- 學位論文
脫鎂葉綠素光動力治療誘發抗藥性乳癌細胞MCF-77/ADR死亡機制
The Mechanism of Photodynamic Therapy-induced Death in Drug-resistant Breast Cancer MCF-7/ADR Cells by Pheophytin
摘要
光動力療法 (Photodynamic therapy，PDT) 已廣泛的被用來治療實體腫瘤及非癌症的疾病，其原理是使對細胞與組織無毒性之光感藥物累積在病變組織中，再利用適當波長之光能活化光感藥物，使其產生具有細胞毒性之活性氧分子(ROS)，進而達到選擇性殺死細胞的功效。我們先前證實利用脫鎂葉綠素a、脫鎂葉綠素b、脫鎂葉綠酸 a、脫鎂葉綠酸 b 作為光感藥物，搭配波長660 nm紅光激發，可對人類肝癌細胞 (HuH-7)、口腔癌細胞 (SCC-4) 及皮膚癌細胞 (A431) 產生光敏毒性。本研究持續探討脫鎂葉綠素對人類抗藥性乳癌細胞 (MCF-7/ADR) 之光動力效應及造成細胞死亡之機制。首先，MTT分析結果發現細胞存活率隨藥物濃度提升、光照能量加強以及共培養時間拉長而下降，脫鎂葉綠素a與脫鎂葉綠素 b在光照能量5.1 J/cm2，共培養時間6小時之半致死劑量分別為128.6及163.9 ng/mL。脫鎂葉綠素b-PDT造成細胞內ROS增加較脫鎂葉綠素a為高；脫鎂葉綠素a-PDT造成ATP下降較脫鎂葉綠素b顯著。相對的，脫鎂葉綠素b-PDT造成LDH的釋放較脫鎂葉綠素a為高。Annexin V/propidium iodide染色結果發現MCF-7/ADR細胞經過PDT後主要以凋亡型態死亡。光感藥物主要累積於粒線體，PDT後造成粒線體損傷，進而使caspase-9及caspase-7活化，並造成PARP蛋白斷裂；在PDT後12小時，可觀察到DNA片段化及細胞週期變化中亞二倍體比例增加。此外，脫鎂葉綠素PDT也會造成抗藥性之p-gp蛋白表現減低；並抑制HSP 70之保護機制。本研究結果推論，脫鎂葉綠素對MCF-7/ADR細胞具有光動力效應，並可透過內源性路徑導致細胞凋亡。
並列摘要
Photodynamic therapy (PDT) is widely used as a treatment option for solid tumor and some non-cancerous diseases. Photosensitizer with relatively low toxicity accumulated in the pathological site can be activated by light at appropriate wavelength. Activated photosensitizer leads to the production of reactive oxygen species (ROS) and eventually results in cancer cell death. Previously, we have demonstrated that photosensitizer such as pheophytin a, pheophytin b, pheophorbide a, and pheophorbide b excited with red light (660 nm) exhibited photodynamic toxicity on human hepatoma carcinoma cells (HuH-7), human oral cancer cells (SCC-4) and human epidermoid carcinoma cells (A431). Here, the photodynamic effect against multidrug resistant human breast carcinoma cells (MCF-7/ADR) by pheophytins and related cell death mechanisms were explored. The results of MTT assay revealed that cell viability decreased with the increase in the pheophytin concentration, light dosage, and incubation time. The IC50 values for pheophytin a and pheophytin b with 5.1 J/cm2 irradiation and 6 hour incubation were 128.6 and 163.9 ng/mL, respectively. Pheophytin a-PDT caused less ROS production than pheophytin b-PDT. The intracellular ATP level after pheophytin a-PDT was lower than that after pheophytin b-PDT. On the other hand, pheophytin a-PDT led to higher lactate dehydrogenase (LDH) release compared to pheophytin b-PDT. Annexin V/propidium iodide double stain has further confirmed that apoptosis has occurred in MCF-7/ADR cells after PDT. Colocalization of pheophytins and rhodamine-123 demonstrated that photosensitizer was mainly accumulated within mitochondria. The damage to the mitochondria led to the activation of caspase-9 and caspase-7, followed by PARP cleavage. DNA fragmentation and the increase of the sub-G1 cell population in cell cycle were observed after pheophytin-PDT. In addition, pheophytin-PDT showed inhibitory effect on multi-drug resistance by down-regulating p-glycoprotein expression and cellular protection by decreasing HSP70 expression in MCF-7/ADR cells. In conclusion, this study demonstrated that both pheophytin a and pheophytin b possess photodynamic activity against MCF-7/ADR cells via intrinsic apoptotic pathway.