臺灣博碩士論文加值系統

G6PD（glucose-6-phosphate dehydrogenase），主要作用於pentose phosphate pathway（ppp）的第一步驟，伴隨產生NADPH，其功能為提供細胞內NADPH及五碳醣（ppp之最終產物）的來源。當細胞處於快速生長分裂時期，G6PD活性會上升，當細胞停止生長時，活性又會下降，故G6PD的活性與細胞的生長能力密切相關。在某些癌症中都有細胞G6PD活性提高的現象。我們實驗室之前的研究發現，經人類G6PD基因轉染的NIH-3T3 細胞，使其高量表現G6PD的活性，結果可使細胞生長加速並產生轉型癌化之現象，若將其注入裸鼠，則可導致腫瘤的發生。所以G6PD在細胞內除了正常的生理功能外，對於細胞癌化過程，似乎也扮演了一個重要的角色。
為研究G6PD對癌細胞的影響，我們試圖由癌細胞株找出G6PD高量表現者，對十數株人類癌細胞株測其G6PD活性，結果發現包括肝癌（Hep3B，Huh-7，HA22T）、肺癌（CL1-0，CL1-5）、子宮頸癌（HeLa，SiHa，CaSki）、胃癌（AGS，SC-M1）、大腸癌（LS174T）等細胞株並無大量表現G6PD的現象；不過在食道癌（CE48T，CE81T，CE146T）則發現其G6PD活性是一般細胞的3~10倍之高。
進一步取得食道癌患者樣本，其腫瘤組織G6PD活性都有上升的情形，而且，似乎G6PD活性也會隨腫瘤由良性至惡性等進展過程而增高。如果這是一明顯而普遍性如此的話，G6PD或許可做為偵測食道癌的一個marker，但這方面還需搜集更多病患樣本來評估其可行性。
接著我們嘗試去研究G6PD在癌細胞表現的調控機制，經由western、northern、Southern blot的結果，發現食道癌細胞的G6PD可能是在transcriptional level大量表現，而且DNA並無明顯的複製(amplification)或重組(rearrangement)現象。
既然G6PD活性與細胞生長有密切的關聯，我們利用DHEA（dehydroepiandrosterone；a G6PD inhibitor）及6-AN [6-aminonicotinamide；a 6PGD (6-phosphogluconate dehydrogense) inhibitor]去抑制癌細胞內G6PD及6PGD的活性，並觀察其對不同癌細胞的生長抑制影響，我們發現單獨處理DHEA或6AN都有抑制細胞增生的效果，且兩者以適當比例混合，能得到更明顯的抑制效果；由in vitro 直接測定藥物對G6PD、6PGD酵素活性的抑制，也可看到兩者結合使用，能更有效降低酵素的活性。由加入ribonucleosides、 deoxyribonucleosides 、NADPH及GSH的實驗中，我們可看到ppp代謝途徑的下游產物都能有不同程度的回復DHEA、6AN造成的生長抑制作用，由此我們推斷細胞內的氧化還原環境及ribose-5-phospate (DNA及RNA合成材料)的含量與癌細胞的生長息息相關，而G6PD在當中扮演相當重要的角色。
利用DHEA及6AN去處理經轉染人類G6PD基因的NIH-3T3 細胞株N2、H6、H7（其G6PD活性分別為正常NIH3T3細胞的1、6、16倍），結果發現，G6PD表現量愈高的細胞株對該藥的敏感性也愈高。所以，我們認為利用DHEA及6AN這兩個酵素抑制劑，應能對G6PD高表現的fibroblasts有不錯的治療效果。
另外我們以nude mice 為animal model，植入H7細胞以誘發腫瘤生長。在接種後一星期起，給予不同劑量組合之DHEA及6AN，並觀察腫瘤的生長情形。結果在單獨處理DHEA（50mg/kgBW）或6AN（10mg、20mg/kgBW），其腫瘤生長延遲天數分別為4.5、9、11天；若以DHEA（50mg/kgBW）配合6AN（10、20 mg/kgBW），其腫瘤生長的抑制效果最好，其腫瘤生長延遲天數分別為11、＞19天（控制組則為2.5天）。所以此種混合DHEA及6AN的方式，在對G6PD高表現的腫瘤，有相當好的治療效果，因此我們認為結合DHEA與6AN不失為一具潛力之抗癌藥物。

G6PD (glucose-6-phosphate dehydrogenase) catalyzes the convert of G6P(glucose-6-phosphate) to 6PG(6-phsphgluconate) which is accompanied with the formation of NADPH in the first step of pentose phoshate pathway (PPP). In this manner, the PPP provides the major source of pentoses in the cells for DNA and RNA synthesis. The cellular level of NADPH is closely relative to the activation of catalase and the synthesis of GSH (glutathione). Previous reports indicated that the activity of G6PD was proportional to the growth rate of normal cells and high G6PD activities were detected in many cancers, including breast cancer, cervical carcinoma, prostatic carcinoma, endometrial carcinoma and lung cancer. Our previous experiments have shown that two G6PD-overexpressing cell lines (H6 and H7), which were NIH-3T3 transformed by a human G6PD gene, form colonies on soft agar and induce tumors in nude mice. These data indicate that G6PD may act as an important role in the process of tumor formation.
In order to examine whether G6PD is highly expressed in established cancer cell lines, the G6PD activity in those cell lines was examined. It was found that the G6PD activities in hepatoma cell lines (Hep3B, Huh-7,HA22T), lung cancer cell lines (CL1-0,CL1-5), cervical cancer cell lines (HeLa, SiHa, CaSki), gastrocarcinoma cell lines (AGS, SC-M1), and colon cancer line (LS174T) were about average and those in esophagus cancer cell lines (CE48T, CE81T, CE146T) were about 3 to 10 folds higher than that of normal level. The G6PD activities in two esophageal cancer samples were also higher than normal tissues as well. In addition, the increase of G6PD appears to be correlated with the malignancy of tumors. Using Southern, northern and western blotting analyses, we found that high level expression of G6PD in the three esophagus cancer cell lines was possibly regulated at the transcriptional level and no obvious DNA recombination was identified in the g6pd gene.
Because the G6PD activity is highly correlated with the cell growth rate, we used G6PD inhibitor (DHEA) and 6PGD inhibitor (6-AN) separately or in combination to test their cytotoxic effects on cancer cells. Our results showed that DHEA or 6-AN could inhibit the cell growth to a certain level, while the most significant inhibition effect was observed by the treatment using both DHEA and 6-AN. The supply of PPP downstream products, such as ribonucleosides, deoxyribonucleosides, NAPDH and GSH, could compensate the DHEA and/or 6-AN effect on cellular growth inhibition. Although, we don't known the exact functions of these chemicals, the intracellular redox environment and the contents of the raw materials for DNA and RNA synthesis may play roles on cancer cell growth.
Two G6PD transfected clones which had a 16-fold (H7) and 6-fold (H6) increase in their intracellular G6PD activity were compared with control cells transfected with a vector alone (N2). The sensitivities of these cell lines to DHEA and 6-AN were corresponding to the intracellular level of G6PD activities. After inoculation of 2×106 H7 cells in nude mice for one week, these mice were treated with DHEA (subcutaneously) and/or 6-AN (intraperitoneally), separately or in combination, then measured the tumor sizes in the following days. Our results show that the delayed intervals for tumor growth were 4.5 days for DHEA (50 mg/kg.BW), 9 days for 6-AN (10 mg/kg.BW), 11 days for 6-AN (20 mg/kg.BW), 11 days for DHEA/6-AN (50 mg/kg.BW＋10 mg/kg.BW) and >19 days for DHEA/6-AN (50 mg/kg.BW＋20 mg/kg.BW) as compared 2.5 days for the untreated control. So it appears that the combination of DHEA and 6-AN is an effective treatment for some tumor cells that overexpress G6PD. Taken together, the combined useage of DHEA and 6-AN may act as a potent anticancer recipe for cancer therapy.

壹、 緒言 1
一、 G6PD的介紹 1
(1) G6PD在細胞的正常生理功能 1
(2) G6PD的酵素與基因結構 2
(3) G6PD之調控機制 4
(4) G6PD與cancer之關係 5
二、 食道癌的發生與治療 6
三、 DHEA及6AN的作用 7
(1)DHEA 7
(2) 6AN 9
四、 研究目的 11
貳、 材料與方法 13
一、 細胞株 13
二、 細胞培養 13
三、 G6PD酵素活性的測定 14
四、 細胞與組織蛋白質的萃取 14
五、 由human及mouse全血分離lymphocytes 15
六、 西方點墨法 (Western Blot) 15
七、 北方點墨法 (Northern Blot) 16
八、 南方點墨法 (Southern Blot) 18
九、 DHEA及6AN in vitro對G6PD活性的抑制 19
十、 DHEA及6AN in vivo對細胞proliferation的抑制作用 19
十一、 DHEA及6AN對nude mice腫瘤生長的抑制作用 20
參、 結果 21
第一部份：G6PD在癌細胞的表現與調控機制 21
(Ⅰ) 腫瘤細胞株的G6PD酵素活性 21
(Ⅱ) 細胞株HeLa、Huh-7、CE48T、CE81T、CE146T
之G6PD蛋白質、mRNA量的表現情形 22
(Ⅲ) 細胞株HeLa、Huh-7、CE48T、CE81T、CE146T
之G6PD的DNA變化 23
(Ⅳ) 老鼠食道組織G6PD並無大量表現 23
(Ⅴ) 人類食道癌患者腫瘤組織的G6PD活性 24
第二部份：DHEA、6AN對癌細胞的生長抑制作用 24
(Ⅰ) DHEA與DHEA-S的比較 24
(Ⅱ) DHEA與6AN結合處理的比例 25
(Ⅲ) DHEA、6AN對細胞生長的抑制作用 25
(Ⅳ) DHEA、6AN對細胞生長抑制的可能途徑 25
(Ⅴ) DHEA、6AN對G6PD-overexpressing cells的生長抑制作用 27
(Ⅵ) DHEA、6AN in vivo對腫瘤生長的抑制作用 27
肆、 討論 29
圖表 36
伍、 參考資料 59

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