Effect of Photofrin-mediated photocytotoxicity on a panel of human pancreatic cancer cells

Abstract

Background and objective: Pancreatic cancer is a leading cause of cancer-related deaths in men and women. Early clinical studies suggest that photodynamic therapy (PDT) might be a useful modality in the management of this deadly disease. In this study, the photocytotoxicity of Photofrin-mediated PDT on different human pancreatic cancer cells (BxPc-3, HPAF-II, Mia PaCa-2, MPanc-96, PANC-1 and PL-45) was examined.

Materials and methods: After co-incubating cancer cells with Photofrin (0-10 μg/ml) for 4h, the cells were irradiated with 0-6J/cm(2) of 630 nm light. The effect of Photofrin PDT on the survival of cells were examined using tetrazolium-based colorimetric assay and clonogenic assay. PDT-induced apoptosis was analyzed by flow cytometry. Expressions of apoptosis-related proteins were determined by western blot analysis.

Results: Photofrin PDT strongly inhibited the survival of pancreatic cancer cells. A small portion of cells (<15%) underwent apoptosis 24h after PDT at LD50. Cleavage of caspase-3, caspase-8, caspase-9 and PARP after PDT were also confirmed. BxPc-3, Mia PaCa-2, MPanc-96, and PANC-1 cells were more sensitive and HPAF-II and PL-45 cells less sensitive.

Conclusion: Photofrin PDT can induce apoptosis and inhibit survival of human pancreatic cancer cells.

Keywords: Apoptosis; Pancreatic cancer cells; Photodynamic therapy; Photofrin.

Figure 1

MTS assay. Dose escalation study of PDT effect on cell viability was performed at drug dose levels of 0—10 μg/ml and light dose levels of 0—6 J/cm². Tetrazolium compound MTS assay was carried out 24 h after PDT. Solid line: light control; dash line: 3 J/cm² and dot line: 6 J/cm².

Figure 2

Clonogenic assay. Poorly differentiated human pancreatic cancer cell lines were subjected to Photofrin PDT at the following drug and light doses — Mia PaCa-2: 5 μg/ml and 3 J/cm²; MPanc-96: 5 μg/ml and 3 J/cm² and PL-45: 5 μg/ml and 6 J/cm². Control (drug only) and treated cells were grown in a fresh medium immediately after PDT. Clonogenic assay was examined after two weeks. There was no colony formation in Photofrin PDT groups.

Figure 3

Flow cytometric assay. PDT-induced apoptosis was detected by PI staining and flow cytometric analysis at 24 h after PDT. Pancreatic cancer cell lines were subjected to Photofrin PDT at the following drug and light doses respectively — BxPc-3: 2 μg/ml and 3 J/cm²; HPAF-II: 5 μg/ml and 3 J/cm²; Mia PaCa-2: 5 μg/ml and 3 J/cm²; MPanc-96: 2 μg/ml and 3 J/cm²; Panc-1: 2 μg/ml and 3 J/cm² and PL-45: 5 μg/ml and 6 J/cm². Representative light and drug controls are shown on the top panel. The percentage of apoptotic cells is indicated.

Figure 4

Western blot analysis. PDT-induced apoptosis was confirmed by detecting the cleaved caspase-3, caspase-8, caspase-9, and PARP fragments (arrows) and by the disappearance of the uncleaved precursors of these proteins in pancreatic cancer cells.