Figure 1.
The fluorescence intensity of talaporfin sodium in cultured ESCC cells.
The fluorescence intensity of talaporfin sodium in cultured ESCC cells (10000 cells) incubated with the indicated concentrations of talaporfin sodium for 24 h was measured by flow cytometry. As shown, talaporfin sodium was almost equally incorporated to the various ESCC cells in vitro.
Figure 2.
Cytotoxic effect of t-PDT on ESCC cells.
Cell viability at 48-PDT was assessed using the WST-1 assay. t-PDT induced a talaporfin sodium dose-dependent cell death in ESCC cells (white square in the figures) regardless of differentiation grade or 5-FU resistance. (A) TE-5 (derived from poorly differentiated ESCC), (B) TE-8 (derived from moderately differentiated ESCC), (C) TE-10 (derived from highly differentiated ESCC), (D) TE-11 (derived from moderately differentiated ESCC), (E) TE-5R (5-FU-resistant cells derived from parental TE-5 cells) and (F) TE-11R (5-FU-resistant cells derived from TE-11 cells). A viability of 100% was defined as the amount of absorption at 450 nm found in untreated (non-irradiated and absence of treatment with talaporfin sodium) cells. Each point represents the mean ± S.D. from experiments conducted at least in triplicate. *P<0.01 vs untreated (non-irradiated and absence of treatment with talaporfin sodium) cells (n = 3).
Figure 3.
Induction of apoptosis in TE-11R cells treated with t-PDT.
(A) TE-11R cells were pretreated with talaporfin sodium (30 µg/mL) for 24 h, and then irradiated (10 J/cm2). Phase-contrast images were taken at 2 or 4 h after t-PDT. The images of untreated cells are also shown. Arrows indicate perinuclear vacuolization and cell shrinkage suggesting apoptosis. Scale bar, 100 µm. (B) Flow cytometric analysis of apoptosis in TE-11R cells treated with or without talaporfin sodium (30 µg/mL) for 24 h with or without subsequent laser irradiation (10 J/cm2). Cells were stained with FITC-labelled annexin V and propidium iodide (PI) 4 h after irradiation. A representative experiment out of three is shown.
Figure 4.
Generation of ROS in TE-11R cells treated with t-PDT.
(A) TE-11R cells were treated with the indicated concentrations of talaporfin sodium for 24 h and received irradiation subsequently. Intracellular ROS levels at 4 h after irradiation treatment were determined by DCF assay. The intracellular ROS level was significantly increased by t-PDT in a talaporfin sodium dose-dependent manner. * P<0.01 vs untreated (non-irradiated and absence of treatment with talaporfin sodium) cells (n = 3).
Figure 5.
Formation of DNA double-strand breaks in TE-11R cells treated with t-PDT.
TE-11R cells were treated with the indicated concentrations of talaporfin sodium for 24 h with or without subsequent laser irradiation (10 J/cm2). (A) The expression of γ-H2AX was evaluated by fluorescence microscopy at 24 h after the irradiation. Under the non-irradiated conditions, γ-H2AX expression was not observed (upper panels), whereas a talaporfin sodium dose-dependent phosphorylation of γ-H2AX was found in the irradiated groups (lower panels). A representative experiment out of three is shown. Scale bar, 50 µm. (B) Phase contrast image was shown at 24 h after the irradiation. Scale bar, 50 µm.
Figure 6.
Inhibition of anchorage-independent cell growth due to t-PDT.
Soft-agar colony-formation assays demonstrated the activity of anchorage-independent cell growth in TE-11R cells. The histograms show the average number (A) or size (B) of colonies per high-power field. Assays were performed in triplicate. t-PDT blocked colony formation completely, although talaporfin sodium alone or irradiation alone had no effect. *P<0.01 vs untreated (non-irradiated and absence of treatment with talaporfin sodium) cells (n = 3).
Figure 7.
t-PDT suppress tumor formation in vivo.
(A) Tumor response in mice treated with t-PDT at the indicated doses of talaporfin sodium. t-PDT was performed when tumor volume reached about 50–150 mm3. Each point represents the average response rate (relative tumor volume on tumor size in day 0) of seven mice. Talaporfin sodium dose-dependent tumor reduction was shown in xenografted ESCC tumors treated with t-PDT. *P<0.01, **P<0.001, ***P<0.0001 vs the control groups at the indicated time points (n = 7). (B) Hematoxylin and eosin and immunohistochemical (Ki67) staining. Scale bar, 1mm. Ki67 positive cells indicate the viable and proliferative ESCC cells.