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標題: | 鐵螯合酶在具光動力抗性之口腔癌細胞中扮演重要分子基礎角色 The Ferrochelatase Plays a Key Role in Molecular Basis of Oral Cancer Cells with Resistance to Photodynamic Therapy |
作者: | Ya-Shin Tsai 蔡雅心 |
指導教授: | 陳信銘 |
關鍵字: | 口腔癌,五胺基酮戊酸,原紫質九,δ-胺基乙醯丙酸合成?,亞鐵螯合?,抗光性, Oral cancer,5- aminolevulinic acid,Protoporphyrin IX,Aminolevulinic acid synthase,Ferrochelatase,PDT-resistance, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 口腔癌指發生在口腔內部之惡性腫瘤的疾病,大多數為黏膜上鱗狀細胞癌化形成的鱗狀細胞癌。目前對於口腔癌的治療,仍以手術切除為最快速且有效的方法,但是手術後往往會造成病患的臉部外觀缺損。不僅影響病患的生理功能,也可能會導致心理壓力。光動力治療(photodynamic therapy, PDT)是一種治療癌症的新方式,其治療範圍侷限於腫瘤組織上,因此經光動力治療後的病灶部位不會有明顯的疤痕及顏面缺損,在臨床上已有不錯的治療成效,對癌症患者來說是一大福音,但是光動力治療時間通常需達數周至數個月,藥物費用較為昂貴,以及因細胞特性及生長環境的差異,甚至需要經過數次治療,若產生如抗藥性般的抗光性反應,如何恢復對光動力治療具抵抗性的細胞治療效果,以及縮短治療時間是研究光動力治療中很重要的一環。
五胺基酮戊酸(5-Aminolevulinic acid, ALA)本身非光敏物質,但被細胞吸收經代謝路徑後,形成內生性光感物質原紫質九(protoporphyrin XI, PpXI),文獻指出腫瘤細胞內δ-胺基乙醯丙酸合成酶(δ-Aminolevulinic acid synthase, ALAS)和紫質膽質原脫氨基脢(porphobilinogen deaminas, PBGD)活性比正常細胞高,而亞鐵螯合酶(ferrochelatase, FECH)活性則比正常細胞低,使PpXI具選擇性地大量累積在癌細胞中,受光激發後會產生具細胞毒性的單態氧(1O2)及活性氧分子(reactive oxygen species, ROS),進而殺死腫瘤。 許多研究指出位在細胞膜上的腺嘌呤核苷三磷酸結合卡匣G2 (ATP-binding cassette super-family G member 2, ABCG2)會把癌細胞內的PpIX排出細胞外,使ALA-PDT效果下降。另外,可將粒線體中PpIX轉化形成血紅素(heme)的第一型血紅素氧化酵素(Heme Oxygenase-1, HO-1)亦會降低ALA-PDT療效,由此可知癌細胞中ALAS1、PBGD、FECH、ABCG2、HO-1蛋白表現量皆與PpIX生成量相關,進而影響光動力治療成效。本篇研究主要探討長期接受光動力治療效果不彰,產生具有抵抗性的口腔癌細胞株,其與無抗性的癌細胞間有哪些特殊差異,造成在光動力治療下仍然存活,並研究使用抑制劑是否能再次強化光動力治療的成效。 實驗顯示,Ca9-22和SAS兩種細胞分別創建出的抗性細胞,與母代細胞在同焦耳數能量下照光,其細胞存活率高於母代細胞。加入ALA處理後,抗性細胞中累積PpIX減少,ROS生成下降。之後探討PpIX生成路徑的變化,不論是母代還是抗性細胞其PBGD、ABCG2和HO-1表現量皆無明顯差異;而抗性細胞中ALAS1、FECH表現量高於母代細胞。加入不同劑量的FECH抑制劑N-methyl Protoporphyrin IX (NMPP),隨NMPP濃度上升,經ALA處理的抗性細胞中累積PpIX量亦上升,受光動力照射後細胞凋亡上升。 由研究結果得知,Ca9-22和SAS抗性細胞FECH蛋白表現較多,NMPP作用下可抑制FECH活性,使得癌細胞中PpIX累積量上升,增加ALA-PDT治療效果。 Oral cancer is malignant tumor that develops in the tissues of the mouth or throat. Most of oral cancers are squamous cells carcinomas. Surgery is still the quickest and the most effective method to treat oral cancer, but it will cause irreversible defects on patient’s face after operation. It not only affects patient’s physiological function, but also leads to psychological stress. Photodynamic therapy (PDT) is a novel treatment for tumor, which involving selective delivery of a photosensitive dye into target cells.The advantages of PDT are that it leaves less significant scars or surgical defects and has no long-term side effects. However, PDT takes a longer time to treat, costs a lot and the effectiveness is different depending on cell types and growth environment. If tumor increases resistance to PDT owing to repeat treatment, how to recover PDT efficiency and shorten the treatment period of the PDT-resistant cells are important issues to be resolve. 5- aminolevulinic acid (ALA) is not itself fluorescent, the metabolic precursor of heme, which is metabolised into endogenous fluorescent protoporphyrin IX (PpIX) in the heme biosynthesis pathway. ALA-induced PpIX accumulation has been shown that aminolevulinic acid synthase (ALAS) and porphobilinogen deaminase (PBGD) has higher activity in various malignant tissues than in normal tissues; the opposite was found for ferrochelatase (FECH). ATP-binding cassette G2 (ABCG2), which is located on the cell membrane, transports PpIX from the intracellular to the extracellular region. In addition, Heme Oxygenase-1 (HO-1), which converts mitochondrial PpIX into heme, can reduce the efficiency. The aim of the present study was to investigate whether the molecular mechanisms for the variation in PpIX levels, and whether potential inhibitors and natural substances could inhibit the expression of target protein and recuse the efficacy of ALA-PDT in PDT-resistant cell. We created cell lines resistant to ALA-PDT derived from human oral cancer. After administration ALA, the resistant cells resulted in the lower PpIX and the lower sensitivity to ALA-PDT. The protein expression of enzymes involved in heme synthesis and degradation was determined by western blot analysis. The resistant cells expressed higher level of FECH, which catalyzes insertion of ferrous iron into PpIX. After red light exposed, the resistant cells decreased oxidative stress. In conclusion, the resistant cells created by Ca9-22 and SAS expressed higher level of FECH which reduced the accumulation of PpIX. Inhibition of FECH by N-methyl protoporphyrin (NMPP) led to increased of PpIX accumulation and ROS generation, and enhanced therapeutic effect of ALA-PDT in resistant cells. We suggested that FECH played an important role in ALA-PDT resistance. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21268 |
DOI: | 10.6342/NTU201903549 |
全文授權: | 未授權 |
顯示於系所單位: | 口腔生物科學研究所 |
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