The methylene blue (MB) used for PDT was acquired from Aldrich (Milwaukee, WI, USA), and RPMI-1640 medium was purchased from Hyclone (Logan, UT, USA). Antibodies against PARP-1, Bcl-2, Bcl-xL, Mcl-1, and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-phospho-JNK, anti-phospho-p38, and anti-phospho-ERK were purchased from Cell Signaling (Beverly, MA, USA). Benzyl carbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk) was purchased from Biomol (Plymouth Meeting, PA, USA), 2′7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) was purchased from Molecular Probes (Eugene, OR, USA), and N-acetylcysteine (NAC) and all other chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Human lung adenocarcinoma cells (A549 cells) were obtained from the American Type Culture Collection (Rockville, MD, USA). The A549 cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 U/ml) at 37ºC in a humidified incubator with 5% CO₂ and 95% air. When the cells were subconfluent, the medium was replaced with fresh medium and 0.1–2.0 μg/ml of MB was added to the culture medium for 1 h. The cells were then irradiated with 650 nm light produced by a dye laser (Red Diode Laser) at 30–120 Joules (J).

For the morphological evaluation of cell death, approximately 5×10⁵ A549 cells were plated into 60-mm cell culture dishes overnight. For the trypan blue exclusion assay, trypsinized cells were pelleted and resuspended in 0.2 ml of medium, 0.5 ml of 0.4% trypan blue solution, and 0.3 ml of phosphate-buffered saline solution (PBS). The samples were mixed thoroughly, incubated at room temperature for 15 min, and examined under a light microscope. At least 300 cells were counted for each survival determination.

For western blot analyses, A549 cells were lysed with 1X Laemmli lysis buffer (2.4 M glycerol, 0.14 M Tris, pH 6.8, 0.21 M SDS, 0.3 mM bromophenol blue) and boiled for 10 min. Protein content was measured with the BCA Protein Assay Reagent (Pierce, Rockford, IL, USA). The samples were diluted with 1X Laemmli lysis buffer containing 1.28 M β-mercaptoethanol, and equal amounts of protein were loaded on 8–12% SDS-polyacrylamide gels. Proteins were separated by SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% nonfat dry milk in PBS-Tween-20 (0.1%, v/v) for 1 h. The membrane was incubated with primary antibody (diluted according to the manufacturer's instructions) at room temperature for 1.5 h. Horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG was used as the secondary antibody. Immunoreactive protein was visualized by the chemiluminescence protocol (ECL, Amersham, Arlington Heights, IL, USA). To ensure equal protein loading, each nitrocellulose membrane was stripped and reprobed with an anti-actin antibody after the experiment was completed.

To assess for DNA fragmentation after MB and/or PDT for 24 h, ~1×10⁶ treated A549 cells were lysed for 30 min on ice in buffer containing 10 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA and 0.5% Triton X-100. Lysates were vortexed and cleared by centrifugation at 12,000 × g for 30 min. Fragmented DNA in the supernatant was extracted with an equal volume of a mixture of neutral phenol:chloroform:isoamyl alcohol (25:24:1) and analyzed electrophoretically on 1.5% agarose gels containing 0.1 g/ml EtBr. The cells were fixed on slide glass through the application of 4% paraformaldehyde for 30 min at room temperature. After washing with PBS, 300 nM 4′, 6′-diamidino-2-phenylindole (DAPI) was added to the fixed cells for 10 min, after which they were examined by fluorescence microscopy. Apoptotic cells were identified by condensation and fragmentation of nuclei. All DAPI staining experiments were performed in duplicate.

The generation of ROS was measured by staining with 2′,7′-dichlorofluorescein diacetate (DCF-DA). Briefly, A549 cells were seeded in six-well plates (1×10⁵ cells per well), allowed to attach overnight and treated with MB and/or PDT. The cells were stained with 20 μM DCF-DA for 30 min at 37ºC and fluorescence was detected by a fluorescence microscope.

To monitor MMP, JC-1 dye was used as previously described (14). In cells with undamaged mitochondria, the aggregated dye appears as red fluorescence, whereas in cells with altered MMP that are undergoing apoptosis, the dye remains as monomers in the cytoplasm and emits diffuse green fluorescence. The red/green fluorescence ratio is dependent on MMP. After A549 cells were treated with MB and PDT, they were stained with a JC-1 MMP detection kit for 10 min and analyzed by flow cytometry. The fluorescence intensity was measured with the FACScan flow cytometer (Beckman Coulter, Inc., Hialeah, FL, USA).

All experiments were repeated three or more times. The results are represented as means ± standard deviations (SDs). The difference between two mean values was analyzed using Student's t-test and was considered statistically significant at p<0.05.

To evaluate the effects of MB-PDT on cell viability, A549 cells were treated with various concentrations of MB and/or PDT (30 and 60 J) for 24 h and a trypan blue assay was performed. As shown in Fig. 1, while MB alone had little effect on cell viability, MB treatment followed by PDT significantly decreased cell viability as determined by trypan blue exclusion (Fig. 1A) and DAPI staining (Fig. 1B). These results suggest that PDT enhances the toxicity of MB in A549 cells.

To address the significance of caspase activation in MB-PDT-induced apoptosis, we examined caspase activity after MB-PDT. Treatment of A549 cells with various concentrations of MB and PDT resulted in a dramatic increase in the cleavage of PARP, a caspase substrate, and in the cleavage of procaspase-3 (Fig. 2A) in a time-dependent manner (Fig. 2B). In order to confirm that the activation of caspases is a key step in MB-PDT-induced apoptosis, the A549 cells were pretreated with z-VAD-fmk (25 μM), a cell-permeable caspase inhibitor, followed by MB-PDT for 24 h. As shown in Fig. 2C, MB-PDT-induced apoptosis was prevented by pretreatment with z-VAD-fmk, as indicated by the reduced PARP cleavage. We also found that z-VAD-fmk prevented the dose-dependent increase in the accumulation of apoptotic DNA after treatment with MB-PDT (Fig. 2D). These results suggest that MB-PDT-induced cell death is associated with caspase activation.

We further examined whether MB-PDT-induced apoptosis is associated with the modulation of apoptosis regulatory proteins. As shown in Fig. 3A, exposure of A549 cells to MB and PDT led to a slight decrease in Bcl-xL. However, levels of Bcl-2 and Mcl-1 in A549 cells were markedly reduced by MB and PDT in a dose-dependent manner. We assessed MMP in order to determine the role of mitochondrial damage in MB-PDT-induced apoptosis of A549 cells. As shown in Fig. 3B, we found that exposure of A549 cells to MB and PDT significantly reduced MMP in a dose-dependent manner. The proportion of cells with a loss of MMP substantially increased with MB treatment (1 and 2 μg/ml) plus PDT (60 J).

In order to investigate whether a MAPK signaling pathway was involved in cell death in A549 cells treated with MB-PDT, we examined the phosphorylation of p38, JNK and ERK in A549 cells after MB and/or PDT. As shown in Fig. 4A, p38 activity was increased slightly at 2 h after MB application (2 μg/ml) and the increased p38 activity was sustained after 8 h of treatment with MB alone, whereas a much stronger and prolonged activation of p38 was observed after combined treatment with MB and PDT. To further confirm the involvement of the p38 signaling pathway in MB-PDT-induced apoptosis, we applied specific inhibitors of JNK (SP600125), p38 (SB203589), and ERK (PD98059). Pretreatment of A549 cells with the p38 inhibitor SB203589 before MB-PDT increased cell viability (Fig. 4B) and reduced proteolytic cleavage of PARP (Fig. 4C). Taken together, these results suggest that the prolonged activation of the MAPK p38 pathway plays an important role in MB-induced photosensitization.

ROS are byproducts of the normal metabolism of oxygen, and they are generated by various environmental stresses such as drugs, UV light and heat shock. ROS play an important role in apoptosis under both physiologic and pathologic conditions (15,16). Therefore, we examined whether ROS generation was involved in MB-induced photosensitization of A549 cells by measuring the intracellular hydrogen peroxide level in A549 cells after MB-PDT. As shown in Fig. 5A, the combination of MB and PDT markedly increased intracellular hydrogen peroxide levels in A549 cells. We next investigated whether ROS generation was directly associated with MB-PDT-induced apoptosis. Pretreatment with NAC, a well-known antioxidant, markedly increased cell viability after MB-PDT (Fig. 5B). Moreover, apoptosis induced by MB-PDT was markedly attenuated by pretreatment with NAC (Fig. 5C). Taken together, these results indicate that MB-induced photosensitization and MB-PDT-induced apoptosis are mediated by the generation of ROS.