Fig 1.
AG–4 induces anti-proliferative effect.
(A) Time-dependent effect in U937 cells. U937 cells were treated with AG–4 (0–50 μM) for 24, 48 or 72 hours. Cell death was assessed by MTS-PMS assay. Each data point represents the mean±SEM of at least three independent experiments in duplicate. (B) Effect on other cells. Human cancer cell lines (Raji, MCF–7, HCT–15) and healthy human PBMC were incubated with AG–4 (0–50 μM) for 48 h. Cell death was assessed by MTS-PMS assay. Each data point represents the mean±SEM of at least three independent experiments in duplicate.
Fig 2.
AG–4 induces redox imbalance in U937 cells.
(A) Effect of AG–4 on ROS generation. U937 cells were treated with AG–4 (5.4 μM, 0–3 h), stained with CM-H2DCFDA and ROS was measured by flow cytometry. For the inhibition of ROS generation, cells were co-treated with NAC (2.5 mM) and AG–4 (5.4 μM, 3 h) and ROS was similarly quantified. Data represent mean GMFC±SEM of three independent experiments (***p<0.001, as compared with control). (B) Effect of antioxidant on survival of U937 cells. Cells were co-incubated with AG–4 (0–50 μM) and NAC (2.5 mM) for 48 h and MTS-PMS assay was performed. Each point corresponds to the mean ± SEM of at least three experiments in duplicate. (C) Effect of antioxidant on AG–4 induced apoptosis. Cells were treated with AG–4 (5.4 μM, 48 h) in presence or absence of NAC (2.5 mM). They were co-stained with Annexin V-FITC and PI followed by analysis for phosphatidylserine externalization using flow cytometry as described in materials and methods. Histograms represent percentage apoptotic cells and have been derived from at least three experiments (***p<0.001, compared to control cells; @@@ p<0.001, compared to only AG–4 treated cells). (D) Effect of AG–4 on level of non-protein thiols. U937 cells treated with AG–4 (5.4 μM, 0–3 h) were labelled with CMFDA and analysed for fluorescence. Data are expressed as mean GMFC±SEM of three independent experiments (*p<0.05, ***p<0.001, as compared with control).
Fig 3.
Involvement of mitochondrial pathway in AG–4 induced apoptosis.
(A) Loss of mitochondrial membrane potential. Cells were incubated with AG–4 (5.4 μM, 0–48 h) and loaded with JC–1 for flow cytometric analysis of mitochondria transmembrane potential. Data is a representative of three different experiments. (B) Effect on intracellular Ca2+. Cells preloaded with Fluo–4 AM were incubated with AG–4 (5.4 μM). The flow cytometric measurement of free cytosolic Ca2+ levels was seen as a fluorescent signal. Data are expressed as mean GMFC±SEM of three independent experiments (***p<0.001, as compared with control). (C) Altered expression levels of pro- and anti-apoptotic proteins. Whole cell extracts were made from control and AG–4 (5.4 μM, 0–48 h) treated cells and subjected to western blot analysis for Bcl–2, Bcl-xl, Bax, Bad. Analysis was confirmed with three different sets of extracts. β-actin served as a loading control. Histogram shows the time dependent decrease in Bcl–2/Bax ratio (*p<0.05, ***p<0.001, as compared with control). (D) Contribution of Bax in AG–4 induced cytotoxicity. Cells were transfected with siBax for 48 h followed by treatment with AG–4 (0–50 μM, 48 h). Cell viability was determined by MTS-PMS assay. Results are expressed as IC50 (mean ± SEM) from three independent experiments (***p<0.001, compared to only AG–4 treated cells). (E) Contribution of Bax in AG–4 induced apoptosis. Cells were transfected with siBax for 48 h followed by treatment with AG–4 (0–50 μM, 48 h). The percentage of apoptotic cells was determined by Annexin V and propidium iodide dual staining. Results are expressed as mean ± SEM from three independent experiments (***p<0.001, compared to control cells; @@@ p<0.001 compared to only AG–4 treated cells). (F) Effect on cytochrome c release. Cytoplasmic and mitochondrial fractions were prepared from control and AG–4 treated (5.4 μM, 0–48 h) cells using mitochondria/cytosol fractionation kit as described in materials and methods and cytochrome c was analyzed by Western blotting. Data shown are from one of the three experiments.
Fig 4.
Activation of Caspase -8, -9, -3 by AG–4.
Activity of caspase–8 (A), -9 (B), -3 (C) was measured in control and AG–4 treated (5.4 μM, 0–48 h) U937 cells using colorimetric tetrapeptide substrates. Results are expressed as mean±SEM from three independent experiments. (D) Effect of caspase inhibitor on cell viability. U937 cells were incubated with AG–4 (0–50 μM) along with pan caspase inhibitor, Z-VAD-fmk (20 μM) for 48 h and MTS-PMS assay was performed. Each point corresponds to the mean±SEM of at least three experiments in duplicate. (E) Effect of caspase inhibitor on AG–4 induced apoptosis. Cells were treated with AG–4 (0–50 μM, 48 h) with or without Z-VAD-fmk (20 μM). They were co-stained with Annexin V-FITC and PI followed by analysis using flow cytometry. Histograms represent percentage of apoptotic cells and have been derived from at least three experiments (***p<0.001, compared to control cells; @@@ p<0.001, compared to only AG–4 treated cells). (F) AG–4 enhances PARP cleavage. PARP cleavage was evaluated by Western blotting analysis in extracts of control and AG–4 (5.4 μM, 0–48 h) treated cells. Data was confirmed with three different sets of experiments.
Fig 5.
Effect of AG–4 on cell cycle progression and DNA degradation.
(A) Flow cytometric analysis of cell cycle phase distribution of U937 cells treated with AG–4 (5.4 μM, 0–48 h). The percentage of sub G0/G1 cells was assessed using propidium iodide staining. Histograms depict percentage of cells in various phases of cell cycle. The figure is one representative of three independent experiments. (B) Analysis of TUNEL positivity. Control and AG–4 treated (5.4 μM, 48 h) U937 cells were stained as described in Materials and Methods. Cells were examined under light microscope (100X). Presence of DNA nicking is indicated by arrows. The figure is a representative profile of at least three experiments.
Fig 6.
AG–4 alters expression levels of autophagic proteins and induces LC3 conversion.
Whole cell extracts were made from control and AG–4 (5.4 μM, 0–48 h) treated cells and subjected to western blotting for (A) Atg proteins (B) LC3 processing. For the inhibition of autophagy, cells were pre-treated with known autophagy inhibitor, 3-MA (10 mM, 4 h) followed by treatment with AG–4 (5.4 μM, 48 h). (C) Expression of LC3 mRNA. Cells were treated with AG–4 and LC3 mRNA expressionwere examined by quantitative real-time PCR. (D) U937 cells were treated with AG–4 (5.4 μM, 0–48 h) [with or without co-treatment with Bafilomycin A1 (50 nM), E64d (10 μg/ml) plus pepstatin A (10 μg/ml) or Chloroquine (5 μM)]. Lysates were similarly prepared and subjected to western blotting. β-actin was used to ensure equal loading. The figure is a representative profile of three experiments. (E) Control and AG–4 treated U937 cells were stained with Cyto-ID and quantified by flow cytometry.
Fig 7.
Detection of AVO in U937 cells.
Control and AG–4 treated (5.4 μM, 0–48 h) U937 cells in the presence or absence of 3-MA (10 mM, 4 h) were stained with AO (1 μg/ml, 15 min) followed by flow cytometry for quantification or fluorescence microscopy. (A) Microphotograph of AVO. Detection of green and red fluorescence in AO stained cells was performed using a fluorescence microscope (60 X). At least 20 microscopic fields were observed for each sample. (B) Flow cytometric quantification of AVO. Histograms represent the percentage of cells with AVO and have been derived from at least three experiments (*p<0.05, ***p<0.001, compared to control cells; @@@ p<0.001, compared to only AG–4 treated cells).
Fig 8.
(A) TEM microphotographs of autophagosomes. Representative electron micrographs of control and AG–4 treated (5.4 μM, 0–48 h) U937 cells. Black arrows indicate autophagosomes and black arrow heads indicate autophagolysosomes including residual digested material. The figure is a representative profile of three experiments. (B) Contribution of autophagy in AG–4 induced cytotoxicity. Cells were pre-treated with 3-MA (10 mM, 4 h) or transfected with siAtg 5 for 72 h followed by treatment with AG–4 (0–50 μM, 48 h). Cell viability was determined by MTS-PMS assay. Results are expressed as IC50 (mean ± SEM) from three independent experiments (***p<0.001, compared to only AG–4 treated cells). (C, D) Effect of anti-oxidant on AG–4 induced autophagy. Cells were treated with AG–4 (5.4 μM, 48 h) in presence or absence of NAC (2.5 mM) followed by flow cytometry for quantification of AVO or immunoblotting for Atg 5 expression levels. (C) Histograms represent the percentage of cells with AVO and have been derived from at least three experiments (***p<0.001, compared to control cells; @@@ p<0.001, compared to only AG–4 treated cells). (D) The figure is a representative profile of at least three experiments.
Fig 9.
AG–4 induced apoptosis and autophagy are dependent on each other.
(A, C) Effect of inhibitors on AG–4 induced Annexin V positivity and AVO formation. Cells were treated with Z-VAD-fmk (20 μM), 3-MA (10 mM, 4 h) or both Z-VAD-fmk and 3-MA or transfected with siAtg 5 or siBax followed by treatment with AG–4 (5.4 μM, 48 h). (A) Histograms depict percentage of apoptotic cells and are presented as the mean ± SEM from three independent experiments (***p<0.001, as compared with control; @@@p<0.001, as compared with only AG–4 treated cells). (C) Histograms depict percentage of cells with AVO and are presented as the mean ± SEM from three independent experiments (***p<0.001, as compared with control, @@@p<0.001, as compared with only AG–4 treated cells). (B, D) Effect of inhibitors on apoptotic and autophagic proteins. Cells were treated with Z-VAD-fmk (20 μM), 3-MA (10 mM, 4 h) or both Z-VAD-fmk and 3-MA or transfected with siBax or siAtg 5 followed by treatment with AG–4 (5.4 μM, 48 h). Whole cell lysates were prepared and subjected to immunoblot analysis using specific antibodies against Bax or Atg 5. Analysis was confirmed with three different sets of experiments. (E) Effect of simultaneous inhibition of apoptosis and autophagy on AG–4 induced cytotoxicity. Cells were treated with Z-VAD-fmk (20 μM) and 3-MA (10 mM, 4 h) followed by treatment with AG–4 (0–50 μM, 48 h). Cell viability was determined by MTS-PMS assay. Results are expressed as IC50 (mean ± SEM) from three independent experiments (***p<0.001, compared to only AG–4 treated cells).
Fig 10.
AG–4 inhibits PI3K/Akt/mTOR pathway.
(A) Control and AG–4 treated (5.4 μM, 0–48 h) cells were analyzed by western blot for phosphorylated and total PI3K expression. The results shown are representative of three experiments. Histograms represent densitometric analysis of relative phosphorylation levels of PI3K. (B) Control and AG–4 treated (5.4 μM, 0–48 h) cells were analyzed by western blot for Akt pathway proteins. Analysis was confirmed with three different sets of extracts. (C) Control and AG–4 treated (5.4 μM, 0–48 h) cells were analyzed by western blot for mTOR pathway proteins. The figure is a representative profile of three experiments. (D, E, G) Effect of inhibitors on AG–4 induced cytotoxicity, Annexin V positivity and AVO formation. Cells were treated with LY294002 (20 μM, 1 h); Rapamycin (20 nm, 1 h) or transfected with siAkt, simTOR followed by treatment with AG–4 (D) Cell viability was assessed by MTS-PMS assay. Data are presented as IC50 (mean ± SEM) from three independent experiments (*p<0.05, **p<0.01, as compared with only AG–4 treated cells). (E) Histograms depict percentage of apoptotic cells and are presented as the mean ± SEM from three independent experiments (***p<0.001, as compared with control; @@p<0.01 & @p<0.05, as compared with only AG–4 treated cells). (G) They were then analysed for AVO by AO staining. Histograms depict percentage of cells with AVO and are presented as the mean ± SEM from three independent experiments (***p<0.001, as compared with control; @@@p<0.001, @@p<0.01 & @p<0.05, as compared with only AG–4 treated cells). (F, H) Effect of inhibitors on apoptotic and autophagic proteins. Cells were treated with LY294002 (20 μM, 1 h), Rapamycin (20 nm, 1 h) or transfected with siAkt, simTOR followed by treatment with AG–4 (5.4 μM, 0–48 h). Whole cell lysates were prepared and subjected to immunoblot analysis using specific antibodies against Bax or Atg 5. Analysis was confirmed with three different sets of experiments.