- 學位論文
脫鎂葉綠素光動力治療誘發人類乳腺癌 MCF-7 細胞死亡之機制探討
The Mechanism of Photodynamic Therapy-induced Death in Human Mammary Gland Adenocarcinoma MCF-7 Cells by Pheophytin
摘要
光動力療法 (Photodynamic therapy, PDT) 為使光感藥物選擇性的累積於腫瘤或病變組織，施以特定波長之光能激發光化學反應，進而產生具更細胞毒性的活性氧分子 (ROS)，對腫瘤細胞或病變組織造成傷害，而達到病灶改善或治療的目的。本研究首先以 MTT 試驗探討不同濃度之光感藥物-脫鎂葉綠素 a 與脫鎂葉綠素 b 以及不同培養時間，結合紅光發光二極體 (660 nm，10 mW/cm2) 進行光照後，對人類乳腺癌 MCF-7 細胞之 PDT 效用，結果發現 200 ng/mL 脫鎂葉綠素 a 或 b 作用 2 小時後，施以 2.55 J/cm2 光照，能使 MCF-7 細胞存活率下降至 50 % 左右；將光照能量提升至 5.10 J/cm2，存活率則下降至 30 % 以下；隨著光感藥物作用時間增長到 6 小時，細胞致死率可達 90 % 左右。進一步以乳酸脫氫酶 (LDH) 釋放分析MCF-7 細胞細胞膜完整性是否被破壞，結果發現 500 ng/mL 光感藥物作用 2 小時，PDT 後會造成LDH 釋放率顯著增加；隨著培養時間增長到 6 小時，LDH 釋放率提高到 30 % 左右，而脫鎂葉綠素 a 造成細胞壞死現象又較脫鎂葉綠素 b 為顯著。接著進行 ATP 含量分析 PDT 是否會消耗細胞內 ATP，結果發現隨著光感藥物培養時間增長至 6 小時，細胞內 ATP 含量隨之降低，其中又以脫鎂葉綠素 a 所消耗之 ATP 含量較脫鎂葉綠素 b 顯著。另外，量測細胞內之 ROS 含量分析 PDT 後是否產生氧化性損傷，結果發現 MCF-7 細胞經過脫鎂葉綠素 PDT 後，細胞內 ROS 水平顯著的提升。經Annexin V/Propidium iodide 螢光染色後，發現脫鎂葉綠素 PDT 造成MCF-7 細胞產生凋亡反應，隨著共培養時間拉長及光照劑量增加，壞死反應亦趨顯著。西方點墨法偵測凋亡相關蛋白的表現，結果發現 PDT 後 Bax/Bcl-2 比例增加，procaspase-9、procaspase-7 及 PARP 因被活化而表現量下降；TUNEL 分析與DNA電泳分析發現 PDT 後細胞核內的 DNA片段化現象明顯增加，而脫鎂葉綠素 a 造成DNA片段化現象又較脫鎂葉綠素 b 為顯著。綜上所述，脫鎂葉綠素 a 及 b 能對人類乳腺癌 MCF-7 細胞引發光毒性，且在 PDT 後會透過內源性細胞凋亡路徑引發細胞死亡。本研究的發現可作為 PDT 應用於動物實驗及臨床研究之重要參考依據。
並列摘要
Photodynamic therapy (PDT) is an alternative anticancer treatment in which direct tumor-cell killing results from selective accumulation of photosensitizers in the tumor sites and phototoxicity occurs when light-activated photosensitizers transfer the energy to oxygen nearby to produce singlet oxygen. In this study, we first demonstrated the phototoxicity of pheophytin a and pheophytin b on human mammary gland adenocarcinoma MCF-7 cells using MTT assay by combining different concentrations of photosensitizer and incubation time as well as light doses (660 nm, 10 mW/cm2) generated from light emitting diodes (LEDs). When MCF-7 cells were incubated with pheophytin a or b at 200 ng/mL for 2 hr and treated with 2.55 J/cm2 irradiation, cell viability decreased to 50%. The viability of MCF-7 cells lowered to 30% with the irradiation of 5.10 J/cm2. Cell viability declined to around 10% as the incubation time increased to 6 hr under the same treatment condition. We further analyzed PDT resulted damage to cell membrane by lactate dehydrogenase (LDH) release assay. A significant LDH release was found when MCF-7 cells were incubated with the photosensitizer at 500 ng/mL for 2 hr. As the incubation time lengthened to 6 hr, LDH release increased to 30%. The cell death resulted from pheophytin a-PDT was higher than that from pheophytin b-PDT. Intracellular level of ATP was next analyzed to determine whether the intracellular level of ATP was consumed by PDT. The results showed that the intracellular ATP level decreased as the incubation time with photosensitizer increased to 6 hr. The depletion of ATP resulted from pheophytin a-PDT was higher than that from pheophytin b-PDT. Furthermore, the ROS production was measured to explore if the photooxidative effect was produced after PDT. ROS level was significantly increased when MCF-7 cells were treated with pheophytin-PDT. The staining of Annexin V/Propidium iodide revealed that pheophytin a- or b-PDT induced apoptic responses in MCF-7 cells. As the light dose and pre-incubation time increased, the percentage of necrotic cells increased. The apoptotic related proteins were further detected by western blotting. The Bax/Bcl-2 ratio increased after pheophytin a- or b-PDT. Caspase-9 and caspase-7 activation were found, followed by PARP cleavage. DNA fragmentation was also observed afterwards after PDT by TUNEL assay and DNA electrophoresis. DNA fragmentation resulted from pheophytin a-PDT was higher than that from pheophytin b-PDT. In conclusion, III both pheophytin a and pheophytin b possess phototoxicity against human mammary gland adenocarcinoma MCF-7 cells. Cell death was caused by PDT via intrinsic apoptic pathway. The results may be applied in animal studies and clinical therapies in the future.