雄激素在乳腺癌发生发展中作用的研究
摘要
乳腺癌是女性最常见的恶性肿瘤,在我国,其发病率一直呈上升趋势。影响乳腺癌发生发展的因素很多,性激素是其病因学研究较多的一个方面。
早在1895年第一次行双侧卵巢切除术治疗晚期乳腺癌时,人们就开始探讨性激素,特别是雌激素对乳腺癌发生发展的作用。从上世纪50年代以来,关于雌激素、雌激素受体、雌激素受体阻断、芳香化酶抑制等方面的研究报道已经很多。流行病学资料显示乳腺癌的危险因素大多与体内激素代谢的改变有关。年轻时的初次全程怀孕、多次生产以及充足的母乳喂养都是防止肿瘤进展的独立保护因素。与之相反,初潮早、绝经晚、绝经后应用激素替代治疗都与乳腺癌危险增加有关,而且这种增加与长期暴露在较高的性激素,特别是雌激素的环境下有关,然而雄激素是否也对乳腺癌的发生发展起作用还有争议,不同的研究有不同的、甚至相反的结论。
多数的流行病学调查显示雄激素水平升高与绝经后妇女乳腺癌风险增加有关,如Missmer等最新报道认为循环睾丸酮浓度最高1/4者与最低1/4者相比,前者罹患乳腺癌的风险要高2倍。Eliassen对绝经前妇女的研究也发现循环睾丸酮和乳腺癌的发病风险升高有关联。与流行病学资料结果不同,关于雄激素与乳腺癌细胞株增殖关系的研究因很多变量而影响了结果。如雄激素的类型与剂量,特定细胞株的选用等,这些变量都会使雄激素的作用表现不一,有些研究显示促增殖效应,有些就显示为抗增殖效应。动物实验也显示出两种相反的研究结果。而临床当中应用芳香化酶抑制剂治疗乳腺癌取得良好效果,其作用机理就是阻止肾上腺来源的雄激素在靶细胞内转化为雌激素,这从侧面证实了雄激素促进人乳腺癌生长的作用。因此雄激素对乳腺癌的作用到底如何还需要深入研究。
本研究课题应用酶联免疫吸附试验检测人乳腺癌组织及癌旁乳腺组织中睾丸酮的含量,比较它们的差异,并进一步分析癌组织中睾丸酮的含量与乳腺癌临床生物学特征的关系,结合睾丸酮对人乳腺癌MCF-7细胞株增殖的影响,及睾丸酮对MCF-7荷瘤裸鼠肿瘤生长影响的研究,探讨睾丸酮对乳腺癌发生发展的作用,以期为乳腺癌的治疗和预防提供一个新的指标和思路。
主要研究内容和结果如下:
第一部分雄激素在乳腺癌组织中的表达及意义
目的:探讨雄激素睾丸酮在乳腺癌组织、癌旁2cm及癌旁5cm乳腺组织中的表达情况,并探讨乳腺癌组织中睾丸酮的水平与乳腺癌临床生物学特征的关系。
方法:应用酶联免疫吸附试验检测30例乳腺癌患者乳腺癌组织、癌旁2cm乳腺组织及癌旁5cm乳腺组织中睾丸酮的含量,进行比较研究,并分析肿瘤组织中睾丸酮含量与乳腺癌的临床生物学特性(月经状况、肿瘤大小、淋巴结状况、TNM分期、组织学分级、病理类型、ER/PR/AR/ Her-2状态等)的关系。
结果:
1乳腺癌组织及癌旁乳腺组织中雄激素含量的比较乳腺癌组织中睾丸酮的含量与癌旁2cm和癌旁5cm乳腺组织中睾丸酮含量的比较差异无统计学意义(P=0.215)。绝经前乳腺癌患者乳腺癌组织中睾丸酮的含量低于绝经后患者(P=0.037)。绝经后患者乳腺癌组织中睾丸酮的含量高于癌旁5cm乳腺组织中睾丸酮的含量(P=0.031)。绝经前乳腺癌患者乳腺癌组织中睾丸酮的含量与癌旁乳腺组织中的含量比较差异无统计学意义(P=0.443)。
2乳腺癌组织雄激素含量与乳腺癌临床生物学特征的关系
2.1乳腺癌组织中的睾丸酮含量与体重大小不相关,r=-0.158,P=0.402;与年龄不相关,r=0.292,P=0.117;与体重指数(BMI)不相关,r=-0.046,P=0.811。
2.2乳腺癌组织中睾丸酮的含量与肿瘤组织学分级有关,组织学分级高者睾丸酮含量明显升高,III级者的睾丸酮含量高于II级者和I级者(P=0.001)。而I级与II级患者肿瘤中睾丸酮含量比较差异无统计学意义(P=0.286),这两者与III级比较差异均有统计学意义(P=0.001,P=0.001)。
2.3乳腺癌组织中睾丸酮的含量与肿瘤大小、淋巴结状况、TNM分期、病理类型无关,P值均大于0.05。
2.4乳腺癌组织中睾丸酮含量与ER、PR、AR、Her-2免疫组化的表达无关,P值均大于0.05。
3乳腺癌组织中AR的表达与ER、PR、Her-2的表达比较
乳腺癌组织中AR的阳性率为86.67%,ER的阳性率为66.67%,两者比较显示正相关(P=0.008);PR的阳性率为60%,与AR比较显示正相关(P=0.018);Her-2的阳性率为80%,与AR比较无关(P=0.388)。
4乳腺癌组织中AR的表达与临床病理指标的关系
乳腺癌组织的AR表达与患者的月经状况、病理类型、肿瘤大小、淋巴结转移、TNM分期、分级等无关,P值均大于0.05。
结论:
1.绝经后女性乳腺癌患者肿瘤组织中睾丸酮的含量明显高于绝经前女性乳腺癌肿瘤组织中的含量,绝经后乳腺癌患者肿瘤组织中睾丸酮含量明显高于癌旁乳腺组织中的含量,提示睾丸酮在绝经后妇女乳腺癌的发生发展中有一定作用。
2.乳腺癌组织中睾丸酮的含量与其组织学分级有关,组织学分化III级的睾丸酮含量明显高于I级和II级,表明睾丸酮含量的增加与组织学分化差、恶性程度高的乳腺癌类型有关。
3.乳腺癌组织中睾丸酮含量的检测可能有助于乳腺癌恶性程度的判断。有助于乳腺癌复发转移风险及预后的判断,有可能提供一个新的内分治疗途径。
第二部分雄激素对乳腺癌MCF-7细胞株增殖的影响
目的:研究雄激素对乳腺癌MCF-7细胞增殖的影响,探讨雄激素在乳腺癌发生发展方面的可能作用机制。
方法:采用MTT法检测雄激素睾丸酮对MCF-7乳腺癌细胞株增殖的影响,采用流式细胞术(Flow cytometry,FCM)分析睾丸酮作用后的乳腺癌细胞周期、凋亡率的情况,并检测分析其中CyclinD1和AR蛋白的表达情况。
结果:
1不同浓度睾丸酮对MCF-7乳腺癌细胞株增殖的影响高浓度睾丸酮(10-5 mol /L)作用后,MCF-7乳腺癌细胞株的存活率明显下降,抑制率明显增加,24h、48h、72h的抑制率分别为12.46%、22.21%和44.72%。稍低浓度睾丸酮(10-9 mol /L)可促进MCF-7细胞的生长。10-7 mol /L与10-11 mol /L的睾丸酮既不抑制MCF-7细胞的生长,也不促进其生长。
2不同浓度睾丸酮对MCF-7细胞周期和凋亡的影响
2.1作用24h时,高浓度睾丸酮(10-5 mol /L)组的细胞凋亡率升高,并可促使MCF-7乳腺癌细胞从G1期向S期及G2/M过渡,与对照相比差异有统计学意义(P值均小于0.05)。稍低浓度睾丸酮(10-9 mol /L)的细胞凋亡率与对照比差异无统计学意义(P>0.05)。但细胞G0/G1期比率下降,S期及G2/M期比率升高(P值均小于0.05)。
2.2作用48h时,高浓度睾丸酮(10-5 mol /L)组细胞凋亡率显著升高,但其可促使MCF-7细胞从G1期向S期及G2/M过渡,与对照相比,高浓度睾丸酮组细胞的G0/G1期比率显著下降,S期及G2/M期比率升高(P值均小于0.05)。10-9 mol /L睾丸酮组的细胞凋亡率和细胞周期与对照相比差异均无统计学意义(P值均大于0.05)。
2.3 10-7 mol /L、10-11 mol /L浓度的睾丸酮对MCF-7细胞的细胞周期及凋亡率均无明显影响(P值均大于0.05)。
3睾丸酮对MCF-7细胞株中CyclinD1和AR蛋白表达的影响
3.1 MCF-7细胞经10-5 mol /L睾丸酮处理24h后,CyclinD1蛋白表达量较对照组增加(P <0.05),经10-9 mol /L睾丸酮处理后,CyclinD1蛋白表达量亦有所增加(P <0.05)。而AR蛋白的表达量均无明显变化(P值均大于0.05)。
3.2 MCF-7细胞经10-5 mol /L睾丸酮处理48h后, CyclinD1蛋白表达量较对照组增加(P <0.05),而AR蛋白表达量较对照组有所下降但差异无统计学意义(P >0.05)。经10-9 mol /L睾丸酮处理后,CyclinD1蛋白表达量较对照组无明显变化(P >0.05),而AR蛋白表达量有所升高,但差异仍无统计学意义,可是与10-5 mol /L睾丸酮处理后的AR蛋白表达量比较明显升高(P <0.05)。
结论:
1.睾丸酮对乳腺癌MCF-7细胞的增殖有双向作用。稍低浓度(10-9 mol /L)时表现出促进增殖的作用,更高浓度(10-5 mol /L)时表现出抑制增殖的作用,提示睾丸酮对乳腺癌作用的复杂性。
2.稍低浓度(10-9 mol /L)睾丸酮可促使乳腺癌MCF-7细胞株表达CyclinD1,加速细胞周期,促进增殖。提示睾丸酮在乳腺癌的发生发展方面确实能发挥促进作用。
3.更高浓度睾丸酮可抑制乳腺癌细胞株的增殖、促进其凋亡,可能和其促使乳腺癌细胞过度表达CyclinD1有关。提示大剂量雄激素可能在乳腺癌的治疗方面可能起到较好效果。
4.高浓度睾丸酮可下调乳腺癌细胞株中AR的表达,稍低浓度睾丸酮可上调AR的表达。提示大剂量雄激素可能抑制AR阳性的乳腺肿瘤。
5.针对睾丸酮对乳腺癌细胞的作用研究,可以部分解释第一部分研究中睾丸酮可能促使肿瘤生长的推测及睾丸酮含量高者肿瘤恶性程度高的现象。
第三部分雄激素对MCF-7乳腺癌荷瘤裸鼠肿瘤生长的影响
目的:观察不同剂量丙酸睾丸酮对MCF-7乳腺癌荷瘤裸鼠肿瘤生长的影响,探讨其可能的机制。
方法:构建MCF-7乳腺癌荷瘤裸鼠模型,再用不同剂量的丙酸睾丸酮(50mg/kg,400mg/kg)及化疗药物进行干预,检测肿瘤的生长情况。用微粒子发光法检测裸鼠血清睾丸酮的含量,用流式细胞术及免疫组化法检测肿瘤中的CyclinD1和AR的表达。
结果:
1成功构建MCF-7乳腺癌荷瘤裸鼠模型裸鼠接种MCF-7乳腺癌细胞株后均有肿瘤结节形成,成瘤率达100%,在7d左右开始成瘤,瘤体逐渐增大,移植瘤呈圆形、卵圆形或结节状,瘤体表面光滑。
2.肿瘤生长状况
至实验结束时,对照组裸鼠的肿瘤体积为0.2072±0.1100cm3,化疗组裸鼠的肿瘤(0.1795±0.0905cm3)生长相对缓慢,抑瘤率为13.39%。但与对照相比差异无统计学意义(P=0.868)。给予低剂量丙酸睾丸酮组裸鼠的肿瘤体积(0.5613±0.1872cm3)明显增大,与对照组和化疗组相比差异有统计学意义(P值分别为0.047,0.034),促瘤率为170.84%。给予高剂量丙酸睾丸酮组裸鼠的肿瘤生长复杂,3只的肿瘤逐渐缩小,2只的肿瘤逐渐增大,至实验结束时平均体积为0.3366±0.4635cm3,与对照组、化疗组、低剂量丙酸睾丸酮组比较差异均无统计学意义(P值分别为0.443,0.353,0.191)。
3流式细胞学检查结果
3.1雄激素受体蛋白的表达:各组肿瘤中AR的表达差异无统计学意义,P=0.177。
3.2 CyclinD1蛋白的表达:低剂量丙酸睾丸酮组裸鼠肿瘤的CyclinD1的表达量为403.28±31.23,高于其在对照组(358.47±36.44,P=0.035)和化疗组(352.94±19.14,P=0.020)的表达。
4免疫组化检查结果
免疫组化检测肿瘤中AR蛋白表达结果显示,AR蛋白主要表达在细胞核,各组比较没有明显差异。CyclinD1在低剂量睾丸酮组肿瘤中呈阳性表达,可见胞核有明显棕黄色颗粒;其在其他各组呈弱阳性表达。
5血清睾丸酮水平的检测结果
用药结束后,处死动物时分别检测了各组实验鼠血清睾丸酮水平。对照组血清睾丸酮含量为1.40±0.23ng/ml ,稍低于化疗组的含量(2.38±0.17ng/ml),但差异没有统计学意义,P=0.07。50mg/kg丙酸睾丸酮用药组血清睾丸酮含量明显升高,达到13.50±1.58ng/ml,明显高于对照组和化疗组(P值分别为0.000和0.000)。而400mg/kg丙酸睾丸酮用药组血清睾丸酮含量亦明显升高,达到14.85±0.33ng/ml,明显高于对照组、化疗组和低剂量睾丸酮用药组(P值分别为0.000、0.000和0.019)。
结论:
1.低剂量丙酸睾丸酮(50mg/kg)可刺激MCF-7乳腺癌荷瘤裸鼠肿瘤的生长,和对照组相比差异有统计学意义。表明外源性低剂量雄激素有促进乳腺癌生长的作用。
2.高剂量丙酸睾丸酮(400mg/kg)可抑制MCF-7乳腺癌荷瘤裸鼠肿瘤的生长,但作用结果不完全一致,具体机制有待进一步研究。
3.低剂量雄激素促进MCF-7乳腺癌荷瘤裸鼠肿瘤的生长,其肿瘤内CyclinD1的表达明显增加,而AR表达无明显变化。表明雄激素促进乳腺癌的生长可能是通过促进CyclinD1的表达引起的。
Breast cancer is the most common type of malignant tumors in women. Its incidence has increased gradually in our country. There are many factors to affect the genesis and development of breast cancer. Sex hormone is one of the factors which have been extensively investigated.
Beginning with the report of bilateral oophorectomy for advanced breast cancer in 1895, sex hormone, especially estrogen, was focused on to explore its effect on the growth and development of breast cancer. In the 1950s, there has been an enormous number of reports on estrogens, estrogen receptors(ER), ER blockade, aromatization. Most of the established epidemiological risk factors for breast cancer are related to alteration in endogenous hormone metabolis. A young age at first full-term pregnancy, high parity and prolonged duration of breast feeding all independently protect against tumor development. On the contrast, early menarche, late menopause and use of postmenopausal hormone therapy have been known to be associate with increased risk of breast cancer, and the increase is associated with higher sex hormone, especially estrogen. It is still unknown whether androgen is involved in the growth and development of breast cancer. Different studies always show different, or even contrary results.
In most epidemiological studies on postmenopausal women, elevation of androgen level are found to be associated with the increase of breast cancer. Missmer’s report showed those with the highest fourths testosterone have about 2 times of breast cancer risk than those with the lowest fourths. Eliassen also found the relationship between the blood level of testosterone with the risk of breast cancer in premenopausal women. Different from epidemiological investigations, studies on androgens and breast cancer cell line proliferation have multiple variables that affect the outcome of those studies. The type and the dose of androgens, the particular cell lines used and other variables have demonstrated different effects. Some show stimulating effects, some show antiproliferation effects. Studies on animals also show contrary outcomes. But the fact that aromtase inhibitors, blocker of the transform of estrogen from adrenal androgen, which have shown good effect on breast cancer confirms the stimulating effects of androgen on breast cancer. So the role of androgen on breast cancer still needs further explored.
In the present study, we used enzyme-linked immunosorbent assay (ELISA) to measure the testosterone concentration in breast cancer and breast tissue, and analyzed the difference between them, detected the relationship between the breast cancer testosterone concentration and clinicobiological features. Then we investigated the influence of testosterone on MCF-7 breast cancer cell line and breast cancer xenograft (MCF-7) in nude mice to detect the role of androgen on the growth and development of breast cancer, to offer a new marker or idea for the treatment and prognosis of breast cancer.
Part I The significance of androgen in breast cancer tissue Objective: To detect the concentration of testosterone in breast cancer, breast tissue 2cm and 5cm away from the tumor, and analyze the relation between the testosterone concentration and clinicobiological features.
Methods: ELISA was performed to examine the concentration of testosterone in breast cancer, breast tissue 2cm and 5cm away from the tumor in 30 breast cancer patients. Relationship was analyzed between them and clinicobiological features(menopause status, tumor size, nodal status, TNM stage, histological grade, histological type, ER/PR/AR status). Results:
1 The difference of testosterone concentration between breast cancer and tissue 2cm and 5cm away from tumor.
There was no significance in testosterone concentration between breast cancer and breast tissue 2cm and 5cm away from tumor(P=0.215). The testosterone concentration in premenopausal patients was lower than those in postmenopausal patients(P=0.037). In postmenopausal patients, the testos- terone concentration in breast cancer was higher than those in tissue at 5cm from tumor(P=0.031). No significant difference was found between breast cancer and the breast tissue in premenopausal patients(P=0.443).
2 The relationship between testosterone concentration in breast cancer tissue and clinicobiological features
2.1 There were no correlation between testosterone concentration in breast cancer tissue with weight (r=-0.158, P=0.402), age (r=0.292, P=0.11), and Body Mass Index(BMI) (r=-0.046, P=0.811).
2.2 Testosterone concentration in breast cancer tissue was related to histological grade. Testosterone concentration was higher in those with worse grade. Those with grade III had much more testosterone than those with grade I,II (P=0.001). No difference was found between grade I and grade II (P=0.286).
2.3 No relationship were found between testosterone concentration and tumor size, nodal status, TNM stage, pathological type (P>0.05 for all).
2.4 No relationship were found between testosterone concentration and the expression of ER, PR, AR, Her-2 (P>0.05 for all).
3 The relationship between AR and ER, PR, Her-2
Positive rate of AR in breast cancer was 86.67%, ER was 66.67%, PR was 60%. There were significant relationship between the expression of AR and ER (P=0.008), PR(P=0.018). No significance was found between AR and Her-2(P=0.388) whose positive rate was 80%.
4 Relationship between the expression of AR and clinibobiological features No relationship were found between the expression of AR and menopausal status, tumor size, pathological type, nodal status, TNM stage and histological grade (P>0.05 for all).
Conclusions
1 Testosterone concentration in breast cancer tissue of postmenopausal patients is higher than that in premenopausal patients. Testosterone concentration in breast cancer tissue is higher than that in breast tissue 5cm away from tumor in postmenopausal women, which suggests that testosterone may play a certain role in the genesis and development of breast cancer.
2 Testosterone concentration in breast cancer tissue is related to histological grading. Testosterone concentration is higher in those with grade III than those with grade I,II. The more involved axillary nodes is companied with the higher testosterone level in primary tumor. All of above suggests that the higher testosterone concentration, the higher malignant of the breast cancer. 3 The testosterone concentration in breast tumors may contribute to the diagnosis of the malignant status, to the diagnosis of recurrence and metastasis risk and prognosis. It may provide a novel endotherapy approach for breast cancer.
Part II The influence of androgen on MCF-7 breast cancer cell line
Objective: To study the influence of androgen on MCF-7 breast cancer cell line and explore the mechanism that how androgen affect the genesis and growth of breast cancer.
Methods: MTT was used to measure the proliferation of MCF-7 after stimulated with testosterone. Flow cytometry(FCM) was used to analyze the breast cancer cell cycle, apoptosis rate and the expression of CyclinD1 and androgen receptor(AR).
Results:
1 Influence of testosterone at different concentration on the proliferation of MCF-7 breast cancer cell line
The survival rate of MCF-7 cells exposed to 10-5 mol /L testosterone decreased significantly and inhibition rate increased markedly. The inhibitory rate was 12.46%,22.21%,44.72% in 24, 48, 72 h respectively. Cultures of MCF-7 cells exposed to 10-9 mol /L testosterone had cell growth compared with control cultures. Cultures of MCF-7 cells exposed to 10-7 mol /L and 10-11 mol /L testosterone showed neither proliferation nor inhibition in any time.
2 Influence of testosterone at different concentration on the MCF-7 cell cycle and apoptosis
2.1 After 24 h, high concentration testosterone(10-5 mol /L) increased the apoptosis rate of MCF-7 cells and impeded the progression of G1 cells into S phase and G2/M phase compared with control cultures(P<0.05 for all). No difference of apoptosis rate was found between the 10-9 mol /L testosterone pretreatment group and control group, but low concentration(10-9 mol /L) testosterone decreased the proportions of G1 phase cells and increase the proportions of S phase and G2/M phase cells(P<0.05 for all).
2.2 After 48h, 10-5 mol /L testosterone induced apoptosis of MCF-7 cells increasely, but it also decreased the proportions of G1 phase and increase the proportions of S phase and G2/M phase (10-5 mol /L). No difference was found between 10-9 mol /L testosterone group and control group.
2.3 10-7 mol /L and 10-11 mol /L testosterone showed little influence on cell cycle and apoptosis of MCF-7 cells(P>0.05 for all).
3 Influence of testosterone on the expression of CyclinD1 and AR of MCF-7 cells
3.1 After 24h, Cultures of MCF-7 cells exposed to 10-5 mol /L and 10-9 mol /L testosterone exhibited increased expression of CyclinD1 compared with control cultures (P<0.05 , both). There was no difference of AR in all groups. (P>0.05 for all).
3.2 After 48h, Cultures of MCF-7 cells exposed to 10-5 mol /L testosterone exhibited increased expression of CyclinD1 compared with control cultures (P<0.05). 10-9 mol /L testosterone showed no influence on CyclinD1. AR was found increased slightly(P>0.05) in 10-9 mol /L testosterone group. There was a significant difference of AR between 10-5 mol /L testosterone group and 10-9 mol /L testosterone group(P<0.05).
Conclusions:
1 Testosterone has bidirective effect on breast cancer cells. Lower concentration testosterone (10-9 mol /L) may stimulate MCF-7 cells proli- feration. Higher concentration testosterone(10-5 mol /L) may inhibite MCF-7 cells proliferation, which shows the complex effect of testosterone on breast cancer.
2 Lower concentration testosterone(10-9 mol /L) may increases the expression of CyclinD1 and impeds cell cycle, improves proliferation, which suggests that testosterone play a positive role on the genesis and development of breast cancer.
3 Higher concentration testosterone may inhibite the proliferation of MCF-7 cells, induce apoptosis which maybe relate to the overexpression of CyclinD1. High dose androgen may be useful for the treatment to breast cancer.
4 Higher concentration testosterone may down-regulate the expression of AR in breast cancer cells whereas lower concentration testosterone may up-regulate it, which suggests that high dose androgen may inhibite the proliferation of breast caner with positive AR.
5 This study may partly explain the hypothesis that higher concentration of testosterone may promote the proliferation of breast cancer and the pheno- menon that the more testosterone in tumor the worse malignance of tumor.
Part III Influence of androgen on the growth of human breast cancer xenograft (MCF-7) in nude mice
Objective: To investigate the effect of testosterone propionate (TP) at different dose on the growth of MCF-7 xenograft tumor in nude mice and to explore its may-be mechanism.
Methods: To establish the transplanted MCF-7 tumors in nude mice. Then to investigate the growth of transplanted tumors intervented by testosterone propionate at different dose (50mg/kg, 400mg/kg) and chemotherapic agent. Microparticle cheniluminescance immunoassay was performed to measure the level of serum testosterone of nude mice. FCM and immunohistochemical staining (IHC) was performed to examine the expression of CyclinD1 and AR in the transplanted tumors.
Results:
1 To establish the transplanted MCF-7 tumors in nude mice successfully About in 7 days, transplanted tumor by MCF-7 cells could be found in nude mice. The tumor all developed gradually into a smooth, circle or oval nodules.
2 Growth status of transplanted tumors
The volume of transplanted tumors in chemotherapy group were slightly nonsignificantly decreased compared with control group (P=0.868). The volume of transplanted tumor in low dose TP group increased significantly compared with control group and chemotherapy group(P=0.047, P=0.034 respectively). The growth of transplanted tumors in high dose TP group was complex. 3 transplanted tumors was gradually reduced, 3but 2 transplanted tumors became bigger obviously. There was no significant difference between high dose TP group and the other groups (P>0.05 for all).
3 Results of the expression of AR and CyclinD1 by FCM
3.1 No significant difference of AR expression was found among the four groups(P>0.05 for all) .
3.2 The expression of CyclinD1 in transplanted tumor in low dose TP group (403.28±31.23) was higher than those in control group (358.47±36.44,P=0.035) and chemotherapy group (352.94±19.14,P=0.020). 4 Results of the expression of AR and CyclinD1 by IHC AR protein was mainly expressed in the cell nucleus. No difference of AR expression was found among the four groups. Positive expression of CyclinD1 was found in low dose TP group and weakly expression in other groups.
5 Results of serum testosterone in nude mice transplanted by breast cancer xenograft (MCF-7).
Testosterone level in control group (1.40±0.23ng/ml) was non- significantly lower than that in chemotherapy group (2.38±0.17ng/ml, P=0.07). The testosterone level in low dose TP (50mg/kg) group (13.50±1.58ng/ml) was obviously increased compared with control group and chemotherapy group (P=0.000, P=0.000). The highest testosterone level was found in high dose TP (400mg/kg) group (14.85±0.33ng/ml), which was significantly higher than the other three groups(P=0.000, P=0.000, P=0.019 respectively).
Conclusion:
1 Low dose TP can stimulate the MCF-7 transplanted tumor which suggests than low dose androgen may promote the growth of breast cancer.
2 High dose TP shows inconsistent effect on the MCF-7 transplanted tumor which still needs further approach.
3 The growth of transplanted tumor in low dose TP with higher expression of CyclinD1 which means that the positive effect of androgen on breast cancer maybe trigger by CyclinD1.
早在1895年第一次行双侧卵巢切除术治疗晚期乳腺癌时,人们就开始探讨性激素,特别是雌激素对乳腺癌发生发展的作用。从上世纪50年代以来,关于雌激素、雌激素受体、雌激素受体阻断、芳香化酶抑制等方面的研究报道已经很多。流行病学资料显示乳腺癌的危险因素大多与体内激素代谢的改变有关。年轻时的初次全程怀孕、多次生产以及充足的母乳喂养都是防止肿瘤进展的独立保护因素。与之相反,初潮早、绝经晚、绝经后应用激素替代治疗都与乳腺癌危险增加有关,而且这种增加与长期暴露在较高的性激素,特别是雌激素的环境下有关,然而雄激素是否也对乳腺癌的发生发展起作用还有争议,不同的研究有不同的、甚至相反的结论。
多数的流行病学调查显示雄激素水平升高与绝经后妇女乳腺癌风险增加有关,如Missmer等最新报道认为循环睾丸酮浓度最高1/4者与最低1/4者相比,前者罹患乳腺癌的风险要高2倍。Eliassen对绝经前妇女的研究也发现循环睾丸酮和乳腺癌的发病风险升高有关联。与流行病学资料结果不同,关于雄激素与乳腺癌细胞株增殖关系的研究因很多变量而影响了结果。如雄激素的类型与剂量,特定细胞株的选用等,这些变量都会使雄激素的作用表现不一,有些研究显示促增殖效应,有些就显示为抗增殖效应。动物实验也显示出两种相反的研究结果。而临床当中应用芳香化酶抑制剂治疗乳腺癌取得良好效果,其作用机理就是阻止肾上腺来源的雄激素在靶细胞内转化为雌激素,这从侧面证实了雄激素促进人乳腺癌生长的作用。因此雄激素对乳腺癌的作用到底如何还需要深入研究。
本研究课题应用酶联免疫吸附试验检测人乳腺癌组织及癌旁乳腺组织中睾丸酮的含量,比较它们的差异,并进一步分析癌组织中睾丸酮的含量与乳腺癌临床生物学特征的关系,结合睾丸酮对人乳腺癌MCF-7细胞株增殖的影响,及睾丸酮对MCF-7荷瘤裸鼠肿瘤生长影响的研究,探讨睾丸酮对乳腺癌发生发展的作用,以期为乳腺癌的治疗和预防提供一个新的指标和思路。
主要研究内容和结果如下:
第一部分雄激素在乳腺癌组织中的表达及意义
目的:探讨雄激素睾丸酮在乳腺癌组织、癌旁2cm及癌旁5cm乳腺组织中的表达情况,并探讨乳腺癌组织中睾丸酮的水平与乳腺癌临床生物学特征的关系。
方法:应用酶联免疫吸附试验检测30例乳腺癌患者乳腺癌组织、癌旁2cm乳腺组织及癌旁5cm乳腺组织中睾丸酮的含量,进行比较研究,并分析肿瘤组织中睾丸酮含量与乳腺癌的临床生物学特性(月经状况、肿瘤大小、淋巴结状况、TNM分期、组织学分级、病理类型、ER/PR/AR/ Her-2状态等)的关系。
结果:
1乳腺癌组织及癌旁乳腺组织中雄激素含量的比较乳腺癌组织中睾丸酮的含量与癌旁2cm和癌旁5cm乳腺组织中睾丸酮含量的比较差异无统计学意义(P=0.215)。绝经前乳腺癌患者乳腺癌组织中睾丸酮的含量低于绝经后患者(P=0.037)。绝经后患者乳腺癌组织中睾丸酮的含量高于癌旁5cm乳腺组织中睾丸酮的含量(P=0.031)。绝经前乳腺癌患者乳腺癌组织中睾丸酮的含量与癌旁乳腺组织中的含量比较差异无统计学意义(P=0.443)。
2乳腺癌组织雄激素含量与乳腺癌临床生物学特征的关系
2.1乳腺癌组织中的睾丸酮含量与体重大小不相关,r=-0.158,P=0.402;与年龄不相关,r=0.292,P=0.117;与体重指数(BMI)不相关,r=-0.046,P=0.811。
2.2乳腺癌组织中睾丸酮的含量与肿瘤组织学分级有关,组织学分级高者睾丸酮含量明显升高,III级者的睾丸酮含量高于II级者和I级者(P=0.001)。而I级与II级患者肿瘤中睾丸酮含量比较差异无统计学意义(P=0.286),这两者与III级比较差异均有统计学意义(P=0.001,P=0.001)。
2.3乳腺癌组织中睾丸酮的含量与肿瘤大小、淋巴结状况、TNM分期、病理类型无关,P值均大于0.05。
2.4乳腺癌组织中睾丸酮含量与ER、PR、AR、Her-2免疫组化的表达无关,P值均大于0.05。
3乳腺癌组织中AR的表达与ER、PR、Her-2的表达比较
乳腺癌组织中AR的阳性率为86.67%,ER的阳性率为66.67%,两者比较显示正相关(P=0.008);PR的阳性率为60%,与AR比较显示正相关(P=0.018);Her-2的阳性率为80%,与AR比较无关(P=0.388)。
4乳腺癌组织中AR的表达与临床病理指标的关系
乳腺癌组织的AR表达与患者的月经状况、病理类型、肿瘤大小、淋巴结转移、TNM分期、分级等无关,P值均大于0.05。
结论:
1.绝经后女性乳腺癌患者肿瘤组织中睾丸酮的含量明显高于绝经前女性乳腺癌肿瘤组织中的含量,绝经后乳腺癌患者肿瘤组织中睾丸酮含量明显高于癌旁乳腺组织中的含量,提示睾丸酮在绝经后妇女乳腺癌的发生发展中有一定作用。
2.乳腺癌组织中睾丸酮的含量与其组织学分级有关,组织学分化III级的睾丸酮含量明显高于I级和II级,表明睾丸酮含量的增加与组织学分化差、恶性程度高的乳腺癌类型有关。
3.乳腺癌组织中睾丸酮含量的检测可能有助于乳腺癌恶性程度的判断。有助于乳腺癌复发转移风险及预后的判断,有可能提供一个新的内分治疗途径。
第二部分雄激素对乳腺癌MCF-7细胞株增殖的影响
目的:研究雄激素对乳腺癌MCF-7细胞增殖的影响,探讨雄激素在乳腺癌发生发展方面的可能作用机制。
方法:采用MTT法检测雄激素睾丸酮对MCF-7乳腺癌细胞株增殖的影响,采用流式细胞术(Flow cytometry,FCM)分析睾丸酮作用后的乳腺癌细胞周期、凋亡率的情况,并检测分析其中CyclinD1和AR蛋白的表达情况。
结果:
1不同浓度睾丸酮对MCF-7乳腺癌细胞株增殖的影响高浓度睾丸酮(10-5 mol /L)作用后,MCF-7乳腺癌细胞株的存活率明显下降,抑制率明显增加,24h、48h、72h的抑制率分别为12.46%、22.21%和44.72%。稍低浓度睾丸酮(10-9 mol /L)可促进MCF-7细胞的生长。10-7 mol /L与10-11 mol /L的睾丸酮既不抑制MCF-7细胞的生长,也不促进其生长。
2不同浓度睾丸酮对MCF-7细胞周期和凋亡的影响
2.1作用24h时,高浓度睾丸酮(10-5 mol /L)组的细胞凋亡率升高,并可促使MCF-7乳腺癌细胞从G1期向S期及G2/M过渡,与对照相比差异有统计学意义(P值均小于0.05)。稍低浓度睾丸酮(10-9 mol /L)的细胞凋亡率与对照比差异无统计学意义(P>0.05)。但细胞G0/G1期比率下降,S期及G2/M期比率升高(P值均小于0.05)。
2.2作用48h时,高浓度睾丸酮(10-5 mol /L)组细胞凋亡率显著升高,但其可促使MCF-7细胞从G1期向S期及G2/M过渡,与对照相比,高浓度睾丸酮组细胞的G0/G1期比率显著下降,S期及G2/M期比率升高(P值均小于0.05)。10-9 mol /L睾丸酮组的细胞凋亡率和细胞周期与对照相比差异均无统计学意义(P值均大于0.05)。
2.3 10-7 mol /L、10-11 mol /L浓度的睾丸酮对MCF-7细胞的细胞周期及凋亡率均无明显影响(P值均大于0.05)。
3睾丸酮对MCF-7细胞株中CyclinD1和AR蛋白表达的影响
3.1 MCF-7细胞经10-5 mol /L睾丸酮处理24h后,CyclinD1蛋白表达量较对照组增加(P <0.05),经10-9 mol /L睾丸酮处理后,CyclinD1蛋白表达量亦有所增加(P <0.05)。而AR蛋白的表达量均无明显变化(P值均大于0.05)。
3.2 MCF-7细胞经10-5 mol /L睾丸酮处理48h后, CyclinD1蛋白表达量较对照组增加(P <0.05),而AR蛋白表达量较对照组有所下降但差异无统计学意义(P >0.05)。经10-9 mol /L睾丸酮处理后,CyclinD1蛋白表达量较对照组无明显变化(P >0.05),而AR蛋白表达量有所升高,但差异仍无统计学意义,可是与10-5 mol /L睾丸酮处理后的AR蛋白表达量比较明显升高(P <0.05)。
结论:
1.睾丸酮对乳腺癌MCF-7细胞的增殖有双向作用。稍低浓度(10-9 mol /L)时表现出促进增殖的作用,更高浓度(10-5 mol /L)时表现出抑制增殖的作用,提示睾丸酮对乳腺癌作用的复杂性。
2.稍低浓度(10-9 mol /L)睾丸酮可促使乳腺癌MCF-7细胞株表达CyclinD1,加速细胞周期,促进增殖。提示睾丸酮在乳腺癌的发生发展方面确实能发挥促进作用。
3.更高浓度睾丸酮可抑制乳腺癌细胞株的增殖、促进其凋亡,可能和其促使乳腺癌细胞过度表达CyclinD1有关。提示大剂量雄激素可能在乳腺癌的治疗方面可能起到较好效果。
4.高浓度睾丸酮可下调乳腺癌细胞株中AR的表达,稍低浓度睾丸酮可上调AR的表达。提示大剂量雄激素可能抑制AR阳性的乳腺肿瘤。
5.针对睾丸酮对乳腺癌细胞的作用研究,可以部分解释第一部分研究中睾丸酮可能促使肿瘤生长的推测及睾丸酮含量高者肿瘤恶性程度高的现象。
第三部分雄激素对MCF-7乳腺癌荷瘤裸鼠肿瘤生长的影响
目的:观察不同剂量丙酸睾丸酮对MCF-7乳腺癌荷瘤裸鼠肿瘤生长的影响,探讨其可能的机制。
方法:构建MCF-7乳腺癌荷瘤裸鼠模型,再用不同剂量的丙酸睾丸酮(50mg/kg,400mg/kg)及化疗药物进行干预,检测肿瘤的生长情况。用微粒子发光法检测裸鼠血清睾丸酮的含量,用流式细胞术及免疫组化法检测肿瘤中的CyclinD1和AR的表达。
结果:
1成功构建MCF-7乳腺癌荷瘤裸鼠模型裸鼠接种MCF-7乳腺癌细胞株后均有肿瘤结节形成,成瘤率达100%,在7d左右开始成瘤,瘤体逐渐增大,移植瘤呈圆形、卵圆形或结节状,瘤体表面光滑。
2.肿瘤生长状况
至实验结束时,对照组裸鼠的肿瘤体积为0.2072±0.1100cm3,化疗组裸鼠的肿瘤(0.1795±0.0905cm3)生长相对缓慢,抑瘤率为13.39%。但与对照相比差异无统计学意义(P=0.868)。给予低剂量丙酸睾丸酮组裸鼠的肿瘤体积(0.5613±0.1872cm3)明显增大,与对照组和化疗组相比差异有统计学意义(P值分别为0.047,0.034),促瘤率为170.84%。给予高剂量丙酸睾丸酮组裸鼠的肿瘤生长复杂,3只的肿瘤逐渐缩小,2只的肿瘤逐渐增大,至实验结束时平均体积为0.3366±0.4635cm3,与对照组、化疗组、低剂量丙酸睾丸酮组比较差异均无统计学意义(P值分别为0.443,0.353,0.191)。
3流式细胞学检查结果
3.1雄激素受体蛋白的表达:各组肿瘤中AR的表达差异无统计学意义,P=0.177。
3.2 CyclinD1蛋白的表达:低剂量丙酸睾丸酮组裸鼠肿瘤的CyclinD1的表达量为403.28±31.23,高于其在对照组(358.47±36.44,P=0.035)和化疗组(352.94±19.14,P=0.020)的表达。
4免疫组化检查结果
免疫组化检测肿瘤中AR蛋白表达结果显示,AR蛋白主要表达在细胞核,各组比较没有明显差异。CyclinD1在低剂量睾丸酮组肿瘤中呈阳性表达,可见胞核有明显棕黄色颗粒;其在其他各组呈弱阳性表达。
5血清睾丸酮水平的检测结果
用药结束后,处死动物时分别检测了各组实验鼠血清睾丸酮水平。对照组血清睾丸酮含量为1.40±0.23ng/ml ,稍低于化疗组的含量(2.38±0.17ng/ml),但差异没有统计学意义,P=0.07。50mg/kg丙酸睾丸酮用药组血清睾丸酮含量明显升高,达到13.50±1.58ng/ml,明显高于对照组和化疗组(P值分别为0.000和0.000)。而400mg/kg丙酸睾丸酮用药组血清睾丸酮含量亦明显升高,达到14.85±0.33ng/ml,明显高于对照组、化疗组和低剂量睾丸酮用药组(P值分别为0.000、0.000和0.019)。
结论:
1.低剂量丙酸睾丸酮(50mg/kg)可刺激MCF-7乳腺癌荷瘤裸鼠肿瘤的生长,和对照组相比差异有统计学意义。表明外源性低剂量雄激素有促进乳腺癌生长的作用。
2.高剂量丙酸睾丸酮(400mg/kg)可抑制MCF-7乳腺癌荷瘤裸鼠肿瘤的生长,但作用结果不完全一致,具体机制有待进一步研究。
3.低剂量雄激素促进MCF-7乳腺癌荷瘤裸鼠肿瘤的生长,其肿瘤内CyclinD1的表达明显增加,而AR表达无明显变化。表明雄激素促进乳腺癌的生长可能是通过促进CyclinD1的表达引起的。
Breast cancer is the most common type of malignant tumors in women. Its incidence has increased gradually in our country. There are many factors to affect the genesis and development of breast cancer. Sex hormone is one of the factors which have been extensively investigated.
Beginning with the report of bilateral oophorectomy for advanced breast cancer in 1895, sex hormone, especially estrogen, was focused on to explore its effect on the growth and development of breast cancer. In the 1950s, there has been an enormous number of reports on estrogens, estrogen receptors(ER), ER blockade, aromatization. Most of the established epidemiological risk factors for breast cancer are related to alteration in endogenous hormone metabolis. A young age at first full-term pregnancy, high parity and prolonged duration of breast feeding all independently protect against tumor development. On the contrast, early menarche, late menopause and use of postmenopausal hormone therapy have been known to be associate with increased risk of breast cancer, and the increase is associated with higher sex hormone, especially estrogen. It is still unknown whether androgen is involved in the growth and development of breast cancer. Different studies always show different, or even contrary results.
In most epidemiological studies on postmenopausal women, elevation of androgen level are found to be associated with the increase of breast cancer. Missmer’s report showed those with the highest fourths testosterone have about 2 times of breast cancer risk than those with the lowest fourths. Eliassen also found the relationship between the blood level of testosterone with the risk of breast cancer in premenopausal women. Different from epidemiological investigations, studies on androgens and breast cancer cell line proliferation have multiple variables that affect the outcome of those studies. The type and the dose of androgens, the particular cell lines used and other variables have demonstrated different effects. Some show stimulating effects, some show antiproliferation effects. Studies on animals also show contrary outcomes. But the fact that aromtase inhibitors, blocker of the transform of estrogen from adrenal androgen, which have shown good effect on breast cancer confirms the stimulating effects of androgen on breast cancer. So the role of androgen on breast cancer still needs further explored.
In the present study, we used enzyme-linked immunosorbent assay (ELISA) to measure the testosterone concentration in breast cancer and breast tissue, and analyzed the difference between them, detected the relationship between the breast cancer testosterone concentration and clinicobiological features. Then we investigated the influence of testosterone on MCF-7 breast cancer cell line and breast cancer xenograft (MCF-7) in nude mice to detect the role of androgen on the growth and development of breast cancer, to offer a new marker or idea for the treatment and prognosis of breast cancer.
Part I The significance of androgen in breast cancer tissue Objective: To detect the concentration of testosterone in breast cancer, breast tissue 2cm and 5cm away from the tumor, and analyze the relation between the testosterone concentration and clinicobiological features.
Methods: ELISA was performed to examine the concentration of testosterone in breast cancer, breast tissue 2cm and 5cm away from the tumor in 30 breast cancer patients. Relationship was analyzed between them and clinicobiological features(menopause status, tumor size, nodal status, TNM stage, histological grade, histological type, ER/PR/AR status). Results:
1 The difference of testosterone concentration between breast cancer and tissue 2cm and 5cm away from tumor.
There was no significance in testosterone concentration between breast cancer and breast tissue 2cm and 5cm away from tumor(P=0.215). The testosterone concentration in premenopausal patients was lower than those in postmenopausal patients(P=0.037). In postmenopausal patients, the testos- terone concentration in breast cancer was higher than those in tissue at 5cm from tumor(P=0.031). No significant difference was found between breast cancer and the breast tissue in premenopausal patients(P=0.443).
2 The relationship between testosterone concentration in breast cancer tissue and clinicobiological features
2.1 There were no correlation between testosterone concentration in breast cancer tissue with weight (r=-0.158, P=0.402), age (r=0.292, P=0.11), and Body Mass Index(BMI) (r=-0.046, P=0.811).
2.2 Testosterone concentration in breast cancer tissue was related to histological grade. Testosterone concentration was higher in those with worse grade. Those with grade III had much more testosterone than those with grade I,II (P=0.001). No difference was found between grade I and grade II (P=0.286).
2.3 No relationship were found between testosterone concentration and tumor size, nodal status, TNM stage, pathological type (P>0.05 for all).
2.4 No relationship were found between testosterone concentration and the expression of ER, PR, AR, Her-2 (P>0.05 for all).
3 The relationship between AR and ER, PR, Her-2
Positive rate of AR in breast cancer was 86.67%, ER was 66.67%, PR was 60%. There were significant relationship between the expression of AR and ER (P=0.008), PR(P=0.018). No significance was found between AR and Her-2(P=0.388) whose positive rate was 80%.
4 Relationship between the expression of AR and clinibobiological features No relationship were found between the expression of AR and menopausal status, tumor size, pathological type, nodal status, TNM stage and histological grade (P>0.05 for all).
Conclusions
1 Testosterone concentration in breast cancer tissue of postmenopausal patients is higher than that in premenopausal patients. Testosterone concentration in breast cancer tissue is higher than that in breast tissue 5cm away from tumor in postmenopausal women, which suggests that testosterone may play a certain role in the genesis and development of breast cancer.
2 Testosterone concentration in breast cancer tissue is related to histological grading. Testosterone concentration is higher in those with grade III than those with grade I,II. The more involved axillary nodes is companied with the higher testosterone level in primary tumor. All of above suggests that the higher testosterone concentration, the higher malignant of the breast cancer. 3 The testosterone concentration in breast tumors may contribute to the diagnosis of the malignant status, to the diagnosis of recurrence and metastasis risk and prognosis. It may provide a novel endotherapy approach for breast cancer.
Part II The influence of androgen on MCF-7 breast cancer cell line
Objective: To study the influence of androgen on MCF-7 breast cancer cell line and explore the mechanism that how androgen affect the genesis and growth of breast cancer.
Methods: MTT was used to measure the proliferation of MCF-7 after stimulated with testosterone. Flow cytometry(FCM) was used to analyze the breast cancer cell cycle, apoptosis rate and the expression of CyclinD1 and androgen receptor(AR).
Results:
1 Influence of testosterone at different concentration on the proliferation of MCF-7 breast cancer cell line
The survival rate of MCF-7 cells exposed to 10-5 mol /L testosterone decreased significantly and inhibition rate increased markedly. The inhibitory rate was 12.46%,22.21%,44.72% in 24, 48, 72 h respectively. Cultures of MCF-7 cells exposed to 10-9 mol /L testosterone had cell growth compared with control cultures. Cultures of MCF-7 cells exposed to 10-7 mol /L and 10-11 mol /L testosterone showed neither proliferation nor inhibition in any time.
2 Influence of testosterone at different concentration on the MCF-7 cell cycle and apoptosis
2.1 After 24 h, high concentration testosterone(10-5 mol /L) increased the apoptosis rate of MCF-7 cells and impeded the progression of G1 cells into S phase and G2/M phase compared with control cultures(P<0.05 for all). No difference of apoptosis rate was found between the 10-9 mol /L testosterone pretreatment group and control group, but low concentration(10-9 mol /L) testosterone decreased the proportions of G1 phase cells and increase the proportions of S phase and G2/M phase cells(P<0.05 for all).
2.2 After 48h, 10-5 mol /L testosterone induced apoptosis of MCF-7 cells increasely, but it also decreased the proportions of G1 phase and increase the proportions of S phase and G2/M phase (10-5 mol /L). No difference was found between 10-9 mol /L testosterone group and control group.
2.3 10-7 mol /L and 10-11 mol /L testosterone showed little influence on cell cycle and apoptosis of MCF-7 cells(P>0.05 for all).
3 Influence of testosterone on the expression of CyclinD1 and AR of MCF-7 cells
3.1 After 24h, Cultures of MCF-7 cells exposed to 10-5 mol /L and 10-9 mol /L testosterone exhibited increased expression of CyclinD1 compared with control cultures (P<0.05 , both). There was no difference of AR in all groups. (P>0.05 for all).
3.2 After 48h, Cultures of MCF-7 cells exposed to 10-5 mol /L testosterone exhibited increased expression of CyclinD1 compared with control cultures (P<0.05). 10-9 mol /L testosterone showed no influence on CyclinD1. AR was found increased slightly(P>0.05) in 10-9 mol /L testosterone group. There was a significant difference of AR between 10-5 mol /L testosterone group and 10-9 mol /L testosterone group(P<0.05).
Conclusions:
1 Testosterone has bidirective effect on breast cancer cells. Lower concentration testosterone (10-9 mol /L) may stimulate MCF-7 cells proli- feration. Higher concentration testosterone(10-5 mol /L) may inhibite MCF-7 cells proliferation, which shows the complex effect of testosterone on breast cancer.
2 Lower concentration testosterone(10-9 mol /L) may increases the expression of CyclinD1 and impeds cell cycle, improves proliferation, which suggests that testosterone play a positive role on the genesis and development of breast cancer.
3 Higher concentration testosterone may inhibite the proliferation of MCF-7 cells, induce apoptosis which maybe relate to the overexpression of CyclinD1. High dose androgen may be useful for the treatment to breast cancer.
4 Higher concentration testosterone may down-regulate the expression of AR in breast cancer cells whereas lower concentration testosterone may up-regulate it, which suggests that high dose androgen may inhibite the proliferation of breast caner with positive AR.
5 This study may partly explain the hypothesis that higher concentration of testosterone may promote the proliferation of breast cancer and the pheno- menon that the more testosterone in tumor the worse malignance of tumor.
Part III Influence of androgen on the growth of human breast cancer xenograft (MCF-7) in nude mice
Objective: To investigate the effect of testosterone propionate (TP) at different dose on the growth of MCF-7 xenograft tumor in nude mice and to explore its may-be mechanism.
Methods: To establish the transplanted MCF-7 tumors in nude mice. Then to investigate the growth of transplanted tumors intervented by testosterone propionate at different dose (50mg/kg, 400mg/kg) and chemotherapic agent. Microparticle cheniluminescance immunoassay was performed to measure the level of serum testosterone of nude mice. FCM and immunohistochemical staining (IHC) was performed to examine the expression of CyclinD1 and AR in the transplanted tumors.
Results:
1 To establish the transplanted MCF-7 tumors in nude mice successfully About in 7 days, transplanted tumor by MCF-7 cells could be found in nude mice. The tumor all developed gradually into a smooth, circle or oval nodules.
2 Growth status of transplanted tumors
The volume of transplanted tumors in chemotherapy group were slightly nonsignificantly decreased compared with control group (P=0.868). The volume of transplanted tumor in low dose TP group increased significantly compared with control group and chemotherapy group(P=0.047, P=0.034 respectively). The growth of transplanted tumors in high dose TP group was complex. 3 transplanted tumors was gradually reduced, 3but 2 transplanted tumors became bigger obviously. There was no significant difference between high dose TP group and the other groups (P>0.05 for all).
3 Results of the expression of AR and CyclinD1 by FCM
3.1 No significant difference of AR expression was found among the four groups(P>0.05 for all) .
3.2 The expression of CyclinD1 in transplanted tumor in low dose TP group (403.28±31.23) was higher than those in control group (358.47±36.44,P=0.035) and chemotherapy group (352.94±19.14,P=0.020). 4 Results of the expression of AR and CyclinD1 by IHC AR protein was mainly expressed in the cell nucleus. No difference of AR expression was found among the four groups. Positive expression of CyclinD1 was found in low dose TP group and weakly expression in other groups.
5 Results of serum testosterone in nude mice transplanted by breast cancer xenograft (MCF-7).
Testosterone level in control group (1.40±0.23ng/ml) was non- significantly lower than that in chemotherapy group (2.38±0.17ng/ml, P=0.07). The testosterone level in low dose TP (50mg/kg) group (13.50±1.58ng/ml) was obviously increased compared with control group and chemotherapy group (P=0.000, P=0.000). The highest testosterone level was found in high dose TP (400mg/kg) group (14.85±0.33ng/ml), which was significantly higher than the other three groups(P=0.000, P=0.000, P=0.019 respectively).
Conclusion:
1 Low dose TP can stimulate the MCF-7 transplanted tumor which suggests than low dose androgen may promote the growth of breast cancer.
2 High dose TP shows inconsistent effect on the MCF-7 transplanted tumor which still needs further approach.
3 The growth of transplanted tumor in low dose TP with higher expression of CyclinD1 which means that the positive effect of androgen on breast cancer maybe trigger by CyclinD1.
引文
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