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標題: | 探討長期高胰島素培養對於三陰性乳癌細胞放射線感受性的影響 Investigating the Effects of Chronic Insulin Treatment on the Susceptibility of Triple-Negative Breast Cancer Cells to Ionizing Radiation |
作者: | 薛涵禧 Han-Xi Xue |
指導教授: | 郭靜穎 Ching-Ying Kuo |
關鍵字: | 三陰性乳腺癌,高胰島素血症,脂肪酸氧化,放射線抗性,DNA損傷反應, Triple-negative breast cancer,hyperinsulinemia,fatty acid oxidation,radioresistance,DNA damage response, |
出版年 : | 2023 |
學位: | 碩士 |
摘要: | 代謝症候群會增加人們罹患肥胖、第二型糖尿病、心血管疾病的風險,是目前全世界棘手的健康問題。患有代謝症候群的人們時常伴隨著高胰島素血症的發生,而高胰島素血症也會促進乳腺癌進程。多項研究指出肥胖會使乳腺癌病患對放射線治療產生抗性且病患也會有預後較差之情形。三陰性乳腺癌(TNBC)約佔所有乳腺癌患者的15-20%,因其缺乏有效之治療靶點,故目前以手術搭配化學治療與放射線治療為主。然而,代謝症候群和乳腺癌病患產生放射線抗性之原因與詳細機制目前仍不清楚。
實驗室於過往研究中建立了一個飲食誘導肥胖的EO771乳腺癌同源小鼠模型,透過生化檢測觀察到,相較於正常飲食之小鼠,由高脂肪飲食所誘導之肥胖小鼠血中胰島素含量較高,顯示高胰島素血症的發生。另外,高脂肪飲食不但會誘使小鼠腫瘤的生長,也增加小鼠腫瘤對放射線治療的抗性。為了進一步探討由肥胖所誘導之高胰島素血症與放射線抗性的關聯與詳細機制,將多個TNBC細胞株長期培養於高胰島素環境(CI)中,以觀察其DNA損傷反應以及對放射線抗的感受性。首先,我們確認這些CI細胞對額外之胰島素刺激已不再有反應。另外,CI細胞皆表現較佳之生長能力且對放射線具有抗性。除此之外,我們發現MDA-MB-231-CI細胞經放射線照射過後,DNA雙股斷裂有減少的情形。因此,我們推測CI TNBC細胞產生放射線抗性的機制,很有可能是因為在放射線照射後較少的DNA損傷產生。 接著,我們進一步利用轉錄體分析觀察到CI細胞於粒線體代謝與脂肪酸氧化相關代謝路徑有上調的趨勢。後續也證明MDA-MB-231-CI細胞會增加其粒線體活性,且伴隨著細胞內及粒線體中活性氧化物質之積累。最後,透過藥物Etomoxir抑制MDA-MB-231-CI細胞中脂肪酸氧化之活性則會促進放射線誘導的DNA雙股斷裂增加並且提升MDA-MB-231-CI細胞對於放射線的敏感性。 綜上所述,CI TNBC細胞可能藉由粒線體與脂肪酸氧化之活性而產生放射抗性,而CI TNBC細胞與粒線體及脂肪酸氧化活性之關聯,以及其造成放射線抗性之詳細機制仍需進一步研究,以期待能更清楚了解高胰島素血症所造成乳腺癌細胞產生放射線抗性之分子機制。 Metabolic syndrome is a growing public health concern worldwide. It is associated with obesity, type 2 diabetes and is characterized by hyperinsulinemia which has been demonstrated to promote breast cancer progression. In addition, some studies have shown that obesity promotes breast cancer radioresistance and has poor therapeutic outcome. Triple-negative breast cancer (TNBC) is a subtype of breast cancer that accounts for 15-20% of all cases and is characterized by low expression of estrogen, progesterone, and HER2 receptors, resulting in limited therapeutic strategies. The mainstay treatment for TNBC remains surgery followed by cytotoxic chemotherapy and radiotherapy. However, the link between metabolic syndrome and radioresistance in breast cancer remains unclear. According to our previous study, we established a high-fat diet (HFD)-induced obesity EO771 syngeneic breast cancer model. Through biochemical analysis, we observed that mice fed with HFD had higher fasting insulin level in mice comparing to those fed with control diet, indicating the development of hyperinsulinemia. We also showed that HFD not only promoted tumor growth but also exhibited radioresistance after radiotherapy. To investigate the relationship and the underlying mechanism between obesity-associated hyperinsulinemia and tumor radioresistance, several chronic insulin (CI)-treated TNBC cell lines have been established for characterizing the DNA damage response and radiosensitivity. First, we confirmed that these CI TNBC cells, were no longer responsive to additional insulin stimulation. In addition, these CI cells exhibited higher cell proliferation rate and were more radioresistant. Besides, we observed that MDA-MB-231-CI cells had less DNA double-strand breaks after irradiation, suggesting that these CI TNBC cells were resistant to irradiation possibly due to less DNA damage production. Furthermore, we conducted transcriptomic analysis and observed an upregulation of metabolic pathways related to mitochondrial metabolism and fatty acid oxidation in CI cells. Subsequently, we confirmed that MDA-MB-231-CI cells exhibited increased mitochondrial activity, accompanied by the accumulation of intracellular and mitochondrial reactive oxygen species. Finally, by inhibiting fatty acid oxidation activity with a CPT1 inhibitor, etomoxir, we found an increase in DNA double-strand breaks in MDA-MB-231-CI cells following radiation exposure. Additionally, etomoxir resensitized MDA-MB-231-CI cells to radiation. In summary, CI TNBC cells might develop radioresistance through the activation of mitochondrial and fatty acid oxidation activities. The association between CI TNBC cells, mitochondrial function, fatty acid oxidation activity, and the detailed mechanisms underlying radioresistance require further investigation to acquire a clearer understanding of the molecular mechanisms by which hyperinsulinemia contributes to radioresistance in breast cancer cells. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/89222 |
DOI: | 10.6342/NTU202302229 |
全文授權: | 同意授權(限校園內公開) |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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