癌症是造成現代人死亡重要的疾病之一，在癌細胞生長的過程中，會偏好以糖解作用獲得能量，並產生大量的乳酸使其周遭的為環境酸化，德國科學家 Otto Warburg 在1920年代發現了這個現象，後來被科學家稱為瓦氏效應 (Warburg effect)。實驗室先前統整多個生物資料庫 (The Human Protein Atlas、Oncomine、cBioportal)發現，磷酸丙糖異構酶1(Triosephosphate isomerase 1, TPI1) 在大部分的腫瘤細胞都是過度表現解有較差的預後；實驗室也從肺癌病患檢體透過免疫組織染色法發現，磷酸丙糖異構酶1在早期肺癌病人，大部分都表現在細胞核內，而晚期的肺癌病人，磷酸丙糖異構酶1大部分表現在細胞質中。因此我利用短髮夾核糖核酸 (shRNA) 的技術，從中研院購買會導致細胞磷酸丙糖異構酶1默化的病毒進行細胞感染，將實驗室兩株肺癌惡性程度不同的細胞株 A549 以及 H1299 中的磷酸丙糖異構酶1默化 (A549 較良性，H1299 較惡性)，並探討其在癌細胞中扮演的角色，以及對其能量代謝的影響。從細胞生長密度、MTT 及細胞群落形成法 (Colony formation) 的實驗結果可以發現，磷酸丙糖異構酶1默化的細胞株，不論是生長速度明顯下降。接著看細胞爬行能力是否改變，從傷口癒合能力實驗 (Wound healing migration assay) 細胞遷移追蹤 (Cell migration tracking) 實驗觀察到，磷酸丙糖異構酶1的默化會造成細胞爬行變慢，也影響到細胞爬行的方向。由於磷酸丙糖異構酶1是參與在糖解作用其中的酵素，因此接著分析兩株肺癌細胞株其培養液的酸鹼值及對葡萄糖依賴度。從測試培養液 pH 值的實驗結果發現，A549磷酸丙糖異構酶1默化其培養液酸化程度較慢，H1299 則沒有明顯影響。從細胞生長密度及 MTT 實驗也發現 A549 磷酸丙糖異構酶1默化株，細胞死亡的時間較控制組來的慢。說明磷酸丙糖異構酶1默化對於培養液的酸化及葡萄糖依賴程度有下降的現象。再來以西方點墨法分析參與在糖解作用及細胞生長的相關蛋白，發現參與在糖解作用關鍵酵素 Hexokinase Ⅰ 及 Hexokinase Ⅱ 的表現量在兩株細胞有所差異，另一個關鍵酵素 PFK1 則都是有明顯的下降，而細胞週期相關蛋白，Cyclin B1 皆有明顯下降的現象，推測可能是導致細胞生長變慢的原因。

Analysis The Correlation between Triosephosphate Isomerase 1 (TPI1) and Malignancy of Lung Cancer

Author: Shu-Feng Yeh
Advisor: Dr. Wen-Tsan Chang
Department of Biochemistry and Molecular Biology

SUMMARY
Cancer is one of the most important diseases that cause death. In previous study found that many cancer cells produce increased level of lactate, even when oxygen abounds, and change its metabolism from Tricarboxylic acid cycle (TCA cycle) to aerobic glycolysis. This phenomenon is called “Warburg Effect”. One of the enzyme in glycolysis: Triosephosphate isomerase 1 (TPI1) is a isomerase that catalyzes the isomerization of glyceraldehydes 3-phosphate (G3P) and dihydroxy-acetone phosphate (DHAP). TPI1 plays an important role in glycolysis and is essential for efficient energy production. Our previous study found that TPI1 associates with lung cancer malignancy. The location of TPI1 is found in nucleus in early stage lung cancer patients, while in cytosol late stage lung cancer patients. In my study, I found that TPI1 knockdown shows a lower proliferative and colony formation ability by the MTT and colony formation assay. TPI1 knockdown can decrease cancer cells glucose addiction and culture medium acidification. It means TPI1 knockdown can reduce cancer Warburg effect level. In wound healing and cell migration tracking assay, the rate of migration in TPI1 knockdown cells decrease, and the accumulated distance becomes shorter. In the western blot result, the expression of the rate-limiting key enzyme in glycolysis, PFKL, declined. Cell cycle related protein expression level, the western blot result shows that Cyclin B1 is declined.
Key words: Warburg effect, Triosephosphate isomerase 1, Lung cancer, Glycolysis
INTRODUCTION
Under sufficient oxygen condition, normal cells metabolize glucose through oxidative phosphorylation for gaining energy. If under hypoxia condition, cells prefer to a anaerobic glycolysis to digest glucose. In cancer cell, even under regular oxygen condition, they still use glycolysis for glucose metabolism. This phenomenon was found by German scientist Otto Warburg in 1920. The different metabolic pathway of gaining energy between normal and cancer cells in oxidative condition may be a good target for curing cancer. Triosephosphate isomerase 1 (TPI1) is a isomerase that catalyzes the isomerization of glyceraldehydes 3-phosphate (G3P) and dihydroxy-acetone phosphate (DHAP). Clinical significance of TPI1, Triosephosphate isomerase deficiency, is a disorder characterized by a shortage of red blood cells (anemia), movement problems, increased susceptibility to infection, and muscle weakness that can affect breathing and heart function.
MATERIALS AND METHOD
To establish the TPI1 knockdown stable clone, we use shRNA lentivirus system as tool. We infect shTPI1 into A549 and H1299 cancer cell line. The proliferation rate and colony formation ability was tested by MTT assay, colony formation assay and taking pictures with microscope. We treat with different glucose concentration and observe medium acidification to exam Warburg effect level. We also use western blot to detect the change of related protein expression.
RESULT AND DISCUSSION
In the MTT assay and colony formation assay, TPI1 knockdown cell decrease proliferation and colont formation ability. In wound healing assay and single cell migration tracking, TPI1 knockdown cell shows a lower migration. We also find different protein expression level in PI1 knockdown cell.
CONCLUSSION
TPI1 knockdown cause cell morphology change, decrease cell proliferation, migration and colony formation, degree of glucose dependence decreased in A549 TPI1 knockdown cell line and related protein expression change.

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