研究人類葡萄糖-6-磷酸去氫?（G6PD）在癌細胞的表現 及抗癌藥物對 G6PD-過度表現之纖維母細胞的影響

Abstract

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 , CE8lT , 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 、 20mg/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-Mi), 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 tmhe 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 2×10^6 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 (10mg/kg.BW), 11 days for 6-AN (20 mg/kg.BW), 11 days for DHEA/6-AN (50mg/kg.BW+10mg/kg.BW) and >19 days for DHEA/6-AN (50 mg/kg.BW+20mg/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.