雌激素受體β拮抗劑減少粒線體生合成及促進MCF-7乳癌細胞生長

Estrogen receptor β agonists attenuate mitochondrial biogenesis and promote cell growth in MCF-7 breast cancer cells

張項詒(Hsiang-Yi Chang)

指導教授 ： 高淑慧

臺北醫學大學/醫學院/醫學科學研究所/碩士(2009年)

https://doi.org/10.6831/TMU.2009.00143

摘要

乳癌是女性常見的疾病，66%的患者具有雌激素受體(Estrogen receptor, ER) 陽性的特徵。雌激素進入細胞後，和雌激素受體結合，並進入細胞核，成為轉錄因子，啟動一連串的基因並轉錄出生長因子使腫瘤生長。雌激素受體有兩類分別為ERα和ERβ，迄今對於ERβ的調控功能尚未了解，有研究指出乳癌組織中的ERβ若位於細胞質則病患其存活率。而ERβ被證實可以進入粒線體中，但其功能尚未完全了解，我們推測在乳癌細胞，ERβ agonist可能調降粒線體的生合成功能(biogenesis)，抑制粒線體的呼吸作用，並且降低三羧基酸循環(TCA cycle)所產生的ATP，使乳酸過度堆積，進而促進腫瘤生長。本研究中，我們首先發現ERβ大多位於粒線體當中，當以 ERβ的agonists diarylpropionitrile (DPN)和17β-estradiol (E2)處理MCF-7乳癌細胞，DPN和E2可促進細胞生長。在細胞增生的實驗中(MTS assay)發現相對控制組，E2有2.6倍; DPN有1.6倍的增生率。此外E2和DPN也會降低粒線體的生合成，E2組粒線體mRNA降低為76.7%，DPN則降低為76.4%。並且降低由粒線體基因編碼(mitochondrial DNA encoded) 的粒線體呼吸鏈蛋白質Complex IV subunit I。進一步的實驗發現粒線體膜電位，DPN處理促使膜電位下降24.8%， E2組則是下降13.5%。在活性氧分子(Reactive oxygen species, ROS)的產生方面，添加oligomycin會引發細胞內產生大量的ROS。添加DPN或E2能減緩oligomycin ROS的生成，在DPN組ROS下降24.9%，E2組則能下降29.6%。在ATP的偵測中，E2會降9.8%，DPN則會降低9.5%的ATP含量。同時我們也發現當細胞在E2和DPN的作用下，會引發hypoxia-induciable factor 1α(HIF1-α)和pyruvate dehydrogenase kinase(PDK)的蛋白質表現量上升，並使TCA cycle酵素pyruvate dehydrogenase(PDH)表現量下降，醣解作用酵素phosphofructokinase的表現量增加也代表細胞更依賴醣解作用產生的能量，並且提高了無氧發酵作用酵素lactate dehydrogenase(LDH)的活性。當我們以ERβ shRNA處理細胞後，則會降低由E2或DPN所增加的LDH活性，因此我們可以推論MCF-7腫瘤細胞在ERβ agonists作用後可以抑制粒線體的功能，使細胞偏好無氧發酵產生能量進而促進腫瘤生長。
關鍵字：乳癌、雌激素受體β、粒線體

關鍵字

乳癌 ； 雌激素受體β ； 粒線體

並列摘要

Breast cancer is prevalent among women and estrogens are involved in two-thirds of breast tumors. Estrogens bind with estrogen receptors (ERs) in the cytoplasm, and are translocated to nucleus as transcription factors to activate the expression of a number growth factors and promote tumor growth. Up till now, two ERs have been identified: Estrogen receptor α (ERα) and β (ERβ). The role of ERα in the estrogen-mediated pathway has been carefully studied, whereas the function of ERβ is still unclear. Previous studied have demonstrated that the expression of cytoplasmic estrogen receptor β (ERβ) indicates the poor survival and prognosis. ERβ has also been found to translocate to mitochondria, but its mechanism remains unclear. We hypothesize that ERβ agonists can attenuate mitochondrial biogenesis, suppress mitochondrial respiration, inhibit ATP production, enhance glycolysis and lactate accumulation, and promote tumor proliferation in breast cancer. In this study, MCF-7 breast cancer line was treated with ERβ agonists, diarylpropionitrile (DPN) and 17β-estradiol (E2), and the expression of major ERβ was detected in mitochondria of MCF-7. E2 and DPN were found to promote cell growth. In cell proliferation MTS assay, E2 or DPN could enhance cell proliferation by 2.6 and 1.6 times, respectively. In addition, E2 or DPN were found to downregulate mitochondrial ND-1 mRNA level to 76.7% and 76.4%, respectively. The expressions of respiratory complex proteins were also decreased by DPN or E2 treatment. Moreover, the effect of E2 and DPN on mitochondrial membrane potential was assessed. DPN attenuated mitochondrial membrane potential by 24.8% and 13.5% by E2,
as compared with the control group. In assay for the reactive oxygen species (ROS), we found that DPN and E2 could reduce the oligomycin-induced ROS generation. A 9.8% decrease in ATP production by E2 and 9.5% decrease by DPN were also found. Furthermore, hypoxia-inducible factor 1α (HIF1-α) and pyruvate dehydrogenase kinase (PDK) protein levels were increased by DPN or E2, but the expression of pyruvate dehydrogenase (PDH) was decreased. The activities of both glycolytic enzyme phosphofructokinase (PFK) and lactate dehydrogenase (LDH) were increased, indicating that the means the cell relied more on glycolysis for supply of energy. By use of ERβ shRNA, the LDH activity was reduced by DPN or E2 treatment. In conclusion, we demonstrated that ERβ agonists inhibit mitochondrial function and cancer cells therefore derive most of their energy from anaerbolic glycolysis to maintain cell growth. Key words：breast cancer、estrogen receptor β (ERβ)、mitochondria

並列關鍵字

endometriosis ； estrogen receptor β (ERβ) ； mitochondria

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