A Photodynamic Pathway to Apoptosis Induced by Hypericin in Human Myeloma ARH-77 Cell Line: Possible Relevance to Photodynamic Therapy.

Hypericin, a photosensitizing plant pigment from Hypericum perforatum, is a protein tyrosine kinase inhibitor that has been exploited in models for anti-tumor and anti-viral activity. In this study, we investigated the effects of hypericin on the human myeloma cell line ARH-77 as a model to determine whether hypericin-induced cell death is available. The cells were incubated with hypericin at concentrations ranging from 0.001 to 10 microg/ml in RPMI at 37°C in 5% CO2 atmosphere for 4 h. Then, the cells were irradiated at 532 nm (fluence=24 J/cm2) using a dye laser pumped by an argon laser (Orion). After 72 h exposure, the IC50 of hypericin was 0.005 microg/ml as determined by the MTT assay. Hypericin exerted phototoxic effect on ARH-77 cells, while it did not produce toxic effect in the absence of irradiation. After 72 h exposure to 0.005 microg/ml photoactive hypericin, apoptosis was assessed by morphological changes, DNA fragmentation and flow cytometric analysis using Annexin V and propidium iodide staining. Most of the cells were accumulated in the late stage of apoptosis and these cells were brightly stained and fragmented nuclei and cytoplasmic blebbing were observed. A decrease in the number of apoptotic cells was detected when protein kinase C was inhibited by addition of staurosporine to photoactive hypericin induced ARH-77 cells. From these results, we demonstrated that exposing myeloma cell line ARH-77 to photoactive hypericin inhibits cell growth in a dose dependent manner, induces apoptotic cell death by protein kinase C activation, and provides a rationale for potential applications in vivo.