葉綠素衍生物光動力治療誘發人類肝癌Huh-7細胞死亡之機制探討

The Mechanism of Photodynamic Therapy-Induced Death in Human Hepatoma Carcinoma Huh-7 Cells by Chlorophyll Derivatives

曹心薇(Hsin-Wei Tsao)

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/cycu200700907

摘要

光動力療法（Photodynamic therapy，PDT）為癌症患者提供另一創新之醫療概念；其原理是利用光感物質來標定腫瘤細胞，再配合特定波長之光源照射病變組織，進而選擇性的殺死癌細胞。具有不傷害周圍正常組織，以達到治療癌症的優勢。先前本實驗室已證實以菠菜所萃取出之葉綠素衍生物pheophytin a （A3），pheophorbide a （A4）、pheophytin b （B3） 及pheophorbide b（B4）可作為光感物質，導致人類肝癌細胞（HuH-7）死亡，本研究將持續探討PDT對HuH-7導致細胞死亡之作用機制。在研究結果發現四種葉綠素衍生物均會標定於HuH-7細胞之粒線體上，再給予紅光（660 nm）刺激執行PDT後產生大量之ROS，而這些ROS造成HuH-7細胞同時進行凋亡與壞死。針對凋亡之路徑探討結果發現，PDT造成HuH-7細胞的粒腺體膜電位喪失，使Cytochrome c從粒線體釋放至細胞質，Procaspase-9會被活化成Caspase-9，最後再活化Caspase-3。Caspase-3被活化後會將細胞核內的PARP切斷，使PARP失去修復DNA之功能，導致DNA斷裂，造成HuH-7細胞進行凋亡。

關鍵字

葉綠素衍生物 ； 細胞凋亡 ； 細胞壞死 ； 人類肝癌細胞 ； 光動力治療

並列摘要

Photodynamic therapy (PDT) is an innovative treatment for cancer therapy. The advantage of PDT is specific targeting to the tumor sites without damaging the normal tissue nearby, in which a photosensitizing drug selectively accumulates in tumors and is subsequently activated by visible light of an appropriate wavelength matched to the absorption spectrum of the photosensitizer. Previously we have demonstrated that PDT using chlorophyll derivatives including pheophytin a (A3), pheophytin b (B3), pheophorbide a (A4) and pheophorbide b (B4) has excellent phototoxicity against human hepatocellular carcinoma cells (HuH-7). The objective of the present study was to investigate the mechanism of photodynamic mediated cell death in HuH-7 cells caused by these photosensitizers. Our findings indicated that chlorophyll derivatives could target the mitochondria in HuH-7 cells. With combination of the irradiation of light emitting diodes (LEDs, 660 nm, 10 mW/cm2), the generation of reactive oxygen species and a collapse of mitochondrial membrane potential were observed. The loss of mitochondrial membrane potential led to the release of cytochrome c from the mitochondria to the cytosol, followed by activation of Caspase-9 and Caspase-3. The activation of Caspase-3 resulted in poly(ADP-ribose) polymerase (PARP) cleavage and led to DNA fragmentation in HuH-7 cells. Our results suggest that PDT with chlorophyll derivatives induce apoptosis of HuH-7 cells via the mitochondrial mediated pathway.

並列關鍵字

photodynamic therapy ； human hepatocellular carcinoma ； chlorophyll derivative ； necrosis ； apoptosis

參考文獻

[2] Ackroyd R.; C. Kelty; N. Brown; and M. Reed.. The history of photodetection and photodynamic therapy. Photochem. Photobiol., 2001, 74, 656-69.

[3] Dennis, EJ.; Dolmans, GJ.; Fukumura Dai and Rakesh K. Jain. Photodynamic therapy for cancer. Nature, 2003, 3, 380-387.

[4] Egger, Norman G.; Schoenecker Jr, James A.; Gourley, William K.; Motamedi, Massoud; and Anderson, Karl E. Photosensitization of experimental hepatocellular carcinoma with protoporphyrin synthesized from administered δ-aminolevulinic acid: studies with cultured cells and implanted tumors. J. Hepatol., 1997, 26, 913-920

[5] Reiners Jr., J.J.; Caruso, J.A.; Mathieu, P.; Chelladurai, B.; Yin, X.-M.; and Kessel, D. Release of cytochrome c and activation of pro-Caspase-9 following lysosomal photodamage involves bid cleavage. Cell Death Differ., 2002, 9, 934-944.

[6] Otake M; Nishiwaki M; Kobayashi Y; Baba S; Kohno E; Kawasaki T; Fujise Y; and Nakamura H. Selective accumulation of ALA-induced PpIX and photodynamic effect in chemically induced hepatocellular carcinoma. Br. J. Cancer, 2003, 89, 730-736.