探討Gleevec在人類大腸癌細胞中TRAIL所誘發細胞凋亡之訊息傳遞路徑

Abstract

癌症的治療在研發出多種細胞毒殺的化學治療藥物之後雖然大有進展，但腫瘤細胞對這些藥物所衍生出來的抗藥性仍舊是治療上的主要障礙。過去研究發現，TRAIL會選擇性的引起腫瘤細胞的死亡，而同時對於正常組織細胞幾乎沒有毒性；在本論文中，為了研究TRAIL合併其他臨床使用藥物的潛力，我們將探討Gleevec與TRAIL合併使用之下，對癌症細胞所引起的效果以及分子機制。Gleevec是臨床上用來治療慢性骨髓性白血病的首選抗癌藥物，其作用是可以有效抑制蛋白酪氨酸激酶c-Abl 及Bcr-Abl的作用。本篇研究中，我們比較了不同之人類癌症細胞株的毒性測試後，發現K562 (人類白血病細胞株)合併使用Gleevec 和TRAIL可以增加對細胞的毒性，然而，在人類大腸癌細胞 HCT116和SW480卻出現相反的結果。在上述兩株細胞中，0.1-1 μM 濃度下的Gleevec可以保護TRAIL所引發的細胞凋亡；但是在其他的細胞株中 (HT29, PC3, LNCaP, HaCaT 以及p53-null HCT116 細胞) 就沒有此現象存在。此外我們發現Gleevec可以減輕TRAIL所引起JNK和p38的活化，進而減緩對細胞的毒性。在HCT116細胞中，我們發現利用c-Abl 和 p73的小型干擾RNA可同時減弱TRAIL所誘發的細胞毒性和其所誘發激酶的活性，而此時Gleevec的保護效果就無法顯現。但是Gleevec的作用並不存在於對壓力誘導劑anisomycin所引發的激酶活化及細胞毒性。總結來說，我們認為在人類大腸癌細胞中TRAIL所產生的毒性是需經由p38 及 JNK的調控來增加蛋白凋亡酶的活化。經由實驗結果我們認為TRAIL的細胞毒性亦同時需要c-Abl及p73的參與，且對下游p38 及 JNK的調控是必要的步驟。這些結果提供了一個新的經由c-Abl/p73路徑來調控TRAIL引起酵素激酶JNK及p38活性及細胞凋亡的機制，以及未來在臨床上合併使用TRAIL和Gleevec發展治療人類大腸癌時應多加考慮的因素。

Even though there are many advances in the cancer therapy following the introduction of cytotoxic chemotherapeutic drugs, the development of drug combination to decrease cancer resistance and side effect still needs more efforts. TRAIL has been shown to selectively induce tumor cell death, and has minimal toxicity against normal tissues. In seeking to develop combination validity of TRAIL, we elucidate the effects of Gleevec in TRAIL-induced cancer cell death. Gleevec, a clinical drug effectively used for chronic myelogenous leukemia, is a tyrosine kinase inhibitor against c-Abl and Bcr-Abl. Studying cell viability changes in various human cancer cells, we found Gleevec can induce the increased cytotoxicity in TRAIL-treated K562 leukemia cells, but achieve an opposite effect in HCT116 and SW480 colon cancer cells. In both cancer cells, TRAIL-induced cell apoptosis is reduced by Gleevec in a concentration range of 0.1-1μM. No alteration of cell toxicity following TRAIL and Gleevec co-treatment is observed in HT29, PC3, LNCaP, HaCaT and p53-null HCT116 cells. JNK and p38 MAPK inhibitors effectively block TRAIL-induced toxicity. Accordingly Gleevec can attenuate TRAIL-induced JNK and p38 activation and cell apoptosis. In addition, siRNA targeting knockdown of c-Abl and p73 reduce TRAIL-induced cytotoxicity and render HCT116 cells less responsive to stress kinase activation. Moreover, in the downregulation condition of p73, Gleevec no longer induces cytoprotective effect. However, Gleevec does not affect cell death and stress kinase activation caused by anisomycin. All together it is suggested that in addition to activation of classical caspase cascade, TRAIL-induced apoptosis in human HCT116 colon cancer cells requires p38 and JNK activation. We propose both kinase activations induced by TRAIL depend on c-Abl activity and its downstream targeting effector p73. Our results provide a novel mechanism for stress kinase activation by death receptors, and additional concern in developing combination cancer therapy with TRAIL and Gleevec in the future.