AICAR和15dPGJ2在人類大腸癌細胞中誘導TRAIL和TNFα所引發的細胞毒性之訊息傳導路徑

Abstract

癌症的治療在我們研發出多種細胞毒殺化學治療藥物之後雖然大有進展，但腫瘤細胞對這些藥物發展出來的抗藥性仍舊是治療上的主要障礙。過去的研究發現，TRAIL會選擇性地引起腫瘤細胞死亡，而同時對正常組織細胞幾乎沒有毒性；因此單獨使用TRAIL，或是合併其他抗癌藥物在治療癌症極具潛力。在這個研究中，我們探討在人類大腸癌細胞株(HCT116)，AICAR (AMPK活化劑)與15dPGJ2 (PPAR-γ活化劑) 增加TRAIL所引起細胞凋亡相關的效果與分子機制。我們發現AICAR與15dPGJ2會強化TRAIL與TNFα引發的細胞凋亡。AICAR的作用需要AMPK，但不會改變TRAIL/TNFα的受體蛋白表現量。AICAR可經由參與多種細胞凋亡的調控階段而促進腫瘤細胞的凋亡。AICAR與TRAIL或TNFα一起作用時會促進caspases 8, 9 及 3的活化，會抑制抗細胞凋亡蛋白Bcl-2，會增加Bid的分裂, 會使粒腺體表面電位差減少、會促進p38與JNK活化、也會抑制NF-kB活性。這些研究結果顯示AMPK可能與許多由活化TRAIL所引發的細胞凋亡訊息傳遞有關。相反地，我們的結果顯示15dPGJ2誘導TRAIL所引發的細胞毒性來自於增加DR5受體的轉錄，而此作用與PPAR-γ無關。CHOP是這個作用的媒介分子。利用CHOP的小型干擾RNA (small interfering RNA)可減弱15dPGJ2對DR5的促進調控而逆轉對TRAIL的致敏性。此外，DR5的表現是經由ROS，鈣離子，PKC與PKR所媒介。我們也發現15dPGJ2可以引發GRP78與XBP1表現，顯示15dPGJ2也會產生內質網的壓力。總結來說，我們結果提供了癌症治療的新方法，即可利用AICAR或15dPGJ2合併TRAIL的使用來增加療效。AICAR的作用是經由AMPK，牽涉到粒腺體有關的細胞凋亡機制；另一方面，15dPGJ2的致敏效果是經由CHOP媒介的DR5調控，而非PPAR-γ相關的機制；ROS，鈣離子與PKC的活性會引起CHOP基因的轉錄。

Even though there have been many advances in the therapy of cancer following the introduction of cytotoxic chemotherapeutic drugs, the development of drug-resistance remains a major obstacle in the treatment of tumors. TRAIL has been shown to be selectively cytotoxic in inducing tumor cell death, and has minimal or no toxicity against normal tissues. Thus TRAIL-mediated tumor cell death, either alone or in combination with other anticancer therapy, is considered as new strategy with great potential for anticancer effect. In this study we investigated the combinatorial effects and molecular mechanisms of AICAR (a pharmacological activator of AMPK) and 15dPGJ2 (an activator of PPARγ) in sensitization of TRAIL-induced apoptosis of human colon cancer HCT116 cells. We found AICAR and 15dPGJ2 can potentiate TRAIL- and/or TNFα-induced cell apoptosis. The action of AICAR requires AMPK, is independent of protein expression of cytokine receptors, and may occur at various stages of apoptotic pathways. AICAR co-treatment with TRAIL or TNFα enhances activation of caspases 8, 9 and 3; down-regulates the antiapoptotic protein BcL-2; increases the cleavage of Bid and loss of mitochondrial membrane potential; potentiates activation of p38 and JNK; and inhibits NFκB activity. These results imply that AMPK might be involved in the regulation of multiple signaling pathways, and in turn exerts sensitized TRAIL apoptosis. In contrast, our results revealed that the sensitization of TRAIL cytotoxicity by 15dPGJ2 is resulting from the transcriptional upregulation of death receptor 5 (DR5), but independent of PPARγ. We demonstrated CHOP as a mediator of DR5 induction by15dPGJ2. CHOP small interfering RNA attenuated the DR5 up-regulation due to 15dPGJ2 and reversed its sensitization effects with TRAIL. Moreover, DR5 expression through CHOP up-regulation is mediated by ROS, calcium, PKC and PKR. Furthermore, we also found that 15dPGJ2 is able to induce GRP78 and XBP1 expression, indicating the contribution of ER stress. In summary, we have developed a novel strategy of combining AICAR and 15dPGJ2 with TRAIL for the treatment of colon cancer cells. The sensitization effect of AICAR is through AMPK pathway, and involves mitochondria-dependent apoptotic cascades. In contrast, the sensitization effect of 15dPGJ2 is through PPARγ independent pathway, but depends on CHOP-mediated DR5 upregulation. The increase of ROS, calcium and PKC activity are contributed to CHOP gene transcription.