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. 2016 Apr 26;7(17):23468-81.
doi: 10.18632/oncotarget.8048.

Activation of autophagy flux by metformin downregulates cellular FLICE-like inhibitory protein and enhances TRAIL- induced apoptosis

Uddin Md Nazim  1 , Ji-Hong Moon  1 , Ju-Hee Lee  1 , You-Jin Lee  1 , Jae-Won Seol  1 , Seong-Kug Eo  1 , John-Hwa Lee  1 , Sang-Youel Park  1
Affiliations

Activation of autophagy flux by metformin downregulates cellular FLICE-like inhibitory protein and enhances TRAIL- induced apoptosis

Uddin Md Nazim et al. Oncotarget. .

Abstract

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily. TRAIL is regarded as one of the most promising anticancer agents, because it can destruct cancer cells without showing any toxicity to normal cells. Metformin is an anti-diabetic drug with anticancer activity by inhibiting tumor cell proliferation. In this study, we demonstrated that metformin could induce TRAIL-mediated apoptotic cell death in TRAIL-resistant human lung adenocarcinoma A549 cells. Pretreatment of metformindownregulation of c-FLIP and markedly enhanced TRAIL-induced tumor cell death by dose-dependent manner. Treatment with metformin resulted in slight increase in the accumulation of microtubule-associated protein light chain LC3-II and significantly decreased the p62 protein levels by dose-dependent manner indicated that metformin induced autophagy flux activation in the lung cancer cells. Inhibition of autophagy flux using a specific inhibitor and genetically modified ATG5 siRNA blocked the metformin-mediated enhancing effect of TRAIL. These data demonstrated that downregulation of c-FLIP by metformin enhanced TRAIL-induced tumor cell death via activating autophagy flux in TRAIL-resistant lung cancer cells and also suggest that metformin may be a successful combination therapeutic strategy with TRAIL in TRAIL-resistant cancer cells including lung adenocarcinoma cells.

Keywords: TRAIL; autophagy flux; cancer; metformin.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Metformin enhances TRAIL-induced apoptosis in lung adenocarcinoma cells
A549, Calu-3 and HCC-15cells were pretreated with metformin at different concentrations (0, 1, 2, and 4 mM) for 12 h followed by treatment with 200 ng/ml of TRAIL protein for an additional 2 h. A. and E. Cell morphology photographed under light microscope in A549 and Calu-3 Cells (×100); B. and G. Cell viability determined with crystal violet assay in A549 and HCC-15 Cells; C. and H. Bar graph showing the average density of crystal violet dye in A549 andHCC-15 Cells; D. and F. Cell viability determined with trypan blue dye exclusion assays in A549 and Calu-3 Cells. * p <0.05 ** p<0.01, *** p < 0.001: significant differences between control and treatment group; Met: metformin; TRAIL: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand.
Figure 2
Figure 2. Metformin induces autophagy flux and enhanced apoptosis mediated by TRAIL
A549 adenocarcinoma cells were pretreated with metformin at different concentrations (0, 1, 2, and 4 mM) for 12 h. A. and B. Cells were harvested and analyzed by Western blotting to determine the expression levels of DR-4, DR-5, LC3-II; C. Western blot and Representative immunocytochemistry of A549 cells after treatment with metformin for 12 h to determine p62 protein levels; D. TEM shows the ultrastructure of cells treated with 4 mM metformin for 12 h. Arrows indicate autophagosomes, including residual digested material and empty vacuoles; E. Ac-cas3and Ac-cas8 expression levels determined by western blot analysis. A549 cells were pre-treated with metformin for 12 h and then exposed to 200 ng/ml TRAIL for an additional 1 h. β-actin was used as loading control. *** p < 0.001: significant differences between control and treatment group; Met: Metformin; Ac-cas3: Activated caspase 3; Ac-cas8: Activated caspase 8; TRAIL: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand.
Figure 3
Figure 3. Metformin enhances TRAIL-induced tumor cell death is blocked by autophagy inhibitor
A549 adenocarcinoma cells were also pretreated with chloroquine for 1 h followed by treatment with metformin (4 mM) for 12 h. After that, cells were treated with 200 ng/ml of TRAIL protein for an additional 2 h. A. Cell morphology photographed under light microscope (×100); B. Cell viability measured by crystal violet assay; C. Bar graph showing average density of crystal violet dye; D. Treated cells measured by trypan blue dye exclusion assays. ** p<0.01, *** p < 0.001: significant differences between metformin and treatment group. Met: Metformin; TRAIL: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand; CQ: Chloroquine.
Figure 4
Figure 4. Autophagy inhibitor blocks TRAIL mediated tumor cell death by metformin via regulating autophagy flux
A549 adenocarcinoma cells were pretreated with chloroquine for 1h followed by treatment with metformin (4 mM) for 12 h. Cells were harvested and analyzed by Western blotting. A. and B. Expression levels of DR-4, DR-5, LC3-II, and p62; C, E. and F. Representative immunocytochemistry of A549 cells after treatment with metformin (12 h) and chloroquine to determine p62 protein levels and surface expression of DR-4 and DR-5 ; D. Ac-cas3 and Ac-cas8 expression levels based on western blot analysis. A549 adenocarcinoma cells were pretreated with chloroquine for 1 h followed by treatment with metformin (4 mM) for 12 h. After that, A549 cells were treated with 200 ng/ml TRAIL protein for an additional 1h. β-actin was used as loading control. Met: Metformin; Ac-cas3: Activated caspase 3; Ac-cas8: Activated caspase 8; TRAIL: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand; CQ: Chloroquine.
Figure 5
Figure 5. Metformin enhances TRAIL-induced tumor cell death is blocked by genetic autophagy inhibitor
A549 adenocarcinoma cells were pretreated with ATG5 siRNA-1and ATG5 siRNA-2 or negative control NC siRNA for 24 h followed by treatment with 4 mM metformin for 12 h with or without 200 ng/ml of TRAIL protein for an additional 2 h. A. Cell morphology photographed under light microscope (×100); B. Cell viability measured by crystal violet assay; C. Bar graph showing average density of crystal violet dye; D. Treated cells measured by trypan blue dye exclusion assays. ** p<0.01, *** p < 0.001: significant differences between metformin and each treatment group. Met: Metformin; TRAIL: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand; siATG5: ATG5 small interfering RNA; NC: Negative control.
Figure 6
Figure 6. Genetic autophagy inhibitor blocks TRAIL-induced tumor cell death by metformin via regulating autophagy flux
A549 adenocarcinoma cells were pretreated ATG5 siRNA or negative control (NC) siRNA for 24 h followed by treatment with 4 mM metformin for 12 h. Cells were harvested and subjected to western blot analysis. A. and B. Protein levels of DR-4, DR-5, LC3-II, p62 and ATG5; C. Protein level of p62 based on immunocytochemistry in A549 cells after treatment with ATG5 siRNA or NC siRNA and metformin for 12h; D. Protein levels of Ac-cas3 and Ac-cas8 based on western blot analysis. A549 adenocarcinoma cells were pretreated with ATG5 siRNA or NC siRNA for 24 h followed by treatment with metformin (4mM) for 12 h with or without 200 ng/ml TRAIL for an additional 1h. β-actin was used as loading control. Met: Metformin; Ac-cas3: Activated caspase 3; Ac-cas8: Activated caspase 8; TRAIL: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand; siATG5: ATG5 small interfering RNA; NC: Negative control.
Figure 7
Figure 7. Metformin enhances TRAIL-induced apoptosis through downregulation of c-FLIP
A549 cells were pretreated with metformin at different concentrations (0, 1, 2, and 4 mM) for 12 h followed by treatment with 200 ng/ml of TRAIL protein for an additional 1 h. A, B. and C. Cells were harvested and analyzed by Western blotting and immunocytochemistry to determine the expression levels of c-FLIP; D. c-FLIP expression levels based on western blot analysis. A549 adenocarcinoma cells were pretreated with chloroquine for 1 h followed by treatment with metformin (4 mM) for 12 h; E. Protein levels of c-FLIP based on western blot analysis. A549 adenocarcinoma cells were pretreated with ATG5 siRNA or NC siRNA for 24 h followed by treatment with metformin (4mM) for 12 h with or without 200 ng/ml TRAIL for an additional 1h. β-actin was used as loading control. Met: Metformin; c-FLIP :cellular FLICE–like inhibitory protein; CQ: Chloroquine; TRAIL: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand; siATG5: ATG5 small interfering RNA; NC: Negative control.
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References

    1. Aggarwal BB. Signalling pathways of the TNF superfamily: a double-edged sword. Nature reviews Immunology. 2003;3:745–756. - PubMed
    1. Pitti RM, Marsters SA, Ruppert S, Donahue CJ, Moore A, Ashkenazi A. Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. The Journal of biological chemistry. 1996;271:12687–12690. - PubMed
    1. Ashkenazi A, Pai RC, Fong S, Leung S, Lawrence DA, Marsters SA, Blackie C, Chang L, McMurtrey AE, Hebert A, DeForge L, Koumenis IL, Lewis D, Harris L, Bussiere J, Koeppen H, et al. Safety and antitumor activity of recombinant soluble Apo2 ligand. The Journal of clinical investigation. 1999;104:155–162. - PMC - PubMed
    1. Bellail AC, Qi L, Mulligan P, Chhabra V, Hao C. TRAIL agonists on clinical trials for cancer therapy: the promises and the challenges. Reviews on recent clinical trials. 2009;4:34–41. - PubMed
    1. Bhardwaj A, Aggarwal BB. Receptor-mediated choreography of life and death. Journal of clinical immunology. 2003;23:317–332. - PubMed

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