Early apoptosis and cell death induced by ATX-S10Na (II)-mediated photodynamic therapy are Bax- and p53-dependent in human colon cancer cells

Figure 1 ATX-S10Na(II)-PDT-mediated cell death and early apoptosis were Bax- and p53-dependent. Cells were treated with, or without, ATX-S10Na(II)-PDT and harvested at each indicated time point following irradiation. A: Phototoxicity was determined by MTT assay 24 h after laser irradiation. Data are expressed as a ratio of cell viability relative to untreated control cells. Data represent the mean ± SD of three independent experiments (^aP < 0.05 vs wild-type cells). B, C: Early apoptotic changes (B) and late apoptotic or necrotic changes (C) were determined by annexin V apoptosis assays at 3 or 6 h after laser irradiation. Data are expressed as a percentage of annexin V-positive and PI-negative (B) or annexin V-positive and PI-positive cells (C). Data represent the mean ± SD of three independent experiments (^bP < 0.05 vs wild-type cells). D: Cell cycle distributions were determined by flow cytometry at 3 or 6 h after laser irradiation. Data are expressed as a percentage of sub-G₁ fraction. E: Morphological changes in response to ATX-S10Na(II)-PDT were determined by transmission electron microscopy assays at 3 or 6 h following laser irradiation. Typical subcellular changes in response to PDT are shown (original magnification × 12 000). Scale bar: 15 μm, N: nuclei.

Figure 2 Induction of apoptosis by ATX-S10Na (II)-PDT was caspase-dependent. Cells were treated with, or without, ATX-S10Na (II)-PDT, harvested and lysed at 6 h after laser irradiation. Induction of caspase activity was assayed. A: caspase-3; B: caspase-8; C: caspase-9. Data represent the mean ± SD of three independent experiments (^aP < 0.05 vs wild-type cells).

Figure 3 Low levels of Bcl-2 and Bcl-x_L played a role in ATX-S10Na(II)-PDT. Cells were treated with, or without, ATX-S10Na(II)-PDT, harvested and lysed at 3 or 6 h after laser irradiation. Western blot analysis was performed. Each protein level was quantified by using NIH Image J program. Data represent the mean ± SD of three independent experiments.