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標題: 在皮膚細胞探討Imiquimod誘導DNA損傷之研究
Study of Imiquimod Induced DNA Damage in Skin Cells
作者: 江欣宜
Hsin-Yi Jiang
關鍵字: 無;no
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由於 DNA 是生物重要遺傳指令分子,維持 DNA 的完整性對細胞乃至整個 生物個體可說是極為重要的一環。外在環境的因素或是由生物體內源性所產生的 內生性破壞,都有可能造成 DNA 的損傷。目前在許多的癌症用藥上,會針對癌 細胞造成 DNA 損傷,例如形成 DNA 加合物(DNA adducts),或是 DNA 鏈斷 裂而導致基因體的不穩定性。這些 DNA 損傷反應會導致 DNA 的修復、衰老, 或凋亡。Imiquimod 是化學合成的核酸類似物,並且是類鐸受體 7 (Toll-like receptor 7,TLR7)、類鐸受體 8 (Toll-like receptor 8,TLR8)的配合體(ligand), 亦是免疫調節劑,目前在臨床上用來治療基底細胞癌(basal cell carcinoma,BCC)、 日光角化症等皮膚腫瘤和病毒疣。近期研究指出Imiquimod可直接毒殺腫瘤細胞或 透過活化細胞型免疫反應(cellular-mediated immune response)清除腫瘤細胞,且 被認為可以調控細胞自噬以及細胞凋亡的進行。然而 Imiquimod 是否能造成 DNA 損傷的機制仍不明瞭,在我們的研究中證明,從皮膚細胞的 DNA 鏈斷裂、 磷酸化 H2AX 的蛋白表現增加,以及在小鼠背部細胞形成磷酸化 H2AX 的 foci 中可得知 Imiquimod 的確能造成 DNA 損傷,且此現象在 in vitro 和 in vivo 都 得到相同的結果。且此結果不依 TLR7/TLR8 。實驗室證實了Imiquimod 所誘導 的 DNA 損傷機制是透過磷酸化 ATM/ATR,以及其下游磷酸化 Chk1/Chk2、 Ser15-p53,和 p53 的蛋白表現及基因表現增加之外,也促進 p53 轉至入核的表 現。假如抑制 ATM/ATR,會影響其下游以及 p53 基因表現,和抑制細胞凋亡。 最後,我們發現 Imiquimod 所誘導的細胞凋亡是依賴於 p53,且在 shRNA knock down 或是突變(mutant)p53的皮膚細胞中發現會抑制 Imiquimod 所誘導的細胞 凋亡。有趣的是,Imiquimod 在沒有 TLR7/TLR8 的皮膚癌細胞會形成 DNA 環 丁基環(cyclobutane ring)dimer。這是第一次提到 Imiquimod 有潛力成為一種 DNA損傷劑。此研究完成我們將可提供有關 Imiquimod 誘導 DNA 損傷與 p53 功能和細胞凋亡間相關性的確鑿證據。這一新發現不僅有助理解 Imiquimod 誘導 細胞凋亡的機制,在臨床應用中也有利於我們重新考慮 Imiquimod 的安全性和相 對風險,並謹慎使用於特定的癌症治療上。

DNA is the macromolecules which encode genetic instructions, therefore, maintaining the genomic DNA integrity and stability is very important in all living organisms. DNA damage could be caused by environmental and endogenous factors. Indeed, induction of DNA damage by chemotherapeutic agents is wild-used in clinical cancer therapy. Certain chemotherapeutic agents cause DNA damages, such as DNA adduct, strand break and result in genomic instability. These DNA damage responses lead to DNA repair, senescence or apoptosis. Imiquimod (IMQ), a synthetic Toll-like receptor 7/8 (TLR7/8) ligand, contains both antitumor activity by directly inducing tumor cell death and by activation of cellular-mediated immune response to against tumors. IMQ had been clinical used in viral warts, actinic keratosis, basal cell carcinoma (BCC) and several skin tumors. IMQ also directly induces autophagy and apoptosis in the BCC cells. However, whether the IMQ could induce DNA damage is still unclear. In this study, we demonstrated that IMQ induced DNA double strand break, H2AX expression and H2AX foci accumulation in human skin cell lines and in upper layer of mouse epidermis. These effects were not dependent on TLR7/8 expression. IMQ also induced DNA damage response, including increased phosphor-ATM/ATR and their downstream phosphor-Chk1/Chk2 expression that promoting p53 expression, phosphorylation, nuclear translocalization and up-regulation of p53 target genes. Pharmacological inhibition of ATM/ATR suppressed ATM/ATR-Chk1/Chk2-p53 axis and prevented apoptosis in IMQ-treated cells. Finally, we provided evidence that IMQ induced apoptosis was dependent on p53, those p53 knock-down and p53 mutant skin cancer cells were much resistant to the IMQ induced apoptosis. Interestingly, IMQ also induced cyclobutane ring dimer formation, enhanced XPA expression and nuclear translocalization in TLR7/8-negative skin cell lines. As our knowledge, this is the first time to mention that the IMQ has the potential to be a DNA damage agent. This novel finding may help us to understand and reconsider the safety and relative risk of IMQ application, cautiously apply IMQ to specific cancer population in clinical and provide a promising approach to facilitate the development of novel therapeutic strategies for IMQ-tumor therapy in basic cancer research and in clinical application.
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