芹菜素誘導人類肝癌細胞活性氧自由基產生及細胞內鈣離子增加導致細胞凋亡機制之研究。

Apigenin Induces Production of Reactive Oxygen Species and Increase in Intracellular Calcium Level result in Human Hepatocellular Carcinoma Cell Death by Apoptosis.

廖婉茹(Wan-Lu Liao)

指導教授 ： 麥富德

臺北醫學大學/醫學院/醫學科學研究所/碩士(2012年)

https://doi.org/10.6831/TMU.2012.00066

摘要

肝癌為常見的死因素之一，在肝癌早期發現並治療，其治療效果及癒後恢復更佳，然而肝癌的早期症狀並不明顯，因而無法達到有效的治療，1976年Michael Sporn等學者提出以化學預防為概念，來達到預防、阻斷、抑制，甚至逆轉癌化作用的過程。芹菜素(Apigenin)是一種普遍存在蔬果及許多植物的黃酮類天然化合物，先前文獻指出apigenin具有抗氧化、抗發炎等作用，且對多種癌細胞具有生長抑制或誘發凋亡之作用。本研究主要探討apigenin對人類肝癌細胞株(human hepatoma cell line；HepG2)是否具有引發細胞死亡之作用及其可能的作用機制，並探討將其作為肝癌預防的可能性。經實驗結果發現，apigenin對人類肝癌細胞株HepG2有誘發細胞死亡的作用，且其效果與apigenin劑量與時間有相關性，經80μΜ的apigenin 48小時處理後，HepG2細胞生存率約45%，而對正常小鼠肝細胞(normal mouse liver cells；BNL. CL2)則無影響。在顯微觀察下HepG2細胞型態，未處理的HepG2細胞完整展開且型態為多為多角型狀，但隨著apigenin處理劑量增加，細胞漂浮數目增加且貼附的細胞型態差異性增加且形態不具規則性，利用4',6-diamidino-2-phenylindole (DAPI)染色，發現80μΜ的apigenin 48小時處理後，發現細胞質濃縮情形，此外檢測細胞內二級訊號傳遞者:鈣離子含量，發現HepG2細胞內鈣離子濃度較未處理的細胞高。進一步以流式細胞儀檢測HepG2細胞內活性氧自由基（Reactive oxygen species; ROS）含量變化，發現apigenin處理後，會使活性氧自由基含量增加，且細胞內活性氧自由基含量與apigenin處理劑量呈現相關性，在80μΜ的apigenin 1.5小時處理後，即有明顯過氧化自由基含量增加的現象，而維生素C(ROS inhibitor) 處理後發現對apigenin處理造成HepG2細胞死亡的作用具有抑制的效果，因此推測apigenin可能會藉由誘發細胞內活性氧自由基大量表現促使HepG2死亡，同樣以不同濃度的apigenin 3小時處理後，發現粒腺體膜電位降低與apigenin處理劑量呈現相關性，以80μΜ的apigenin不同時間處理，在3-6小時有最明顯的粒腺體膜電位降低現象發生，最後，以雷射共軛焦顯微鏡觀察apigenin處理是否造成粒腺體上的細胞色素C釋放至細胞質中，由實驗結果得知，經80μΜ的apigenin 12小時處理後，粒腺體上的細胞色素C釋放至細胞質中，根據本研究結果，推測apigenin對人類肝癌細胞株HepG2誘發凋亡，和鈣離子及活性氧化物增加有關，且會造成粒腺體膜電位下降，使細胞通透性增加，細胞色素C 釋放，而啟動下游細胞凋亡路徑。

關鍵字

芹菜素 ； 活性氧自由基 ； 凋亡 ； 粒線體

並列摘要

Liver cancer is one of the major cause of death in the world. If we can find liver cancer earlier, the effect of therapy and recovery will better. But since it is difficult to detect the symptoms of early stage liver cancer, the effect of therapy is not significant enough in these days. Michael Sporn and other scholars(1976) have pointed to the conception of chemoprevention which referred to using chemicals for preventing, blocking, inhibiting, or even reversing the process of carcinogenesis. Apigenin is a natural flavonoid which rich in fruits and vegetables, and many of plant, in previous studys, apigenin had the function of anti-oxidation, anti-inflammation, and it could inhibite the cell growth of some cancer cell line or induce them to apoptosis. This study focused on the cell death level and the mechanism of human hepatoma cell line (HepG2) after apigenin treatmented.
The results demonstrated that apigenin could induce HepG2 cell death, which cell death effect is a dose and time depended manner. To treat apigenin 80μΜ for 48 hours induced the cell viability of HepG2 is 45%, but were no affected on normal mouse liver cells (BNL. CL2). To observe the morphology of HepG2 by microscopic, we found that the morphology of untreated HepG2 cells (control) were fully expanded and most of them had polygonal shape, however, the number of suspension cell increased and the differences of morphology increased and had irregular shape of attached cell with increasing doses of apigenin. We found that the chromatin condensation were observed in apigenin-treated HepG2 cells by DAPI after treated apigenin 80μΜ for 48hours, in addition, we detected the calcium level(intracellular signal transduction)and found that it is higher in apigenin-treated HepG2 cells than control. To detected the Reactive Oxygen Species (ROS) level by flow cytometer and found that the ROS level increased after treated apigenin, the ROS level was a dose depend relationship, and ROS level was significant increased after treated apigenin 80μΜ for 1.5hours. To co-treated Ascorbic Acid (AA) (ROS inhibitor) with apigenin and found that the effort of apigenin caused HepG2 cell death inhibited, therefore, we suggested the possibility of apigenin induced HepG2 cell death by ROS increased. We also found that mitochondrial membrane potential (MMP) decreased significant and it was a dose depend relationship after difference apigenin dose treated for 3 hours. Finally we observed the distribution of cytochrome C by laser confocal microscope and the results indicate that the cytochrome C release to the cytoplasm after treated apigenin 12 hours. according to this study we suggested that apigenin could induce HepG2 cell apoptosis and it was related to calcium and ROS level increased, than result in MMP decreased and permeability increased, cytochrome C released to activate the downstream apoptosis pathway.

並列關鍵字

Apigenin ； Reactive Oxygen Species (ROS) ； apoptosis ； Mitochondria

參考文獻

[2] M. Levrero, "Viral hepatitis and liver cancer: The case of hepatitis C," Oncogene, vol. 25, pp. 3834-3847, 2006.

[3] H. Moriwaki, M. Shimizu, M. Okuno, and R. Nishiwaki-Matsushima, "Chemoprevention of liver carcinogenesis with retinoids: Basic and clinical aspects," Hepatol Res, vol. 37 Suppl 2, pp. S299-302, Sep 2007.

[4] "cancer chemoprevention as adjuvant therapy."

[5] S. Okada, "Cancer chemoprevention as adjuvant therapy for hepatocellular carcinoma," Japanese Journal of Clinical Oncology, vol. 31, pp. 357-358, 2001.

[6] C. M. Croce, "Oncogenes and cancer," N Engl J Med, vol. 358, pp. 502-11, Jan 31 2008.