光動力合併化學治療對口腔癌細胞株之影響

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.