| Abstract: | 橘黴素 (CTN)是一種常見於食物及飼料中的黴菌次級代謝產物，在本篇論文中我們主要利用人類腎臟細胞株 (HEK293)來探討CTN所引起的致毒機制，其中包括了CTN對於細胞週期的影響以及訊息傳遞途徑的活化。隨著CTN處理濃度的提高造成哺乳動物細胞的存活率下降，並使停留在G2/M細胞週期的細胞數目比例增加。由調節點蛋白質的角度觀察，CTN的處理會使細胞內cdc25c的蛋白質表現量下降，而p53和p21的蛋白質表現量上升。此外，我們發現在CTN處理後的HEK293細胞中，α-tubulin的二聚體 (dimer)和多聚體 (polymer)蛋白質含量會隨著CTN處理濃度的增加而降低，但是卻不會在α-tubulin mRNA的層次造成影響。進一步利用免疫細胞化學染色的方法證明得知，CTN的處理除了能使細胞內α-tubulin的分布和結構呈現異常現象之外，也會增加累積在有絲分裂期的細胞數目。綜合以上結果，CTN可能藉由改變G2/M時期的調節點蛋白質的活化程度及干擾微管的正常表現而使細胞週期無法正常進行。
在HEK293細胞中，MAPKs訊號途徑的ERK1/2及JNK磷酸化會隨著暴露CTN時間或劑量的增加而顯著增加，CTN亦能誘增ERK1/2和JNK下游早期活化基因 (egr-1、c-fos、fosb、junb及c-jun)的mRNA含量，並且使細胞核內轉錄因子Egr-1和AP-1的表現量增加；為了進一步探討CTN所誘導的轉錄因子是否具有細胞內的生物功能，我們分別以Egr-1或AP-1的DNA結合序列作為增強子分別建構出含有luciferase報導者基因的質體進行實驗，結果顯示CTN的處理能夠活化含有Egr-1和AP-1 DNA結合序列的啟動區，進而使報導者蛋白luciferase大量產生，此外CTN也能誘導HEK293細胞中的MMP3基因 (啟動區具有AP-1結合序列)或是GADD45B基因 (啟動區具有Egr-1結合序列)的RNA表現量。綜合以上結果顯示，CTN能活化人類細胞株中MAPKs的訊號傳遞途徑，活化MAPKs下游早期活化基因並進而調控Egr-1和AP-1下游基因的表達。
Citrinin (CTN), a frequent natural contaminant of foods and feeds, is a secondary metabolite of certain fungi. To investigate the molecular mechanism of CTN-induced cytotoxicity, human embryonic kidney (HEK293) cells were chosen to identify the effect of CTN on cell cycle arrest and cellular signaling transduction pathway. CTN decreased the survival rate and arrested cell cycle in G2/M phase in a dose-dependent manner. In view of checkpoint proteins, exposure of cells to CTN caused the down-regulation of cdc25c and up-regulation of p53, p21 and cdc2 in a dose-dependent manner. In addition, treatment of cells with CTN decreased the levels of α-tubulin dimer and polymer, but not the level of α-tubulin mRNA. Results of immunocytochemistry further proved that CTN led to the abnormal organization and arrangement of cellular α-tubulin as well as the increase of cell number in M phase. Taken together, our findings suggest that CTN leads to G2/M arrest via interfering the checkpoints of G2/M and the biological function of α-tubulin.
Exposure of HEK293 cells to CTN activated extracellular signal -regulated kinase (ERK) and Jun N-terminal kinase (JNK) of MAPKs signaling pathway in dose- and time-dependent manners. CTN not only increased the mRNA levels of downstream immediate-early genes, including egr-1, c-fos, fosb, junb and junB, but also elevated the protein levels of transcription factors, AP-1 and Egr-1. To investigate whether the CTN-induced transcription factors have biological functions in vivo, DNA binding sequences of Egr-1 and AP-1 were used as enhancer elements to construct the reporter plasmids for luciferase assay. CTN treatment was found to significantly increase the luciferase activity driven by Egr-1 or AP-1 DNA binding sequence. CTN also increased the mRNA levels of MMP3 and GADD45B, which harbor AP-1 and Egr-1 DNA binding sequence in its promoter regions, respectively. Overall, our data indicate that treatment of HEK293 cells with CTN activates the MAPKs pathways, which not only contribute to the induction of immediate-early genes, but also regulat the expression of Egr-1 and AP-1 downstream genes. |
