A human epithelial carcinoma cell line (A431) and malignant melanoma cell line (G361) were maintained in DMEM medium. Both cell lines were cultured in medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin and 100 μg/ml of streptomycin and maintained at 37°C in a humidified incubator with 5% CO₂. Pa was prepared as a 10 mM stock in dimethyl sulfoxide (DMSO; BDH Merck, Darmstadt, Germany). Further dilutions were made in serum-free DMEM.

The PDT irradiation light source was a light-emitting diode (LED; 613–645 nm; Philips Luxeon Lumileds, San Jose, CA, USA). The cells (1×10⁵/well) were pre-incubated with Pa in complete growth medium in the dark for 2 h. For the following experiments, the cells were irradiated at 1.25 J/cm². After 24 h of incubation, the cells were rinsed with phosphate-buffered saline (PBS). For the control group, the cells were incubated in the same medium without Pa or light.

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay was used to assess cell proliferation after Pa-PDT treatment. After the medium was removed, the cells were incubated with an MTT solution (5 mg/ml in PBS) for 3 h, and the absorbance was measured using an auto ELISA plate reader at 570 nm. In addition, we performed cell proliferation assays using a cell proliferation ELISA kit (Roche Applied Science, Indianapolis, IN, USA).

The cells were treated with Pa-PDT for 24 h. The cells were then washed with PBS and harvested in lysis buffer. Samples containing equal amounts of protein were loaded onto each lane of an SDS-polyacrylamide gel for electrophoresis and subsequently transferred onto a polyvinylidene difluoride membrane. The membranes were blocked and then incubated with antibodies. Antibodies against Beclin-1, Bcl-2, Atg5, LC3B and p-mTOR were purchased from Cell Signaling Technology (Beverly, MA, USA); p-Akt, caspase-7, PARP and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Autophagy is characterized by the formation of acidic vesicular organelles (AVOs) (17). A431 and G361 cells were seeded in 6-cm² plates and incubated for 24 h. After treatment with Pa-PDT for 24 h, acridine orange (1 μg/ml) was added to the living cells for 30 min, and the cells were removed from the plate with trypsin-EDTA and collected in phenol red-free growth medium. Green (510–530 nm) and red (650 nm) fluorescence emission from 1×10⁴ cells illuminated with blue (488 nm) excitation light was measured with a FACSCalibur using CellQuest software (Becton-Dickinson).

To observe autophagy formation, skin cells were grown on glass coverslips for 24 h in a humidified incubator in 5% CO₂ and at 37°C. After treatment with Pa-PDT for 24 h, the cells were treated with 0.05 mM monodansylcadaverine (MDC; Sigma-Aldrich Chemical) at 37°C in 5% CO₂ for 10 min. The cells were then fixed with 4% paraformaldehyde in PBS for 10 min. Following incubation, the cells were washed three times with PBS and immediately analyzed under a fluorescence microscope (IX-71; Olympus, Tokyo, Japan). Fluorescence of MDC was measured at the excitation wavelength of 380 nm with an emission filter at 530 nm.

The cells were harvested and fixed with 70% ethanol for 1 h at 4°C for cell cycle analysis. After washing with cold PBS, the cells were incubated with DNase-free RNase and propidium iodide (PI) at 37°C for 30 min. The specific binding of Annexin V-FITC/PI was performed by incubating the cells for 15 min at room temperature in a binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂, pH 7.4) containing saturating concentrations of Annexin V-FITC and PI. Following incubation, the cells were pelleted and analyzed in a FACScan analyzer (Beckman Coulter Inc., Fullerton, CA, USA).

Caspase-3 activity was assessed using a caspase-3 colorimetric assay kit (Clontech, Palo Alto, CA, USA) following the manufacturer’s instructions. The cells seeded in 6-well plates (2×10⁵ cells/well) were treated with or without Pa-PDT for 24 h as previously described. The cells were collected and resuspended in lysis buffer containing 50 mM HEPES, pH 7.4, 0.1% CHAPS, 1 mM DTT, 0.1 mM EDTA and 0.1% Triton X-100. Cell lysates were centrifuged at 12,000 × g for 10 min at 4°C. The supernatants were incubated with the reaction buffer containing 2 mM Ac-DEVD-pNA for 1 h at 37°C. Caspase activity was determined by measuring the absorbance at 405 nm.

The CAM assay was used to examine the inhibition of tumor growth in vivo. The CAM assay was performed as previously described (18). Briefly, fertilized chicken eggs were transferred to an egg incubator maintained at 37°C and 50% humidity and allowed to grow for 10 days. The fertilized chick eggs were sterilized, and a 1-cm² window was cut, using the false air sac technique, on one side of the egg to expose the CAM. Skin cancer cells (2×10⁶) were placed on the exposed CAM, and the windows were sealed with transparent tape. The eggs were incubated in a humidified incubator at 37°C for three days and pre-treated with normal saline or Pa (0.1 μM) prior to light exposure (1.25 J/cm²). At the next day, the excised tumors were fixed in 10% formalin and paraffin-embedded and cell staining was performed with hematoxylin and eosin (H&E).

The statistical analyses were performed with data obtained from three independent experiments. The data are represented as the mean ± SEM. A P-value <0.05 was considered to indicate a statistically significant result.

To test the effect of Pa-PDT, A431 and G361 cells were pre-treated with different concentrations of Pa (0.05 or 0.1 μM) for 2 h in the dark, followed by photoactivation with 1.25 J/cm² of LED. Twenty-four hours after exposure, the cell proliferation was determined using an MTT assay. We found that these cell lines exhibited no significant decrease in cell proliferation due to Pa or light alone (data not shown). However, the cell proliferation in both cell lines was severely decreased by Pa-PDT treatment. In A431 cells, Pa doses of 0.05 and 0.1 μM with light resulted in cell growth inhibition rates of 48.8 and 77.7% at 24 h, respectively (Fig. 1A). Similarly, Pa-PDT also induced a significant cytotoxicity in G361 melanoma cells in a Pa-dose-dependent manner (Fig. 1B). After 48 h PDT treatment, the cell growth slightly recovered in both cells compared with growth at 24 h (Fig. 1). These results demonstrate that Pa-PDT exerts an antiproliferative effect on human skin cancer cells.

To further elucidate the underlying mechanisms of Pa-PDT-induced cell growth inhibition, we assessed the levels of Beclin 1, Bcl-2, ATG5 and LC3B-I/II, which play a crucial role in autophagy. Western blotting revealed that Pa-PDT induced an increase in the expressions of Beclin 1 in a time-dependent manner, whereas the expression of Bcl-2 (Beclin 1 regulatory proteins) was reduced in A431 cells (Fig. 2A). The induction of ATG5 and LC3-II expression began to be clearly observed at 12 h after Pa-PDT treatment (Fig. 2B). However, the G361 cell line exhibited no significant expression of these proteins after Pa-PDT treatment. This finding suggested the possibility that the mechanism of Pa-PDT-mediated cell growth inhibition in A431 cells, but not in G361 cells, was related to the Beclin 1-dependent autophagy.

The mTOR/AKT pathway is a major signaling pathway that regulates autophagy (19). To confirm whether Pa-PDT regulated autophagy in A431 cells, we assessed the influence of Pa-PDT on the activation of mTOR and Akt. In treating A431 cells with Pa-PDT, the levels of p-AKT and p-mTOR decreased at 6 and 12 h (Fig. 2C and D). In G361 cells, Pa-PDT did not affect the expression of p-Akt but slightly decreased the level of phospho-mTOR at 24 h. These results indicate that Pa-PDT inhibits the Akt/mTOR pathway and that these changes induce autophagy in A431 cells.

Autophagy is characterized by the formation of acidic AVOs (20). To confirm the presence of vacuoles in Pa-PDT-treated A431 cells, we performed acridine orange staining to obtain acidic AVOs. As shown in Fig. 3A, the number of AVOs was increased in A431 cells treated with Pa-PDT compared with the control cells. In G361 cells, the number of AVOs was slightly increased. Similar results were also obtained by MDC staining. The fluorescent compound MDC is a specific marker for autolysosomes and is commonly used to stain autophagic vesicles. A431 and G361 cells were treated with PDT for 24 h and analyzed using fluorescence microscopy. As shown in Fig. 3B, in the control cells, MDC-labeled vacuoles were not detected. However, in Pa-PDT-treated A431 cells, MDC-labeled cells were strongly detected, and the number of these cells increased during treatment in a concentration-dependent manner. In G361 cells, MDC-labeled cells were weakly detected. Collectively, these results also suggest that Pa-PDT induced autophagy in A431 cells but not in G361 cells.

It has been suggested that PDT can activate the MAPK pathway and regulate the cell death process (21,22). The activation of MAPKs in Pa-PDT-treated cells was monitored by western blotting. As shown in Fig. 4A, the phosphorylation of ERK1/2 and p38 MAPK was induced after 6 h of Pa-PDT treatment in A431 cells, whereas the activation of JNK was not detected. Similarly, the LC3B-I/II level was increased in a time-dependent manner during Pa-PDT treatment. In G361 cells, the phosphorylation of p38 MAPK was induced at 24 h after Pa-PDT treatment, whereas the phosphorylation of ERK was slightly increased at 12 h (Fig. 4A).

We next examined whether MAPK signaling was involved in Pa-PDT-induced autophagy. A431 cells were treated with an ERK1/2 inhibitor (U0126) in a gradient for 6 h followed by western blot analysis. As expected, ERK phosphorylation by PDT was inhibited by treatment with U0126 in a time-dependent manner. Following U0126 treatment, LC3B-II protein levels decreased in A431 cells, suggesting that ERK1/2 signaling was involved in Pa-PDT-induced autophagy (Fig. 4B). However, the inhibition of ERK phosphorylation by U0126 did not affect the LC3-II levels in G361 cells, suggesting that Pa-PDT induced cell growth inhibition by other pathways in G361 cells. To confirm the effect of U0126 on Pa-PDT-induced autophagy, we performed acridine orange staining to visualize autophagy using fluorescence microscopy. As shown in Fig. 4C, the control cells primarily emitted green fluorescence, indicating a lack of AVO. Pa-PDT strongly increased the intensity of red fluorescence in A431 cells but not in G361 cells. Alternatively, treatment of cells with 20 μM U0126 decreased the formation of Pa-PDT-induced AVO. These results indicate that the ERK1/2 pathway is involved in Pa-PDT-induced autophagy in A431 cells.

To further examine the role of p38 MAPK in Pa-PDT-mediated cytotoxicity, the p38 MAPK-specific inhibitor SB202190 was used. Pa-PDT induced PARP cleavage in A431 and G361 cells. SB202190 inhibited PARP cleavage in both cells. However, U0126 did not block PARP cleavage by Pa-PDT (Fig. 4D). Collectively, these results suggest that Pa-PDT also leads to apoptosis through p38 MAPK activation in A431 and G361 cells.

To determine whether the decrease in viability of A431 and G361 cells was also caused by the induction of apoptosis, we quantified apoptosis by flow cytometry using the Annexin V-FITC/PI double staining assay. As shown in Fig. 5A, a significant number of apoptotic cells (74.2%) was detected in G361 cells after Pa-PDT treatment, whereas <24% apoptotic-positive cells were detected in A431 cells. Consistent with this observation, caspase-3 activity was also strongly detected in G361 cells treated with Pa-PDT compared to A431 cells (Fig. 5B). In addition, the cleaved form of PARP was increased by Pa-PDT treatment in a time-dependent manner in both cells. However, PARP cleavage was stronger in G361 cells than in A431 cells (Fig. 5C).

To investigate potential cross-talk between autophagy and apoptosis induction in response to Pa-PDT, we investigated whether Pa-PDT-induced apoptosis could be inhibited by siRNA against ATG-5. In A431 cells, Pa-PDT-induced LC3B expression was inhibited by ATG-5 siRNA (Fig. 5D). However, Pa-PDT-induced PARP cleavage was not inhibited by ATG-5 siRNA in A431 and G361 cells. In addition, 3MA, an early stage inhibitor of autophagy, did not block Pa-PDT-induced PARP cleavage (data not shown), suggesting that autophagy is unrelated to Pa-PDT-mediated apoptosis.

Additionally, to determine whether Pa-PDT-induced apoptosis affected the autophagy pathway, we examined the expression levels of key autophagy proteins, LC3B, during treatment with the caspase inhibitor zVAD-fmk. Pretreatment of cells with the caspase inhibitor zVAD-fmk did not prevent Pa-PDT-induced expression of LC3B proteins in A431 cells (Fig. 5E). Our data demonstrated that Pa-PDT induces autophagy and/or apoptosis in A431 and G361 cells independently.

To identify the pathobiological characteristics of the transplantation tumors in the CAM, hematoxylin-eosin (H&E) staining and immunohistochemical analysis were performed.

Histological examination demonstrated that Pa-PDT reduced tumor thickness and increased cell death in both cell-xenograft CAM. Specifically, the expression of LC3B protein was more strongly increased by Pa-PDT in A431-implanted tumors. Additionally, TUNEL staining confirmed that TUNEL-positive cells were more frequently observed in G361-implanted tumor sections (Fig. 6A). These results demonstrate that Pa-PDT leads to selective and effective cell death of skin cancer cells through the autophagy and apoptosis pathway.