| Abstract: | Pemetrexed（alimta）目前已作為非小細胞肺癌患者的二線或第一線治療。在臨床上仍然有高比例的非小細胞肺癌病人對於Pemetrexed產生抗藥性或者有復發現象。然而，仍缺乏臨床生物指標能作為預測其藥效反應。以基礎細胞實驗，探討感性與抗性細胞株的抗藥性的分子標記，以及細胞毒性、癌症轉移分子之表現與細胞轉移能力間的關係，尋找生物標記以利於臨床辨識腫瘤轉移的可能性。使用Pemetrexed處理人類一系列非小細胞肺癌細胞，經由MTT分析細胞在Pemetrexed不同濃度時的生長抑制、流式細胞儀分析細胞週期分佈、以西方點墨法分析參與細胞週期蛋白表現量來評量抗腫瘤影響。以及探討對Pemetrexed有感性以及有抗性的NSCLC細胞株其癌症轉移分子的表現，使用Boyden chamber、Gelatin zymography和刮傷癒合試驗觀察細胞移行能力之差異。當非小細胞肺細胞株處理Pemetrexed，從一系列肺癌細胞以MTT細胞毒性結果篩選對Pemetrexed有感性的A549細胞、A549-p53i以及對Pemetrexed有抗性的H1355細胞。並且發現只有A549細胞所表現的p53與nm23-H1蛋白及Lipocalin2 mRNA與蛋白會隨Pemetrexed的濃度增加而上升，但抗性的H1355細胞不受影響。也發現在缺乏表現p53的A549細胞對於Pemetrexed仍有較高的敏感性。最後，我們在進一步將抑制了p53的A549細胞以及將A549細胞抑制Lipocalin2、nm23-H1的表現後，觀察對於Pemetrexed的移行能力的影響。結果在不表現p53的A549肺癌細胞在低濃度Pemetrexed時的細胞轉移能力明顯降低。以及抑制Lipocalin2表現的A549肺癌細胞，其細胞移行能力明顯降低，在處理Pemetrexed達2μM其細胞移行能力降低達顯著性意義，而抑制nm23-H1表現的A549肺癌細胞則移行能力提升並且處理Pemetrexed其移行能力影響不具顯著性意義。本研究也從流氏細胞儀與細胞週期調控蛋白的表現結果得知A549細胞經Pemetrexed影響p53增加促使p21、p27蛋白上升，抑制cyclin A，促使細胞週期停滯於G1/S期，H1355的機制則不同於A549細胞週期的表現。從研究我們也提出Lipocalin2可作為偵測ALIMTA感受性的肺癌細胞之預測基因指標其初步結果。
Pemetrexed（alimtaR）has been approved for second and first-line treatment in non-small cell lung cancer (NSCLC) patients. There are many NSCLC patients resistance to ALIMTA. However, there is still lacking of clinical biomarkers for predicting the therapeutic response of ALIMTA. Clinical implication and future perspectives of molecular markers were in the detection of circulating cancer cells of patients with malignancy. In in vitro, expression of metastasis molecular, migration, invasion and growth inhibition has been observed between durg-sensitive cells and durg-resistant cells following exposure to ALIMTA. Human NSCLC cell lines, with variable expression of the known molecular determinants of ALIMTA sensitivity, were exposed to ALIMTA using different dose. Antitumor effect was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell cycle distribution by flow cytometry, and expression of cell cycle mediators by Western blot. The metastasis was determined by median effect analysis. Migration and invasion by Boyden chamber, wound healing assay, gelatin zymography. When cells were exposed to ALIMTA, the differant cytotoxic effect was observed in drug-sensitive A549 cell lines and drug-resistant H1355 cell lines. As a consequence of the increase in p53 protein, nm23-H1 protein and mRNA were up-regulated expression in following exposure to ALIMTA from A549 cells. ALIMTA treatment determined a significant up-regulation Lipocalin2 in protein and mRNA in A549 cells, and did not significantly affect the levels of Lipocalin2 mRNA in H1355 cells. Further, RNA interference (RNAi)-mediated Lipocalin2 and nm23-H1 down-regulation generated sensitility and metastases to ALIMTA in A549 cells. When p53 expression was inhibited by RNA interference in adenocarcinoma cell line A549 (A549-p53i cells), cells had a highest motility. But Cells lacking of p53 were also more sensitive to ALIMTA treatment by migration assay. Lipocalin2 inhibition would decrease the mobitity of A549 cells. Until 2 μM ALIMTA treatment, the migration would be reduced in lipocalin2 silensing A549 cells. Moreover, nm23-H1 inhibition would induce the mobitity of A549 cells, more resistant to ALIMTA treatment by migration assay. ALIMTA increases p27KIP1 and p21CIP1/WAF1 binding to cyclinA-CDK2 complexes and inhibits cyclinA activity in A549 cells. P21CIP1/WAF1 and p27KIP1 are able to arrest the growth of cells in the G1 phase of the cell cycle. In conclusion ALIMTA could play a role as a genetic determinant of ALIMTA sensitive and resistant in NSCLCs. |
