|Abstract: ||摘要:粒線體功能缺損導致人類肝癌細胞惡化：第八型介白質與ADAM之角色 背景：近年我們在人類肝癌細胞株研究發現：粒線體功能缺損可能改變細胞核基因表現、蛋白質合成及代謝路徑、甚至誘發化療抗性及細胞遷移(Chang et al., 2008 & 2009)。我們以siRNA及抗體實驗證實：粒線體功能缺損時，能藉由上皮生長因子受體(EGFR)受質amphiregulin(AR)過表現和AR自泌循環(autocrine loop)以調控上述癌症惡化現象。此外，粒線體功能缺損引發的AR過表現可能肇因於細胞內鈣離子移動及活性氧分子(ROS)增加；而細胞膜表面ADAM活性則為釋放AR的關鍵(Chang et al., 2009)。然而，人類肝癌細胞因粒線體功能缺損而改變的訊息分子如何調控細胞核基因表現、如何活化ADAM、導致癌症惡化仍是亟待探究的問題。因此，我們先根據粒線體功能缺損時轉錄體變化預測其參與之生物反應路徑，結果顯示：細胞激素訊息傳遞、細胞激素及其受體複合體之重組等可能改變。初步以細胞激素蛋白晶片篩選並以即時定量聚合酶連鎖反應分析發現，無論是抑制粒線體DNA轉錄轉譯或破壞粒線體呼吸功能，均能促進趨化素IL-8過表現及分泌。由於IL-8訊息能活化IL-8受體(CXCR1/CXCR2)、活化ADAM造成EGFR受質釋放、並轉活化EGFR，進而促進與癌細胞增殖、細胞存活、侵襲能力、血管新生等惡化行為相關之基因表現(Tanida et al., 2004)；且肝癌患者腫瘤分期、無病存活率不良與血清IL-8濃度相關(Ren et al., 2003)。故我們假設：人類肝癌細胞粒線體功能缺損時，粒線體調控訊息分子造成細胞核IL-8過表現及分泌，活化IL-8受體與ADAM而釋放過表現的AR、形成AR自泌、轉活化EGFR，導致肝癌惡化。 方法：在人類肝癌細胞株轉染siRNA抑制粒線體DNA轉錄或給予粒線體呼吸鏈酵素抑制劑，分別造成粒線體基因缺損或呼吸功能缺損。評估粒線體缺損造成的細胞功能改變包括耗氧率、ATP消耗、細胞內鈣離子移動及ROS生成等。以即時定量聚合酶連鎖反應及ELISA定量IL-8基因表現及分泌，以immunoblotting分析IL-8受體、EGFR及其下游訊息活化，以siRNA或專一抗體評估粒線體缺損時IL-8、IL-8受體對ADAM活化、細胞增殖、轉移及化療抗性的影響(分別以ADAM activity assay、cell growth assay、wound healing assay、transwell migration assay、transwell invasion assay、LDH assay、cell cycle analysis評估之)。製作一系列IL-8啟動子AP-1、C/EBP或NFκB調控區刪除變異的pGL3冷光酵素報告質體，並以EMSA分析粒線體缺損引發之訊息分子(鈣離子、ROS…)對IL-8基因表現的影響。 預期成果：第一年年，人類肝癌細胞株粒線體功能缺損誘發IL-8基因表現、自泌循環及CXCR1/CXCR2活化，對細胞增殖、化療抗性、遷移及侵襲之影響。第二年，因粒線體功能缺損而改變的訊息因子，活化ADAM、釋放EGFR受質、轉活化EGFR訊息傳遞。第三年，粒線體逆行訊息調控IL-8基因啓動子轉錄因子之機制。綜合而言，本研究將呈現人類肝癌細胞粒線體缺損調控癌症惡化之機轉、釐清IL-8自泌及ADAM活化之角色，期望作為癌症治療策略參考。|
Abstract:Mitochondrial Dysfunction-Induced Cancer Malignance in Human Hepatoma Cells: The Roles of Interleukin-8 and ADAM Background: Our recently studies revealed that mitochondrial dysfunction-induced the changes of nuclear genes expression, protein synthesis, metabolic pathways, as well as chemo-resistance and cell migration in human hepatoma cells. By using of specific RNA interference (siRNA) and a neutralizing antibody, we found that mitochondrial dysfunction-induced amphiregulin (AR) autocrine loop-mediated cancer malignance in HepG2 cells. Meanwhile, the mitochondrial dysfunction induced cytosolic Ca2+ Methods: The siRNA of mitochondrial transcription factor and inhibitors of mitochondrial respiratory enzymes will be used to induce mitochondrial genetic stress and respiratory defect in human hepatoma cells, respectively. The gene expression and secretion of IL-8 will be analyzed by real-time quantitative PCR and ELISA, respectively. The activation of IL-8 receptor, activation of EGFR will be measured by immunoblotting. Mitochondrial dysfunction-induced activation of ADAM, cell proliferation, metastasis, and chemo-resistance will be examined by ADAM activity assay, cell growth assay, wound healing assay, transwell migration/invasion assay, LDH assay, and cell cycle analysis, respectively. The siRNA as well as neutralizing antibody of IL-8, IL-8 receptor, and ADAM will be used to evaluate their roles in cancer malignance. Additionally, the pGL3 luciferase reporter vectors containing the human IL-8 and its mutant promoters will be constructed, and the electrophoretic mobility shift assays will be done to identify the transcription factors in response to mitochondrial dysfunction-induced signaling molecules (such as calcium, and ROS, etc.) in human hepatoma cells. mobilization, and reactive oxygen species (ROS) overproduction may contribute to AR induction. Moreover, a disintegrin and metalloprotease (ADAM) could be one of the important modulators of mitochondrial dysfunction-regulated AR secretion. However, the mitochondria-to-nucleus retrograde signaling in responsible to changes of gene expression, and the molecular mechanisms by which the mitochondrial dysfunction leads to activation of ADAM have yet to be elucidated. Therefore, we were investigated mitochondrial dysfunction-induced changes of transcriptome and annotated their biological functions. We found that interleukin signaling, and interleukin receptor complex assembly may affected by mitochondrial dysfunction. Furthermore, we found that both of mitochondrial respiratory defect and mitochondrial genetic stress upregulated IL-8 induction and secretion in HepG2 cells. Because of previous studies demonstrated that IL-8 signaling positively regulates the activation of IL-8 receptor (CXCR1/CXCR2), and shedding of EGF ligands via activation of ADAM, with subsequent transactivation of the EGFR, as well as transcription of genes implicated in cancer cell proliferation, angiogenesis, and invasion. Additionally, the elevation of serum IL-8 level correlates with tumor stage and poor disease-free survival in patient with hepatocellular carcinoma. Together, we have hypothesized that IL-8 could be one of the important target genes involved in the mitochondrial retrograde signaling and to modulate cancer progression through the activation of IL-8 receptor and ADAM, as well as transactivation of EGFR in human hepatoma cells. Summary: The results of this study will provide important information of the roles of IL-8 signaling play in the mitochondrial dysfunction-modulated human hepatoma malignance. Further characterization of the activation of ADAM to transactivate EGFR confer with IL-8 signaling in human hepatoma is crucial to characterize the potential benefits of combination therapy in the management of this disease.