Cell line and culture conditions
All experiments were performed using exponential growing A549 human lung adenocarcinoma cell line, obtained from NCI-Frederick Cancer DCTD cell line repository. These cells were maintained in humidified incubator with RPMI 1640 medium supplement with 10% fetal bovine serum (FBS) (Gibco/Invitrogen, São Paulo, SP Brazil), added with 2mM of glutamine, 1% of penicillin/streptomycin, and 0.1% of amphotericin B (Sigma-Aldrich, St Louise, MO, USA) at 37oC and 5% CO2.
Drug treatments
A549 cells were seeded in 12-well plates (1 x 104 cells/well). After 24 h of incubation, cells were treated for 48 h with cisplatin (Cis), etoposide (Eto), carboplatin (Carb), paclitaxel (Pac), gemcitabine (Gem) and also with co-treatments of Cis plus Eto and Carb plus Pac. Drugs concentration was chosen through dose-response curves based on the literature and on the peak of blood concentration reached by each drug (data not shown). DMSO was used to dilute all chemotherapeutics except Carb, which was diluted in PBS. All chemotherapeutics were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Analysis of apoptosis by Annexin V FITC and PI staining
Apoptosis was measured 48 h after treatment with Annexin V-FITC plus Propidium Iodide (PI) co-staining according to the manufacturer’s protocol (BD Biosciences; CA, USA). Briefly, cells were collected and washed once with Annexin binding buffer. After this, cells were resuspended in a staining buffer containing 2.5 μL of Annexin V-FITC and 5 μL of PI per sample, for 15 min in the dark. Cells were analyzed by flow cytometry (Attune-AB Applied Biosystems).
Calreticulin (CRT) externalization measurement
After the treatment, cells were collected and washed twice with ice cold PBS and fixed with 4% paraformaldehyde in ice cold PBS for 5 min. Cells were centrifuged at 1200 rpm for 5 min and washed again in ice cold PBS. Then cells were incubated for 30 min at 4oC with staining solution (mix per sample: 200 μL ice cold PBS + 4 μL Fetal Bovine Serum + 1 μL Anti-CRT antibody) [#FMC75, Abcam Cambridge, MA]. Isotype-control IgG1 (BD Biosciences; CA, USA) was used as control (mix per sample: 200 μL ice cold PBS + 4 μL fetal bovine serum + 1 μL isotype-control IgG1). Next, cells were washed twice with ice cold PBS, centrifuged for 5 min at 1200 rpm and resuspended in ice cold PBS. Samples were analyzed by flow cytometry (Attune-AB Applied Biosystems) to identify the percentage of CRT positive cells (i.e. cells that externalized the CRT) and also the intensity of CRT. Levels of CRT externalization were also assessed as to cell size (FSC) and intracellular cell complexity (SSC).
ATP release assay
To measure levels of extracellular ATP released in response to chemotherapeutics supernatants were collected 48 h after treatment. We used the ATP assay kit Sigma-Aldrich (St. Louis, MO, USA) based on luciferin-luciferase conversion, according to the manufacturer’s protocol. Briefly, the content was centrifuged at 1200 rpm for 5 min and 10 μL of cleared supernatants of each condition were transferred to a 96 well plate. Then, 90 μL of ATP reagent was added to each well, followed by incubation for 1 min at room temperature. After this, we analyzed fluorescence emission in aspectromax M3 microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Extracellular HMGB1 measurement
To measure extracellular HMGB1, supernatants were collected and centrifuged at 1200 rpm for 5 min and immediately analyzed by dot blot immunoassay. Serial dilutions of samples (1, 2, 4, and 8 μL) were applied to a nitrocellulose membrane. The membrane was blocked with 5% BSA in TBS-T buffer (0.05% Tween20 in TBS) for 1 h at room temperature and incubated with primary antibody anti-HMGB1 (Abcam Cambridge, MA) 1:1000 dissolved in BSA/TBS-T for 30 min. Then, membrane was washed three times with TBS-T by 5 min and incubated with secondary antibody anti-rabbit 1:1000 (Abcam Cambridge, MA) for 30 min at room temperature. Next, membrane was washed three times with TBS-T (15, 5 and 2 min) and once with TBS (20 mMTris-HCl 150 mMNaCl pH 7.5) by 5 min, followed by incubation with ImmobilonTM to chemiluminescent reaction (Millipore, EUA) for 1 min. Images of the membrane were recorded by the Image Quant imager LAS 500 (Healthcare GE Life Sciences). Quantification was performed using ImageJ software and levels of HMGB1 were corrected by cell number.
Acridine Orange (AO) assay
Autophagy levels were determined by detecting autolysosome formation through the Acridine Orange (AO) staining [22]. AO is a marker of acidic vacuolar organelles that fluoresces green in the whole cell (indicated by BL1 channel in flow cytometry) and red in acidic compartments (indicated by BL3 channel in flow cytometry), mainly in autolysosomes. Treated cells were trypsinized, collected and stained for 15 min at room temperature, in the dark, with AO at 2.7 µM. The percentage of AO-positive cells and the intensity of red/green fluorescence were assessed by flow cytometry (Attune-AB applied biosystems).
Statistical Analysis
All statistics were performed in PASW Statistics 18. Shapiro-Wilk was used to test the normal distribution of results. Data are expressed as means ± standard error of the mean (SEM). Comparisons between groups were performed using the Student´s t-test and the One-Way Analysis of Variance (ANOVA) followed by Tukey post-hoc test for multiple comparison, as appropriated. A ‘p value’ under 0.05 was considered as significant.