探討精胺酸合併維生素C在人類肝癌細胞株HA22T/VGH中抑制五碳醣磷酸代謝途徑並誘發氧化壓力之研究

Study on arginine combined with vitamin C induced oxidative stress mediated by inhibited pentose phosphate pathway in human hepatoma cell line HA22T/VGH

謝寶萱(Bau-Shan Hsieh)

指導教授 ： 張基隆

高雄醫學大學/醫學院/醫學研究所/博士(2010年)

https://doi.org/10.6832/KMU.2010.00108

摘要

肝細胞癌( 又稱肝癌 )是世界上第五位常見癌症，佔癌症死因的第三位，已成為全球主要健康問題，目前常用的化學治療方法效果差、副作用大且易產生抗藥性，有鑑於此，以營養療法治療癌症逐漸受到重視並具良好功效，精胺酸與維生素C是人體重要的營養元素，正常生理濃度下分別具有血管擴張及抗氧化等功能，但是否具有抑制腫瘤生長作用仍待釐清。本研究將利用精胺酸單獨或是合併維生素C處理人類惡性肝癌細胞株HA22T/VGH，並探討誘導細胞凋亡之機制。我們首先利用trypan blue染色法分析單獨精胺酸1,000 μg/ml或是維生素C 100 μg/ml不會影響細胞存活率，但是合併精胺酸1,000 μg/ml與維生素C 100 μg/ml處理細胞24小時候，細胞死亡率達IC50濃度，並活化粒線體凋亡路徑蛋白Bax、cytochrome c、caspase-9，抑制抗凋亡蛋白Bcl-2、Bcl-xL作用；此外死亡受體路徑蛋白FasL、Fas、FADD、caspasse-8也被活化，最後caspase-3增加造成DNA片段化，而加入caspase-8、-9、-3與caspase-family抑制劑皆能有效逆轉精胺酸合併維生素C所誘導的細胞凋亡現象。接下來我們進一步探討精胺酸合併維生素C誘導細胞凋亡機制，以流式細胞儀偵測單獨精胺酸1,000 μg/ml或是維生素C 100 μg/ml作用細胞後不會增加活性氧物質與一氧化氮，但是合併使用下卻會顯著增加、粒線體膜電位下降與細胞內ATP含量減少，抗氧化物麩胱甘肽減少，顯示精胺酸合併維生素C會造成細胞氧化壓力，導因於五碳醣磷酸代謝路徑所需酵素G6PD、6PGD、TAL活性下降，導致終產物NADPH減少而無法維持GSH的恆定。而加入抗氧化物質麩胱甘肽、過氧化氫分解酶、N-acetyl-L-cysteine ( NAC )、6-磷酸葡萄糖及丙酮酸或是一氧化氮合成酶抑制劑( L-NAME )可以有效抑制細胞凋亡。但是加入麩胱甘肽合成抑制劑( BSO )與增加精胺酸合併維生素C則會明顯增加活性氧化物質與一氧化氮含量與提高25 %細胞死亡率。綜合以上實驗結果，我們發現單獨使用精胺酸或是維生素C並不會導致細胞死亡，但在合併使用下會抑制五碳醣磷酸代謝路徑的進行，減少NADPH合成進而影響細胞內氧化還原狀態，產生大量活性氧/氮物質，最終促使氧化壓力的形成，並誘導癌細胞凋亡。此研究結果可提供營養元素治療肝癌動物模式的基礎理論依據。

關鍵字

精胺酸 ； 維生素C ； 五碳醣磷酸代謝途徑 ； 氧化壓力

並列摘要

Hepatocellular carcinoma ( HCC ) is the 5th most common cancer and is the third cause of cancer-related death in the world, which becomes a major global health problem. Now, the commonly used chemotherapies are accompanied by side effects and are easy to develop drug resistance. On the other hand, nutritional therapy has become an alternative choice for the treatment of cancer and shown a good effect. Arginine and vitamin C are essential nutrients for human and they function as a vasodilator and antioxidant, at the physiological concentration ranges. However, it is still not known whether they are suitable to be components of the formular of nutritional therapy or not. This study was aimed to test the possibility of arginine and/or vitamin C treatment on the malignant hepatoma cell line ( HA22T/VGH ). The molecular mechanism would be further explored. Cell viability assay by trypan blue exclusion method showed that arginine at the concentration of 1,000 μg/ml or vitamin C 100 μg/ml was not toxic to HA22T/VGH cells. However combination of both reagents were toxic and be the IC50 concentration. Furthermore, we found this combination induced apoptosis by activation of the mitochondrial apoptosis-related proteins such as Bax, cytochrome c, and caspase-9, and by inhibition of anti-apoptotic proteins of Bcl-2 and Bcl-xL. In addition, the death receptor of FasL, Fas, FADD, and caspasse-8 were also activated. Importantly, activation of the critical caspase-3 caused DNA fragmentation. Interesting, we found this combination-induced apoptosis would be reversed by the caspase-8, -9, -3 and caspase-family inhibitor. The flow cytometry analysis showed arginine or vitamin C did not affect the levels of reactive oxygen species ( ROS ) and reactive nitrogen species ( RNS ), while combined treatment could significantly increased ROS and RNS and change mitochondrial membrane potential. Furthermore, the intracellular ATP and glutathione levels are also decreased. The key enzymes of the pentose phosphate pathway ( PPP ) including glucose 6-phosphate dehydrogenase ( G6PD ), 6-phospho-gluconate dehydrogenase ( 6PGD ), and transketolase ( TAL ) activity were also decreased by this combination treatment. Due to the decrease in NADPH, the GSH homeostasis could not be maintained. Moreover, addition of the antioxidants of glutathione, catalase, or N-acetyl-L-cysteine ( NAC ), or glucose-6-phosphate, pyruvate or nitric oxide synthase inhibitor ( L-NAME ) could significantly inhibit this combination-induced apoptosis. Noticeably, addition of glutathione synthesis inhibitor ( L-buthionine-[S,R]-sulfoximine ; BSO ) which markedly increased the levels of ROS and RNS, elevated about 25 % apoptotic cell death. These finding demostrated that combination treatment of arginine and vitamin C could induce apoptosis of HA22T/VGH through the pentose phosphate pathway and reduce NADPH synthesis resulting in interference in the cellular redox state. The study provides the potential nutrient therapy of combined arginine and vitamin C treatment in hepatoma and the molecular mechanisms were also revealed.

並列關鍵字

arginine ； vitamin C ； pentose phosphate pathway ； oxidative stress

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