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標題: | 探討養分缺乏影響癌細胞代謝及HIF-1alpha表現的分子機制 The molecular mechanisms for nutrient deprivation-induced energy homeostasis and HIF-1α induction in cancer cells |
作者: | Ching-An Wu 巫清安 |
指導教授: | 林琬琬 |
關鍵字: | 瓦氏效應,丙酮酸脫氫?酵素,缺氧可誘發性因子甲型,自噬體,內部核糖體進入位, Warburg effect,PDK,HIF-1α,Autophagy,IRES, |
出版年 : | 2014 |
學位: | 博士 |
摘要: | 由於血流供給不足,缺氧與養分缺乏是固態腫瘤兩個很重要的特徵。與已經被廣泛研究的缺氧相反,養分不足,這另外一個固態腫瘤的特徵對癌細胞的影響則不清楚。在這篇論文中,我們嘗試去探索養分缺乏對癌細胞代謝與缺氧可誘發性因子甲型(Hypoxia-inducible factor-1α)的影響。
在第一部分,我們證明利用Hank's buffered salt solution (HBSS)造成養分缺乏會增加細胞外乳糖(lactate)的產生、細胞質內丙酮酸(pyruvate)的增加,並降低細胞氧氣的消耗,但是並不會影響乳糖脫氫酵素(lactate dehydrogenase)的活性或是葡萄糖的吸收。我們發現養分缺乏會快速的誘發丙酮酸脫氫酶酵素(pyruvate dehydrogenase kinase)之活化以及丙酮酸脫氫酶(pyruvate dehydrogenase)的磷酸化,而抑制ROS或AMPK會抑制這個現象。我們也證明ROS的產生會促進AMPK的活化。更進一步來說,抑制丙酮酸脫氫酶酵素、ROS與AMPK都可以顯著的減少養分缺乏所誘發的乳糖產生,同時促進養分缺乏所誘發的細胞凋亡。綜合來說,我們首次證明養分缺乏會經由ROS/AMPK依賴性的活化丙酮酸脫氫酶酵素這個新的機轉,來促使細胞經由糖解作用來產生ATP,也就產生瓦氏效應,而這個作用會延緩細胞因為養分缺乏而造成的死亡。 在第二個部分,我們的證據顯示,養分缺乏會經由cap非依賴性的內部核糖體進入位(internal ribosome entry site)的轉譯來增加可誘發性因子甲型的蛋白表現。特別的是,藉由si-ATG5、3-methyladenine或chloroquine來抑制自噬體(autophagy)可以顯著抑制養分缺乏所誘發的缺氧可誘發性因子甲型的表現,但是si-Beclin 1卻沒有這效果。此外,很有趣的是,缺氧時也會經由內部核糖體進入位來轉譯缺氧可誘發性因子甲型,但是與養分缺乏不同的是,si-Beclin 1而不是si-ATG5會抑制缺氧所誘發的缺氧可誘發性因子甲型內部核糖體進入位活性。綜合來說,我們首次證明養分缺乏與缺氧時替代性自噬體和缺氧可誘發性因子甲型的cap非依賴性轉譯的關聯性。我們證明Beclin 1非依賴性的自噬體可以正向調控養分缺乏所誘發的缺氧可誘發性因子甲型的內部核糖體進入位活性,而ATG5非依賴性的自噬體則參與缺氧所誘發的缺氧可誘發性因子甲型的內部核糖體進入位活性。 Hypoxia and nutrient deprivation are two important phenomenon in solid cancer due to the poor blood supply. In contrast to well-studied effect of hypoxia on tumor growth, the effect of nutrient deficiencies on cancer is not well defined. In this thesis, we try to explore the effect of nutrient deprivation on tumor cell metabolism and HIF-1α expression. For the part I, we demonstrate that Hank's buffered salt solution (HBSS) starvation increased lactate production, cytoplasmic pyruvate content and decreased oxygen consumption, but failed to change the lactate dehydrogenase (LDH) activity or the glucose uptake. We found that HBSS starvation rapidly induced pyruvate dehydrogenase kinase (PDK) activation and pyruvate dehydrogenase (PDH) phosphorylation, both of which were reversed by inhibition of ROS and AMPK. Our data further revealed the involvement of ROS production in AMPK activation. Moreover, inhibition of PDK, ROS, and AMPK all significantly decreased HBSS starvation-induced lactate production accompanied by enhancement of HBSS starvation-induced cell apoptosis. Taken together, we for the first time demonstrated that a low-nutrient condition drives cancer cells to utilize glycolysis to produce ATP (the Warburg effect) through a novel mechanism involving ROS/AMPK-dependent activation of PDK. Such an event contributes to protecting cells from apoptosis upon nutrient deprivation. For the part II, Our data showed that nutrient deprivation induces a significant HIF-1α protein expression through the internal ribosome entry site (IRES)-dependent translation. Notably inhibition of autophagy by si-ATG5, 3-methyladenine and chloroquine, but not si-Beclin-1, significantly reverses nutrient deprivation-induced HIF-1α responses. Furthermore, it is interesting to note that different from nutrient starvation, si-Beclin 1 but not si-ATG5 can inhibit hypoxia-induced HIF-1α IRES activation. Taken together, we for the first time highlight a link from alternative autophagy to IRES-dependent protein translation of HIF-1α under two unique stress conditions. We demonstrate Beclin 1-independent autophagy is involved to positively regulate nutrient deprivation induced-HIF-1α IRES activity, while ATG5-independent autophagy is involved in the HIF-1α IRES activation caused by hypoxia. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/5377 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 藥理學科所 |
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