仿葉綠素衍生物對癌細胞之光敏毒性研究

The Study of Phototoxic Effect of Chlorophyll Derivatives on Cancer Cells

陳瑩縈(Ying-Ying Chen)

指導教授 ： 婁世亮 ； 李文婷

中原大學/工學院/醫學工程研究所/碩士(2005年)

https://doi.org/10.6840/cycu200500772

摘要

光動力治療 (Photodynamic therapy) 是利用光感物質在受到光激發後，由激態回復到基態的過程中導致細胞內的氧分子活化成單態氧，進而使癌細胞死亡。本論文探討仿葉綠素衍生物 (Chlorophyll Derivatives) - chlorophyll a (A1)、 chlorophyllide a (A2)、 pheophytin a (A3)、pheophorbide a (A4)、 chlorophyll b (B1)、chlorophyllide b (B2)、 pheophytin b (B3)及pheophorbide b (B4) 對肝癌細胞 (Huh-7) 及口腔癌細胞 (SCC-4) 之光敏毒性。利用660 nm雷射二極體光照處理經仿葉綠素衍生物作用之癌細胞，以MTT assay分析細胞活性，找出最佳仿葉綠素衍生物。在固定光照總能量，找出分別對肝癌及口腔癌細胞有抑制效果之最佳藥物濃度。接著，探討在最佳藥物濃度下，不同光照總能量對肝癌及口腔癌細胞之光敏毒性。最後探討藥物引起癌細胞之可能死亡方式。
結果發現，肝癌細胞株方面， 0.25 μg/ml之仿葉綠素衍生物A3結合20.4 J/cm2光照總能量、2.5 μg/ml之A4結合5.1 J/cm2光照總能量、5 μg/ml之A4結合2.55 J/cm2光照總能量、0.25 μg/ml之B3結合20.4 J/cm2光照總能量及3 μg/ml B4結合2.55 J/cm2光照總能量，可明顯抑制肝癌細胞之存活，細胞活性降低為≦10 % 。而口腔癌細胞株方面， 0.25 μg/ml之仿葉綠素衍生物A3結合20.4 J/cm2、5 μg/ml之A4結合2.55 J/cm2、0.25 μg/ml之B3結合20.4 J/cm2及5 μg/ml之B4結合2.55 J/cm2，可明顯抑制口腔癌細胞之存活，細胞活性降低為≦10 %。
利用Tunnel assay分析最佳仿葉綠素衍生物A3、A4、B3及B4之細胞致死機制，發現A4及B4可造成口腔癌及肝癌細胞DNA斷裂，A3及B3則只有少許DNA斷裂，推測A4及B4之致死機制為細胞凋亡，而A3及B3則可能為細胞壞死與細胞凋亡共同造成的。
將最佳仿葉綠素衍生物A3、A4、B3及B4，結合10.2 J/cm2光照總能量處理正常細胞，發現其對不朽化肝細胞之細胞毒性與Huh-7相當，對於角質細胞則無明顯毒性。顯示此四種藥物對口腔癌細胞具選擇性，未來可應用於口腔癌之臨床治療。

關鍵字

光敏毒性 ； 仿葉綠素衍生物 ； 肝癌 ； 口腔癌

並列摘要

Photodynamic therapy (PDT) is an anticancer treatment that combines a photosensitizer, oxygen, and visible light. Phototoxicity occurs when photosensitizers capture light energy and transfer that energy to oxygen to produce singlet oxygen which is responsible for direct tumor-cell killing. The purpose of the study was to investigate the phototoxic effect of chlorophyll derivatives, including chlorophyll a (A1), chlorophyllide a (A2), pheophytin a (A3), pheophorbide a (A4), chlorophyll b (B1), chlorophyllide b (B2), pheophytin b (B3) and pheophorbide b (B4) on human hepatoma cell line (Huh-7) and human oral cancer cell line (SCC-4). The chlorophyll derivatives with phototoxicity for cancer cells were selected by MTT assay after the combination of the treatment with 660 nm diode laser irradiation and chlorophyll derivatives on the cells. The optimal drug concentration for inhibition on oral cancer and hepatoma cell survival was studied by using fixed total light energy. The effect of different total light energy on phototoxicity of oral cancer and hepatoma cells was investigated under the optimal concentration of drugs. Possible mechanism to cause cancer cell death was also explored after PDT.
The result show that the concentration of 0.25 μg/ml of chlorophyll derivative A3 combined with total applied energy of 20.4 J/cm2 , 2.5 μg/ml of A4 with total applied energy of 5.1 J/cm2, 5 μg/ml of A4 with total applied energy of 2.55 J/cm2, 0.25 μg/ml of B3 with total applied energy of 20.4 J/cm2 , and 3 μg/ml of B4 with total applied energy of 2.55 J/cm2 significantly inhibited Huh-7 cell survival, which was lower then 10 %. The concentration of 0.25 μg/ml of chlorophyll derivative A3 combined with total applied energy of 20.4 J/cm2, 5 μg/ml of A4 with total applied energy of 2.55 J/cm2, 0.25 μg/ml of B3 with total applied energy of 20.4 J/cm2, and 5 μg/ml of B4 with total applied energy of 2.55 J/cm2 significantly inhibited SCC-4 cell survival, which was lower than 10 %.
Possible mechanism for causing cancer cell death by chlorophyll derivative A3, A4, B3, and B4 was studied by Tunnel assay. A4 and B4 were found to induce DNA fragmentation in both SCC-4 and Huh-7 cells. Only little DNA fragmentation was found after A3 and B3 PDT. The results suggested A4 and B4 might induce SCC-4 and Huh-7 cell death through apoptosis, whereas A3 and B3 might cause cell death through both apoptosis and necrosis.
Chlorophyll derivative A3, A4, B3, and B4 were shown to induce phototoxic effect on immortalized hepatocytes (THLE-2) but not on keratinocytes after PDT at the total applied energy of 10.2 J/cm2. The data suggests that these four drugs have selectivity on oral cancer cells, which may be applied in clinical treatment for patients with oral cancers in the future.

並列關鍵字

phototoxicity ； chlorophyll derivative ； oral cancer ； hepatoma

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