Figure 6.

PD triggers LMP to release lysosomal contents augmented by autophagic flux burden

(A) Western blot analysis of the SMPD1 and cathepsin B levels in lysosome and cytoplasm fractions after PD treatment. (B–E) HepG2 and Hep3B cells were treated with PD (3 μM, 24 h) in the presence or absence of CA-074 methyl ester (50 μM), a cathepsin B inhibitor. Then the activity of cathepsin B was analyzed by flow cytometry (B), the cell viability was tested by WST-1 assay (C), death cells were analyzed by PI staining assay (D), and the expression of pro-caspase 3, cleaved caspase-3 was determined by western blotting (E). (F–H) The anticancer effects of PD were augmented by starvation and Torin1 treatment. HCC cells were treated with PD (3 μM), serum deprived or Torin1 (1 μM, a specific mTOR inhibitor) as indicated. The cell viability was then tested by WST-1 assay (F), the ability to form colonies was determined by the colony formation assay (G), and caspase 3/7 activity was measured by flow cytometry (H). (I) A proposed model summarizing how PD triggers lysosome leakage, contributing to cell death, which can be amplified by the intracellular autophagic flux burden. All data are represented as mean ± SD, n = 3; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.