- 學位論文
探討肺腺癌細胞珠存活及侵入能力的機制
Mechanisms involved in cell survival and invasion in lung adenocarcinoma cell lines
摘要
癌症的轉移是造成治療不易主因。在此我們選用具有不同侵入能力的CL1-1和CL1-5肺腺癌細胞亞株，探討不同訊息分子在其侵入及存活機制中扮演的角色。我們發現細胞內蛋白質酪胺酸磷酸化程度，CL1-5表現高於CL1-1，且Akt和Erk活性在CL1-5也較高。為了釐清這些分子對癌細胞侵入能力的影響，我們使用抑制劑抑制Akt和Erk活性。結果發現抑制Akt和Erk確實會減少MMP-9的分泌以及CL1-5的侵入能力，且抑制Akt效果優於Erk。因癌細胞轉移除了必須具有較強的侵入能力外，也需要有較強的存活能力。所以，我們接著以迫使細胞懸浮（anoikis），或者再另外加入EGTA減少細胞之間的黏附，以檢測細胞對此種處理所誘發凋亡之敏感性。結果顯示，CL1-5確實比CL1-1有較強抗拒anoikis的能力，而抑制Akt和Erk活性時會加強細胞的死亡比率。然而，CL1-5在誘發anoikis的初期，蛋白質酪胺酸磷酸化會大量增加，這些磷酸化可被激酶抑制劑staurosporine、AG17及AG1024所抑制。此外，在此時期Src、caveolin及Met的酪胺酸磷酸化也會增加。檢測細胞內ROS發現迫使細胞懸浮時會增加ROS，而NADPH oxidase抑制劑DPI和ROS scavenger NAC可以降低細胞懸浮時所產生的ROS及蛋白質酪胺酸磷酸化。另外，DPI和NAC也會降低懸浮細胞存活率。所以侵入強的CL1-5之所以對anoikis具較強的抗性至少部份是因為其細胞內具較高的Akt和Erk活性，而ROS引起的蛋白質酪胺酸磷酸化對抵抗anoikis也扮演重要角色。
並列摘要
Metastasis is a major problem to make the treatment difficult. Here we used lung adenocarcinoma cell lines CL1-1 and CL1-5 which exhibit different extent of invasiveness to study mechanisms involved in cell survival and invasion. We found that higher levels of protein tyrosine phosphorylation, p-Akt and p-Erk were detected in CL1-5. Inhibition of PI3K and MEK resulted in a decrease in MMP-9 secretion and cell invasion. Moreover, greater inhibition was observed by PI3K inhibitors, suggesting that Akt is more important in promoting tumor invasion. It has been known that metastatic tumor cells must survive during dissipation. Therefore, we forced cells to suspend or included EGTA in suspended cell, and examine their sensitity to anoikis. CL1-5 was more resistant to anoikis, but inhibition of PI3K and MEK increased the extent of apoptosis. Suprisingly, levels of protein tyrosine phosphorylation were increased at the initial stage of anoikis in CL1-5. This can be alleviated by several kinase inhibitors, including staurosporine, PP2, AG17 and AG1024. Besides, the tyrosine phosphorylation of Src, caveolin and Met was also increasd at this stage. ROS was also increasd when cells were forced to suspend. Inclusion of NADPH oxidase inhibitor DPI and ROS scavenger NAC reduced ROS production, protein tyrosine phosphorylation, and cell survival. Thus, highly invasive cell line CL1-5 is resistant to anoikis, and the constitutive activation of Akt and Erk may play a part in it, so does the enhanced protein tyrosine phosphorylation induced by ROS.