Benzyl Isothiocyanate 對於引起人類皮膚癌細胞A375.S2 cells的細胞凋亡與抑制生長的影響

Benzyl Isothiocyanate Induces Growth Inhibition and Cell Apoptosis in Human Melanoma A375.S2 Cells

吳律緯(Liu-Wei Wu)

指導教授 ： 黃素華 ； 鍾景光

亞洲大學/健康學院/生物科技學系碩士班/碩士(2009年)

摘要

皮膚癌是一種最常罹患的癌症疾病之一，越來越普遍發生在年輕族群上。近年來已有許多的文獻及報導指出，增加十字花科蔬菜的攝取量，可有效預防罹患癌症的風險並有效降低癌症發生率。Benzyl isothiocyanate (BITC) 是來自十字花科蔬菜中的一種化合物，也證明能夠在許多的癌細胞上誘導細胞週期的停止和癌細胞的凋亡。目前並沒有針對BITC 抑制A375.S2 人類皮膚癌生長之研究，所以在這項研究中，我們去探討BITC 是否能誘導A375.S2 細胞的凋亡。A375.S2 細胞在BITC 藥物治療下，以MTT 試驗去測定細胞的存活率。以DAPI staining 和Comet assay 去偵測DNA 損傷與細胞核質濃縮的現象。利用流式細胞儀去偵測細胞中的粒腺體膜電位的變化、活性氧基群的表現、細胞質中鈣離子濃度的變化、以及細胞週期的影響。最後再利用流式細胞儀去偵測caspase-3 的活性與Annexin V 親和力分析，藉此進一步的確認細胞凋亡的現象。由西方墨點法分析發現 cytochrome c、AIF 與Endo G 由粒線體中釋放出來並造成下游路徑的活化並造成細胞凋亡。在這項研究結果中表示，細胞經藥物BITC 24 小時處理後，細胞的存活率有明顯的下降且IC50 為10±0.5μM。BITC 能夠引起A375.S2 細胞的細胞週期停滯於G2/M期，由Comet assay 和DAPI staining 也可發現有DNA 受損之情形。此外BITC 也會造成細胞的粒線體膜電位下降，增加活性氧基群以及細胞內鈣離子濃度。基於這些觀察，BITC 在人類黑色素瘤A375.S2 細胞上可以誘導細胞凋亡。

關鍵字

芐基異硫氰酸酯, 黑色素瘤, 細胞凋亡

並列摘要

Skin cancer is one of the common carcinomatous diseases. It is becoming increasingly common in younger populations.Recently, many reports indicated that increased intake of cruciferous vegetables may be effective in preventing the risk of cancer and to reduce the incidence of cancer. Benzyl isothiocyanate (BITC), a compound presented in cruciferous vegetables, had shown to induce cell cycle arrest and apoptosis in many cancer cells. There is no report to show BITC inhibited the growth of A375.S2 human skin cancer cells. In this study BITC affecting apoptosis in A375.S2 cells was investigated. MTT assay was used to measure cell viability of A375.S2 cells after BITC treatment. DNA damage was determined by DNA fragmentation assay, DAPI staining and Comet assay. Flow cytometric analysis was performed to investigate the levels of mitochondrial membrane potential (ΔΨm), the production of reactive oxygen species (ROS), intracellular Ca2+ release and cell cycle distribution. Finally, we used the flow cytometry to examine caspase-3 activity and Annexin V affinity assay for apoptosis. In western blotting assay, we found cytochrome c, AIF and Endo G were released from mitochondria and activated downstream pathway to cause cell apoptosis. Our results showed that BITC treatment for 24 h significantly reduced the cell survival with an IC50 of 10±0.5 μM. BITC induced cell cycle arrest at G2/ M phase in A375.S2 cells. Moreover, BITC also caused DNA damage, decreased ΔΨm and increased ROS and intracellular Ca2+ levels. These observations indicate that BITC could induce apoptosis in human melanoma A375.S2 cell.

並列關鍵字

Benzyl Isothiocyanate, Melanoma, Apoptosis

參考文獻

11. Shang-Wen Yuan, G.-W.C., Jing-Gung Chung, The Inhibitory Function of Shikonin on N-acetylation of 2-aminofluorene in Human Malignant Melanoma Cells (A375.S2). Dermatol sinica, 2004. 160.

44. Lu, H.F., et al., Gypenosides induced G0/G1 arrest via inhibition of cyclin E and induction of apoptosis via activation of caspases-3 and -9 in human lung cancer A-549 cells. In Vivo, 2008. 22 (2): p. 215-21.

3. Rebbeck, T.R., et al., Cancer epidemiology, biomarkers & prevention, and cancer prevention research: two journals, a common goal. Cancer Epidemiol Biomarkers Prev, 2008.17(11): p.2903-5.

4. Todd R, W.D., Oncogenes. Anticancer Res, 1999. 19: p. 4729-46.

5. Lin, J.K., Y.C. Liang, and S.Y. Lin-Shiau, Cancer chemoprevention by tea polyphenols through mitotic signal transduction blockade. Biochem Pharmacol, 1999. 58(6): p. 911-5.