| 摘要: | 惡性黑色素瘤是一極易轉移、極具擴散性的惡性腫瘤，雖然化學療法和放射線療法是本病的傳統治療方法，但療效極為有限。本研究目的在探討五倍子中沒食子酸成份對於人類惡性黑色素瘤細胞株(A375.S2)的抑癌機制、細胞凋亡路徑以及其抑制侵襲與轉移能力等機制之研究。本研究以沒食子酸作用A375.S2細胞株，以流式細胞儀來偵測細胞的存活率、細胞週期，以倒立式位相差顯微鏡來觀察細胞型態上的改變，採用DAPI染色法觀察DNA受損程度。結果發現隨著藥物濃度上升和加藥暴露時間的延長，具有劑量及時間依賴效應性抑制癌細胞存活。顯微鏡觀察到以沒食子酸作用過的A375.S2細胞會有細胞皺縮和凋亡小體產生現象，而DAPI染色法偵測到細胞的DNA損傷。以流式細胞計數儀來偵測細胞週期，發現沒食子酸會造成細胞週期G2/M期的阻滯，並有細胞凋亡(apoptosis)的現象產生。
以流式細胞儀來偵測活性氧自由基(ROS)、鈣離子濃度及粒線體膜電位，結果發現內質網的壓力反應促使ROS及鈣離子濃度升高，誘發粒線體膜電位下降，促成細胞的凋亡，此結果可能與粒線體膜上細胞膜孔道打開有關。而後再以西方墨點法來偵測凋亡蛋白表現量的改變，Real-Time PCR來偵測凋亡蛋白mRNA的表現量，發現Fas, Fas-L, Caspase-8, Caspase-9, Caspase-3, Bax, Cytochrome c, endonuclease G (Endo G)及apoptosis-inducing factor (AIF)蛋白表現量增加，而Bcl-2蛋白表現量減少。綜合以上結果，以沒食子酸作用A375.S2細胞所引起細胞凋亡路徑，是通過活化死亡受體Fas觸發FADD/Caspase-8的途徑或是經由上調Bax以及下調Bcl-2啟動粒線體凋亡路徑，並激發Caspase串聯反應。此外，沒食子酸也誘導A375.S2細胞的內質網產生壓力，使得未折疊蛋白質及ROS大量累積，促使鈣離子由內質網釋出，影響到粒線體膜功能失常而釋放出細胞色素c及凋亡相關因子Endo G及AIF導致細胞凋亡，此提供沒食子酸誘導A375.S2細胞凋亡主要是經由Caspase cascade-dependent路徑，以及部份是經由Caspase cascade-independent路徑引發細胞凋亡。
同時以細胞穿透及移動分析試驗，發現A375.S2細胞隨著沒食子酸藥物濃度的增加而細胞穿透與轉移的能力下降。以西方墨點法及Real-Time PCR來偵測侵襲與轉移蛋白表現量及mRNA的改變，結果顯示基質金屬蛋白酶（MMP-2及MMP-9）表現隨著藥物濃度增加而下降，而沒食子酸抑制或延緩A375.S2細胞的侵襲與轉移可經由抑制Ras, ERK1/2, p38與Rho A路徑，來調節MMP-2及MMP-9蛋白表現，顯示沒食子酸具有抑制人類黑色素瘤細胞(A375.S2)侵襲與轉移的能力。
Malignant melanoma is one of malignant cancers characterized with high ability of invasion and metastasis. Although chemotherapy and radiotherapy are commonly used to treat melanoma, the effectiveness of these therapies is very limited. The purpose of this study is to investigate the inhibitory mechanisms of Gallic Acid on human melanoma A375.S2 cell line, including the mechanism of suppressing tumor growth, apoptosis pathway and the ability to inhibit the invasion and metastasis of A375.S2 cell line. A375.S2 cells were exposed to the Gallic Acid, the cell cycle progression and apoptosis ratio were detected by flow cytometry; the morphological changes of the cells were observed under light and electron microscope; DNA damage was observed by DAPI staining. Results showed that Gallic Acid inhibited the growth of A375.S2 cells by inducing apoptosis in a both time-dependent and dose-dependent manner. After a couple of hours, the Gallic Acid-treated A375.S2 cells had been observed typical morphorlogical changes, such as cell shrinkage and apoptotic bodies formation under microscope, and DNA damage detected by DAPI staining. Detecting cell cycle by flow cytometry, 250 μM Gallic Acid was found to induce G2/M phase arrest.
The reactive oxygen species (ROS), calcium concentration and mitochondrial membrane potential (MMP) were examined by using flow cytometry. The results showed that the endoplasmic reticulum stress response promote the release of ROS and the elevation of calcium concentration. These induced the decrease in the levels of mitochondrial membrane potential and followed by dysfunctions of the mitochondria, resulting in apoptosis of A375.S2 cells. Western blotting and Real-Time PCR assays were employed for detecting the mRNA expression and the apoptotsis-associated proteins, such as Fas, Fas-L, Caspase-8, Caspase-9, Caspase-3, Bax, Cytochrome c, apoptosis-inducing factor (AIF) and endonuclease G (Endo G). These protein expression were increased, and Bcl-2 protein expression was decreased. In conclusion, Gallic Acid induced apoptosis of A375.S2 cells through various pathways. By initiating death receptors of FAS/ FASL caused caspase-8 cascade activity; by up-regulation of the proapoptotic proteins(Bax) and down-regulation antiapoptotic proteins(Bcl-2) induced caspase cascade activity. In addition, Gallic Acid also induced A375.S2 cells to produce endoplasmic reticulum stress, leading a large number of unfolded proteins response and ROS accumulation, as well as promoting the release of calcium ion from the endoplasmic reticulum. All these led to the dysfunction of mitochondrial membrane, and then promoted cytosolic release of AIF and Endo G, inducing apoptosis. This has proven that Gallic Acid induced A375.S2 cells apoptosis mainly through the Caspase cascade-dependent pathway, and Caspase cascade-independent as a minor pathway.
Both the invasion and migration ability of A375.S2 cells were found reduced as the concentration of Gallic Acid increased. The amount of invasion and migration protein as well as mRNA expression changes were detected by Western blot and Real-time PCR, the results showed that matrix metalloproteinase (MMP-2 and MMP-9) expression decreased with the increase in drug concentration. Gallic Acid inhibited or delayed the A375.S2 cell invasion and metastasis via inhibition of Ras, ERK1/2, p38, Rho A pathway to regulate MMP-2 and MMP-9 protein expression. Thus, Gallic Acid has the ability to inhibit the invasion and migration of human melanoma cells (A375.S2) . |
