檳榔素誘導人類口腔上皮癌細胞 ( KB ) 細胞週期停滯之分子機制

The Molecular Mechanism of Cell Cycle Arrest Induced by Arecoline in Human Oral KB Epithelial Cells

李柏勳(Po-Hsuen Lee)

指導教授 ： 何元順 博士 ； 鄭景暉 博士

臺北醫學大學/醫學院/醫學檢驗生物技術學研究所/碩士(2002年)

摘要

咀嚼檳榔被認為與口腔癌、口腔白斑症以及口腔黏膜下纖維化的發生有密切的關係。檳榔子中生物鹼的主成分，檳榔素，在細菌及細胞培養的研究中已被發現具有基因毒性，然而關於此成分的致癌機轉仍不甚明瞭。本研究將探討檳榔素對於人類口腔上皮癌細胞(KB細胞)其細胞週期的調控機制，以及活性氧在其中所扮演的角色。在MTT與SRB assay的實驗中，檳榔素會以劑量與時間相關的方式抑制細胞的生長。流式細胞技術的結果並指出檳榔素會造成細胞在G2/M時期停滯。而經過72小時的作用後，以DNA裂片分析及Annexin V-PI染色法偵測到有細胞凋亡的發生。而在西方墨點法的實驗中，檳榔素的刺激會使得細胞內Cyclin B1的蛋白表現以及Cdc2Tyr15磷酸化有增加的趨勢，顯示細胞可能是停滯於G2時期而不是M時期。此外，Wee 1蛋白表現的增加與Cdc25C的分解可能與Cyclin B/Cdc2活性的下降有關，但是Myt 1與Chk1的蛋白表現則沒有觀察到有明顯的變化。利用DCF-DA螢光染劑的偵測，發現細胞內活性氧的含量在檳榔素的刺激下有上升的趨勢。而抗氧化劑，如glutathione及N-acetyl-L-cysteine，可預防檳榔素對細胞所造成的生長抑制、G2時期停滯以及外觀上的改變，然而在superoxide dismutase與catalase的處理下則觀測不到相似的作用。由以上的結果，我們推論檳榔素會抑制KB細胞的生長，藉由磷酸化Cdc2Tyr15的方式，使細胞停滯在G2時期，並在72小時後誘導細胞凋亡的發生。而在這些檳榔素所調控的反應中，活性氧可能扮演了重要的角色。

關鍵字

檳榔素 ； 細胞週期 ； 活性氧

並列摘要

Betel quid chewing has a strong association to the incidence of oral cancer, leukoplakia and oral submucous fibrosis. Arecoline, the main areca alkaloid, has been reported to have genotoxic effects in bacterial test systems and cultured mammalian cells, while the mechanism of carcinogenesis associated with this compound is not well understood. The present study examines the effects of arecoline on cell cycle control and the possible role of reactive oxygen species (ROS) in these arecoline-mediated responses in a human oral KB epithelial cell line. Based on the MTT and SRB assay, arecoline inhibited the growth of KB cells in a dose- and time-dependent manner. Incubation of cells with arecoline caused a G2/M arrest in cell cycle progression as analyzed by flow cytometry. After 72 h treatment, arecoline induced apoptosis as detected in DNA fragmentation assay and Annexin V-PI staining. In Western blotting assay, arecoline treatment increased the protein level of Cyclin B1 and the phosphorylation of Cdc2Tyr15, and might indicate a G2 arrest response instead of a mitotic arrest. Besides, elevation of Wee 1 and degradation of Cdc25C may play a possible role in the inactivation of Cyclin B/Cdc2 complex. Under arecoline treatment, KB cells generated ROS, as determined by flow cytometry using DCF-DA. In addition, antioxidants such as glutathione and N-acetyl-L-cysteine, preventeded the arecoline-induced growth inhibition、G2 arrest and morphological changes of KB cells, while catalase and superoxide dismutase lacked these preventive effects. From these results, we conclude that arecoline inhibits the growth of human oral KB cells by preventing the G2 phase transition, and inhibits the activity of Cdc2 in a phosphorylation- dependent manner. Among these arecoline-regulated responses, ROS might play a critical role.

並列關鍵字

arecoline ； cell cycle ； reactive oxygen species

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