- 學位論文
組蛋白乙醯化修飾於光動力治療所引發促存活分子表達之機制探討及臨床應用
Histone acetylation involves in the increased expression of prosurvival molecules induced by photodynamic therapy: mechanistic investigation and clinical implication
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摘要
光動力治療(Photodynamic therapy, PDT)能藉由產生氧化壓力引發腫瘤毒殺效應，為極有潛力之癌症治療方式。但是近年研究發現，經過光動力治療後之腫瘤細胞可能會經由誘發促存活分子cyclooxygenase-2 (COX-2)的表達，導致腫瘤復發。然而目前對於COX-2是如何在光動力治療後表現的相關機轉尚未明瞭，因此於本研究中，我們將深入探討光動力治療引發促存活分子COX-2轉錄的分子機轉，並藉此發展增進光動力治療之治療模式。本研究首先發現處以光感物質五氨基酮戊酸(ALA)或chlorin e6 (Ce6)之光動力治療皆能活化p38MAPK，並顯著提升人類黑色素瘤A375和老鼠大腸癌C26細胞中組蛋白乙醯化轉酶(histone acetyltransferase) p300HAT的表現及活性，且能觀察到光動力治療後組蛋白乙醯化(Histone acetylation)顯著上升。以組蛋白乙醯化轉酶抑制劑anacardic acid (AA)或p300HAT之shRNA阻止p300HAT於光動力治療後上升，會大幅減低細胞核內組蛋白乙醯化程度，並抑制COX-2表現。使用染色質免疫沉澱(chromatin immunoprecipitation)分析，顯示光動力治療後會吸引更多p300HAT結合至COX-2的啟動子區域，加強組蛋白及轉錄因子NF-κB之乙醯化修飾，提升COX-2的轉錄，而類似的結果能經由處理過PEGylated Liposome Ce6 (PL-Ce6)-PDT之A375或C26小鼠腫瘤模式獲得證實。接著使用組蛋白乙醯化轉酶抑制劑anacardic acid (AA)處理A375和C26細胞，發現能有效提升光動力治療之細胞毒殺效果。進一步於植有C26腫瘤之BALB/c小鼠進行實驗，發現相較於只接受光動力治療，或光動力治療結合COX-2抑制劑組別，組蛋白乙醯化轉酶抑制劑AA結合光動力治療能最為顯著抑制腫瘤生長。實驗更發現除了COX-2外，促存活分子survivin在光動力治療後，同樣也會受到活化的組蛋白乙醯化轉酶調控而加強其表現。最後證實結合組蛋白乙醯化轉酶抑制劑AA與光動力治療之結合型治療能藉由抑制survivin表現，提升caspase-3活性，造成Becline-1裂解而阻礙光動力治療後之細胞自噬，同時加強細胞凋亡發生。綜合以上，顯示光動力治療能藉由p38MAPK路徑活化組蛋白乙醯化轉酶，而促使促存活分子表現，我們藉此發展組蛋白乙醯化轉酶抑制劑結合光動力治療之結合型治療模式，並證實在細胞和動物實驗中都能有效提升光動力之治療效益，希望對於將來臨床應用能提供新的治療方向。
並列摘要
Oxidative stress induced by photodynamic therapy (PDT) mediates the tumoricidal effect, but has also been shown to induce the expression of pro-survival molecules, such as cyclooxygenase-2 (COX-2), which are involved in tumor recurrences after PDT. However, the molecular mechanism is still not fully understood. In this study, we found that PDT-induced p38MAPK activation could significantly up-regulate the activity and expression of histone acetyltransferase p300 (p300HAT) in A375 and C26 cells under ALA or chlorin e6 (Ce6) mediated photodynamic treatment. Colony-formation assay showed PDT-induced cytotoxicity was dramatically elevated in the presence of p300HAT inhibitor, anacardic acid (AA). Further studies showed that the increased p300HAT can transfer the acetyl group to histone H3 and NF-κB p65 subunit to up-regulate the COX-2 expression. The effect was reduced by the shRNA of AA or p300HAT. Using chromatin immunoprecipitation analysis, we found that the acetylation of histone H3 and NF-κB increases their binding to the COX-2 promoter region. These findings were further verified in the in vivo xenograft mouse model bearing murine C26 and human A375 tumors treated with PEGylated Liposome Ce6 (PL-Ce6) mediated PDT. Meanwhile, the combination of PDT and AA resulted in a greater tumor regression in BALB/c mice bearing C26 tumors, compared to those treated with PDT only or combined with COX-2 inhibitor. Finally, we demonstrated that suppression of the PDT-induced p300HAT activity also resulted in the decreased expression of survivin, restoring caspase-3 activity and sensitizing PDT-treated cells from autophagy to apoptosis due to the Becline-1 cleavage. This study demonstrates that the molecular mechanisms involved in histone modification induced by PDT-mediated oxidative stress, suggesting that HAT inhibitors may provide a novel therapeutic approach for improving PDT response.
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