Figure 1.
LUT induces apoptotic cell death specifically in malignant keratinocytes.
A. Detection of metabolic activity by MTT assay, 24 hours after treatment with LUT (10–100 µM) or DMSO (equal amount as highest LUT concentration) in different SCC cell lines. (Representive experiment out of 3, n = 16) B. Flow cytometric analysis of propidium iodide (PI) stained cells, 24 hours after treatment with LUT (50 µM). PI-positive cells = dead cells. (Representive experiment is shown, n = 3) C. Detection of caspase signaling by western blot analysis 24 hours after different concentrations of LUT (10–100 µM). UVB (120 mJ/cm2) was used as a positive control for cell death in NHK. D. Luminescent detection of caspase 8 and caspase 9 activity 24 hours after treatment with 50 µM LUT. Fold increase values against untreated control are shown. (Experiment performed three times in duplicate) E. Measurement of viability by trypan blue exclusion assay, 24 hours after treatment with LUT (50 µM) and zVAD-fmk (50 µM).
Figure 2.
LUT induced apoptosis involves modulation of AKT signaling.
A. Time dependent effect of LUT on AKT phosphorylation (AKT-P; Ser473) and on apoptosis markers (Parp-cleavage and caspase 3 activation) was studied using western blot in MET1 and MET4 cells B. Protein and phosphorylation levels of AKT and its downstream targets in MET1 and MET4 cells treated with LUT (20 and 50 µM) for 24 hours and 48 h obtained using western blot analysis. Numbers, obtained by densitometric analysis of the western blot, indicate the ratio of phosphorylated to total AKT and p70S6 protein. C. MET1 and MET4 cells were pre-treated or not with AI (10 µM) for 1 hour and subsequently treated with LUT as indicated. Densitometric analysis of cleaved caspase 3 relative to actin level is shown. A representative blot of at least 3 independent experiments is shown. D. Cells were treated with AI and LUT for 24 hours as indicated and the amount of apoptotic DNA was determined by the Cell death detection ELISA as described in Materials and Methods. Experiment was performed twice in duplicate E. MET1 and MET4 cells transiently transfected with a construct expressing Myr-AKT-HA, were treated or not with LUT (50 µM) for 24 hours. Fugene HD = transfectant only; Myr-AKT = constitutively active AKT). Densitometric analysis of cleaved caspase 3 relative to actin level is shown. A representative blot of at least 3 independent experiments is shown.
Figure 3.
Expanded lysosomal compartment following LUT-treatment in metastatic SCC cells.
A. First row: Bright field microscopic pictures of MET4 treated or not with LUT (50 μM) for 24 hours. Second and third row: Fluorescent microscopic images of AO stained MET4 cells treated with LUT (50 µM) or left untreated. Red = AVO; Green: non-acidic cell compartment (scale bar = 20 µm). B. Representive flow cytometric quantification of AVO formation (increase in red fluorescence in MET1, MET4 and NHKs following treatment with indicated concentrations of LUT C. Confocal images of LAMP-1 and LAMP-2 stained (green) MET4 cells treated with LUT (50 µM) for 16 h. DAPI (blue) was used for nuclear staining.
Figure 4.
Involvement of autophagy in the response of SCC cells on LUT.
A. Electron micrographs showing the ultrastructure of MET4-cells treated with LUT (20 µM) or not. Arrows indicate autophagosomes (scale bar = 1 µm). B. (left panel) Fluorescent microscopy analysis of MET4 cells transiently overexpressing mRFP-GFP-LC3, treated or not with LUT (10 or 50 µM) for 24 hours (scale bar = 20 µm). (right panel) Quantification of red and green fluorescent punctae of at least 10 cells per condition is shown. C. Protein levels of p62 and LC3-II upon LUT treatment in MET4 cells. One representative blot of at least three independent experiments is shown. Values are the ratio's p62 and LC3-II to actin level of densitometric analysis. D. Cells were pre-treated (1 hour) with an autophagy inhibitor (INH), CQ (50 µM) or apoptosis inhibitor (INH), ZVAD-fmk (50 µM), before LUT treatment (50 µM). Lysates were made after 24 hours and analysed by western blot. A representative blot of at least three independent experiments is shown. Numbers are the ratios of the densitometric analysis for p62/actin and LC3II/actin.
Figure 5.
Inhibition of late phase autophagy increases LUT induced cell death in metastatic SCC.
A. Cells were pre-treated with CQ (0 or 50 µM) and after 1 hour LUT (0, 20 or 50 µM) was added for 24 hours. The amount of apoptotic DNA was determined by the Cell death detection ELISA. Enrichment factor relative to untreated controls (UNTR/-) was calculated. Experiment was performed twice in duplicate. B. Western blot of cell lysates of MET4 cells pre-treated for 1 hour with CQ (50 µM) and/or 3-MA (10 mM) before the addition of LUT (50 µM) for 24 hours. Densitometric analysis of PARP% (cleaved Parp/(total Parp + cleaved Parp) and of cleaved caspase 3 relative to actin level is shown. Representative blot of three independent experiments is shown. C. Trypan blue exclusion assay of MET4 cells treated as indicated and described in B. (Experiment performed twice, n = 4).