檸檬酸(Citrate)是一種有機弱酸，其普遍存在於蔬果類當中，並且被大量應用在食品工業、化妝品和醫藥上面，其作用非常的廣泛。換言之，檸檬酸具有高度的穩定性與安全性。而在生化方面，檸檬酸也扮演了重要的角色，為檸檬酸循環(TCA cycle)的第一代謝產物，並且也參與了脂肪酸(Fatty acid)的合成。但特別的是，檸檬酸在先前許多研究中被認為是糖解作用(Glycolysis)的抑制劑。在瓦式效應(Warburg effect)中，惡性的腫瘤細胞會傾向將其生長代謝方式，由傳統的檸檬酸循環轉變成糖解作用來快速獲取能量。在實驗室之前的研究裡，我們發現穩定默化檸檬酸合成酶的子宮頸癌細胞株有惡性轉型的現象。這種細胞對於一些抗癌藥物，例如依托泊苷(Etoposide)和放射線治療的抵抗性也提高了。從之前實驗結果發現，限制培養基中葡萄糖的含量可以有效的殺死這個細胞株，暗示著這株細胞株的能量代謝途徑的確已經轉移到糖解作用，因此本研究使用糖解作用抑制劑檸檬酸鈉(Sodium citrate)做為抑制惡性腫瘤的藥物。在本實驗裡面發現檸檬酸鈉可以專一且有效地殺死這株細胞株，另外在實驗室之前建立的穩定默化乳酸脫氫酶乙型(Lactate dehydrogenase B)與穩定默化六碳糖激酶1(Hexokinase 1)的子宮頸癌細胞株，其能量代謝傾向走糖解作用的惡性腫瘤細胞，實驗結果同樣看到抑制效果。相反地，若是將檸檬酸鈉處理在之前實驗室建立的穩定默化乳酸脫氫酶甲型(Lactate dehydrogenase A)與穩定默化六碳糖激酶2(Hexokinase 2)的子宮頸癌細胞株，其能量代謝趨向於走氧化磷酸化(Oxidative phosphorylation)，細胞也較不惡性，實驗結果發現這兩種細胞株對於檸檬酸的敏感性較低，暗示著檸檬酸鈉可以專一性殺死較為惡性的細胞。除了處理在實驗室已建立的細胞株外，本實驗也將檸檬酸鈉處理在不同的野生型腫瘤細胞株內，結果發現較為惡性的野生型腫瘤細胞株確實有大量的死亡。透過藥物處理後的西方點墨法發現，糖解作用途徑中的己糖激酶2(Hexokinase 2)和乳酸脫氫酶甲型(LDHA)皆有受到抑制，且從許多文獻中指出，抑制這兩種酵素的表現確實會使惡性的腫瘤細胞造成死亡，因此更加證實檸檬酸鈉會使腫瘤細胞無法進行糖解作用而導致細胞走向細胞凋亡(Apoptosis)。臨床上，檸檬酸鈉與檸檬酸鉀(Potassium citrate)分別扮演著不同功能，然而在本實驗當中，這兩種不同的藥物依然可以達到抑制惡性腫瘤生長的效果，因此，從這個地方可以得知，使惡性腫瘤造成大量死亡的主要是檸檬酸，並非檸檬酸上所攜帶的金屬離子所致。進一步我將檸檬酸處理在動物實驗上，也同樣在高濃度看到了腫瘤抑制效果。綜合以上實驗結果發現，檸檬酸未來可以用來做為一種抗癌藥物，不但成本低廉且穩定性高，而且也可以依造病患的狀況，給予攜帶不同金屬離子的檸檬酸做為治療。

Citrate is an organic weak acid exists in vegetables and fruits. It has been used in food industrial, cosmetic science and biomedicine fields, and is a compound of high stability and safety. Citrate also plays an important role in biochemistry, serving as the first deriviative metabolite in tricaboxylic acid cycle, and a modulator in fatty acid synthesis. Particularly, previous studies indicate that citrate is an inhibitor of glycolysis. Malignant tumor cell has an inclination of shifting its metabolic pathway from traditional TCA cycle to glycolytic pathway, a phenomenon called Warburg effect. In our previous research, we found out that a cervical cancer cell line will undergo malignant transformation after persistent silencing of citrate synthase. This cell line has a higher resistance to Etoposide and radiation treatment, but is much more sensitive to glucose deprivation culture enviornment, suggesting that these cells shift its energy consumption pathway to glycolysis metabolism. In this study I use glycolysis inhibitor, sodium citrate, as an anti-tumor drug. My experiment results suggest that sodium citrate can kill this cell line with high efficiency. This result is also confirmed in treating with stable silencing of Lactate dehydrogenase B or Hexokinase I tumor cell line which are also a kind of malignant tumor cell lines. On the contrary, stable silencing of Lactate dehydrogenase A or Hexokinase II tumor cell line, which uses oxidative phosphorylation as its energy source, shows no such drug sensitivity after sodium citrate treatment. This indicates that sodium citrate can specifically kill these malignant tumor cell lines. In spite of the treatment to our own established cell lines, I also treat sodium citrate with other wild type tumor cell lines, and get the same result. My western data shows that glycolytic enzymes Hexokinase II and LDHA have a lower expression pattern after drug treatment. According to other studies, inhibition of these two enzyme expressions would lead to glycolysis pathway shut down and force cells’apoptosis. Sodium citrate and Potassium citrate play different roles clinically, but have shown the same result in the experiment, suggesting that it is the citrate compound, not the metal ions, that inhibits malignant tumor growth. Followed by previous experiments, we also find out the same result in animal experiments. To summarize the experiment results, we suggest that citrate could be a potential anti-tumor drug with a lower price and highly stability. It is also possible that we can treat the patients with different metal ions compounds according to their condition.

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