Targeting AKT/mTOR and Bcl-2 for Autophagic and Apoptosis Cell Death in Lung Cancer: Novel Activity of a Polyphenol Compound

Abstract

Autophagic cell death (ACD) is an alternative death mechanism in resistant malignant cancer cells. In this study, we demonstrated how polyphenol stilbene compound PE5 exhibits potent ACD-promoting activity in lung cancer cells that may offer an opportunity for novel cancer treatment. Cell death caused by PE5 was found to be concomitant with dramatic autophagy induction, as indicated by acidic vesicle staining, autophagosome, and the LC3 conversion. We further confirmed that the main death induction caused by PE5 was via ACD, since the co-treatment with an autophagy inhibitor could reverse PE5-mediated cell death. Furthermore, the defined mechanism of action and upstream regulatory signals were identified using proteomic analysis. Time-dependent proteomic analysis showed that PE5 affected 2142 and 1996 proteins after 12 and 24 h of treatment, respectively. The crosstalk network comprising 128 proteins that control apoptosis and 25 proteins involved in autophagy was identified. Protein-protein interaction analysis further indicated that the induction of ACD was via AKT/mTOR and Bcl-2 suppression. Western blot analysis confirmed that the active forms of AKT, mTOR, and Bcl-2 were decreased in PE5-treated cells. Taken together, we demonstrated the novel mechanism of PE5 in shifting autophagy toward cell death induction by targeting AKT/mTOR and Bcl-2 suppression.

Keywords: AKT/mTOR; Bcl-2; apoptosis; autophagic cell death; autophagy; lung cancer; polyphenol; proteomics; stilbene compounds.

Figure 1

Effects of PE5 on cell viability and apoptotic cell death in non-small cell lung cancer. (A) PE5 structure and the plant specimen of Paphiopedilum exul. (B) All cells were treated for 24–48 h and analyzed by MTT assay. Graphs showing the percentages of cell viability. (C) Normal cells were similarly treated for 24–48 h and analyzed for the percentages of cell viability. (D) The IC50 and selectivity index (SI) in all cells were calculated for each cell type. (E–G) Cells were seeded and treated for 24 h before adding Hoechst 33342 to stain the cell nucleuses. Images were detected by using a fluorescence microscope, and the percentages of nuclear-fragmented were calculated. (H–I) Apoptotic and necrotic cells were determined using annexin V-FITC/PI staining with flow cytometry. (J–M) Apoptosis-related proteins were measured by western blot analysis. Cells were treated and were detected caspase3, PARP, cleaved caspase3, and cleaved PARP protein levels. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. The relative protein levels were calculated by densitometry. Data represent the mean ± SD (n = 3), (* p < 0.05, ** p < 0.01, compared with the untreated control), and (^# p < 0.05, compared with cisplatin).

Figure 2

Effect of PE5 on autophagy induction and autophagic flux in lung cancer cells. (A,B) the morphological changes of the cells were detected using a microscope and the number of vacuoles per cells were calculated after H460 and H292 cells were treated with PE5. (C) H460 cells were treated with PE5 and stained with monodansylcadaverine (50 μmol/L) and visualized by fluorescence microscopy (Olympus IX51 with DP70). (D) H460 cells were treated with 50 μM PE5 for 24 h and observed by transmission electron microscopy. Arrowheads indicate the autophagosomes and the arrows show the vacuoles. (E) After getting treated with indicated amounts of PE5 for indicated time periods (6–48 h), LC3 proteins were measured by western blot analysis. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. (F,G) Autophagy flux was determined in 50 μM PE5-treated cells in the presence of chloroquine (10 µM). The LC3 proteins were determined by western blot analysis. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. The cells were treated 50 μM PE5 with or without chloroquine (10 µM) for 24–48 h. The level of LC3 expression was analyzed by immunofluorescence staining. Data represent the mean ± SD (n = 3) and (* p < 0.05, ** p < 0.01, compared with the untreated control) (^# p < 0.05, compared with PE5-treated alone or different time).

Figure 3

Effect of PE5 on autophagy regulatory proteins and autophagic cell death. (A,B) H460 cells were treated with PE5 and detected for LC3, p62, ATG5, and ATG7 proteins by western blotting. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. (C) H460 cells were pre-treated with wortmannin (1 µM) (an autophagic inhibitor) and treated with PE5 for 24 h. Expression of LC3 was analyzed by immunofluorescence staining. (D) H460 cells were treated with PE5 in the presence of wortmannin (1 µM) or rapamycin (200 nM). Cell viability was analyzed by MTT assay. (E,F) Cells were transfected with siATG7 and treated with 50 µM PE5 for 24 h. Expression levels of each ATG and LC3 were assessed by western blot analysis. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. Cell viability was assessed using the MTT assay at 48 h. (G) H460 cells were pre-treated with wortmannin (1 µM) (an autophagic inhibitor) or Z-VAD-FMK (20 μM) (apoptosis inhibitor) and treated with PE5 for 24 h. Expression of PARP and cleaved PARP were analyzed by western blot analysis. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. Data represent the mean ± SD (n = 3) and (* p < 0.05, ** p < 0.01, compared with the untreated control) (^# p < 0.05, compared with PE5-treated alone and significantly different from siATG-transfected cells).

Figure 4

Proteomic analysis of PE5-treated cells. (A) Brief method involving proteomic analysis and mass spectrometric analysis. (B) Venn diagram (analyzed by jVenn software; http://jvenn.toulouse.inra.fr/app/example.html, access date: 1 January 2020) showing the different proteins between the control and PE5-treated cells at 12 and 24 h. (C) Gene ontology classification according to the biological process and molecular function terms of the upregulated and downregulated proteins using Panther software (Panther software; http://www.pantherdb.org/, access date: 20 January 2020).

Figure 5

Protein alteration in apoptosis and autophagy pathway. (A) Venn diagram was analyzed by jVenn software; http://jvenn.toulouse.inra.fr/app/example.html, access date: 16 June 2020. This diagram represented the number of different apoptosis and autophagy proteins affected by PE5. (B) Heatmap represented the levels of 128 proteins in regulating apoptosis signaling pathways in the control and 50 μM PE5 groups at 12 and 24 h using the MultiExperiment Viewer (MeV) in the TM4 suite software (http://mev.tm4.org/#/welcome, access date: 22 June 2020). (C) Heatmap represented the levels of 25 proteins in regulating autophagy pathway in the control and 50 μM PE5 groups at 12 and 24 h using the MultiExperiment Viewer (MeV) in the TM4 suite software (http://mev.tm4.org/#/welcome, access date: 22 June 2020).

Figure 6

Networks of functional protein–protein interactions of the top 20 downregulated proteins in response to PE5. (A,B) The functional protein–protein interactions of the top 20 downregulated proteins, and the significant nodes of each network were identified and rebuilt as a network of the signaling pathway in cancer using STITCH database version 5.0 (http://stitch.embl.de/, access date: 24 June 2020). (C,D) The key proteins p-PI3K, PI3K, mTOR, p-mTOR, AKT, p-AKT, Bcl-2, and Bax were determined by western blotting in H460 cells, and the immunoblot signal intensities were quantified by densitometry. Data represent the mean ± SD (n = 3), (* p < 0.05, ** p < 0.01, compared with the untreated control).

Figure 7

Effect of PE5 on patient-derived primary lung cancer cells. (A) The morphology of patient-derived primary cancer cell lines (ELC08 and ELC10) and their molecular characteristics. (B) Graphs showing the percentages of cell viability. All cells were treated for 24–48 h and analyzed by MTT assay. Apoptotic nuclei in the cells treated with PE5, determined by Hoechst 33342 staining and visualized by fluorescence microscopy. Cells were treated with PE5 and stained with monodansylcadaverine (50 μmol/L) and visualized by fluorescence microscopy (Olympus IX51 with DP70). (C) H460 cells were treated with PE5 in the presence of wortmannin (1 µM). Cell viability was analyzed by MTT assay. Data represent the mean ± SD (n = 3), (* p < 0.05, ** p < 0.01, compared with the untreated control).

Figure 8

Schematic display of PE5 inducing autophagic cell death and apoptotic cell death.