【作者】 张燕；

【导师】 祝淑钗；

【作者基本信息】 河北医科大学 ， 肿瘤学， 2009， 博士

【摘要】 乳腺癌作为一种女性常见病,在世界范围内女性恶性肿瘤性疾病中发病率居第一位,死亡率位居第二。据2007年统计,全世界每年约有7.9万新发乳腺癌病例,占全部女性恶性肿瘤总数的21%。全世界乳腺癌年均发病率为30.30/10万,世界人口调整率为32.97/10万。其中近60%的乳腺癌新发病例在发达国家。虽然乳腺癌的发病率在世界范围内差异较大,但整体均呈上升趋势,发病年龄也渐呈年轻化趋势。对于发展中国家的我们也不例外,在一些大城市,乳腺癌已位居妇女癌症中第一位,也成为女性恶性肿瘤的头号杀手。因而提高乳腺癌的疗效和治愈率已成为迫在眉睫的问题。以手术、化疗、放疗和内分泌治疗相结合的综合治疗模式已成为当前乳腺癌的标准治疗。随着全身性疾病观念的确立,全身化疗在综合治疗中所起的作用越来越引起重视,在2008版NCCN指南中,可见绝大篇幅内容涉及化疗的治疗规范。其中以阿霉素为代表的葸环类细胞毒药物仍然扮演着不可获却得角色,它对于保证疗效、改善预后和减少高危患者的复发起着至关重要的作用。然而,临床不得不面对的两个问题是:（1）阿霉素的耐药成为影响疗效的巨大障碍,无论天然耐药还是获得性耐药都直接影响了它作为一线药物的应用。尽管多年来对其耐药机制进行了大量研究,目前仍未见到经济、安全又有效的耐药逆转药物普及于临床。（2）阿霉素的心脏毒性作为剂量限制性毒性也严重影响了其在临床的广泛使用,累积剂量的限制也迫使无法进一步提高剂量强度;它所造成心脏不可逆损伤也严重影响患者治疗后的生活质量。在生活质量要求越来越高的今天,对这些问题的解决要求也就更为迫切。研究已经证实褪黑素与化疗同时应用能明显改善化疗所致的血小板减少症、免疫抑制、有助于肿瘤患者的睡眠从而减轻焦虑,它能显著提高晚期肿瘤患者的生活质量。褪黑素的器官保护作用也是近年来研究的热点,尤其有研究发现褪黑素能减轻葸环类药物所致的心脏氧化损伤,无疑对拓展临床上葸环类药物的应用带来了新的希望。此外很早就有研究报道,褪黑素能够提高顺铂、氟脲嘧啶等多种化疗药物的疗效,更有临床研究发现肺癌、胃肠道癌症患者常规化疗同时伍用褪黑素,其疗效较对照组显著提高。鉴于以上,我们设计了本课题:分别从分子、细胞水平和动物实验水平探讨了褪黑素对阿霉素抗乳腺癌作用及其作用机制。第一部分褪黑素对阿霉素抗乳腺癌作用的影响及其机制目的:探讨褪黑素对ER+的MCF-7和ER-的MDA-MB-231细胞系的抑制作用以及对阿霉素抗乳腺癌作用的影响及其机制。方法:（1）应用四甲基偶氮唑蓝（MTT法）检测不同浓度褪黑素和阿霉素分别对MCF-7和MDA-MB-231细胞的抑制作用,检测生理浓度和药理浓度的褪黑素对阿霉素抗癌作用的影响。（2）应用流式细胞学方法分别检测不同浓度褪黑素、阿霉素以及两药不同浓度联用时对细胞周期分布和凋亡的影响。（3）应用Western blotting法检测生理浓度、药理浓度褪黑素、阿霉素和药理浓度褪黑素联合阿霉素对细胞中P53和bcl-2蛋白的表达。结果:（1）褪黑素对MCF-7和MDA-MB-231乳腺癌细胞均有不同程度的抑制作用,且呈剂量和时间依赖性。阿霉素对MCF-7和MDA-MB-231的IC50值分别为0.62ug/ml和1.0ug/ml,二者相比有显著性差异（p＜0.05）。生理浓度(10^-9mol/L)和药理浓度(10^-5mol/L)褪黑素孵育后可使MCF-7的IC50值由0.62ug/ml分别降为0.59ug/ml和0.42ug/ml,前者与孵育前相比未见显著性差异（P＞0.05）,后者差异显著（P＜0.01）。阿霉素对MDA-MB-231的IC50为1.00ug/ml,生理和药理浓度的褪黑素孵育后分别降为0.85ug/ml和0.47ug/ml,与孵育前相比前者未见显著性差异（P＞0.05）,后者差异显著（P＜0.01）。（2）流式细胞学检测结果:生理浓度的褪黑素即可对MCF-7具有明显的G0/G1期阻滞作用和凋亡促进作用（P＜0.05）,且具有浓度依赖性。而生理浓度褪黑素对MDA-MB-231的周期阻滞和凋亡作用并不明显（P＞0.05）,药理浓度方能显示出明显作用（P＜0.05）。阿霉素对该两种细胞均能抑制增殖和促进凋亡,随浓度增高增殖指数降低（P＜0.05）,而凋亡率不增加（P＞0.05）。生理浓度褪黑素联合阿霉素对MCF-7的作用与相应浓度的单纯阿霉素相比,其增殖指数明显降低（P＜0.05）,而凋亡率并不增加（P＞0.05）。药理浓度褪黑素联合阿霉素与相应浓度单纯阿霉素组比较,使MCF-7增殖指数下降,凋亡率亦明显增加。增殖指数与阿霉素有浓度依赖性（P＜0.05）,而凋亡随浓度未显示出明显变化（P＞0.05）。对MDA-MB-231,生理浓度褪黑素仅与高浓度阿霉素联用时能使其增殖指数降低（P＜0.05）,对凋亡率未见显著影响（P＞0.05）。药理浓度褪黑素与低浓度阿霉素联用对MDA-MB-231增殖指数未见明显影响（P＞0.05）,而凋亡率显著增加（P＜0.05）。进一步研究发现增殖指数随阿霉素浓度增加而降低（P＜0.05）,凋亡率变化不明显（P＞0.05）。（3）两种乳腺癌细胞株中均呈P53蛋白低表达、bcl-2蛋白高表达;生理浓度褪黑素即可显著增高MCF-7细胞P53蛋白并降低BCL-2蛋白表达,并有剂量依赖性（P＜0.01）,而对MDA-MB-231细胞中两种蛋白的表达并无显著影响;药理浓度褪黑素均可显著提高两种细胞的P53蛋白,降低bcl-2蛋白表达（P＜0.01）;药理浓度褪黑素联合褪黑素对两种细胞中两蛋白表达与药理浓度褪黑素组相比均未见显著性差异（P＞0.05）;阿霉素对两种蛋白表达未见明显影响（P＞0.05）。结论:（1）细胞培养发现体外褪黑素对ER+和ER-乳腺癌细胞系均有抑制作用,呈现明显的剂量依赖性,但敏感程度不同,提示除雌激素通路外,褪黑素对乳腺癌的抑制作用可能还存在其他机制;（2）生理浓度褪黑素(10^-9mol/L)对阿霉素的抗癌作用未见明显影响,药理浓度(10^-5mol/l)以上褪黑素表现出对阿霉素明显的增敏作用;（3）阿霉素较低浓度时,凋亡促进作用是褪黑素增敏机制的一部分,随着阿霉素浓度的提高,褪黑素的细胞毒增敏机制占主要地位;（4）药理浓度褪黑素能提高乳腺癌细胞P53蛋白表达并降低bcl-2蛋白表达,P53和bcl-2通路至少是其凋亡机制的一部分;对高浓度阿霉素增敏作用中凋亡通路似乎不是主要的。第二部分:褪黑素逆转人乳腺癌细胞MCF-7/ADM对阿霉素耐药的作用机制。目的:探讨生理浓度(10^-9mol/L)和药理浓度(≥10^-5mol/L)褪黑素对耐阿霉素乳腺癌细胞系MCF-7/ADM的逆转作用及其机制。方法（1）应用四甲基偶氮唑蓝（MTT）法检测经不同浓度的褪黑素孵育后,阿霉素对MCF-7/ADM细胞系半数抑制浓度IC50的改变。（2）在电镜下观察单用阿霉素（20ug/ml）以及药理浓度的褪黑素(10^-5mol/l)孵育后再应用阿霉素（20ug/ml）,MCF-7/ADM细胞超微结构的变化。（3）应用分光光度法测定MCF-7/ADM和MCF-7/s细胞内GSH水平以及经不同浓度褪黑素孵育后MCF-7/ADM和MCF-7/s细胞内GSH水平变化。结果:（1）褪黑素孵育前阿霉素作用于MCF-7/ADM的IC50值为24.41ug/ml。经生理浓度(10^-9mol/l)的褪黑素孵育后阿霉素IC50值为20.92ug/ml,两者经比较未见显著性差异（P=0.356）。经药理浓度分别为10^-5mol/L和10^-3mol/L的褪黑素孵育后阿霉素作用于MCF-7/ADM的IC50值分别成为12.25ug/ml和10.46ug/ml,与孵育前阿霉素作用于MCF-7/ADM的IC50（24.41ug/ml）相比,均具有显著性差异（P＜0.01）。（2）与单纯应用阿霉素（20ug/ml）相比,电镜下均可观察到经药理浓度(10^-5mol/l)的褪黑素孵育后再应用阿霉素（20ug/ml）对细胞的损伤更为严重。（3）经分光光度法测得MCF-7/ADM细胞内GSH水平明显高于MCF-7/s,生理浓度褪黑素并不能使MCF=7/ADM和MCF-7/s细胞内GSH水平明显降低（P=0.965）,而药理浓度的褪黑素可以显著降低MCF-7/ADM和MCF-7/s细胞内GSH水平且具有一定的浓度依赖性（p＜0.01）。结论:生理浓度的褪黑素对MCF-7/ADM的耐药未见明显影响,药理浓度的褪黑素对MCF-7/ADM的耐药性具有较明显逆转作用,其逆转机制可能与褪黑素对细胞内GSH水平的调控有关。第三部分:褪黑素联合阿霉素治疗乳腺癌大鼠的活体内研究目的:探讨体内应用褪黑素对阿霉素抗乳腺癌作用的影响,整体评价褪黑素对阿霉素的抗癌作用的机制。方法:（1）应用SD大鼠亚硝基甲脲腹腔2次注射法制备ER+乳腺癌大鼠模型;（2）成瘤模型鼠随机分为空白对照组、溶媒组、阿霉素组、褪黑素组、褪黑素联合阿霉素组,按要求处理后观察各组大鼠的一般状态、1月存活率;（3）各组部分大鼠处理后处死分别取瘤体、心脏,光镜和电镜下观察各组心脏形态学改变。瘤体称重,免疫组化检测E-钙粘蛋白的表达,电镜下观察各组细胞损伤情况。结果:（1）采用亚硝基甲脲2次腹腔注射法制备SD大鼠乳腺癌模型成功,成瘤率91.5%,成瘤时间为注射结束后16-18周;光镜下观察肿瘤病理切片显示为黏液腺癌,免疫组化示ER表达呈阳性。（2）褪黑素组和褪黑素联合阿霉素组大鼠生活状态好于其它几组,阿霉素组生活状态最差;褪黑素组和褪黑素联合阿霉素组1月存活率分别为14/14和11/14,经比较未见显著性差异（P＜0.05）,并且优于其它几组。阿霉素组（5/16）与空白对照组（4/16）比较未见明显差别（P＞0.05）;（3）空白对照组、溶媒组和褪黑素组瘤体重量未见显著性差别（P＞0.05）,褪黑素联合阿霉素组瘤体重量显著低于阿霉素组和褪黑素组（P＜0.01）:从大体、光镜和电镜下均可观察到褪黑素联合阿霉素组大鼠心脏损伤明显轻于阿霉素组;大体上褪黑素联合阿霉素组和褪黑素组肿瘤包膜清晰,与周围正常组织分界较明显;光镜和电镜下可见褪黑素联合阿霉素组肿瘤细胞损伤程度重于阿霉素组;心肌免疫组化测定褪黑素和褪黑素联合阿霉素组E-钙黏蛋白强表达,而空白对照组、溶媒组和阿霉素组未见E-钙黏蛋白明显表达。结论:（1）亚硝基甲脲2次腹腔注射法能诱发SD大鼠产生ER+乳腺癌,成瘤率高且符合要求,缺点是周期较长;（2）褪黑素能显著改善乳腺癌大鼠的生活质量,抑瘤作用不明显可能与观察期尚短有关（3）褪黑素能显著减轻阿霉素所致心脏毒性,并增强其抑瘤作用;（4）褪黑素可能通过促进E-钙黏蛋白表达而发挥抑制肿瘤侵袭的作用。更多还原

【Abstract】 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.PartⅠ: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.PartⅡ: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.PartⅢStudy 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.更多还原