Salinomycin induced ROS results in abortive autophagy and leads to regulated necrosis in glioblastoma

Glioblastoma is the most frequent malignant brain tumor. Even with aggressive treatment, prognosis for patients is poor. One characteristic of glioblastoma cells is its intrinsic resistance to apoptosis. Therefore, drugs that induce alternative cell deaths could be interesting to evaluate as alternative therapeutic candidates for glioblastoma. Salinomycin (SLM) was identified through a chemical screening as a promising anticancer drug, but its mechanism of cell death remains unclear. In the present work we set out to elucidate how SLM causes cell death in glioblastoma cell lines (both established cell lines and brain tumor stem cell lines), aiming to find a potential antitumor candidate. In addition, we sought to determine the mechanism of action of SLM so that this mechanism can be can be exploited in the fight against cancer. Our data showed that SLM induces a potent endoplasmic reticulum (ER) stress followed by the trigger of the unfolded protein response (UPR) and an aberrant autophagic flux that culminated in necrosis due to mitochondria and lysosomal alterations. Of importance, the aberrant autophagic flux was orchestrated by the production of Reactive Oxygen Species (ROS). Alleviation of ROS production restored the autophagic flux. Altogether our data suggest that in our system the oxidative stress blocks the autophagic flux through lipid oxidation. Importantly, oxidative stress could be instructing the type of cell death in SLM-treated cells, suggesting that cell death modality is a dynamic concept which depends on the cellular stresses and the cellular mechanism activated.


Neurosphere size analysis
For quantification of neurosphere size, cells derived from the dissociation of clonal single neurospheres were seeded in 96-well plates, and the size of the generated secondary spheres was assessed after 10 days. We counted 20 neurospheres per sample, and the neurospheres size media was plotted with 95% confidence intervals (CIs). Images were captured and measured using a deconvolution microscope (Zeiss, Germany).

Flow cytometric analysis of apoptosis
The membrane and nuclear events during apoptosis were analyzed by flow cytometry using FITC-Annexin V and propidium iodide (PI) staining. Following treatment, SF188 or GSC11 cells were harvested and centrifuged for 10 min at room temperature at 3000 rpm. Cells were washed with PBS and resuspended in binding buffer, and then 5 μl Annexin V-FITC (20 μg/ml) and 5 μl PI (50 μg/ml) were added. After incubating in the dark for 15 min, the samples were analyzed by flow cytometry (Becton-Dickinson). The assay was performed with a two-color analysis of FITC-labeled Annexin V binding and the uptake of PI. Living cells (Annexin V−/PI−, Q3), early apoptotic cells (Annexin V+/PI−, Q4), late apoptotic cells (Annexin V+/PI+, Q2), and necrotic cells (Annexin V−/PI+, Q1) were distinguished. Therefore, the total apoptotic proportion included the percentage of cells with fluorescence for Annexin V+/PI− and Annexin V+/PI+.

Quantification of acidic vesicular organelles (AVOs)
After treatment with the tested reagents, cells were stained with acridine orange (1 µg/ml for 15 min) (Sigma-Aldrich). After three rinses with PBS, cells were analyzed by flow cytometry. Green (510-530 nm) and red (> 650 nm) fluorescence emission from 10 4 cells illuminated with blue (488 nm) excitation light was measured with a FACSCalibur (Becton Dickinson, San Jose, CA) using the Cell Quest software.

Transmission electron microscopy
The ultrastructural analysis of cell morphology after the indicated treatments was performed using transmission electron microscopy (TEM). Treated cells were centrifuged (5 minutes at 3000 rpm) and rinsed with PBS. Samples were fixed with a solution containing 3% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3. Samples were then washed and treated with 0.1% Millipore-filtered, cacodylate-buffered tannic acid, postfixed with 1% buffered osmium tetroxide for 30 min and stained in block with 1% Milliporefiltered uranyl acetate. The samples were dehydrated with increasing concentrations of ethanol, infiltrated, and embedded in LX-112 medium. After this, they were polymerized in a 60º C oven for 2 days. Ultrathin sections (65 nm) were cut in a Leica Ultracut microtome, stained with uranyl acetate and lead citrate in a Leica EM Stainer, and examined in a Jeol 1210 transmission electron microscope (Jeol Ltd., Herts, UK).

Supplementary Figure S1: (A) Median-effect doses (IC50) of SLM and TMZ in attached cell lines and neurosphere cultures. IC50 is
the median-effect dose (the dose causing 50% of cells to be affected; here, this is equivalent to 50% survival). The results are expressed as mean values from Figure 1A. (B) GSC231, GSC229 and GSC23 were treated with TMZ or SLM at the indicated concentrations and the number of secondary spheres generated was assessed after 10 days and expressed as percentages of the numbers of spheres in nontreated cells. To confirm that the spheroids were formed by stem cells, we randomly selected at least 15 individual secondary spheres and subjected them to further, long-term (two-month) propagation. (C) Representative micrographs illustrating neurosphere size (the scale is 50-µm). Neurosphere size evaluation in GSC11 cells treated with TMZ or SLM at the indicated concentration (µM). (D) Neurosphere size evaluation in GSC11 cells treated with TMZ or SLM at the indicated concentration (µM). Figure S2: (A) T98G and U251MG cells were seeded at a density of 1 × 10 5 cells per well in 6-well plates. After 24 h of culture, cells were incubated with SLM at the indicated concentrations. Cells were collected 48 hours later and subjected to western blot analyses. Shown is a representative western blot of three independent experiments. (B) Analysis of early apoptotic cells after treatment with either SLM or TMZ at the indicated dosages. GSC11 or SF188 cells were stained with 5 μl Annexin V-FITC (20 μg/ml) and 5 μl PI (50 μg/ml) were added and subjected to flow cytometric analysis. The results are expressed as mean percetange values ± SD from three independent experiments. (C) Analysis of acidic vesicular organelles (AVOs) after treatment with either SLM or TMZ at the indicated dosages. GSC11 or SF188 cells were stained with acridine orange (1 μg/ml) and subjected to flow cytometric analysis. The results are expressed as mean values ± SD from three independent experiments. (D) U251 cells were seeded at a density of 1 × 10 5 cells per well in a 6-well plate. The following day cells were incubated with SLM. Cells were collected at the indicated times. Levels of protein expression were analyzed by western blot using antibodies against LC3-I/II and P62. α-tubulin was used as loading control. The western blot shown is representative of three independent experiments. (E) SF188 and GSC11 cells were seeded at a density of 1 × 10 5 cells per well in a 6-well plate. The next day, cells were incubated with SLM and/or TMZ at the indicated concentrations. Cells were collected 48 hours later, and the levels of protein expression were analyzed by western blot using antibodies against LC3-I/II and P62. GAPDH was used as loading control. The western blot shown is representative of three independent experiments.