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Cancers 2020, 12, 2371 4 of 14 Bottom: Bar graphs show densitometric analysis. Data are presented as mean ± SD, n = 3 independent experiments and analyzed by ANOVA test. Left: SQSTM1 protein levels relative to stain-free blot or TUBA. For FaDu, * p < 0.05 for Bup vs CTL and * p < 0.05 for Omi vs CTL. Middle: MAP1LC3B-II protein levels relative to MAP1LC3B-I for SCC-9 (* p < 0.05 for Bup vs CTL and Omi vs CTL) and FaDu (*** p < 0.001 for Bup vs CTL, * p < 0.05 for Omi vs CTL). Right: AKTser473 protein levels relative to stain-free blot or TUBA, for SCC-9: * p < 0.05 for Bup vs CTL, ** p < 0.01 for Omi vs CTL. For FaDu: **** p < 0.0001 for Bup vs CTL, *** p < 0.001 for Omi vs CTL. (C) Autophagy flux analysis with tandem sensor RFP-GFP-LC3B. PI3Ki increase autophagolysosome (AL) formation. Top: GFP is sensitive to the acidic pH of AL. Red = AL; yellow = autophagosomes (AV). Left: merged stack of confocal images. SCC-9 and FaDu were transduced with the tandem sensor 24 h before the 24 h incubation with inhibitors (1 μM Bup, 5 nM Omi). Cells were stained with 4′,6-Diamidino-2-Phenylindole (DAPI). Scale bar = 50 μm. Right: AL and AV counts per cell of SCC-9 and FaDu using the specialized Image J macro analysis (https://imagejdocu.tudor.lu/plugin/analysis/colocalization_analysis_macro_for_red_ and_green_puncta/start). Data are mean ± SD of six images from two independent experiments (three images by experiment per condition with 15–30 cells per picture). For SCC-9 * p < 0.05 for Bup vs CTL, and for FaDu ** p < 0.01 for Omi vs CTL by unpaired Student’s t-test. Uncropped blots are shown in Figure S2. We used western blot analysis to detect two established markers of autophagy (Figure 1B): microtubule-associated protein 1 light chain 3 beta (MAP1LC3B) and sequestosome 1 (SQSTM1), also known as LC3B and p62, respectively. The cytoplasmic MAP1LC3BI is recruited during AV formation and then cleaved and lipidated to generate MAP1LC3BII. Increased levels of MAP1LC3BII combined with decreased levels of SQSTM1, the autophagosomal cargo protein degraded in the AL, are the hallmarks of autophagic activation [15,16,33]. Consistent with autophagy induction, we observed a decrease in the SQSTM1 levels and an increase in MAP1LC3BII/MAP1LC3BI ratios in the presence of PI3Ki Bup or Omi. We analyzed downstream phosphorylation of AKT at serine 473 to confirm the inhibitor activity towards the PI3K signaling pathway. AKTser473 levels were strongly decreased in HNSCC cell lines by Bup or Omi (Figure 1B). We also analyzed the autophagic flux with a specific reporter, the PremoTM (Thermo Fisher Scientific, Waltham, MA, USA) autophagy tandem sensor RFP-GFP-LC3B [15,28,34] (Figure 1C top). LC3B targets this fluorescence construct to the AV, and differences in pH sensitivity between Green Fluorescent Protein (GFP) (sensitive to acidic pH) and Red Fluorescent Protein RFP track the progression from AV (green + red LC3B = yellow) to AL (red only). Autophagic flux is assessed by the number and color of the vesicles. The number of red LC3B vesicles increased with PI3Ki Bup or Omi (Figure 1C bottom). Altogether, our results indicate that Bup and Omi are associated with a high autophagic flux and autophagy activation in both cell lines. 2.2. PI3Ki and CQ Work in Synergy to Decrease Proliferation of HNSCC Cell Lines We postulate that the activation of autophagy by PI3Ki enables cancer cells to resist treatment. We performed real-time cell proliferation assays with the IncuCyte S3 Live-Cell Analysis System to verify our hypothesis that the combination of PI3Ki and autophagy inhibitors will lead to decreased tumor cell proliferation (Essen Bioscience, Ann Arbor, MI, USA). The SCC-9 cells were incubated with the autophagy inhibitor CQ or PI3Ki at concentrations that were Federal Drug Administration (FDA) approved for human use or in clinical trial and relevant for patient treatment (as evaluated from blood, plasma and bronchoalveolar fluid samples). More precisely, Bup concentrations in the plasma of cancer patients treated with 100 mg/day were evaluated at 1 μg/mL (2.5 μM) [35]. The ratio of plasma/tumor concentration was evaluated as 1 in a glioblastoma study [36]. Omi at 2 mg/day reaches concentrations of 85 ng/mL in blood (170 nM) and 236 pg/mL (0.5 nM) in bronchoalveolar fluid [37]. Autophagy inhibitor concentrations evaluated in cancer patients from clinical trials were 5 μM in the blood and 1 μM for the plasma [38]. CQ inhibits autophagy by raising the lysosomal pH, which leads to inhibition of both fusion of AV with lysosome and lysosomal protein degradation. As a lysosomotropic agent, CQ preferentially accumulates in the lysosome of the cell and can accumulate inPDF Image | Dual Inhibition of Autophagy Pathway as a Therapeutic Strategy
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