- 學位論文
肝醣合成激酶誘導腦瘤細胞的瓦伯效應
Glycogen synthase kinase 3β induces Warburg effect in glioma cells
摘要
癌細胞具有增加葡萄糖吸收，將葡萄糖代謝為乳酸，並抑制粒線體的呼吸，降低三磷酸腺苷（adenosine triphosphate, ATP)生成之傾向，此一現象稱為瓦伯效應(Warburg effect)，由Otto Warburg於1920年代所發現。瓦伯效應可作為癌症之治療標的，其中亦包含腦瘤。肝醣合成激酶(Glycogen synthase kinase 3β, GSK3β)是一種絲胺酸(serine)/酥胺酸(threonin)的激酶，參與細胞及代謝上多種訊息路徑。而GSK3β在瓦伯效應中所扮演之角色至今仍未闡明，本研究利用GSK3β的抑制劑TDTZ-8 (4-benzyl, 2-methyl, 1,2,4-thiadiazolidine, 3,5 dione)以及GSK3β的轉殖細胞株，來探討腦瘤中GSK3β之代謝角色。本研究發現利用TDTZ-8以及轉殖細胞株GSK3βK85R抑制GSK3β的活性，或利用shGSK3β轉殖細胞株抑制GSK3β的表現，皆會抑制缺氧誘導因子(hypoxia inducible factor-1 alpha ,HIF-1α)以及葡萄糖轉運蛋白(glucose transporter-1, Glut-1)的表現，並降低丙酮酸去氫酶(pyruvate dehydrogenase, PDH)、丙酮酸去氫激酶(pyruvate dehydrogenase kinase-1, PDK-1)之磷酸化，而HIF-1α、Glut-1、 PDK-1以及PDH為細胞代謝與瓦伯效應中的主要調控者。此外使用TDTZ-8或利用GSK3βK85R及shGSK3β轉殖細胞株，亦會降低葡萄糖吸收以及乳酸堆積，透過提高粒線體的膜電位，增加ATP的產生與耗氧量。此外使用GSK3β過度表現的GSK3βWT細胞株與持續活化GSK3β的GSK3β S9A細胞株相對於TDTZ-8，則導致腦瘤細胞中HIF-1α、Glut-1、PDK-1及PDH-p的表現量增加，並增加葡萄糖吸收以及乳酸堆積，透過降低粒線體的膜電位，降低ATP的產生與耗氧量。而ATP產量、耗氧量與粒線體膜電位的提高伴隨乳酸堆積的減少，表示細胞的代謝由瓦伯效應之無氧糖解轉為有氧的方式去利用葡萄糖。此結果證明GSK3β的活化，可能誘導瓦伯效應，而GSK3β的抑制則能降低此代謝現象。本研究發現GSK3β的抑制劑 TDZD-8逆轉瓦伯效應，而GSK3β則可能為癌症治療的標靶。
並列摘要
Cancer cells have the tendency to increase uptake of glucose, to increase metabolism of glucose to lactate, and are associated with decreases in mitochondrial respiration and adenosine triphosphate (ATP) production, a phenomenon termed the “Warburg effect.” Much attention has focused on the Warburg effect as a target for cancer therapy, including for gliomas. Glycogen synthase kinase 3β (GSK3β) is a serine/threonine kinase that participates in numerous signaling pathways involved in cellular and metabolic processes. Our objective is to study the roles of GSK3β in Warburg effect, which has not been elucidated previously. Specifically, this study focuses on the metabolic roles of GSK3β in glioma cells; we studied the Warburg phenotype in glioma cells using GSK3β activity inhibitor 4-Benzyl-2-methyl-1,2,4- thiadiazolidine-3,5-dione (TDZD-8) and GSK3β clones. The inhibition of GSK3β activity by TDTZ-8 and GSK3βK85R clones and GSK3β expression by shGSK3β clones decreased the expressions of hypoxia inducible factor-1α (HIF1α), glucose transporter-1 (Glut-1), pyruvate dehydrogenase kinase-1(PDK-1) and decreased pyruvate dehydrogenase (PDH) phosphorylation. HIF1α, Glut-1, PDK-1 and PDH are major regulators of cell metabolism and Warburg effect. TDZD-8, GSK3βK85R and shGSK3β clones concurrently decreased glucose uptake, decreased lactate accumulation, increased ATP production and increased oxygen consumption through increasing mitochondria membrane potential in glioma cells. Furthermore, the overexpression of GSK3β in GSK3βWT clones and the constitutive activation of GSK3β activity in GSK3βS9A clones, increased Glut-1, PDK-1 expressions, PDH phosphorylation, glucose uptake and lactate accumulation, but decreased ATP production and decreased oxygen consumption through decreasing mitochondria membrane potential in glioma cells, the opposite effect to the inhibition of GSK3β activity and expression. The increase in ATP production, oxygen consumption and mitochondria membrane potential together with the decrease in lactate accumulation suggests a change in cellular metabolism from anaerobic Warburg phenotype to aerobic usage of glucose. These results suggest that GSK3β may induce the Warburg effect and its inhibition of its activity may decrease this metabolic phenotype. Our findings support a novel role for GSK3β in cancer metabolism, GSK3β may be a possible target for cancer therapy and TDZD-8, which inhibits GSK3β activity, may reverse the Warburg effect.