Fig 1.
A549cisR cells exhibit defective apoptosis following exposure to cisplatin.
A549Pt and A549cisR cells were treated for 48 hours with 5 μM and 5 μM or 25 μM cisplatin (cisPt), respectively. Cells cultured in 1%FBS were used as negative control, and cells treated with staurosporine (STP, 0.1 μM) were used as positive control. Subsequently, cell suspensions were incubated with Annexin V-FITC/PI or FITC-FMK-specific substrates for the detection of apoptosis or active caspases 3,8 and 9, respectively. The percentage of cells with increased Annexin-V/low PI staining (A), and increased caspases activity (B-D) measured by the Cellometer platform are plotted. Graph bars represent the Mean±SEM from at least three independent experiments. *p<0.05 treatment vs. 1%FBS, # p<0.05 A549Pt vs. A549cisR for the same treatment conditions.
Fig 2.
Chloroquine triggers lysosomal membrane permeabilization in A549cisR cells, promoting the release of lysosomal cathepsins into the cytosol.
A549Pt (A) and A549cisR (B) cells were loaded with FITC-dextran 40 kDa for 16 hours, and treated overnight with different concentrations of chloroquine (CQ) in serum-free media. Cells were then fixed, nuclei were stained with Hoechst, and plate was mounted on Cellomics ArrayScan automated fluorescence imager. Representative immunofluorescence microscopy images are shown. (C-D) A549Pt (C) and A549cisR (D) cells were treated overnight with different concentrations of chloroquine in serum-free media. Next day, cytosolic (cy) and lysosomal (ly) or total protein (T) fractions were separated by ultracentrifugation and lysates were loaded on SDS/PAGE gels. Upon transfer, membranes were probed with specific antibodies against cathepsin D, LAMP-1 (lysosomes) and α-tubulin (cytosolic marker).
Fig 3.
A549cisR cells exhibit an increase in the number of lysosomes.
(A) Untreated A549Pt and A549cisR cells were stained for DAPI (blue) and LAMP-1 (green), and immunofluorescent images were captured using Cellomics (40x objective) in two fluorescent channels. (B) The number of lysosomes was quantified by the Cellomics imager using the biocompartmental analysis algorithm. (C) Cells lysates from untreated A549Pt and A549cisR cells were collected and Western blot analysis was performed for LAMP-1 and α-tubulin. (D) Graph displaying LAMP-1 level adjusted to tubulin level as quantified by WB densitometry. Graph bars represent the Mean±SEM from three independent experiments. *p<0.05 A549Pt vs. A549cisR.
Fig 4.
Chloroquine potentiates cisplatin cytotoxicity against A549cisR lung cancer cells in an LMP-mediated manner.
(A) A549cisR cells treated overnight with cisplatin and chloroquine. Next day, cytosolic (cy), total protein (T) and lysosomal (ly) fractions were separated by ultracentrifugation and lysates were loaded on SDS/PAGE gels. Upon transfer, membranes were probed with specific antibodies against cathepsin D (cath D), LAMP-1 (lysosomes) and α-tubulin (cytosolic marker). (B) A549cisR cells were treated with 1%FBS, chloroquine 100 μM, cisplatin 100 μM, or combination with or without pretreatment with E64 (10 μM). Cell viability was measured with CellTiter-Blue®. X-axis, represent the % cell viability at 48 hours (T48/T1) normalized to the negative control 1%FBS. *p<0.05 treatment condition versus 1%FBS. (C) A549cisR cells were treated with different concentrations of cisplatin (cisPt) and CQ and pre-treated or not with E64 (10 μM) for 2 hours. Combination index (CI) was calculated as the ratio of the actual cell viability to the expected cell viability. Graph bars show the Mean±SEM from three independent experiments. *p<0.05 cells pre-exposed to E64 versus cells not pre-exposed to E64 and treated with the same concentration of cisplatin and chloroquine.
Fig 5.
Treatment with pan-caspase inhibitor partially rescues A549cisR cells against cisplatin and chloroquine.
(A, B) A549cisR cells were treated for 48h with chloroquine (CQ, 50 μM), cisplatin (cisPt, 25 μM) or the drug combination. Staurosporine, STP, (0.1 μM) represents the positive control. Subsequently, cell suspension was incubated with Annexin V-FITC/PI or FITC-FMK-specific substrates for apoptosis (A) and caspase (B) assays, respectively. Next, samples from each condition were mounted on the Cellometer imager to measure the fluorescence of which the values are plotted in graphs A and B. Graph bars represent the Mean±SEM from three independent experiments. *p<0.05 treatment vs. 1%FBS. **p<0.05 chloroquine/cisplatin vs. cisplatin only. (C) A549cisR cells were treated with chloroquine 100 μM, cisplatin 100 μM, pretreated or not with the pan-caspase inhibitor Z-VAD-FMK. Cell viability was measured with CellTiter-Blue®. The X-axis represents the percentage of cell viability at 48 hours (T48/T1) normalized to the negative control 1%FBS. Quantitative data are reported as Mean±SEM from three independent experiments. #p<0.05 1%FBS versus treatment, *p<0.05 cisplatin versus other treatments, **p<0.05 chloroquine/cisplatin versus chloroquine/cisplatin/Z-VAD-FMK.
Fig 6.
Chloroquine blocks autophagy in A549cisR and A549Pt cells.
(A) A549Pt and A549cisR cells were plated in 24-well plates and grown for 24 hours, then treated overnight with different chloroquine concentrations. Next, whole cell lysates were prepared and Western blot analysis was performed for LC3-II, p62 and α-tubulin. (B) Protein level was quantified by Western blot densitometry. Graph bars represent Mean±SEM from three independent experiments. *p<0.05 SF versus treatment.
Fig 7.
A549cisR cells exhibit increased autophagic flux following exposure to cisplatin, an effect that is less pronounced in parental cells.
(A, B) A549Pt and A549cisR cells were plated in 24-well plates and grown for 24 hours, then treated overnight with different cisplatin concentrations in 1% FBS media; whenever present, E64 (10 μM) was added for 2h prior to the cisplatin treatment. Next, whole cell lysates were prepared and Western blot analysis was performed for the indicated proteins. (C) Whole cell lysates of cells treated with different concentration of cisplatin without E64 were extracted and western blot was performed for p62 and α-tubulin. Levels of p62 and α-tubulin were determined by densitometry. The graph on the right panel represents the ratio of p62/α-tubulin normalized to the negative control 1% FBS in A549Pt and A549cisR cells exposed to cisplatin at concentration close to their corresponding IC50 (10 μM and 50 μM respectively). Quantitative data are reported as Mean±SEM from three independent experiments. *p<0.05 A549Pt versus A549cisR. (D) A549Pt and A549cisR cells were treated for 48 hours with CQ (50 μM), cisplatin (5 μM for A549Pt or 25 μM for A549cisR) or a drug combination. 1%FBS was used as a negative control. Following incubation with Cyto-ID green detection reagent, cell suspensions were mounted on a Cellometer imager and fluorescence was measured. Y-value depicts the fluorescence value of cyto-ID that stains autophagosomes. Graph bars represent Mean±SEM from three independent experiments. *p<0.05 1%FBS vs. treatment, **p<0.05 A549Pt vs. A549cisR and #p<0.05 chloroquine/cisplatin combination vs. cisplatin alone. (E) The histogram plot generated by the Cellometer in A549cisR cells following incubation with different treatment conditions.
Fig 8.
Inhibition of autophagy using ATG5 siRNA potentiates the activity of cisplatin against A549cisR cells.
(A) A549cisR cells were treated for 24 hours with 25 μM cisplatin prior to transfection with ATG5 siRNA or NT siRNA control. Following two days incubation, media was removed and cells were treated with different cisplatin concentrations for an additional 24 or 48 hours. At the end of the incubation, cell viability was measured using CellTiter-Blue® reagent. Experiments were performed in 1%FBS media. The X-axis represents the percentage of cell viability at 24 hours (T24/T1) normalized to the negative control 1%FBS. Error bars represent SEM from three independent experiment. p-value corresponding to the difference between ATG5 and NT siRNA-transfected cells was obtained by 2-way ANOVA. p-value corresponding to the individual difference between ATG5 and NT siRNA-transfected cells treated with the same concentration of cisplatin was calculated by the Sidak’s post-test analysis, with * annotating a p-value <0.05. (B, C) A549cisR cell lysates were prepared either immediately at the end of the siRNAs (ATG5 or NT) transfections or two days following the end of the transfection (annotated as 2d). Western Blot analysis was performed for the detection of ATG5, LC3, p62 and α-tubulin proteins.