1926 年，Otto Warburg 提出一個理論即使在氧氣充足的情形下某些特定的腫瘤細胞產生能量的方式是依賴糖解作用而不是氧化磷酸化，這種現象被稱為瓦氏效應(Warburg effect)。在腫瘤細胞中造成糖解作用增加具有潛力的機制為粒線體缺失、缺氧、致癌信息和改變的代謝酵素，而延胡索酸酶(Fumarate hydratase, FH)屬於其中的一員。在先前的研究中，檸檬酸循環中的酵素延胡索酸酶(FH)在遺傳上的突變與遺傳性平滑肌瘤性腎細胞癌(Hereditary leiomyomatosis renal cellcarcinoma, HLRCC)有關。當延胡索酸酶(FH)突變會造成延胡索酸(fumarate)累積，過量的延胡索酸會抑制脯氨酸羥化酶(proline hydroxylase, PHD),而脯氨酸羥化酶(proline hydroxylase, PHD)會抑制缺氧誘導因子-1 (HIF-1)表現，進而造成腫瘤血管新生以及腫瘤細胞偏好糖解作用。在腎臟癌症中，延胡索酸酶被認為是一種抑癌基因(tumor suppressor)，但在其他細胞株中的研究卻很少。在此次的研究中，我首先在子宮頸癌細胞中建立穩定沉默延胡索酸酶的細胞株並分析野生型子宮頸癌細胞(HeLa)和穩定沉默延胡索酸酶細胞株(HeLa-shFH)之間的差異。在正常情況下，抑制延胡索酸酶對細胞的生長、爬行能力和粒線體膜電位並沒有明顯的影響。持續性沉默FH 表現的穩定細胞株對葡萄糖攝取能力大於野生型HeLa細胞株。我嘗試利用2-去氧葡萄糖(2-deoxyglucose, 2-DG)抑制糖解作用及限制葡萄糖的來源並研究其對沉默延胡索酸酶細胞株的影響。我發現在處理不同葡萄糖濃度(0,1,4.5 mg/ml)或是在處理2-去氧葡萄糖(2-deoxyglucose, 2-DG)的狀況之下，HeLa-shFH 的細胞株顯示出比野生型細胞株具有更高的敏感性。並且在處理2-deoxyglucose 的沉默延胡索酸酶細胞株相較於野生型子宮頸癌細胞，其細胞死亡情形偏向細胞凋亡(apoptosis)而非細胞壞死(necrosis)。處理電子傳遞鏈complex I 抑制物，Rotenone，以及將細胞培養在不含L-glutamine 的培養液中，穩定沉默延胡索酸酶細胞株和野生型細胞株具有相同的影響。證明沉默延胡索酸酶細胞株對葡萄糖有依賴性。

In 1926, Otto Warburg proposed the theory that certain cancer cells generate ATP dependent on glycolysis pathway rather than oxidative phosphorylation even in the presence of ample oxygen, the phenomenon was called Warburg effect.Mitochondrial defects, hypoxia, oncogenetic signals and altered metabolic enzymes are potential mechanisms leading to increase in glycolysis in cancer cells. Fumarate hydratase (FH) is the component of altered metabolic enzyme. In previous studies, germline mutation of the Kerbs cycle enzyme fumarate hydratase (FH) is related hereditary leiomyomatosis and renal cell cancer (HLRCC).And mutation of FH leads to accumulation of fumarate. The excess metabolite, fumarate inhibits the proline hydroxylases that suppress the expression of hypoxia inducible factor 1 (HIF-1), a transcription factor implicated in tumor angiogenesis and tumor-cell glycolysis. In renal cancer, FH is considered a tumor suppressor gene but rarely studies in other cell line. In this study, I established FH stable knockdown cell lines in HeLa cell and analyzed the differences between wide type cells and stable knockdown cell lines. Suppression of FH has no apparent impact on cell growth, migration ability and mitochondrial membrane potential in normal condition. HeLa-shFH cell lines increase glucose uptake. Then I try to inhibit glycolyis pathway by treatment of 2-deoxyglucose or limit glucose source in HeLa-shFH cell lines and investigate the effect of HeLa-shFH cell lines. I found HeLa-shFH cell lines appear to be more sensitive in low and no glucose condition or in 2-DG existence condition than wild type cell lines. Particularly, in the treatment of 2-deoxyglucose HeLa-shFH cell line prefer apoptosis to necrosis than HeLa cell. HeLa-shFH cell lines and wild type cell lines have the same effect by treatment of complex I inhibitor, Rotenone or cultured in no L-glutamine medium. I demonstrate that HeLa-shFH cell lines depend on glucose.

Alam, N., Bevan, S., Churchman, M., Barclay, E., Barker, K., Jaeger, E., Nelson, H.,
Healy, E., Pembroke, A., and Friedmann, P. (2001). Localization of a gene (MCUL1)
for multiple cutaneous leiomyomata and uterine fibroids to chromosome 1q42. 3-q43.
The American Journal of Human Genetics 68, 1264-1269.
Alam, N., Rowan, A., Wortham, N., Pollard, P., Mitchell, M., Tyrer, J., Barclay, E.,
Calonje, E., Manek, S., and Adams, S. (2003). Genetic and functional analyses of FH
mutations in multiple cutaneous and uterine leiomyomatosis, hereditary
leiomyomatosis and renal cancer, and fumarate hydratase deficiency. Human
molecular genetics 12, 1241.
Balss, J., Meyer, J., Mueller, W., Korshunov, A., Hartmann, C., and von Deimling, A.
(2008). Analysis of the IDH1 codon 132 mutation in brain tumors. Acta
neuropathologica 116, 597-602.
Brahimi-Horn, M., and Pouyssegur, J. (2004). The hypoxia-inducible factor and tumor
progression along the angiogenic pathway. International review of cytology 242,
157-213.
Christofk, H., Vander Heiden, M., Harris, M., Ramanathan, A., Gerszten, R., Wei, R.,
Fleming, M., Schreiber, S., and Cantley, L. (2008). The M2 splice isoform of pyruvate
kinase is important for cancer metabolism and tumour growth. Nature 452, 230-233.
Czy ewska, J., Guzi ska-Ustymowicz, K., Kemona, A., and Bandurski, R. (2008). The
expression of matrix metalloproteinase 9 and cathepsin B in gastric carcinoma is
associated with lymph node metastasis, but not with postoperative survival. Folia
Histochemica et Cytobiologica 46, 57-64.
Doorbar, J. (2007). Papillomavirus life cycle organization and biomarker selection.
Disease Markers 23, 297-313.
Fantin, V., St-Pierre, J., and Leder, P. (2006). Attenuation of LDH-A expression
uncovers a link between glycolysis, mitochondrial physiology, and tumor
maintenance. Cancer Cell 9, 425-434.
Feron, O. (2009). Pyruvate into lactate and back: From the Warburg effect to
symbiotic energy fuel exchange in cancer cells. Radiotherapy and Oncology 92,
329-333.
Funasaka, T., Hu, H., Hogan, V., and Raz, A. (2007). Down-regulation of
phosphoglucose isomerase/autocrine motility factor expression sensitizes human
fibrosarcoma cells to oxidative stress leading to cellular senescence. Journal of
Biological Chemistry 282, 36362.
Gatenby, R., and Gillies, R. (2004). Why do cancers have high aerobic glycolysis?
Nature reviews cancer 4, 891-899.
Gottlieb, E., and Tomlinson, I. (2005). Mitochondrial tumour suppressors: a genetic
and biochemical update. Nature reviews cancer 5, 857-866.
Isaacs, J., Jung, Y., Mole, D., Lee, S., Torres-Cabala, C., Chung, Y., Merino, M.,
Trepel, J., Zbar, B., and Toro, J. (2005). HIF overexpression correlates with biallelic
loss of fumarate hydratase in renal cancer: novel role of fumarate in regulation of HIF
stability. Cancer Cell 8, 143-153.
Kim, J., and Dang, C. (2005). Multifaceted roles of glycolytic enzymes. Trends in
biochemical sciences 30, 142-150.
Kim, J., Tchernyshyov, I., Semenza, G., and Dang, C. (2006). HIF-1-mediated
expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular
adaptation to hypoxia. Cell metabolism 3, 177-185.
Ko, Y., Pedersen, P., and Geschwind, J. (2001). Glucose catabolism in the rabbit VX2
tumor model for liver cancer: characterization and targeting hexokinase. Cancer
letters 173, 83-91.
Krammer, P. (1998). CD95 (APO-1/Fas)-mediated apoptosis: live and let die.
Advances in Immunology 71, 163-210.
Kurtoglu, M., Gao, N., Shang, J., Maher, J., Lehrman, M., Wangpaichitr, M., Savaraj,
N., Lane, A., and Lampidis, T. (2007). Under normoxia, 2-deoxy-D-glucose elicits
cell death in select tumor types not by inhibition of glycolysis but by interfering with
N-linked glycosylation. Molecular cancer therapeutics 6, 3049.
Launonen, V., Vierimaa, O., Kiuru, M., Isola, J., Roth, S., Pukkala, E., Sistonen, P.,
Herva, R., and Aaltonen, L. (2001). Inherited susceptibility to uterine leiomyomas and
renal cell cancer. Proceedings of the National Academy of Sciences 98, 3387.
Liu, H., Hu, Y., Savaraj, N., Priebe, W., and Lampidis, T. (2001). Hypersensitization
of Tumor Cells to Glycolytic Inhibitors. Biochemistry 40, 5542-5547.
Liu, H., Savaraj, N., Priebe, W., and Lampidis, T. (2002). Hypoxia increases tumor
cell sensitivity to glycolytic inhibitors: a strategy for solid tumor therapy (Model C).
Biochemical pharmacology 64, 1745-1751.
Maher, J., Krishan, A., and Lampidis, T. (2004). Greater cell cycle inhibition and
cytotoxicity induced by 2-deoxy-D-glucose in tumor cells treated under hypoxic vs
aerobic conditions. Cancer chemotherapy and pharmacology 53, 116-122.
Mattevi, A., Bolognesi, M., and Valentini, G. (1996). The allosteric regulation of
pyruvate kinase. FEBS letters 389, 15-19.
Moreno-Sanchez, R., Rodriguez-Enriquez, S., Marin-Hernandez, A., and Saavedra, E.
(2007). Energy metabolism in tumor cells. FEBS journal 274, 1393-1418.
Munoz, N., Bosch, F., De Sanjose, S., Tafur, L., Izarzugaza, I., Gili, M., Viladiu, P.,
Navarro, C., Martos, C., and Ascunce, N. (1992). The causal link between human
Nature reviews cancer 4, 891-899.
Gottlieb, E., and Tomlinson, I. (2005). Mitochondrial tumour suppressors: a genetic
and biochemical update. Nature reviews cancer 5, 857-866.
Isaacs, J., Jung, Y., Mole, D., Lee, S., Torres-Cabala, C., Chung, Y., Merino, M.,
Trepel, J., Zbar, B., and Toro, J. (2005). HIF overexpression correlates with biallelic
loss of fumarate hydratase in renal cancer: novel role of fumarate in regulation of HIF
stability. Cancer Cell 8, 143-153.
Kim, J., and Dang, C. (2005). Multifaceted roles of glycolytic enzymes. Trends in
biochemical sciences 30, 142-150.
Kim, J., Tchernyshyov, I., Semenza, G., and Dang, C. (2006). HIF-1-mediated
expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular
adaptation to hypoxia. Cell metabolism 3, 177-185.
Ko, Y., Pedersen, P., and Geschwind, J. (2001). Glucose catabolism in the rabbit VX2
tumor model for liver cancer: characterization and targeting hexokinase. Cancer
letters 173, 83-91.
Krammer, P. (1998). CD95 (APO-1/Fas)-mediated apoptosis: live and let die.
Advances in Immunology 71, 163-210.
Kurtoglu, M., Gao, N., Shang, J., Maher, J., Lehrman, M., Wangpaichitr, M., Savaraj,
N., Lane, A., and Lampidis, T. (2007). Under normoxia, 2-deoxy-D-glucose elicits
cell death in select tumor types not by inhibition of glycolysis but by interfering with
N-linked glycosylation. Molecular cancer therapeutics 6, 3049.
Launonen, V., Vierimaa, O., Kiuru, M., Isola, J., Roth, S., Pukkala, E., Sistonen, P.,
Herva, R., and Aaltonen, L. (2001). Inherited susceptibility to uterine leiomyomas and
renal cell cancer. Proceedings of the National Academy of Sciences 98, 3387.
Liu, H., Hu, Y., Savaraj, N., Priebe, W., and Lampidis, T. (2001). Hypersensitization
of Tumor Cells to Glycolytic Inhibitors. Biochemistry 40, 5542-5547.
Liu, H., Savaraj, N., Priebe, W., and Lampidis, T. (2002). Hypoxia increases tumor
cell sensitivity to glycolytic inhibitors: a strategy for solid tumor therapy (Model C).
Biochemical pharmacology 64, 1745-1751.
Maher, J., Krishan, A., and Lampidis, T. (2004). Greater cell cycle inhibition and
cytotoxicity induced by 2-deoxy-D-glucose in tumor cells treated under hypoxic vs
aerobic conditions. Cancer chemotherapy and pharmacology 53, 116-122.
Mattevi, A., Bolognesi, M., and Valentini, G. (1996). The allosteric regulation of
pyruvate kinase. FEBS letters 389, 15-19.
Moreno-Sanchez, R., Rodriguez-Enriquez, S., Marin-Hernandez, A., and Saavedra, E.
(2007). Energy metabolism in tumor cells. FEBS journal 274, 1393-1418.
Munoz, N., Bosch, F., De Sanjose, S., Tafur, L., Izarzugaza, I., Gili, M., Viladiu, P.,
Navarro, C., Martos, C., and Ascunce, N. (1992). The causal link between human
papillomavirus and invasive cervical cancer: a population-based case-control study in
Colombia and Spain. International Journal of Cancer 52, 743-749.
Parsons, D., Jones, S., Zhang, X., Lin, J., Leary, R., Angenendt, P., Mankoo, P., Carter,
H., and Siu, I. (2008). An integrated genomic analysis of human glioblastoma
multiforme. Science 321, 1807.
Pastorino, J., Shulga, N., and Hoek, J. (2002). Mitochondrial binding of hexokinase II
inhibits Bax-induced cytochrome c release and apoptosis. Journal of Biological
Chemistry 277, 7610.
Pedersen, P. (2007). Warburg, me and Hexokinase 2: multiple discoveries of key
molecular events underlying one of cancers¡¦ most common phenotypes, the
¡§Warburg Effect¡¨, ie, elevated glycolysis in the presence of oxygen. Journal of
bioenergetics and biomembranes 39, 211-222.
Pelicano, H., Martin, D., Xu, R., and Huang, P. (2006). Glycolysis inhibition for
anticancer treatment. Oncogene 25, 4633-4646.
Pollard, P., Briere, J., Alam, N., Barwell, J., Barclay, E., Wortham, N., Hunt, T.,
Mitchell, M., Olpin, S., and Moat, S. (2005a). Accumulation of Krebs cycle
intermediates and over-expression of HIF1 in tumours which result from germline
FH and SDH mutations. Human molecular genetics 14, 2231.
Pollard, P., Wortham, N., Barclay, E., Alam, A., Elia, G., Manek, S., Poulsom, R., and
Tomlinson, I. (2005b). Evidence of increased microvessel density and activation of
the hypoxia pathway in tumours from the hereditary leiomyomatosis and renal cell
cancer syndrome. The journal of pathology 205, 41-49.
Rempel, A., Mathupala, S., Griffin, C., Hawkins, A., and Pedersen, P. (1996). Glucose
catabolism in cancer cells: amplification of the gene encoding type II hexokinase.
Cancer research 56, 2468.
Samudio, I., Fiegl, M., and Andreeff, M. (2009). Mitochondrial uncoupling and the
Warburg effect: molecular basis for the reprogramming of cancer cell metabolism.
Cancer research 69, 2163.
Scaduto, R., and Grotyohann, L. (1999). Measurement of mitochondrial membrane
potential using fluorescent rhodamine derivatives. Biophysical journal 76, 469-477.
Schmitz, I., Kirchhoff, S., and Krammer, P. (2000). Regulation of death
receptor-mediated apoptosis pathways. The International Journal of Biochemistry &
Cell Biology 32, 1123-1136.
Selak, M., Armour, S., MacKenzie, E., Boulahbel, H., Watson, D., Mansfield, K., Pan,
Y., Simon, M., Thompson, C., and Gottlieb, E. (2005). Succinate links TCA cycle
dysfunction to oncogenesis by inhibiting HIF-[alpha] prolyl hydroxylase. Cancer Cell
7, 77-85.
Soldani, C., and Scovassi, A. (2002). Poly (ADP-ribose) polymerase-1 cleavage
during apoptosis: an update. Apoptosis 7, 321-328.
Swietach, P., Vaughan-Jones, R., and Harris, A. (2007). Regulation of tumor pH and
the role of carbonic anhydrase 9. Cancer and Metastasis Reviews 26, 299-310.
Taylor, R., and Turnbull, D. (2005). Mitochondrial DNA mutations in human disease.
Nature Reviews Genetics 6, 389-402.
Thompson, C. (2009). Metabolic enzymes as oncogenes or tumor suppressors. New
England Journal of Medicine 360, 813.
Tsutsumi, S., Hogan, V., Nabi, I., and Raz, A. (2003). Overexpression of the autocrine
motility factor/phosphoglucose isomerase induces transformation and survival of
NIH-3T3 fibroblasts. Cancer research 63, 242.
Warburg, O. (1956). On the origin of cancer cells. Science 123, 309-314.
Wentzensen, N., Vinokurova, S., and Doeberitz, M. (2004). Systematic review of
genomic integration sites of human papillomavirus genomes in epithelial dysplasia
and invasive cancer of the female lower genital tract. Cancer research 64, 3878.
Wu, M., Wu, R., Hung, C., Cheng, T., Tsai, W., and Chang, W. (2005). Simple and
efficient DNA vector-based RNAi systems in mammalian cells. Biochemical and
biophysical research communications 330, 53-59.
Yanagawa, T., Funasaka, T., Tsutsumi, S., Watanabe, H., and Raz, A. (2004). Novel
roles of the autocrine motility factor/phosphoglucose isomerase in tumor malignancy. Endocrine-Related Cancer 11, 749.