Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)可以專一性的毒殺腫瘤細胞，且不會影響正常細胞，所以被認為是一個具有研究價值的抗癌藥物。TRAIL所引起的凋亡作用主要是透過與腫瘤細胞表面DR4和DR5等死亡受器的結合，但是存在於腫瘤表面的DcR1與DcR2等干擾受器(decoy receptor)則會降低TRAIL對腫瘤細胞的毒殺作用。因此若腫瘤細胞對TRAIL產生抗藥性，則會大幅限制了TRAIL在臨床上的治療效果，而目前有許多文獻指出，多種癌細胞株對TRAIL具有抗藥性，且此抗藥性的表現與干擾受器的數量並沒有明顯的相關性。在目前的研究中發現clusterin的活化對於保護癌細胞抵抗TRAIL所引起的細胞凋亡扮演著重要的角色。在本實驗室先前研究分析了八株人類非小型肺癌細胞株對於human recombinant TRAIL的敏感程度，發現對於TRAIL較具敏感性的A549和H460其clusterin的表現量分別為中等與較弱，然而對於TRAIL較不具敏感性的H1355其clusterin的表現量則相對高很多。為了更了解clusterin在TRAIL抗藥性中所扮演的角色，我們分別在clusterin表現量中等和較弱的A549和H460肺癌細胞株轉染Clu ORF/3xflag CMV 10.0載體，或者在H460細胞株的培養液中額外給予human recombinant clusterin，發現增加clusterin的表現會降低細胞株對TRAIL所誘發的細胞凋亡比率；接著，在clusterun表現量較高且對TRAIL有抗藥性的H1355肺癌細胞株，我們利用clusterin siRNA短暫抑制clusterin的表現，發現會增加細胞對TRAIL的敏感性，另外我們也在A549穩定大量表現clusterin的stable clone再轉染clusterin shRNA並挑選出會明顯抑制clusterin表現的stable clone，發現當clusterin表現量被抑制後，其對於TRAIL的敏感性也會增加。綜合以上結果推論，不管是在A549、H460或H1355非小型肺癌細胞株，當clusterin表現較高時確實會促使細胞對TRAIL產生抗藥性。在本實驗之中，我們想了解clusterin在肺癌細胞株中所扮演的角色。使用TRAIL處理A549及H460細胞株後，發現在過度表現clusterin的A549及H460細胞株中，其caspase 3表現量很低。在TRAIL receptor方面，我們也發現在A549及H460細胞株中過度表現clusterin會增加DcR-1的表現量。綜合本實驗的結果推論，在A549和H460非小型肺癌細胞株中clusterin可能是經由調控TRAIL receptor DcR-1而使細胞對TRAIL產生抗藥性；另外在過度表現clusterin的A549及H460細胞株中，使用TRAIL處理，其caspase 3表現量很低，所以在A549和H460非小型肺癌細胞株中clusterin可能也會經由調控抗細胞凋亡機制而使細胞對TRAIL產生抗藥性。
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent as it selectively kills tumor cells but spares normal cells. Resistance to TRAIL by tumor cells limits its therapeutic use in clinic. TRAIL-induced apoptosis is mediated by binding with death receptors, DR4 and DR5, but the decoy receptors ( DcR-1 and DcR-2 ) on the surface of tumor cells collapse TRAIL-mediated apoptosis. Recently, several articles have demonstrated that some tumor cell lines exhibit TRAIL-resistance and this resistance may be not associated with decoy receptors. Recent investigations have indicated that clusterin activation was an important factor for cells to protect apoptosis from TRAIL. Previously, we analyzed the sensitivity of eight human non-small lung cancer ( NSCLC ) cell lines to TRAIL. We found the protein expressions of clusterin are medium and lower in TRAIL- sensitive cell lines ( A549 and H460 ). However, TRAIL- resistant cell lines ( H1355 ) express higher levels of clusterin. In order to investigate the role of clusterin in TRAIL-resistant cell lines, we separately transfected Clu ORF/3xflag CMV 10.0 plasmid into A549 and H460 cell lines in which endogenous clusterin expressions are medium and lower than other lung cancer cell lines tested. We found that overexpression of clusterin can reduce TRAIL-induced apoptosis. The H1355 cells which express a large amount of clusterin were resistant to TRAIL. Inhibition of clusterin expression by transiently transfected clusterin siRNA in H1355 cells resulted in enhanced apoptotic rate to TRAIL. In this research, we investigated the role of clusterin gene in lung cancer cells. After treated A549 and H460 cell lines with TRAIL, stable clones with clusterin overexpression expressed lower caspase 3. As to TRAIL receptors, overexpressed-clusterin could induce DcR-1 expressions in A549 and H460. In brief, TRAIL resistance in non-small cell lung cancer cell lines might result from overexpressed-clusterin through TRAIL receptor DcR-1 up-regulation. Moveover, in stable clones with overexpressed-clusterin, TRAIL treatment decreased induce caspase 3 expression suggesting that clusterin might also regulate antiapoptosis-signaling pathway to cause TRAIL resistance.
