to observe the changes in morphology and ultrastructures in the cell line MCF-7/ADM incubated only with adriamycin (20ug/ml) and the cells incubated with pharmacological concentrations of melatonin(10-5mol/l) plus adriamycin(20ug/ml).Spectrophotometry was applied to determine the level of intracellular GSH in cell line MCF-7/ADM and MCF-7/s,as well as the variation of intracellular GSH in two cell lines be incubated with different concentrations of melatonin.Results:MTT method showed that the IC50 value of cell line MCF-7/ADM for adriamycin before culturing with melatonin was 24.41ug/ml and the IC50 of cell line MCF-7/ADM for adriamycin incubated with physiological concentrations of melatonin(10^-9mol/L) was 20.92ug/ml,with no significant difference.The IC50 value were 12.25ug/ml and 10.46ug/ml about the cell line MCF-7/ADM cultured with 10^-5mol/L and 10^-3mol/L melatonin,respectively,between them having significant difference(P＜0.01). The damage of cell line MCF-7/ADM incubated with pharmacological concentrations of melatonin(10^-5mol/L) plus adriamycin(20ug/ml) was more severe than that of the cell line cultured only with adriamycin(20ug/ml).The level of intracellular GSH in cell line MCF-7/ADM was higher than that in the cell line MCF-7/s,which was not decreased significantly by physiological (10^-9mol/L) concentrations of melatonin(p=0.695),but was significantly decreased by the pharmacological concentrations of melatonin(≥10^-5mol/L). The down-regulating effect of melatonin on the level of intracellular GSH in cell lines MCF-7/ADM and MCF-7/s was dose-depended(p＜0.01).Conclusion:The physiological concentrations of melatonin have no influence on the drug resistance of the cell line MCF-7/ADM.The pharmacological concentrations of melatonin have reverse effect on the drug resistance of the cell line MCF-7/ADM.The reverse mechanism is probably related with the regulation of melatonin on the level of intracellular GSH in tumor cells.Partâ…¢Study on melatonin plus adriamycin treat breast carcinoma rat in vivo.Objective:To investigate the effect of melatonin on antitumor action of adriamycin in vivo,To evaluate the influence of melatonin plus adriamycin on breast cancer wholly.Method:(1) The N-nitroso-N-methylurea(NMU)-induced ER+ rat mammary tumor model was made by 2 times intraperitoneal injection.(2) mammary tumor tats were divided into Blank,Dissolvant,Adriamycin, and Melatonin plus adriamycin groups,to observed the life status and one month survival rates.(3) To observe the morphology change of tumor and heart of rats of groups under light microscope and electron microscope.To weigh the tumor and detect E-cadherin expression by immunohistochemistry method.To observe the damage of tumor cells and myocardium cells under electron microscope.Results:(1) The method of 2 times N-nitroso-N-methylurea(NMU) intraperitoneal injection is successful.The achievement ratio was 91.5%.The time of tumor emergence was 16-18 weeks after injection.The pathological section showed mucinous adenocarcinoma under light microscope and ER-positive by immunohistochemistry method.(2) The life status of Melatonin and Melatonin plus adriamycin groups was better than that of the others,and that of Adriamycin group seemed worst.The one month survival rate of melatonin and melatonin plus adriamycin groups were respective 14/14 and 11/14,there were no significant difference between two groups (P＜0.05),and better than the others.but no significant difference exist between Adriamycin and Blank group.(3) There was no difference of tumor weight among Blank,Dissolvant and Melatonin(P＞0.05),the tumor weight of Melatonin plus adriamycin group was lower than that of Adriamycin and Melatonin group(P＜0.01).The damage of heart of rat in Melatonin plus adriamycin was more remissive than that of Adriamycin group by appearance, light microscope and electron microscope.The peplos of tumor in Melatonin plus adfiamycin and Melatonin groups seemed clearly and the demarcation was obviously between tumor and normal tissue.The damage of tumor cells in Melatonin plus adriamycin group was more severe than that in Adriamycin group.The expression of E-cadherin showed obviously expression in MLT and Melatonin plus adriamycin group,there was no expression of E-cadherin in Blank,Dissolvant and Adriamycin groups.Conclusion:(1) The method of 2 times intraperitoneal injection of N-nitroso-N-methylurea(NMU) is successful.The achievement ratio can meet requirement.The cycle time of tumor emergence seems too long.(2) melatonin can improve the quality of life of tumor rats.That the oncostatic effect seems not so obviously is related with shorter observation time.(3) melatonin alleviates heart damage induced by adriamycin and has sensitization on adriamycin.(4) melatonin may inhibit tumor invasion by decreasing the expression of E-cadherin.