正常細胞在進行能量代謝時，在氧氣充足的情況主要是經由糖解作用、檸檬酸循環和粒線體氧化磷酸化進行呼吸作用產生ATP、二氧化碳和水，又稱為有氧呼吸。而在缺氧的情況下則會經由糖解作用產生乳酸和ATP，又稱為無氧呼吸。而腫瘤細胞以及生長快速的細胞不論在有氧或是無氧的情況下皆偏好使用糖解作用產生ATP和乳酸，此現象即稱為瓦氏效應(Warburg effect)。在腫瘤治療方面目前主要是以外科手術、放射線治療和化學治療為主，放射線治療主要是藉由破壞癌細胞的DNA使其進行細胞凋亡，化學治療主要是利用抗癌藥物抑制腫瘤細胞DNA生合成，抑制細胞微管增生以及針對特定目標蛋白抑制使得細胞凋亡。
然而化學治療雖然可以毒殺腫瘤細胞，但是也會影響其他正常細胞(如腸道表皮細胞和骨髓細胞)的生長，同時也會篩選出具有抗藥性的癌細胞，使日後治療的難度提高。而目前有文獻指出可以針對腫瘤細胞偏好使用糖解作用的特性，使用抑制能量代謝的抗癌藥物進行治療。
在本篇研究中首先我先分析實驗室擁有的六株癌細胞(HeLa, MCF7, MDA-MB-231, PC3, A549, H1299)的生長速率發現其生長速率最快的是H1299而生長速率最慢的是MCF7，並以西方點墨法分析這六株癌細胞p53及相關蛋白的表現量，發現PC3和H1299的p53蛋白幾乎不表現。接下來我利用糖解作用抑制劑2-去氧葡萄糖(2-deoxyglucose, 2-DG)處理六株癌細胞和穩定抑制檸檬酸合成酶HeLa細胞並分析其生長速率，發現2-DG對於HeLa、 PC3、MDA-MB-231的影響較大，對A549、MCF7和H1299的影響較小，而2-DG對穩定抑制檸檬酸合成酶HeLa細胞的生長速率影響不大。接下來我利用第二型糖尿病用藥每幅敏(metformin) 處理六株癌細胞和穩定抑制檸檬酸合成酶HeLa細胞並分析其生長速率，由實驗結果發現metformin對細胞生長影響不大，僅在高濃度(10, 20mM)才會使細胞生長較慢，而對穩定抑制檸檬酸合成酶HeLa僅在20mM對生長速率的影響較為明顯。
由於2-DG和metformin單獨處理細胞對細胞生長影響不大，並且有文獻指出將2-DG和metformin共同處理細胞會顯著抑制腫瘤細胞生長，故我利用2-DG和metformin個10和20mM處理六株癌細胞和穩定抑制檸檬酸合成酶HeLa細胞，由實驗結果發現共同處理2-DG和metformin對細胞生長影響具有加成作用，並誘導細胞進行細胞凋亡，而這種情形在高度利用糖解作用的穩定抑制檸檬酸合成酶HeLa中更為顯著。
綜合以上實驗結果，發現共同處理2-DG和metformin對於影響細胞生長具有加成性且會誘導細胞進行凋亡，這對於以細胞能量代謝作為癌症治療標的具有很大的參考價值。

Summary
As tumor gets energy by energy metabolism, it relies on glycolysis to get abundant adenosine triphosphate (ATP), lactate and macromolecular for growth. This phenomenon calls “Warburg effect.” The ways for tumor therapy are including surgery, radiation therapy and chemotherapy. Surgery is used to treat the patient by removing tumors. The radiation therapy is used to kill tumor cell by breaking the DNA of the tumor cell. The chemotherapy is using anticancer drug to kill cancer by inhibiting the DNA synthesis of tumor, inhibiting the microtuble proliferation and inhibiting the target protein. However, tumor cell has multiple drug resistance (MDR) after treating anticancer drugs, and it may decrease the efficacy of the chemotherapy. As the result, it is important to take anticancer drugs targeting another pathway such as tumor energy metabolism to treat cancer.
In the research, I use different dosage of 2-deoxy glucose (2-DG) and metformin to treat HeLa, MCF7, MDA-MB-231, PC3, A549, H1299 and HeLa that is stable knockdown the citrate synthase (CS). I found that after treating these drugs, the inhibition of the tumor cell growth is not clear. And I cotreated 10 and 20 mM 2-DG and metformin on these tumor cell lines. I found the inhibition of the tumor cell growth is significant. I analyzed the cell apoptosis of these tumor cell lines after cotreating 2-DG and metformin by flow cytometer. I found that it will induce cell apoptosis after cotreating drugs. As the result, I found that there is additive effect of inhibiting tumor cell growth after cotreating 2-DG and metformin.
Key words: energy metabolism, tumor therapy, 2-DG, metformin

INTRODUCTION
The differentiated tissue metabolizes glucose to get ATP and pyruvate. And pyruvate enters TCA cycle and mitochondrial oxidative phosphorylation to get more ATP and carbon dioxide. When there is lack of oxygen, tissue metabolizes glucose by glycolysis and gets ATP and lactate. This is called anaerobic respiration. The proliferative cell and cancer cell prefer to use glycolysis to get ATP and produce lactate. This is called aerobic glycolysis and Warburg effect. (Vander Heiden et al., 2009)


There are several ways to treat tumor, including surgery, radiation therapy and chemotherapy. Doctors take surgery to remove tumor. Radiation therapy breaks DNA of tumor cell to make cancer cell apoptosis. Chemotherapy is treated by using anticancer drugs to kill cancer cell by inhibiting the DNA synthesis of tumor, inhibiting the microtuble proliferation and inhibiting the target protein. However, the anticancer drug may kill the other fast growing cells such as bone marrow cell (Vander Heiden, 2011). The cancer cell may develop multiple drug resistance (MDR) mechanism to against anticancer drug, and it will decrease the efficacy of chemotherapy (Vander Heiden, 2011).
2-deoxy glucose (2-DG) is the analog of glucose. It can inhibit glycolysis after phosphorylated by hexokinase. It is known to make tumor cell apoptosis (Aft et al., 2002). Metformin known is treated type 2 diabetes is known to inhibit the growth of tumor cell(He et al., 2012; Lee et al., 2012). It is reported that metformin cotreated with other anticancer drugs can increase the sensitivity of chemotherapy (Hirsch et al., 2009; Iliopoulos et al., 2011).
The research is to find the effect of inhibiting the tumor cell growth after treating glycolysis inhibitor, 2-DG, and metformin individually and together.

Materials and Methods

Cell culture: Cancer cell lines (HeLa, MCF7, MDA-MB-231, PC3, A549, H1299) are incubated with DMEM and RPMI supplemented with 10% FCS and 1% antibiotics and antimitotic at 37˚C and 5% CO2.
Reagents: 2-DG and metformin were purchased from Sigma-Aldich. DMEM was purchased from Invitrogen.
Flow cytometry analysis: Flow cytometry analysis was using FL1 and FL3 of FAS can purchased from BD

Results and Discussion
I used different dosage of 2-deoxy glucose (2-DG) and metformin to treat HeLa, MCF7, MDA-MB-231, PC3, A549, H1299 and HeLa that is stable knockdown the citrate synthase (CS). I found that after treating these drugs individually, the inhibition of the tumor cell growth is not clear. I found that 10 and 20 mM 2-DG can inhibit the growth of cancer cell significantly. 20 mM metformin could inhibit tumor cell little significantly. And then I cotreated 10 and 20 mM 2-DG and metformin on these tumor cell lines. I found the inhibition of the tumor cell growth is significant. I analyzed the cell apoptosis of these tumor cell lines after cotreating 2-DG and metformin by flow cytometer. I found that it will

induce cell apoptosis after cotreating the two drugs. As the result, I found that there is additive effect of inhibiting tumor cell growth after cotreating 2-DG and metformin. The results of my research indicated that anticancer drug could be treated through tumor energy metabolism.

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