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. 2023 Oct 2;13:1156763. doi: 10.3389/fonc.2023.1156763

Figure 3.

Figure 3

5-ALA-PDT causes cancer cell death via necroptosis and pyroptosis, but not apoptosis. (A) 4T1 cells were treated with 5-ALA (50 μM) (PDT) or left untreated (C) for 4 hours, and then exposed to light (618–630 nm/wavelength) for 1.5 mins. At 0.5, 1, 2, and 4 hours after light exposure, cell lysates were collected for western blot analysis using antibodies against phosphorylated MLKL (p-MLKL), MLKL, caspase-1, cleaved caspase-1 (c-caspase-1), caspase-3, cleaved caspase-3 (c-caspase-3) and GAPDH. (B) 4T1 or (C) DLD-1 cells were treated with vehicle control (DMSO), caspase-1 inhibitor (Z-WEHD-FMK, 20 μM), NLRP inhibitor (MCC950, 20 μM), RIPK1 inhibitor (Nec-1s, 20 μM), RIPK3 inhibitor (Dabrafenib, 10 μM) or caspase-3 inhibitor (Z-DEVD-FMK, 20 μM) in combination with 5-ALA treatment (50 μM (4T1) and 250 μM (DLD-1)) (PDT +) or left untreated (PDT -) for 4 hours. Cells were then exposed to light (618–630 nm/wave) for 1.5 mins. At 4 hours post light exposure, the cell viability was measured using the CCK-8 assay kit. Mean ± SD relative cell viability to control from 4 independent experiments. *p<0.01 by Two-way ANOVA. (D) 4T1 cells treated with or without 5-ALA-PDT were subjected to TUNEL staining followed by flow cytometry analysis. Mean ± SD from 3 independent experiments. *p<0.01 by the student’s t test. (E) Apoptotic 4T1 cells treated with or without 5% DMSO for 24 hours were determined by western blot analysis using antibodies against caspase-3, cleaved caspase-3 and GAPDH, and by TUNEL staining followed by flow cytometry analysis. Mean ± SD from 3 independent experiments. *p<0.01 by the student’s t test.