Mimulone-induced autophagy through p53-mediated AMPK/mTOR pathway increases caspase-mediated apoptotic cell death in A549 human lung cancer cells

Abstract

Anticancer properties and mechanisms of mimulone (MML), C-geranylflavonoid isolated from the Paulownia tomentosa fruits, were firstly elucidated in this study. MML prevented cell proliferation in a dose- and time-dependent way and triggered apoptosis through the extrinsic pathway in A549 human lung adenocarcinoma cells. Furthermore, MML-treated cells displayed autophagic features, such as the formation of autophagic vacuoles, a primary morphological feature of autophagy, and the accumulation of microtubule-associated protein 1 light chain 3 (LC3) puncta, another typical maker of autophagy, as determined by FITC-conjugated immunostaining and monodansylcadaverine (MDC) staining, respectively. The expression levels of LC3-I and LC3-II, specific markers of autophagy, were also augmented by MML treatment. Autophagy inhibition by 3-methyladenine (3-MA), pharmacological autophagy inhibitor, and shRNA knockdown of Beclin-1 reduced apoptotic cell death induced by MML. Autophagic flux was not significantly affected by MML treatment and lysosomal inhibitor, chloroquine (CQ) suppressed MML-induced autophagy and apoptosis. MML-induced autophagy was promoted by decreases in p53 and p-mTOR levels and increase of p-AMPK. Moreover, inhibition of p53 transactivation by pifithrin-α (PFT-α) and knockdown of p53 enhanced induction of autophagy and finally promoted apoptotic cell death. Overall, the results demonstrate that autophagy contributes to the cytotoxicity of MML in cancer cells harboring wild-type p53. This study strongly suggests that MML is a potential candidate for an anticancer agent targeting both autophagy and apoptotic cell death in human lung cancer. Moreover, co-treatment of MML and p53 inhibitor would be more effective in human lung cancer therapy.

Figure 1. The molecular structure of MML and its cytotoxic effects on various cancer cell lines.

(A) Molecular structure of the mimulone. Various human cancer cells, non-small cell lung cancer A549 (B), breast cancer MCF7 (C), colon cancer HCT116 (D) and osteosarcoma U2OS (E) cells were treated with the MML as indicated concentrations (0-80 µM) for 12 h or 24 h and then cell viability was measured by MTT assay. Bar graphs indicate the percentage of viability. (F) A549 cells were treated with the MML at various concentrations (0-80 µM) for 24 h and viable cells were counted by trypan blue staining. Live cells (non-stained cells) were calculated using hemocytometer and bar graph indicates the percentage of viable cells. All data were expressed as mean ± SEM of three independent experiments. *p<0.05 and **p<0.01 compared with control.

Figure 2. MML induces caspase-mediated apoptosis in A549 cells.

(A) A549 cells were treated with the MML as indicated concentrations (0–60 µM) for 24 h and then stained with FITC- conjugated Annexin V and PI. Apoptotic cells were measured by flow cytometer. (B) A549 cells were treated with 60 µM of MML for a time dependent manner. Apoptotic cells were then stained with Annexin V and PI and detected using flow cytometer. (C) Bar graph indicates the percentage of apoptotic cells (dose-dependent manner; top, time-dependent manner; bottom). Apoptotic population was evaluated by Annexin V positive, PI negative (AV+/PI-, white bar) and Annexin V and PI double positive (AV+/PI+, black bar) apoptotic cells. (D) Cells were treated with the MML as indicated concentrations (0–60 µM) for 24 h and Western blot analysis was performed to check apoptosis marker, caspase−3, −8, −9, Bid and PARP-1/2, respectively. GAPDH was used as loading control of Western blot analysis. (E) Cells were pretreated with or without each caspase inhibitor, Z-VAD-FMK (30 µM), Z-DEVD-FMK (30 µM), Z-IETD-FMK (30 µM) and Z-LEHD-FMK (30 µM) for 1 h and then treated with MML (60 µM) for 24 h. Apoptotic cells were stained with Annexin V and PI and then populations were detected by flow cytometer. (F) Cells were treated with caspase inhibitors and MML under the same conditions as the flow cytometric analysis and then cell viability was measured by MTT assay. Bar graphs indicate the percentage of cell viability (top) and the percentage of apoptotic cells (bottom), respectively. Apoptotic population was evaluated by Annexin V positive, PI negative (AV+/PI-, white bar) and Annexin V and PI double positive (AV+/PI+, black bar) apoptotic cells. All data were expressed as mean ± SEM of three independent experiments. *p<0.05 and **p<0.01 compared with control; ^# p<0.05 compared with MML-treated group.

Figure 3. MML treatment induces autophagy in A549 cells.

(A) A549 cells were treated with 60 µM of MML for 24 h and then morphological images were captured by phase contrast microscope (400×, P.C.). Arrows indicate the endogenous autophagosome-like vacuoles (left) and bar graph indicates the number of vacuoles per cell. Cells were treated with MML (60 µM, 24 h) and then cells were stained with MDC and LC3 antibody, respectively. MDC (bright color, middle) images were captured by fluorescence microscope and LC3 (green, right) fluorescent images were detected by confocal microscope (bar; 10 µm). Bar graph indicates the fluorescent intensity of LC3 FITC. (B) Cells were treated with MML (60 µM) for different time periods (0–24 h) and LC3 immunofluorescence staining was performed to detect autophagosomes. Nuclei were stained with DAPI. Images were captured using confocal microscope (bar; 10 µm). (C) A549 cells were treated with various concentrations of MML (0–60 µM) for 24 h. Western blot analysis was then performed with antibodies against ATG7, Beclin-1 and LC3, respectively. GAPDH was used as loading control. Bar graph indicates densitometry analysis of LC3-II/GAPDH ratio. A549 cells were treated with different concentrations of MML (0–60 µM) for 24 h (D) or 60 µM of MML for the different time course (E) and then immunoblot analysis was carried out using antibodies against p62 and LC3, respectively. GAPDH was used as loading control. (F) A549 cells were pre-incubated with or without chloroquine (CQ, 25 µM) for 1 h, then treated with MML (60 µM) for 24 h. Western blot analysis was performed using antibodies as indicated above. GAPDH was used as loading control of Western blotting. All data were expressed as mean ± SEM of three independent experiments. *p<0.05 and **p<0.01 compared with control.

Figure 4. Inhibition of autophagy by 3-MA suppresses MML-induced apoptotic cell death.

(A) A549 cells were pre-incubated with or without 3-MA (10 mM) for 3 h and then incubated with MML (60 µM) for 24 h. Cells were stained with MDC (bright color) or LC3 (green) antibody, respectively. Nuclei were stained with DAPI (blue). Fluorescent images were obtained using confocal microscope (bar; 10 µm). (B) Cells were pretreated with or without 3-MA for 3 h before treatment of MML (60 µM) for 24 h. Western blot analysis was performed with antibodies against LC3, PARP-1/2 and caspase-3, respectively. GAPDH was used as loading control. Bar graphs indicate the ratio of cleaved caspase-3/GAPDH and LC3-II/GAPDH, respectively. (C) A549 cells were pretreated 3 h with or without 3-MA and treated with MML (60 µM) for 24 h and cells were stained with FITC-conjugated Annexin V/PI and then measured by FACS. Bar graph indicates the percentage of apoptotic cells. Percentage of apoptotic cells was evaluated by Annexin V positive and PI negative (AV+/PI-, white bar) and Annexin V and PI double positive (AV+/PI+, black bar) apoptotic cells. (D) Cells were pretreated with or without CQ for 1 h and then incubated with MML for 24 h. Immunoblotting analysis was performed using antibodies as indicated before. Cells were treated with CQ and MML under the same conditions as mentioned before and then cell viability was evaluated by MTT assay (bottom). Bar graph indicates the percentage of cell viability. All data were expressed as mean ± SEM of three independent experiments. *p<0.05 and **p<0.01 compared with control; ^# p<0.05 compared with MML-treated group.

Figure 5. Knockdown of Beclin-1 gene suppresses MML-induced apoptotic cell death.

(A) Control and Beclin-1 knockdown cells were treated with MML (60 µM) for 24 h and then LC3 immunofluorescence staining was carried out for detecting autophagosomes. Nuclei were stained with DAPI. Images were captured by confocal microscope (bar; 10 µm). (B) Knockdown cells (shControl, shBeclin-1) were treated with MML (60 µM) for 24 h. Western blot analysis was performed with antibodies against Beclin-1, LC3, PARP-1/2 and caspase-3, respectively. GAPDH was used as loading control. Bar graphs indicate the ratio of LC3-II/GAPDH and cleaved caspase-3/GAPDH, respectively. (C) Control and Beclin-1 knockdown cells were treated with MML (60 µM) for 24 h and then cell viability was measured by MTT assay. Bar graph represents the percentage of cell viability. All data were expressed as mean ± SEM of three independent experiments. *p<0.05 and **p<0.01 compared with control; ^# p<0.05 compared with MML-treated group.

Figure 6. MML-induced autophagy is associated with the decrease in p53 levels.

(A) A549 cells were co-treated with or without pifithrin-α (PFT-α, 20 µM) and MML (60 µM) for 24 h. After that, immunoblot analysis was performed with antibodies against p53, LC3, PARP-1/2 and caspase-3, respectively. Bar graphs indicate the ratio of LC3-II/GAPDH and the cleaved caspase-3/GAPDH, respectively. (B) A549 cells were co-treated with or without PFT-α (20 µM) and MML (60 µM) for 24 h and then immunofluorescence staining was performed using LC3 (green) antibody. Nuclei were stained with DAPI (blue). Fluorescent images were obtained using confocal microscope (Bar; 10 µm). (C) Cells were co-treated with or without PFT-α (20 µM) and MML (60 µM) for 24 h and apoptotic cells were stained with FITC-conjugated Annexin V/PI and analyzed by flow cytometry. Bar graph indicates the percentage of apoptotic cells. Percentage of apoptotic cells was evaluated by Annexin V positive and PI negative (AV+/PI-, white) and Annexin V and PI double positive (AV+/PI+, black) apoptotic cells. All data were expressed as mean ± SEM of three independent experiments. *p<0.05 and **p<0.01 compared with control; ^# p<0.05 compared with MML-treated group.

Figure 7. MML induces autophagy through p53-mediated AMPK/mTOR pathway.

(A) A549 cells were treated with or without MML (60 µM) for 24 h and Western blot analysis was performed with antibodies as indicated above (p53, p-AMPK-α, AMPK-α, p-ACC, ACC, p-mTOR, mTOR, LC3, PARP-1/2, caspase-3). (B) A549 cells were treated with MML (60 µM) for the different time period as indicated above. Immunoblot analysis was performed with antibodies as indicated before. Bar graphs show the densitometry analysis of p53, p-AMPK and mTOR (bottom). Each graph indicates the ratios of p53/GAPDH, p-AMPK/AMPK, p-mTOR/mTOR and LC3 II/GAPDH, respectively. (C) A549 cells were treated with or without compound C (10 µM) for 1 h and treated with MML (60 µM) for 24 h. Immunoblotting analysis was performed with antibodies as indicated above. Cells were treated with same conditions as indicated before and then cell viability was measured by MTT assay. Bar graph represents the percent of cell viability (bottom). (D) Control cells and shp53 knockdown cells were treated with MML (60 µM) for 24 h and then Western blot analysis was performed with antibodies as indicated before. Cell viability was measured by MTT assay under the same conditions as mentioned above. Bar graph indicates the percent of cell viability (bottom). All data were expressed as mean ± SEM of three independent experiments. **p<0.01 compared with control; ^# p<0.05 compared with MML-treated group.