DataSheet_1_Autolysosomal activation combined with lysosomal destabilization efficiently targets myeloid leukemia cells for cell death.doc

IntroductionCurrent cancer research has led to a renewed interest in exploring lysosomal membrane permeabilization and lysosomal cell death as a targeted therapeutic approach for cancer treatment. Evidence suggests that differences in lysosomal biogenesis between cancer and normal cells might open a therapeutic window. Lysosomal membrane stability may be affected by the so-called ‘busy lysosomal behaviour’ characterized by higher lysosomal abundance and activity and more intensive fusion or interaction with other vacuole compartments.MethodsWe used a panel of multiple myeloid leukemia (ML) cell lines as well as leukemic patient samples and updated methodology to study auto-lysosomal compartment, lysosomal membrane permeabilization and lysosomal cell death.ResultsOur analyses demonstrated several-fold higher constitutive autolysosomal activity in ML cells as compared to human CD34+ hematopoietic cells. Importantly, we identified mefloquine as a selective activator of ML cells' lysosomal biogenesis, which induced a sizeable increase in ML lysosomal mass, acidity as well as cathepsin B and L activity. Concomitant mTOR inhibition synergistically increased lysosomal activity and autolysosomal fusion and simultaneously decreased the levels of key lysosomal stabilizing proteins, such as LAMP-1 and 2.DiscussionIn conclusion, mefloquine treatment combined with mTOR inhibition synergistically induced targeted ML cell death without additional toxicity. Taken together, these data provide a molecular mechanism and thus a rationale for a therapeutic approach for specific targeting of ML lysosomes.</p

Impact of Salinomycin-treatment on PHH synthesis function.

<p>Functionality of PHH after treatment with Salinomycin was analyzed by urea formation and albumin synthesis. (<b>A</b>) 24 hours of Salinomycin exposure at varying concentrations was not accompanied by impaired urea formation at all. (<b>B</b>) In contrast, treatment for 48 hours resulted in a dose-dependent decrease of urea formation at days 1, 3 and 5 without reaching statistical significance. (<b>C+D</b>) Albumin synthesis was markedly impaired at day 1 after stimulation with Salinomycin for 24 and 48 hours, respectively. Further incubation led to continuous recovery of albumin synthesis in the groups with 24 hours of drug exposure. In contrast, treatment for 48 hours resulted only in moderate recovery of albumin synthesis (n = 3). * p<0.05, ** p<0.005.</p

Salinomycin-mediated inhibition of autophagic flux relates to accumulation of dysfunctional mitochondria and increased ROS-production.

<p>(<b>A</b>) Inhibition of basic and HBSS-activated autophagic flux in HepG2 cells stably expressing LC3-GFP treated with 3 MA (2 and 10 mM), LY (2 and 10 mM), nocodazole (2 and 10 µM) or ACH (0.8 and 4 mM) for 7 h. (<b>B</b>) Inhibition of autophagic flux in HepG2 cells treated with Sal (0.5, 2 and 8 µM) for 24 h (left) with representative histograms (right). (<b>C</b>) Flow-cytometric analysis of total mitochondrial mass using MitoTracker Green (MTR green) reveals accumulation of dysfunctional mitochondria. (<b>D</b>) Evidence of increased ROS-production using CM-H2DCFDA (left) with representative histograms (right). All experiments are presented as mean ± SD of n = 3 independent experiments. *p<0.05, **p<0.01.</p

Salinomycin inhibits HepG2 and Huh7 autophagic flux.

<p>HepG2 and Huh7 cells were exposed to different concentrations of Salinomycin (0.5, 2 and 8 µM) for different time points and analyzed for activity of autophagy. (<b>A</b>) Immunoblot analysis of LC3-I and LC3-II-isoforms (up) with densitometry quantitative analysis (down) in HepG2 cells revealed Sal-induced LC3-II-accumulation due to inhibition of autophagic flux as demonstrated by reduced LC3-II-accumulation after addition of ACH. (<b>B</b>) Basic and PP242-activated autophagic flux in Huh7 cells (black bars). Treatment with autophagy inhibitors 3MA (2 and 10 mM), ACH (5 and 20 mM) or CQ (5, 20 µM) for 24 h counteracts PP242 activation of autophagic flux. (<b>C</b>) Inhibition of autophagic flux in Huh7 cells after shRNA-mediated knockdown of ATG7. (<b>D+E</b>) Decreased accumulation of autophagic compartments after the blockage of autophagolysosomal degradation by ACH indicates reduced autophagic flux in HepG2 (<b>D</b>) and Huh7 (<b>E</b>) cells treated with Sal for 24 h. Next to the bar graphs representative histograms are depicted. All experiments are presented as mean ± SD of n = 3 independent experiments.*p<0.05; **p<0.01, ***p<0.001.</p

Assessment of apoptosis-induction in PHH by Salinomycin.

<p>Cultured PHH were exposed to increasing concentrations of Salinomycin (1, 2, 5 and 10 µM) for 24 hours. Apoptosis was detected by M30 Cytodeath kit. Treatment with Fas Ligand (FasL) served as a positive control. Staining of PHH with DAPI (blue) and M30 Cytodeath kit (red) revealed no evidence for induction of apoptosis in PHH even after treatment with high concentrations of Salinomycin. In contrast, FasL clearly induced apoptosis in PHH.</p

Impact of Salinomycin-treatment on <i>in vitro</i> morphology of PHH.

<p>After one day of incubation cultured PHH were exposed to increasing concentrations of Salinomycin (1, 2, 5 and 10 µM) at varying time of exposure (24 and 48 hours, respectively). (<b>A</b>) Impairment of <i>in vitro</i> morphology without subsequent recovery were only detectable after treatment with high concentrations of Salinomycin (10 mM) whereas treatment with up to 5 µM Salinomycin resulted in morphological recovery after lapse of the agent. (<b>B+C</b>) Cell viability was assessed by MTS-assay. Salinomycin treatment for 24 (<b>B</b>) and 48 (<b>C</b>) hours led to significantly impaired cell viability at day 1 and 3. Upon further incubation recovery of the cells was observed as indicated by increasing production of the coloured formazaan-product (n = 6). (<b>D+E</b>) Cell damage of PHH as represented by AST release was only detectable immediately after drug exposure (day 1) to 10 µM Salinomycin for 24 (<b>D</b>) and 48 (<b>E</b>) hours, respectively. This difference did not reach statistical significance (n = 3). Ongoing incubation was accompanied by barely measurable AST release. * p<0.05, ** p<0.005.</p

Salinomycin impairs HCC cell survival.

<p>HepG2 and Huh7 cells were exposed to increasing concentrations of Salinomycin (1, 2, 5 and 10 µM) for 24 hours. (<b>A</b>) Cell viability was assessed by MTS-assay; high concentrations of Salinomycin led to significantly decreased viability of both cell lines (n = 5). (<b>B</b>) HCC cells revealed significantly reduced proliferation after exposure to Salinomycin as demonstrated by decreased ³H-Thymidine uptake. (<b>C</b>) Low concentrations of Salinomycin (left panel, solid line) led to weak increase of apoptotic cells compared to untreated cells (dotted line in overlay). High concentrations markedly induced apoptosis (right panel, solid line). Results are shown as representative scatter-grams of Annexin-V⁺-cells or summarized as mean ± SD of n = 4 independent experiments (<b>D</b>). *p<0.05, **p<0.01.</p