葉綠素衍生物光動力治療誘發人類舌癌SCC-4細胞死亡之機制探討

The Mechanism of Photodynamic Therapy-induced Death in Human Tongue Squamous Carcinoma SCC-4 Cells by Chlorophyll Derivatives

施炎輝(Yen-Hui Shih)

指導教授 ： 李文婷

中原大學/工學院/醫學工程研究所/碩士(2007年)

https://doi.org/10.6840/CYCU.2007.00657

摘要

光動力療法（Photodynamic therapy，PDT）乃利用光感藥物可選擇性的累積於特定腫瘤組織，並吸收特定波長光能引發光化學反應之特性，進而產生活性氧分子對於腫瘤組織造成專一損害性，以達到癌症治療效果。先前研究顯示四種葉綠素衍生物 pheophytin a（A3）、pheophytin b（B3）、pheophorbide a（A4）、pheophorbide b（B4），能夠對於人類舌癌鳞狀細胞（SCC-4）產生光損害性，然而對於細胞死亡機制卻不清楚，因此本實驗針對四種葉綠素衍生物，個別進行探討其在 PDT 後誘發 SCC-4 死亡之機制。以 MTT 試驗探討不同藥物濃度經過紅光發光二極體（660 nm，10 mW/cm2）照射對細胞之致死率，結果發現光照能量 5.1 J/cm2，A3 及 B3 在濃度 250 ng/mL，A4 及 B4 在濃度 125 ng/mL 時，SCC-4 細胞致死率皆達 50 % 以上。在相同藥物濃度條件下，若光照能量增加為 10.2 J/cm2，SCC-4 細胞致死率則增加至 75 % 以上。藉由 Annexin V/PI 染色，觀察 PDT 後之早期反應，發現 A3 及 B3 大部分只有累積 Annexin V 染劑，具有細胞凋亡前期特性，而 A4 及 B4 則發現大量 Annexin V 與PI，具有細胞壞死現象。並且 PDT 後皆造成細胞形態損害與粒腺體膜電位喪失現象。此外，利用西方點墨法觀察，發現四種葉綠素衍生物在經過 PDT 後，皆造成細胞色素 c 由粒腺體釋放至細胞質中，並且發現 caspase-9 活化作用及 PARP 斷裂，其中 A3、A4 還造成caspase-8 及 caspase-3 活化作用。在 PDT 後 12 小時，皆發現具有染色體濃縮與 DNA 斷裂之現象，亦具有 LDH 釋放之壞死作用，而 PDT 後 24 小時 DNA 斷裂作用更顯著。本實驗結果了解 A3、B3、A4、B4-PDT，對於 SCC-4 細胞在早期皆具有凋亡訊號產生，並且具有壞死與凋亡共存作用，引發細胞死亡。此結果應能夠進一步應用於未來動物實驗之參數，並作為臨床治療條件之重要參考依據。

關鍵字

細胞壞死 ； 光動力療法 ； 細胞凋亡 ； 光敏藥物 ； 葉綠素衍生物

並列摘要

Photodynamic therapy (PDT) is an anticancer treatment in which direct tumor-cell killing results from selective accumulation of photosensitizers in the tumor sites and phototoxicity occurs when the photosensitizers are activated by specific wavelengths and the energy of photons are transferred to oxygen nearby to produce reactive oxygen species. Previously, we have demonstrated that chlorophyll derivatives such as pheophytin a (A3), pheophytin b (B3), pheophorbide a (A4), and pheophorbide b (B4) could cause phototoxicity in human tongue squamous carcinoma cells (SCC-4). The mechanism of cell death caused by chlorophyll derivatives- PDT remained unclear. The objective of the study was to explore the mechanism of death of SCC-4 cells mediated by chlorophyll derivatives combined with the irradiation of 660 nm light emitting diodes (LEDs). The viability of cells treated with different concentrations of drugs and various radiant exposures of LEDs was measured by MTT assay. Significant inhibition on the survival of SCC-4 cells (< 50 %) was observed when an irradiation dose of 5.1 J/cm2 combined with the concentration of 0.25 μg/ml of A3, 0.125 μg/ml of A4, 0.25 μg/ml of B3, and 0.125 μg/ml of B4 were applied. The inhibition on cell survival increased to higher than 75% when the radiant exposure of 10.2 J/cm2 was applied under the same drug concentration. The staining of Annexin V/PI revealed that A3 and B3 seemed to induce apoptic response in most of SCC-4 cells by being stained with Annexin V only, whereas A4 and B4 seemed to induce necrotic response by being stained with both Annexin V and PI. The damage to cellular morphology and the loss of mitochondrial membrane potential were found after PDT. According to the results of western blot, the PDT treatment initiated the release of cytochrome c from the mitochondria to the cytosol, thereby activated caspase-9 activation, followed by PARP cleavage. Activation of caspase-8 and caspase-3 was also found in A3 and A4-mediated PDT. DNA fragmentation and LDH release were observed in 12 hours after PDT. The findings suggest that both apoptosis and necrosis are induced in the early stage with A3, B3, A4 and B4 mediated PDT in SCC-4 cells, and apoptosis mediated cell death then occurs in the late stage. The results may be applied in the animal studies and clinical treatments in the future.

並列關鍵字

photodynamic therapy ； apoptosis ； necrosis ； photosensitizer ； chlorophyll derivative

參考文獻

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