探討缺氧誘導因子-1α在口腔癌經光動力治療的表現

Abstract

口腔癌的排名有逐年上升的趨勢，年增率高居癌症第一位，死亡率過半。癌症治療的方式多樣化，目地在延長病患壽命，甚至進一步治癒癌症。以手術切除常因造成顏面傷殘及病患心理壓力而放棄，為尋求減少侵犯性治療，進行新興的光動力治療。光動力治療(Photodynamic therapy, PDT)是新興的癌症治療的方式，光動力治療已運用在其它不易手術部位的癌療。光動力治療的藥物也有進一步的改善，先前我們採用新研發的二代光感藥物(photosensitizer) 5-氨基酮戊酸(5-aminolevulinic acid, ALA)，ALA光動力治療(5-aminilevulinic acid-based photodynamic therapy, ALA-PDT)證實可用於口腔癌的治療，但路徑仍有待進一步研究，而接受光動力治療時所需要的三要件中的氧氣扮演了重要角色。在細胞中對於氧氣的調節扮演重要的角色的缺氧誘導因子-1α（Hypoxia inducible factor-1α, HIF-1α）蛋白是我們想去探討的因子。HIF-1α的過度表現促進腫瘤細胞的生長與血管增生，也影響癌症的療效，如放射線治療與化學治療效果的降低。光動力治療會產生氧化壓力的機轉，過程中細胞環境中的氧會被消耗，造成缺氧狀態，是否是誘發HIF-1α的過度表現的主因，仍有待進一步證實，缺氧誘導因子-1α（Hypoxia inducible factor-1α, HIF-1α）於人類許多癌腫瘤中有過度表現。目前已經發現HIF-1α蛋白之過度表現與許多腫瘤的侵犯、淋巴結轉移、臨床分期、病人存活率及預後都有顯著相關性，而HIF-1α在光動力的治療中是扮演什麼樣的角色，過去的研究中發現其會進一步促進VEGF的生成，其在光動力治療的情況下是否會影響光動力治療的效果。 本研究利用西方墨點法及免疫組織化學染色法，探討HIF-1α於接受光動力治療的惡性口腔癌細胞CA9-22和SAS之間的差別及13例口腔鱗狀細胞癌（OSCC）病患之間的差別。並以HIF-1α和COX-2的抑制劑，以及活性氧分子抑制劑的治療來觀察其蛋白質表現的情況，後續並加入不同的藥物觀察癌細胞治療效果，探討ALA-PDT的影響。 研究中發現其HIF-1α會隨時間增加而增高，誘導HO-1隨時間的增加及VEGF的上升，對細胞凋亡產生抗性，我們發現，以ALA-PDT來處理SAS及CA9-22細胞，可以造成PARP的水解，證實ALA-PDT引起人類口腔癌細胞死亡的方式為細胞凋亡(apoptosis)。先前研究中發現以4J的ALA-PDT處理CA9-22及40J的ALA-PDT處理SAS並不會改變細胞週期。研究以人類口腔癌細胞株CA9-22和SAS探討ALA-PDT中HIF-1α對治療的影響。研究中發現其HIF-1α會隨時間的增加而增高，同時HO-1和VEGF約隨時間的增長而增高，並證實VEGF會受到HIF-1α的調控，ALA-PDT可能因缺氧狀態及單重態氧自由基，產生兩者互相競爭抗恆而影響療效。光動力治療會誘發HIF-1α的表現，HIF-1α會誘發VEGF和HO-1的表現，以ALA-PDT加上HIF-1α抑制劑及COX-2抑制劑可以減少VEGF和HO-1的生成，並增加光動力治療的療效。 HIF-1α有較高表現的口腔癌細胞中顯示對於ALA-PDT有較高的抗性，主要因其誘導相關的蛋白質表現，並對細胞產生保護機制，在光動力治療合併抗缺氧誘導因子-1α可以增強光動力治療的效果。

In Taiwan, the problem of oral cancer in increasing, the increasing rate was the first of the cancer increasing rate. The death patient rate was over half of patient. The methods of treatment cancer were multiple various methods. The aids are elongation the life of patient and cure the disease. The main strategy of the treatment for oral cancer is surgery, but the facial defect resulting from surgery. Due to reduce the invasive treatment, photodynamic therapy (PDT) has been used in the treatment of many cancers, it is the potential therapy, and its advantage is that there is no significant facial defect. The Photodynamic therapy (PDT) is a kind of new developing treatment for cancer. 5-Aminolevulinic acid (ALA) is a novel photosensitizer for photodynamic therapy. We previously showed ALA-PDT can induce cytotoxicity in oral cancer cells. The mechanism for the ALA-PDT induced cytotoxicity is still unknown. Oxygen is the important quench of the three fundamental elements. The expression of hypoxia-inducible factor-1α (HIF-1α) is the regular of oxygen. Overexpressions of HIF-1α have induced the tumor growth and angiogenesis, and affect the treatment result of the cancer therapy, such as radiotherapy and chemotherapy. The PDT has induced the reactive oxygen species (ROS), in the procedure, the O2 was decreased and due to the hypoxia situation. We want to know if the situation was the main cause. That needs us to research. Overexpression of hypoxia-inducible factor-1α (HIF-1α) has been demonstrated in a variety of human cancers and found to be significantly associated with the tumor invasion, lymph node metastasis, clinical stage, survival rate, and prognosis of these cancers. What role is the HIF-1α in the ALA-PDT? In the past study, we found the expression of VEGF was induced with expression of HIF-1α during hypoxia. How to expression of VEGF in the ALA-PDT? In this study, the immunohistochemistry, Western blot analysis, and MTA assay will be used to investigate if the expression of HIF-1α correlates with the varied treatment results of PDT. And the expression of HIF-1α in 13 specimens of oral squamous cell carcinoma (OSCC). We were calculated and compared between the specimens. Then we treated the cell lines with ALA-PDT plus the inhibitor of the HIF-1α and the COX-2, free radical inhibitor and multiple drugs to research the expression of protein. In our study, we found the expressions of HIF-1α were increased with time follow, and induced the expressions of HO-1 and VEGF, then reduced the ALA-PDT-induced apoptosis. In CA9-22 and in SAS cell lines, we used the ALA-PDT treatment could induced the apoptosis. Apoptosis induced by ALA-PDT was both time- and dose-dependent. Pretreatment of cells with imidazole and histidine (singlet oxygen inhibitor) reduced the ALA-PDT-induced apoptosis. These results indicated that singlet oxygen was an important mechanism for ALA-PDT-induced apoptosis in cells lines. In the IHC, the expressions of HIF-1α had no significant finding with gender, age, tumor size, involved depth, tumor region and pathology. The expressions of HIF-1α in nucleus had significant different findings about the resistance to the photodynamic therapy. There is higher resistance to ALA-PDT in the lesion with over expression of HIF-1α. The HIF-1α protein maybe induce the target gene and protein expression to protect the cell survive. PDT combination with anti-HIF-1α treatment enhance the response.