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

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.