The Study Of Role Of Androgen On The Genesis And Development Of Breast Cancer

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 features2.1 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-2Positive 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).Conclusions1 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 lineObjective: 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 lineThe 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 cells3.1 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 miceObjective: 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 tumorsThe 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 FCM3.1 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.