A study on the photodynamic properties of chlorophyll derivatives using human hepatocellular carcinoma cells

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. The objective of this study was to investigate the effects of PDT using chlorophyll derivatives such as pheophytin a (phe a), pheophytin b (phe b), pheophorbide a (pho a) and pheophorbide b (pho b) as the photosensitizers, and the 660 nm light-emitting diodes (LEDs) irradiation on human hepatocellular carcinoma cells (HuH-7). The drug concentration-dependent inhibition of HuH-7 cell viability was studied under LEDs irradiation (10 mW cm(-2)) at radiant exposure of 5.1 and 10.2 J cm(-2) by MTT assay. Significant inhibition of the survival of HuH-7 cells (<10%) was observed when an irradiation dose of 10.2 J cm(-2) combined with the concentration of 0.5 microg ml(-1) of phe a, 0.125 microg ml(-1) of pho a, 0.25 microg ml(-1) of phe b, and 0.125 microg ml(-1) of pho b were applied. The results from Annexin V-propidium iodide staining revealed that phe a, phe b, pho a and pho b could induce cell death in HuH-7 cells predominantly via a necrotic process. The results from immunoblot analyses exhibited that chlorophyll derivative-mediated PDT initiated cytochrome c release, caspase-9 and caspase-3 activation, followed by poly ADP-ribose polymerase (PARP) cleavage. Thus, apoptosis also occurred in HuH-7 cells after PDT treatment, and the execution of the apoptotic process may be initiated from the loss of mitochondrial function. Our findings demonstrate that both apoptosis and necrosis can be induced in HuH-7 cells after PDT using phe a, phe b, pho a and pho b and LEDs.