Figure 1.
Gefitinib inhibits cell growth and induces appearance of MDC-labeled organelles in breast cancer cells.
(A) Example images obtained with IN Cell 1000 showing SKBR3 cells treated for 72 h with 10 µM gefitinib and stained in situ with DRAQ5 (stains nuclei in viable and dead cells), ETH (stains nuclei in dead cells with compromised plasma membrane) and MDC (stains acidic organelles). Based on differential staining and morphological features image recognition software identifies different cell populations. Left image: nuclear imaging masks shown in blue and red indicate viable and dead cells, respectively; cytoplasm of viable cells with MDC-labeled organelles is shown in yellow and cytoplasm of cells without MDC-labeled organelles is shown in light blue. Middle image: magnification of an area, as marked in the left image, showing transparent imaging masks outlining nuclei, cytoplasm and MDC-labeled organelles, as recognized by the HCA Investigator software; DRAQ5 staining is shown in blue, ETH staining is shown in red and MDC staining is shown in green. Right image: a high magnification image showing MDC-labeled organelles outlined in yellow within the cells’ cytoplasm; some of the MDC-labeled structures, indicated by the red arrows, represent multiple closely grouped organelles. (B) Representative images of indicated cells cultured in the presence of 0.5% DMSO (vehicle) or 10 µM gefitinib for 24 h (BT474) or 72 h (SKBR3, JIMT-1, MCF7-GFPLC3) stained in situ with DRAQ5 (blue), ETH (red) and MDC (green). Images in (A) and (B) were pseudo-colored and overlaid using the Investigator software. (C) Quantitation of different cell populations and autophagic organelles in BT474, SKBR3, JIMT-1 and MCF7-GFPLC3 cells by HCA. Cells were treated with vehicle or increasing concentrations of gefitinib for the indicated time. Numbers of viable cells in culture are normalized to vehicle-treated controls. Dead cells are shown as a percent difference in the content of dead (ETH-positive) cells between drug-treated minus vehicle-treated cultures. The proportion of viable cells with MDC-labeled organelles (puncta) in culture is shown as a percent difference in cells with >1 MDC-labeled organelle between drug-treated minus vehicle-treated cultures. Each data point represents a mean±SD from 3 replicate wells. HCA screenings were repeated 2 - 3 times for each cell type with consistent results; representative experiments are shown.
Figure 2.
TEM images of breast cancer cells treated with gefitinib or tamoxifen.
(A) SKBR3 cells treated for 48 h with 0.5% DMSO (vehicle) or 20 μM gefitinib. (B) MCF7-GFPLC3 cells treated for 48 h with vehicle, 20 μM gefitinib or 10 μM tamoxifen. High magnification images in (A) and (B) show details of the cytoplasmic organelles containing membranous and degraded cellular material in double or single membrane bound vesicles indicated by arrows. Representative images are shown.
Figure 3.
Early responses of breast cancer cells to gefitinib.
(A) MCF7-GFPLC3 cells treated for 3 h with vehicle (0 µM gefitinib) or indicated doses of gefitinib. Average GFPLC3, lysotracker red (LTR) and MDC TOA per cell (top graph) and the average distance between GFPLC3, LTR or MDC-labeled organelles (puncta) (bottom graph) were normalized to the respective measurements in vehicle-treated cells expressed as 1. (B) Representative TEM images of MCF7-GFPLC3 cells treated for 3 h with vehicle (top left image) or 10 µM gefitinib (top right image). A higher resolution image of the marked area in gefitinib-treated cells is shown on the bottom. Arrows indicate electron dense organelles engulfing double membrane vesicles, representing autolysosomes. M: mitochondrion. (C) SKBR3 cells treated with indicated doses of gefitinib for 3 h. The average MDC TOA/cell is normalized to vehicle-treated cells expressed as 1. Each data point in (A) and (C) represents a mean±SD from 3 replicate wells. These experiments were repeated 3 times with consistent results. Representative experiments are shown. (D) Western blot analysis of lysates derived from SKBR3 and BT474 cells treated for 3 h and from MCF7-GFPLC3 cells treated for 4 h with increasing concentrations of gefitinib (Gef). B-Actin was used as loading control.
Figure 4.
Gefitinib induces autophagic flux.
(A - D) Autophagic flux assays performed in SKBR3 cells. (A) Western blot analysis of p62 expression in lysates derived from cells treated for 3 h with vehicle (0 µM gefitinib) or increasing concentrations of gefitinib (Gef) (B) Western blot analysis of LC3 levels in lysates derived from cells treated for 3 h with increasing concentrations of gefitinib in the absence or presence of 5 nM bafilomycin A1 (BAF). (C) HCA of TOA in cells treated for 72 h with increasing concentrations of gefitinib in the absence or presence of 10 mM 3-MA added for the last 3 h of treatment. The results are normalized to the vehicle control expressed as 1. Each bar represents the mean±SD from 3 replicate wells. Asterisks indicate statistically significant differences (p<0.05) between cells treated with gefitinib or 3-MA alone and cells treated with 3-MA in the presence of gefitinib. The results shown are representative of two experiments. (D) Western blot analysis of LC3-I and LC3-II levels in lysates derived from cells treated for 24 h with vehicle, 5 µM gefitinib, 5 mM 3-MA and the combination of gefitinib and 3-MA at the corresponding concentrations. Representative blots in (A), (B) and (D) are shown. (E - G) Autophagic flux assays performed in MCF7-GFPLC3 cells. (E) Western blot analysis of autophagic markers in lysates derived from cells treated for 18 h with increasing concentrations of gefitinib. Cleaved GFP is marked as GFP. (F) Western blot analysis of cleaved GFP levels in lysates derived from cells treated with vehicle or 10 µM gefitinib in the absence or presence of 10 mM 3-MA for 3 h (top panel), in the absence or presence of 50 nM bafilomycin A1 (BAF) for 24 h (middle panel), and in the absence or presence of 10 mg/ml lysosomal inhibitors (LYI; pepstatin A and E-64d) added for the last hour of treatment (bottom panel). Tubulin was used as loading control. Representative blots in (E) and (F) are shown. (G) HCA data showing the average GFPLC3 TOA/cell in MCF7-GFPLC3 cells treated for 3 h with increasing concentrations of gefitinib in the absence or presence of indicated autophagy inhibitors added for the last hour of treatment. The results are normalized to vehicle control expressed as 1. Each data point represents the mean±SD from 3 replicate wells and the results shown are representative of two experiments. 3-MA was used at 5 mM, bafilomycin A1 (BAF) was used at 5 nM and LYI were used at 10 µg/ml. Asterisks indicate statistically significant differences (p<0.05) between cells treated with gefitinib or autophagy inhibitors alone and cells treated with autophagy inhibitors in the presence of gefitinib.
Figure 5.
Effects of siRNA mediated EGFR silencing on downstream signaling and autophagy.
(A) Following a transfection with EGFR siRNA, SKBR3 cells were treated for 72 h with vehicle or 5 µM gefitinib. (B) Following a double knockdown with the EGFR siRNA MCF7-GFPLC3 cells were treated with vehicle or 4 µM gefitinib for 24 h. Representative experiments are shown.
Figure 6.
Effects of BECN1 or ATG7 knockdown on autophagy and viability in SKBR3 and MCF7-GFPLC3 cells.
Following a knockdown with the indicated siRNA, cells were treated with vehicle or gefitinib (Gef) for 72 h. (A and D) Validation of BECN1 and ATG7 knockdown at the protein levels and analysis of LC3 and cleaved GFP by Western blotting. HCA analysis of TOA (B and E) and viability (C and F) in SKBR3 and MCF7-GFPLC3 cells following BECN1 or ATG7 knockdown (mean±SD, n = 3 wells). The average TOA and % viability are normalized to scrambled siRNA pretreated and vehicle-treated cells expressed as 1 or 100%, respectively. Cell viability was calculated as the proportion of viable cells in the total cell population. A single asterisk in (B), (E) and (F) indicates a statistically significant difference (p<0.05) between cells transfected with BECN1 or ATG7 and cells transfected with a scrambled non-silencing siRNA for the corresponding gefitinib concentration. A double asterisk in (C) indicates a statistically significant difference (p<0.05) between cells with knockdown genes and cells transfected with a scrambled non-silencing siRNA for a corresponding gefitinib concentration in addition to a statistically significant difference when compared to the vehicle-treated cells within each indicated knockdown.
Figure 7.
3-MA sensitized SKBR3 but not MCF7-GFPLC3 cells to cell death in the presence of gefitinib.
Flow cytometric analysis of apoptosis in SKBR3 (A and B) and MCF7-GFPLC3 (C and D) cells treated for 72 h with vehicle or 10 µM gefitinib (Gef), in the absence or presence of 5 mM 3-MA. Camptothecin (CPT) at 5 µM was used as an inducer of apoptosis (positive control). (A and C) Analysis of the sub-G0/G1 apoptotic cell fraction. The inserted histograms show representative DNA profiles of cells treated with the indicated agents where a sub-G0/G1 cell fraction is indicated with a marker. Arrows in (C) indicate S-G2/M cell cycle block. (B and D) Analysis of apoptosis in cells stained with Annexin V-Alexa647 and PI. The inserted representative dot plots show distribution of cell populations treated with the indicated agents where apoptotic Annexin V-positive cells are marked as “A” in a rectangular region. Bar graphs represent the data (mean±SD) from 3 independently stained samples and show fold change relative to the vehicle-treated controls expressed as 1. Asterisks indicate a statistically significant difference (p<0.05) between cells treated with the vehicle and indicated agents. A double asterisk indicates a statistically significant difference (p<0.05) between cells treated with gefitinib in the presence of 3-MA and single-agent treated cells in addition to a statistically significant difference when compared to the vehicle-treated cells. Representative experiments are shown.
Figure 8.
HCQ and bafilomycin A1 sensitize gefitinib-treated cells to cell death.
(A) BT474, JIMT-1 and MCF7-GFPLC3 cells were treated for 24 h, 72 h and 168 h, respectively, with vehicle (0 µM gefitinib) or increasing concentrations of gefitinib (Gef) in the absence or presence of HCQ (20 µM). The HCA (BT474 and JIMT-1) and flow cytometric data (MCF7-GFPLC3) are expressed as percentage of viable ETH- or PI-excluding cells, respectively, in the total population normalized to vehicle-treated cells expressed as 100%. (B) Western blot analysis of caspase activation in lysates derived from cells treated as indicated in (A). (C) SKBR3 cells were treated for 72 h and (D) MCF7-GFPLC3 cells were treated for 120 h with vehicle, 10 µM gefitinib (Gef), increasing concentrations of bafilomycin A1 (BAF) and the combination of gefitinib and bafilomycin A1 (Gef+BAF). Left graphs: absolute numbers of viable (trypan blue excluding) cells in cultures treated with the indicated agents relative to cultures treated with vehicle expressed as 100%. Middle graphs: flow cytometric analysis of the sub-G0/G1 cell fraction in cells treated with the indicated agents (mean±SD, n = 3). Right graphs: flow cytometric analysis of apoptosis and viability based on Annexin V-Alexa647 and PI staining. PI+AN-: necrotic cells; AN+: apoptotic cells; PI-AN-: viable cells. Each data point in (A), (C) and (D) represents a mean±SD from 3 replicate samples. Asterisks indicate a significant difference (p<0.05) between cells treated with gefitinib and the combination of gefitinib and HCQ in the same category. The results shown are representative of 2 experiments for each cell type. (E) Western blot analysis of caspase activation in lysates derived from SKBR3 and MCF7-GFPLC3 cells treated for 72 h and 120 h, respectively, with vehicle or gefitinib (Gef) in the absence or presence of 10 nM bafilomycin A1 (BAF).
Figure 9.
In vivo efficacy of HCQ alone and in combination with gefitinib.
Mice bearing JIMT-1 tumors were treated (oral gavage) with the indicated agents (n = 6 mice per treatment group). Increase in tumor volume was plotted relative to the volume on the first day of treatment (indicated by arrows) expressed as 100%. (A) Efficacy of HCQ used at different doses. (B) Efficacy of the gefitinib and HCQ combination. An asterisk represents a significant (p<0.05) difference between vehicle and combination treated animals on the last day of treatment.
Figure 10.
Gefitinib-induced autophagy is a reversible process.
(A) Representative images of MCF7-GFPLC3 cells treated for 3 h with vehicle or 10 µM gefitinib (top images) obtained with IN Cell 1000 Analyzer. After the indicated treatment, cell cultures were washed twice with medium and then incubated in the absence of drug for the following 5 h (bottom images). GFPLC3-labeled autophagosomes appear as green fluorescent puncta in cellular cytoplasm. (B) MCF7-GFPLC3 cells were treated for 3 h with vehicle (0 µM) or increasing concentrations of gefitinib and washed as described in (A). Quantitation of GFPLC3 TOA/cell by HCA in MCF7-GFPLC3 cells; the data shown are normalized to vehicle-treated cells expressed as 1 (mean±SD, n = 3 replicate wells). (C) Clonogenic assay performed in MCF7-GFPLC3 cells treated for 3 h with vehicle (Veh) or 10 µM gefitinib (Gef) followed by drug removal and incubation in media for 17 days. The results are expressed as % plating efficiency representing the proportion of cells that are able to form colonies. Bar graphs represent colony count from 3 wells (mean±SD). A representative experiment is shown.