The mechanistic role of extracellular Ca2+ influx in Beauvericin- induced cell deaths in Xenopus oocytes

白殭菌素(Beauvericin, BEA)為首先發現於生長在昆蟲身上的黴菌Beauveria bassiana所分泌的毒素，能導致昆蟲死亡。在in vitro實驗中BEA能引起昆蟲、鼠科、人類腫瘤細胞株死亡。目前研究發現在哺乳動物細胞株中，BEA引起大量的細胞內鈣離子濃度增加，導致細胞凋亡(apoptosis)與細胞壞死(necrosis)的反應。由於BEA引起細胞內鈣離子增加的機制還不清楚，而且細胞內鈣離子增加的來源仍然沒有定論，所以本論文之目的在於探討BEA引起胞內鈣離子增加的機制與鈣離子的來源，並探討這些內流鈣離子與BEA產生細胞毒性的相關性。本實驗要研究的問題是BEA是否會增加細胞內鈣離子濃度，而增加的鈣離子之來源為何處，以及去除鈣離子來源能否抑制BEA所產生的細胞毒性。 我們的研究方法以非洲爪蟾(Xenopus laevis)卵細胞為材料，使用two-electrode voltage clamp記錄給予BEA後產生的電流，利用卵細胞內生性鈣離子調控之氯離子通道為指標，觀察細胞內鈣離子濃度的變化。接著改變細胞外溶液與給予各種抑制劑，找出增加的鈣離子之來源。另外，我們將卵細胞放在不同濃度BEA和不同溶液中培養，觀察卵細胞的存活情形，研究抑制BEA細胞毒性的可能方式。 本論文實驗結果顯示BEA的確能夠增加細胞內鈣離子濃度，進而活化爪蟾卵細胞內生性鈣離子調控之氯離子通道，而且BEA引起增加的鈣離子之來源是胞外鈣離子。另外，我們也發現胞外鈣離子的存在對於BEA引發細胞毒性有其貢獻，但其存在也不是絕對需要。所以，我們根據結果推測BEA產生細胞毒性的機制是透過胞外鈣離子內流，增加細胞內鈣離子濃度，繼而活化鈣離子相關的細胞死亡途徑，造成細胞死亡。Beauvericin (BEA) was first identified in an insect-pathogenic fungus, Beauveria bassiana. In vitro studies demonstrated that BEA induced significant cell deaths in insect, murine, and human tumor cell lines. In mammalian cell lines, BEA is known to induces a significant increase in intracellular Ca2+ concentration ([Ca2+]i) that leads to a combination of cellular apoptotic and necrotic responses. However, the source of [Ca2+]i increase remains inconclusive. Since the the mechanism of BEA-induced [Ca2+]i increase is not clear, we investigate the mechanism of BEA-induced [Ca2+]i increase and the sources of [Ca2+]i increase. We also investigate the correlation between Ca2+ influx and BEA cytotoxic effects. The goal of this thesis is to demonstrate whether BEA is able to increase [Ca2+]i , where [Ca2+]i increase comes from, and whether this [Ca2+]i increase is correlated with BEA-induced cytotoxic effects. Two-electrode voltage clamp method was used to record BEA-induced currents in Xenopus laevis oocytes. BEA was found to activate endogenous Ca2+_activated Cl- currents in Xenopus oocytes, suggesting a significant [Ca2+]i increase by the mycotoxin. BEA-induced currents in oocytes were blocked by the removal of extracellular Ca2+, indicating extracellular Ca2+ influx as the source of BEA [Ca2+]i effect. In addition, BEA was capable of induce potent cytotoxic effects in oocytes, the effect of which was significantly diminished in the absence of extracellular Ca2+. Based on our results, we conclude that the mechanism of BEA-induced [Ca2+]i can be attributed to a mobilization of Ca2+ influx from external bath solution, which in turn activates Ca2+_activated Cl- channels in Xenopus oocytes. In addition, BEA-induced Ca2+ influx contributes to, but is not prerequisite to, the cytotoxic effects of BEA that eventually lead to cell deaths. According to our result, we speculate that the mechanism of BEA-induced cell deaths in Xenopus oocytes involves an increase [Ca2+]i through extracellular Ca2+ influx, which eventually activates Ca2+-sensitive cell death pathway.中文摘要 ……………………………………………………………… 1 英文摘要 ……………………………………………………………… 2 第一章、導論 …………………………………………………………4 第二章、材料與方法 …………………………………………………15 第三章、結果 …………………………………………………………18 第四章、討論 …………………………………………………………26 圖表 ……………………………………………………………………35 參考文獻 ………………………………………………………………4