多環芳香烴受體於組蛋白乙醯化之角色探討

Abstract

組織蛋白去乙醯酶(Histone deacetylase, HDAC)為一種催化組織蛋白乙醯基去除的酵素，目前發現 HDAC家族中主要有11個成員，文獻指出HDAC 與細胞的增殖、分化、移行、血管新生、抗性有關，但是各個成員在細胞中仍扮演許多未知的角色。另一方面，研究顯示許多癌症(如：肝癌、大腸癌、血癌、皮膚癌)或是其他疾病的患者體內皆有異常的乙醯化組蛋白表現，案例中發現組織蛋白的乙醯化似乎與病況有所關連。近幾年已開發許多影響HDAC活性之藥物，如Belinostat、Trichostatin A (TSA)、Vorinostat (SAHA)等，希望藉由影響組織蛋白乙醯化程度以達到治療疾病之目的，而目前SAHA已發展成特定皮膚淋巴癌之臨床用藥。有鑑於近年來 HDAC 抑制劑的發展，本研究利用尚未發表之HDAC抑制劑AzP處理胃癌細胞株，觀察抑制HDAC後對於癌症細胞的影響，並探討可能涉及之分子機制。 首先，依據細胞存活率實驗結果，建立往後處理組所使用AzP最高劑量為5 μM。在細胞移行實驗 (cell transwell migration assay)結果發現加入AzP處理組之細胞移行能力約為對照組的一半效果。；由西方點墨法實驗結果發現處理AzP後AGS細胞內之磷酸化之蛋白激脢Cα (protein kinase Cα, PKCα)、乙醯化組蛋白和多環芳香烴受體 (aryl hydrocarbon receptor, AhR)表現量上升。使用細胞膜萃取之實驗結果得知，AzP會增加磷酸化PKCα在膜上之分布，說明AzP會增加PKCα的活性。此外，核質分離試驗與RT-PCR實驗結果得知，AzP誘導AhR轉錄並增加AhR在細胞核中表現。由啟動子活性試驗結果得知，AzP影響AhR基因啟動子範圍約在轉錄起始位置上游250至100之間。先前研究指出，PKC的磷酸化與胞內鈣離子濃度增加有關，藉由文獻提出之結論，我們推測AzP可能影響細胞內鈣離子濃度改變而造成PKCα活化。因此，進一步藉由鈣離子通道抑制劑 NiCl2和鈣離子鏊合劑EGTA來抑制鈣離子進入胞內，觀察AzP所引起之PKCα磷酸化表現是否受到抑制，結果指出NiCl2和EGTA確實抑制AzP引起之磷酸化PKCα表現量上升現象，證實AzP是透過改變鈣離子濃度而影響PKCα。在另一方面，參考先前文獻研究結果說明PKCα會影響轉錄因子NF-κB表現，而NF-κB又扮演著AhR生成之角色，在本實驗加入NF-κB抑制劑BAY-11-7082發現AzP所誘導之AhR生成並無受到影響，說明AzP並非藉由改變NF-κB來影響AhR生合成。接著利用RNA干擾方式改變細胞內AhR表現量，以觀察AhR在表觀遺傳可能牽涉之角色，結果指出當AhR表現量下降會造成乙醯化組蛋白表現亦下降，由結果得知AhR與組蛋白乙醯化過程有關。最後，利用蛋白－蛋白交互作用試驗發現，當處理AzP後會增加AhR與組蛋白2B之間的作用，但此現象並沒有在組蛋白4中有相同結果。 綜合上述實驗結果，證實AzP透過PKCα路徑而增加AhR轉錄發生，而增加之AhR同時涉及組蛋白乙醯化過程。但目前對於AhR影響組蛋白修飾過程之相關研究仍是十分缺乏，因此在未來這部分值得我們更深入研究。

Histone deacetylases (HDACs) are a class of enzymes that remove acetyl groups from histones, especially from the ε-amino group of lysine, leading the condensation of chromatin and repressing gene expression. The functions of HDACs, including migration, angiogenesis, cell differentiation, proliferation and apoptosis, have been published in many studies. It has been widely demonstrated that the abnormal acetylation of histones, causing from highly active of HDACs, would be related to many diseases, such as cancers and Rubinstein-Taybi Syndrome. In recent years, the medical developments of HDAC inhibitors are becoming common; in the view of this, we used gastric cancer cells treated a novel HDAC inhibitor, AzP, to observe the influences of this compound in vitro. In this study, the results showed that AzP increases histone acetylation and protein kinase Cα (PKCα) phosphorylation through affects sodium/calcium exchanger and raises the cytoplasmic and membrane fraction which indicates that PKCα activity increase; on the other hand, AzP induces the aryl hydrocarbon receptor (AhR) both in mRNA level and protein expression which exists in cytoplasm and nucleus. Besides, in the view of present studies, those studies indicated that PKCα would affect transcriptional factor, NF-κB, expression, and NF-κB plays a role in AhR biosynthesis. Thus, we used NF-κB inhibitor to inhibit NF-κB activity, and found that AzP-induced AhR expression was not changed, meaning that AzP did not affect AhR expression via NF-κB. Moreover, the result of promoter activity assay showed that AzP might influence the 250 to 100 bps upstream from start codon. Interestingly, we also found that repressing of AhR expression by RNA interference leads histone acetylation decreased in AzP-treated group, indicating that AhR plays a role in histone acetylation. Furthermore, the result of immunoprecipitation pointed out AzP increases protein-protein interaction between AhR and histone 2B, suggesting that AhR might has unclear functions in histone acetylation.