非小細胞肺癌細胞轉殖突變Akt基因對玫瑰樹鹼抗藥性的影響

Abstract

拓樸異構酶抑制劑ellipticine是具有抗癌效果的化學治療藥劑之ㄧ，但它在肺癌化學治療效應報導不多。本研究發現ellipticine可抑制人類非小細胞肺癌(NSCLC)A549細胞株的生長。但轉殖突變Akt-Ser473質體至細胞後，由ellipticine所誘導產生的細胞凋亡會被抑制。更多證據顯示ellipticine可調節細胞內部生長存活相關因子Akt與p53共同移動至細胞核，而當轉殖突變Akt-Ser473質體後，p53與Akt的核移動則會被抑制。此外，轉殖突變Akt-Thr308質體雖會抑制細胞凋亡的發生，但其效應較Akt-Ser473不明顯。此外，轉殖野生型p53進入p53缺失的H1299細胞株後，也會有類似的效果﹔但是若轉殖突變p53，則不會有這種效果。因此，可以看出ellipticine可促進細胞p53與Akt移動至細胞核及Akt於Ser-473位點的磷酸化，得以調控ellipticine所引發的細胞凋亡。有更多的證據顯示A549細胞株經由ellipticine所引發的細胞凋亡與細胞自噬的形成有關，這點可使用acridine orange染色來證明。當轉殖突變Akt質體後，ellipticine所誘導的細胞自噬體聚集也會降低，此外，使用細胞自噬抑制劑也有相同的效果。因此本研究顯示ellipticine所誘導產生的細胞凋亡是與Akt及p53移動至細胞核及細胞自噬形成有關。Topoisomerase II inhibitor ellipticine and its analogues were reported as promising antitumor agent. In this work, we showed that the growth of human non-small-cell-lung-cancer (NSCLC) epithelial cells A549 was inhibited by ellipticine. The induced apoptotic cell death disappeared upon transfection with dominant-negative Akt-Ser473 construct and the phenotype reversed. More evidence indicated that ellipticine regulated through nucleus translocalization of endogenous survival signals Akt and p53, and the effects were blocked by dominant-negative Akt-Ser473 and the growth of suppression reverted. On the other hand, the apoptotic phenotype was also be reverted by Akt-Thr308 with less dominant effect. The same effects took place by transfecting into p53-null H1299 cells with wild-typed p53, but not in those with mutated p53. Thus, the apoptotic death caused by ellipticine in A549 cells was determined by modulating subcellular distribution of Akt and its phosphorylation at serine-473 , which was assisted by p53. The effect is distinctive among the topoisomerase II inhibitors. More evidence indicated that the onset of apoptotic death in A549 cells is associated with autophagy, that was confirmed by acridine orange staining. Further more, the autophagesome aggregation caused by ellipticine was blocked by dominant-negative Akt-Ser473 as well as autophagy inhibitor. Altogether, our work corroborates that nuclear translocalization of Akt and p53 are essential in bringing the onset of autophagy in ellipticine-induced apoptosis in A549 cells.