Figure 1.
GNA inhibits growth and induces cell death in cancer cells.
A, The chemical structure of GNA. B, GNA inhibits growth and inhibits cell death in cancer cells. A549 and HeLa cells were treated with various concentrations of GNA for the indicated periods of time, and cell proliferation was analyzed by the MTT assay. C, Untreated cells (control) or cells treated with 3 µM GNA for 24 hours were directly observed under a phase contrast microscope (left) and cell vacuolization was quantitated (right); at least 600 cells were observed. Means ± SEM, n = 3.
Figure 2.
GNA increase autophagic markers in A549 and HeLa cells.
A, Representative images of MDC staining. A549 cells were treated with 3 µM GNA for 24 hours, then stained with 10 µM monodansylcadaverine (MDC) and observed under a phase contrast microscope. B and C, Acridine orange staining. Cells were exposed to the indicated concentrations of GNA for 24 hours, then stained with 1 µg/ml of acridine orange (B, C) and observed under a phase contrast microscope (B) or analyzed by flow cytometry (C). Cells that were treated with 50 µg/ml of rapamycin were used as a positive control. The results shown are representative of at least 2 independent experiments. D, Representative electron microscopy images of GNA-treated A549 cells. The cells were treated with 3 µM GNA for the indicated periods of time and analyzed by electron microscopy. Arrows indicate the vacuoles contacting with vesicles that were densely filled with multi-lamellar structures.
Figure 3.
GNA triggers the formation of autophagic markers in A549 and HeLa cell.
A, Effects of GNA on the distribution of GFP-LC3 punctuates. GFP-LC3/HeLa cells were treated with various concentrations of GNA or 50 µg/ml of rapamycin for 24 hours, then detected by fluorescence microscopy. The percentage of cells with at least 5 GFP-LC3 punctuate dots was calculated; at least 600 cells were observed, Means ± SEM, n = 3, ** means p<0.01, one-way ANOVA. B, Effects of GNA on LC3 protein. A549 cells were treated with various concentrations of GNA for 24 hours or 3 µM GNA for the indicated periods of time, then analyzed by western blotting using anti-LC3 antibodies. GAPDH protein was used as the loading control. The bar graph shows the band intensities of LC3-II relative to those of GAPDH. Mean± SEM, n = 3, *means p<0.05, ** p<0.01, one-way ANOVA. C, Effects of GNA on Beclin1 protein. A549 cells were treated with 3 µM GNA for the indicated periods of time, then analyzed by western blotting using anti-Beclin1 antibodies. GAPDH protein was used as the loading control. The bar graph shows the band intensities of Beclin 1 relative to those of GAPDH. Mean± SEM, n = 3, *means p<0.05, ** p<0.01, one-way ANOVA. D, Effects of GNA on P70S6K phosphorylation. A549 cells were treated with 3 µM GNA for the indicated periods of time, then analyzed by western blotting using anti-P70S6K and anti-p-P70S6K antibodies. GAPDH protein was used as the loading control. The bar graph shows the band intensities of p-P70S6K relative to those of GAPDH. Mean± SEM, n = 3, *means p<0.05, ** p<0.01, one-way ANOVA.
Figure 4.
GNA inhibits the fusion between autophagosomes and autolysosomes.
A, Effect of GNA on the flux of autophagy. GFP-LC3/HeLa cells were cultured in the absence (control) or presence of 3 µM of GNA for the indicated periods of time, and the level of cleaved GFP was analyzed by western blotting using an anti-GFP antibody; 100 mM trehalose was used as a positive control. GAPDH protein was used as the loading control. The bar graph shows the band intensities of GFP-LC3-II or cleaved GFP relative to those of GAPDH. Mean± SEM, n = 3, *means p<0.05, ** p<0.01, one-way ANOVA. B, Dose- and time-dependent effects of GNA on p62 degradation. A549 cells were treated with various concentrations of GNA for 24 hours or 3 µM GNA for the indicated periods of time, and the p62 protein levels were analyzed by western blotting. GAPDH protein was used as the loading control. The bar graph shows the band intensities of p62 relative to those of GAPDH. Mean± SEM, n = 3, ** p<0.01, one-way ANOVA. C, Inhibition of the fusion between autophagosomes and lysosomes upon treatment with GNA. GFP-LC3/Hela cells were treated with 3 µM GNA for the indicated periods of time and subsequently stained with 75 nM LysoTracker Red (LTR) for 15 min. Representative fluorescence microscopy images are shown. D, Inhibition of lysosome acidification upon treatment with GNA. A549 cells were treated with 3 µM GNA for the indicated periods of time or 500 µM of Bafilomycin A1 for 8 hours and subsequently stained with 1 mM LysoSensor Green DND-189 for 15 min. Representative fluorescence microscopy images are shown.
Figure 5.
The knockdown of Beclin 1 decreases GNA-induced cancer cell death.
A, RNA oligonucleotides against Beclin 1 (Beclin 1 siRNA) effectively repress Beclin 1 expression. A549 cells were transfected with siRNAs against Beclin 1 or negative control, and the protein level of Beclin1 was analyzed by western blotting. GAPDH protein was used as the loading control. The bar graph shows the band intensities of Beclin1 relative to those of GAPDH. Mean± SEM, n = 3, ** p<0.01, one-way ANOVA. B, A549 cells were transfected with siRNAs against Beclin 1 or negative control. The cells were then treated with 3 µM GNA for the indicated periods of time, stained with 2 µM Hoechst 33342 and observed under a fluorescent microscope; the percentage of dead cells was quantified by Trypan blue staining. Mean± SEM, n = 3, *means p<0.05, ** p<0.01, one-way ANOVA.
Figure 6.
GNA induces apoptosis-related changes in protein levels.
A, A549 cells were transfected with siRNAs against Beclin 1 or negative control. The cells were then treated with 3 µM GNA for the indicated periods of time. The relative protein level was analyzed by western blotting. GAPDH protein was used as the loading control. The bar graph shows the band intensities of indicated protein relative to those of GAPDH. Mean± SEM, n = 3, *means p<0.05, ** p<0.01, one-way ANOVA. B, A549 cells were transfected with siRNAs against Beclin 1 or negative control. The cells were then treated with 3 µM GNA for the indicated periods of time, and the cells were stained with annexin V/propidium iodide and subjected to flow cytometry analysis. The percentage in the upper left quadrant indicates the proportion of cells labeled with propidium iodide (dead cells), whereas the percentage in the upper right quadrant indicates the population of cells labeled by both annexin V and propidium iodide (late apoptotic and dead cells).
Figure 7.
GNA inhibited autophagy in vivo.
A549 cells were injected s.c. (2×106/mouse) into 30–40-day-old BALB/cA male nude mice. After established tumors had formed (∼50 mm3), the mice were i.v. injected with or without 16 mg/kg GNA twice a week for three weeks. At 24 hours after the last i.v. injection, the mice were sacrificed and the tumors were removed. A, Representative electron microscopy images obtained from the tumors. N, nucleus, E, empty vesicle, M, membranous body. Arrows indicate autophagosomes with contents. B, After preparing tissue homogenate from the tumors, the LC3 and p62 levels were analyzed by western blotting. The results shown are representative of at least 3 independent experiments.