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標題: | 探討氯化鈷藥物處理對光動力治療口腔癌細胞之影響 The Effect of Cobaltous Chloride Treatment on Oral Cancer Cell Treated with Photodynamic Therapy |
作者: | Po-Ya Chang 張博雅 |
指導教授: | 陳信銘 |
關鍵字: | 口腔癌,光動力治療,氯化鈷,HIF1α,ABCG2,PpIX,薑黃素, Oral Cancer,ALA-PDT,Cobaltous Chloride,HIF1α,ABCG2,PpIX,Curcumin, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 口腔癌列屬106年臺灣癌症十大死亡率中第五名,對國人健康造成危害。現今治療方法以手術切除為主,但手術可能造成大範圍的顏面缺損和口腔功能缺失,也會使病患產生心理壓力。為了尋求更好的治療方法,近年來也投入研究新興的治療方式,包含光動力療法(Photodynamic Therapy;PDT)、冷凍治療等,以避免手術切除的負面影響。
5-氨基酮戊酸 (5-Aminolevulinic Acid;ALA)為光動力治療的一種常用藥,ALA進到細胞內部後,經過一連串代謝路徑形成protopophyrin(PpIX),成為PDT當中的光感物質,受光激發後產生單態氧及自由基殺死癌細胞。在臨床上ALA-PDT已被用於治療口腔癌和癌前病變,治療效果不錯且治療後病灶部位不會有明顯的傷口,對腫瘤周邊正常組織影響較小。但是治療所需時間長、需要照射次數多、藥物價格較高,先前研究也指出不同腫瘤的大小、位置、深度等因素都可能影響到ALA-PDT的療效。此外,腫瘤快速增長或PDT後造成的腫瘤血管異常都有可能導致缺氧(hypoxia),使PDT過程中所需的氧含量不足,也會影響口腔癌治療效果。 缺氧狀態下,會活化缺氧誘導因子1α(Hypoxia-inducible factor-1α;HIF1α),與Hypoxia-Response Element (HRE) 結合引起訊息傳遞,活化下游基因使癌細胞產生血管新生、侵襲性、細胞凋亡、轉移、化療抗性及放療抗性等特性。許多研究指出HIF1α也會促使ATP-binding cassette sub-family G member 2 (ABCG2)、Heme oxygenase 1 (HO1)表現量增加,可能減少細胞內的PpIX累積,造成治療不利。 本研究主要探討HIF1α是如何影響到口腔癌細胞PDT效果,並以輔助抑制劑或天然藥物降低其影響,希望透過合併療法提升治療效率,縮短治療時間和成本。 研究結果顯示,除了缺氧環境以外,光動力治療本身亦會引發細胞內HIF1α活化。使用氯化鈷(CoCl2)模擬缺氧細胞內HIF1α大量表現時,光動力治療效果和PpIX累積量都會下降,p-ERK、p-AKT、ABCG2、Nrf2、HO1等蛋白表現量則有顯著提升,使用HIF1α抑制劑YC-1(5-[1-(phenyl-methyl)-1H-indazol-3-yl] -2-furanmethanol)抑制HIF1α之後得到上述蛋白表現量下降的結果。推測HIF1α可能是透過EGFR下游p-ERK、p-AKT訊息傳遞路徑調控Nrf2,促使ABCG2和HO1轉錄,降低細胞內PpIX累積,影響PDT治療效率。以不同抑制劑和天然藥物處理後,發現除了YC-1以外薑黃素(curcumin)和MAPK/ERK抑制劑(PD98059)提高PpIX累積量和PDT效果最好。且Curcumin也能抑制因HIF1α過度表現而增加的ABCG2、Nrf2、HO1表現。 由本研究結果得知,處於缺氧狀態或是HIF1α表現量高的細胞於PDT前先以低毒性的抑制劑或天然藥物處理,可以明顯增加PpIX的生成,進而降低ALA-PDT後細胞存活率,提升治療效果。 Oral cancer was ranked fifth among the top 10 cancer deaths in Taiwan in 106 years, threats to people's lives. Surgical resection is the mainly treatment nowadays, but it may cause a wide range of facial defects and loss of oral function, which will also cause psychological stress to patients. To avoid the negative effects of traditional treatment and find a better way, researchers get into emerging treatment in recent years, including photodynamic therapy (PDT), cryotherapy, etc. 5-Aminolevulinic acid (ALA) is a commonly used drug for PDT. Through a series of metabolic pathways, it forms a protoporphyrin (PpIX)--the photosensitizer in ALA-PDT. PpIX is excited by light, produces singlet oxygen and free radicals to kill cancer cells. Clinically, ALA-PDT has been used to treat oral cancer and oral potential malignant disorder without obvious wound after treatment. The treatment effect is good and causes little effect on the normal tissue. However, PDT treatment takes times for repeating irradiation, and the price of ALA is high. Previous studies have also pointed out that different tumor size, location, depth and other factors may affect the efficacy of ALA-PDT. In addition, rapid tumor growth and PDT-mediated tumor vascular abnormalities may lead to hypoxia--which may cause insufficient oxygen content in the PDT process, and affect the therapeutic effect. Under hypoxic condition, a transcription factor called “hypoxia-inducible factor 1α (HIF1α)” is activated to protect cell. HIF1α binds to Hypoxia-Response Element (HRE) to cause signal transduction, and activates downstream genes causeing angiogenesis, invasiveness, apoptosis, metastasis, chemotherapy and radiotherapy resistance in cancer cells. Many studies have pointed out that HIF1α also promotes the increase of ATP-binding cassette sub-family G member 2 (ABCG2) and Heme oxygenase 1 (HO1) expression, which may reduce the accumulation of PpIX in cells, resulting in low PDT effectiveness. This study focused on how HIF1α affects PDT in oral cancer cells, and used inhibitors or natural drugs to reduces the effects. Aim to improve treatment efficiency and shorten treatment time and cost with combination therapy. Results of the study show that in addition to the hypoxic environment, photodynamic therapy itself also triggered the activation of HIF1α in cells. Cobalt chloride (CoCl2) was used to simulate HIF1α expression in hypoxic cells. We observed that PDT effect and the cumulative amount of PpIX decreased, and the expression levels of p-ERK, p-AKT, ABCG2, Nrf2, and HO1 were increased when we add CoCl2. There was a significant decrease in the expression of the above-mentioned proteins after inhibiting HIF1α by inhibitor YC-1. It is speculated that HIF1α may regulate Nrf2 through p-ERK and p-AKT signal transduction pathway, promote ABCG2 and HO1 transcription. These changes reduce intracellular PpIX accumulation, and affect the efficiency of PDT treatment. After combination treatment with different inhibitors and natural drugs, curcumin and PD98059 were found to increase PpIX accumulation and PDT best except YC-1. Also, curcumin reduced the expression of ABCG2, Nrf2, and HO1. From the results of this study, cells in hypoxia or high HIF1α expression were treated with low toxicity inhibitors before PDT, which significantly increased the production of PpIX, thereby reducing the cell survival rate after ALA-PDT and improving treatment effect. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21250 |
DOI: | 10.6342/NTU201903551 |
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顯示於系所單位: | 口腔生物科學研究所 |
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