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Title: | 光動力合併化學治療對口腔癌細胞株之影響 Effect of photodynamic therapy plus chemotherapy on oral cancer cell lines |
Authors: | Chien-Yang Yeh 葉建陽 |
Advisor: | 江俊斌(Chun-Pin Chiang),郭英雄(Ying-Shiung Kuo) |
Keyword: | 光動力治療,五-氨基酮戊酸,環氧化酵素-2抑制劑,口腔癌,口腔癌細胞株, photodynamic therapy,5-aminolevulinic acid,cyclooxygenase-2 inhibitor,oral cancer,oral cancer cell line., |
Publication Year : | 2005 |
Degree: | 碩士 |
Abstract: | 背景和目的︰以往的研究顯示環氧化酵素-2 (Cyclooxygenase-2, COX-2) 在口腔癌細胞中有過度表現的情形。而COX-2 本身會透過血管生成,細胞增殖及轉移等方式,來促進口腔癌細胞的擴展。5-氨基酮戊酸之光動力治療(ALA-PDT),在針對口腔癌前病變與口腔癌的治療上,已有不錯的療效。Celebrex為一種選擇性的COX-2 抑制劑,目前已被當成一種化學預防性的藥物,應用在不同類型的癌症治療上。在本研究中,我們探討是否Celebrex的給予,對於ALA-PDT在口腔癌細胞株的生長抑制上,能有加強的效果。以及Celebrex的給予,是否能夠增強ALA-PDT所誘導之口腔癌細胞之凋亡。
材料和方法︰在這項實驗中,我們使用兩株不同的口腔癌細胞株,SAS和Ca9-22。將個別SAS和Ca9-22的口腔癌細胞株,分成四個組別,分別以單獨照光、給予Celebrex並照光、ALA-PDT、給予Celebrex合併ALA-PDT處理,以西方墨點法,探討口腔癌細胞株中COX-2的表現;以MTT法,評估口腔癌細胞株活性;以Hoechst 33258螢光染色,研究口腔癌細胞株凋亡細胞的形態;以DNA laddering 分析法,研究口腔癌細胞株凋亡所引起的DNA片斷碎裂等。 結果︰ 在沒有任何外界刺激的情況之下,Ca9-22細胞有COX-2的表現,而SAS細胞則無。當以ALA-PDT處理時,可以發現SAS細胞的COX-2會被誘發而表現,Ca9-22細胞的COX-2則會有增強表現的情形,且COX-2之表現,有劑量與時間依存的情形。若SAS細胞給予Celebrex,再以ALA-PDT處理時,則會減少SAS細胞中COX-2之表現。以MTT細胞活性分析,和未做任何治療的控制組比較,可以發現SAS與Ca9-22細胞,在48小時的培養後,給予Celebrex的組別,會有明顯的細胞生長抑制之情形(p<0.05)。當SAS和Ca9-22細胞在單獨照光和給予Celebrex合併照光之後,並培養4小時的組別裡,縱使光照的能量增強,兩者皆不會有顯著細胞生長抑制的情形。在ALA-PDT和Celebrex合併ALA-PDT的治療組別,則有顯著的細胞生長抑制之情形(p<0.05)。這個協同增強生長抑制的效果,是發生在Celebrex合併ALA-PDT治療的組別上。以Hoechst 33258螢光染色觀察顯示,在光照 (SAS, 40 J/cm2; Ca9-22, 4 J/cm2) 後的72小時,可以發現兩株口腔癌細胞,有ALA-PDT所誘導的細胞凋亡之現象產生。不過更多細胞凋亡的情形,發生在Celebrex合併ALA-PDT的治療組別上。DNA laddering 分析亦顯示Celebrex合併ALA-PDT治療的組別,會比單獨作ALA-PDT治療的組別,有較早產生細胞凋亡、較大量產生細胞凋亡、以及光照劑量較少即有大量細胞凋亡產生之情形。 結論︰ 由於Celebrex的給予,確實能增強ALA-PDT對SAS與Ca9-22口腔癌細胞生長抑制的效果,並且亦能增加ALA-PDT對SAS與Ca9-22所誘導的細胞凋亡,我們認為光動力治療(ALA-PDT)合併COX-2抑制劑治療,對口腔癌細胞株之毒殺上,有協同增強的治療成效。 Background and purpose: Previous studies showed overexpression of cyclooxygenase-2 (COX-2) in oral cancers. COX-2 may promote oral cancer progression via an increase in tumor angiogenesis, cell proliferation, and metastasis. 5-Aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) has been used to treat oral premalignant and malignant lesions with promising results. A selective COX-2 inhibitor – Celebrex has been used as a chemopreventive drug for different types of cancer. In this study, we tested whether addition of Celebrex to ALA-PDT could increase the inhibitory effect of ALA-PDT on the growth of oral cancer cell lines and could enhance the ALA-PDT-induced apoptosis of oral cancer cell lines. Materials and methods: Two oral cancer cell lines, SAS and Ca9-22, were treated with light alone, Celebrex plus light, ALA-PDT alone, and Celebrex plus ALA-PDT. Western blot, MTT assay, Hoechst 33258 staining, and DNA laddering analysis were used to study the COX-2 expression, cell viability, morphology of apoptotic cells, and apoptosis-induced DNA fragmentation in SAS and Ca9-22 cells, respectively. Results: The COX-2 expression was found in Ca9-22 cells but not in SAS cells without external stimulation. The COX-2 expression could be induced in SAS cells and up-regulated in Ca9-22 cells by ALA-PDT in a time- and dose-dependent manner. Addition of Celebrex to the cultures decreased the ALA-PDT-induced COX-2 expression in SAS cells. Compared to the control group without any treatment, a significant inhibition of growth of SAS and Ca9-22 cells was found by the MTT assay after co-incubation with Celebrex for 48 hours (P<0.05). When SAS or Ca9-22 cells were treated with a series of increasing light doses and then cultured for 4 hours, compared to the control group no significant inhibition of cell growth was found in the light only and Celebrex plus light groups, whereas a significant inhibition of cell growth was found in ALA-PDT alone and Celebrex plus ALA-PDT groups (P<0.05). A synergic inhibition of cell growth was found when cells were treated with Celebrex plus ALA-PDT. ALA-PDT-induced apoptosis of SAS and Ca9-22 cells could be observed by fluorescence microscopy with the aid of Hoechst 33258 staining 72 hours after treatment with ALA-PDT with the light dose of 40 and 4 J/cm2, respectively. More apoptotic SAS and Ca9-22 cells were found in the Celebrex plus ALA-PDT group than in the ALA-PDT alone group. DNA laddering analysis showed that apoptosis-induced DNA fragmentation occurred earlier, was induced with less light dose, and was more abundant in the Celebrex plus ALA-PDT group than in the ALA-PDT alone group. Conclusion: Because addition of Celebrex to ALA-PDT could increase the inhibitory effect of ALA-PDT on the growth of SAS and Ca9-22 cells and could enhance the ALA-PDT-induced apoptosis of SAS and Ca9-22 cells. We conclude that COX-2 inhibitor combined with ALA-PDT have the synergic effect on the killing of oral cancer cell lines. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24491 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 臨床牙醫學研究所 |
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