The Anti-tumor and Anti-angiogenic Effects of Photodynamic Therapy with Pheophorbide a on Breast Cancer in vitro and in vivo

Breast cancer is conventionally treated by surgery and radiotherapy, supported by adjuvant chemotherapy or hormonotherapy. However, adverse side effects and drug resistance may be resulted. In this study, the anti-proliferative effect and underlying mechanisms of Pheophorbide a (Pa), a photosensitizer isolated from Scutellaria barbata, on human breast tumor cells were investigated. The results showed that the IC50 value of Pa on human breast tumor MCF-7 cells was 0.5 µM when incubated with the cells for 24 hours after photodynamic therapy with Pa (Pa-PDT). Mechanistic studies demonstrated that Pa was localized in the mitochondria and reactive oxygen species were found to be released after Pa-PDT on MCF-7 cells. As evidenced by the results of cell death detection ELISA and chromatin condensation detected by Hoechst 33342 staining, apoptosis was the major mechanism for the tumor cell death. Mitochondrial membrane depolarization and cytochrome c release revealed the role of mitochondria in the apoptotic mechanism. Increased expression of tumor suppressor protein p53, cleavage of caspase-9 and Poly (ADP-ribose) polymerase indicated the involvement of caspase-dependent pathway. On the other hand, apoptosis-inducing factor release demonstrated the mediation of caspase-independent mechanism.;Since angiogenesis plays an important role in tumor growth and metastasis, the anti-angiogenic mechanism and potential application of Pa-PDT was also studied. PDT with 0.1 to 0.5 µM of Pa resulted in dose-dependent inhibition of growth, migration and tube formation in human microvascular endothelial cells, namely HMEC-1 cells. Matrix metalloproteinase-2 released from HMEC-1 cells into media was monitored by gelatin zymography, and was found to be down-regulated. Therefore, Pa-PDT was found to inhibit several key steps of the angiogenic process.;The preference of vascular versus cellular targeting for PDT is highly dependent upon the relative distribution of photosensitizer (PS) in these compartments, which can be manipulated by controlling the drug- light intervals. We have established an MCF-7 breast cancer xenograft model for comparing the therapeutic outcomes of these PDT strategies. The fractionated PDT with two equal half doses (1.25 mg/kg) induced the most effective MCF-7 tumor growth delay, followed by the same total dose given as a single dose (2.5 mg/kg) at 15 min (short DLI) and 3 h (long DLI) before light administration. Although vascular endothelial growth factor (VEGF) was found to express in vascular endothelial cells after short DLI and fractionated PDT, there were extensive vascular damages as detected by CD31 staining at 24 h after treatment. Moreover, it should be noted that neovessel formation was enhanced after long DLI PDT, which suggests the tendency for tumor regrowth. Detection of apoptosis with the TUNEL assay showed that the fractionated PDT caused the highest extent of cell apoptosis, followed by the short DLI and long DLI, at day 14 after Pa-PDT. The fractionated PS dosing Pa-PDT could offer a new insight for optimizing the PDT therapy in clinical applications.;To summarize, Pa-PDT led to apoptosis of MCF-7 breast cancer cells via both caspase-dependent and -independent pathways. Apart from targeting tumor cells, Pa-PDT also inhibited angiogenesis which is a process related to cancer metastasis. We compared the cellular-targeting and vascular-targeting strategies of Pa-PDT and found that the combination of them could lead to the greatest extent of tumor regression. These results provide a basis for understanding and developing Pa-PDT as a cure for breast cancer.