α-Solanine induces ROS-mediated autophagy through activation of endoplasmic reticulum stress and inhibition of Akt/mTOR pathway

Abstract

α-Solanine is a glycoalkaloid found in species of the nightshade family including potato. It was primarily reported to have toxic effects in humans. However, there is a growing body of literature demonstrating in vitro and in vivo anticancer activity of α-solanine. Most of these studies have shown activation of apoptosis as the underlying mechanism in antitumor activity of α-solanine. In this study, we report α-solanine as a potential inducer of autophagy, which may act synergistically or in parallel with apoptosis to exert its cytotoxic effect. Induction of autophagy was demonstrated by several assays including electron microscopy, immunoblotting of autophagy markers and immunofluorescence for LC3 (microtubule-associated protein 1 (MAP1) light chain-3) puncta. α-Solanine-induced autophagic flux was demonstrated by additionally enhanced--turnover of LC3-II and--accumulation of LC3-specific puncta after co-incubation of cells with either of the autophagolysosome inhibitors--chloroquine and--bafilomycin A1. We also demonstrated α-solanine-induced oxidative damage in regulating autophagy where pre-incubation of cells with reactive oxygen species (ROS) scavenger resulted in suppression of CM-H2DCFDA (5 (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester) fluorescence as well as decrease in LC3-II turnover. α-Solanine treatment caused an increase in the expression of endoplasmic reticulum (ER) stress proteins (BiP, activating transcription factor 6 (ATF6), X-box-binding protein 1, PERK, inositol-requiring transmembrane kinase/endonuclease 1, ATF4 and CCAAT-enhancer-binding protein (C/EBP)-homologous protein) suggesting activation of unfolded protein response pathway. Moreover, we found downregulation of phosphorylated Akt (Thr308 and Ser473), mammalian target of rapamycin (mTOR; Ser2448 and Ser2481) and 4E-BP1 (Thr37/46) by α-solanine implying suppression of the Akt/mTOR pathway. Collectively, our results signify that α-solanine induces autophagy to exert anti-proliferative activity by triggering ER stress and inhibiting Akt/mTOR signaling pathway.

Figure 1

α-Solanine-induced autophagy in A549 cells. (a) Chemical structure of α-solanine. (b) Cytotoxic effect of α-solanine. Cells were treated with α-solanine for 48 h at indicated concentration and cell viability was measured by SRB assay. (c) A549 cells were treated with 10 μM α-solanine for indicated time points. Cell lysates were analyzed by immunoblotting for autophagic and apoptotic markers. (CL, cleaved; FL, full length). (d) Bar graphs representing densitometric quantification of the western blot data (mean±S.E.) of three independent experiments. (e) A549 cells were treated with 10 μM α-solanine and were fixed at different time points. Cells were then reacted with anti-LC3 antibody and were analyzed by confocal microscopy after incubation with Alexafluor 488 tagged anti-rabbit IgG. (f) Bar graph representing average number of typical LC3 puncta/cell. Data are means±S.E. from minimum 25 cells for each experiment; *P=0.2482, ***P<0.0001, compared with untreated control

Figure 2

α-Solanine-induced autophagic flux. A549 cells were treated with α-solanine for 24 h without or with 5 μM CQ (2 h pre-incubation). Cellular LC3 was analyzed by western blot assay (a and c) and by confocal microscopy (e). (b and d) Western blot analysis of A549 cell lysates treated with α-solanine (10 μM, 24 h) in presence or absence of 100 nM BafA1 (2 h pre-incubation) using anti-LC3 antibody

Figure 3

α-Solanine treatment caused autophagosome–lysosome fusion. A549 cells were treated with α-solanine at 10 μM for 24 h and immunostained with anti-LC3 (marker for autophagosome) and anti-LAMP2 (marker for lysosome) antibodies. Fluorescently labeled cells were analyzed under confocal microscope

Figure 4

Ultrastructural study of A549 cells treated with α-solanine. Representative electron microscopic images of A549 cells treated without (a and d) or with (b, c and e) α-solanine (10 μM, 24 h) showing accumulation of autophagic vacuoles (marked with arrows) in treated cells. (f) Electron micrograph showing cristae and electron-dense mitochondrial matrix in vehicle-treated cells. (g) Lack of mitochondrial electron density and cristae was evident after α-solanine treatment

Figure 5

α-Solanine-induced intracellular ROS to trigger autophagy. (a) A549 cells were treated with 10 μM α-solanine in presence or absence of 5 mM NAC. Cells were then stained with DCFDA and MitoSOX Red dye before being examined under confocal microscope. (b) Flow cytometric analysis of DCFDA-stained A549 cells for ROS accumulation. (c) Mean fluorescence intensities of at least 25 microscopically examined cells for each treatment were plotted and analyzed statistically (c); ***P<0.005. (d) Mean DCFDA fluorescence of α-solanine-treated A549 cells was analyzed by flow cytometry before and after exposure to NAC. (e) α-Solanine-treated cell lysates (with or without pre-incubation with NAC) were analyzed by western blot assay using specific antibodies. (f) Densitometric analysis of LC3-II levels relative to GAPDH. **P<0.05; ***P<0.005. (g) Increased lipid peroxidation in α-solanine-treated cells. (h) Effect of α-solanine on MMP of A549 cells. Cells were treated with 10 μM α-solanine, stained with 2 μM JC1 and examined under microscope. (i) α-Solanine caused release of cytochrome c from mitochondria. A549 cells were treated with 10 μM α-solanine for 24 h, immunostained with cytochrome c and examined under confocal microscope

Figure 6

α-Solanine activated UPR pathway. (a) A549 cells were exposed to 10 μM α-solanine and harvested at indicated time points. Cell lysates were subjected to western blot assay to determine activation of UPR pathway and band intensities were quantified. (b) ER calcium ion release in α-solanine (10 μM, 24 h) treated cells was investigated by fluorescence microscopy after staining with 5 μM Fluo-4AM. (c) A549 cells were transfected with non-targeting or PERK siRNA and treated with α-solanine for 24 h. The corresponding proteins were evaluated by immunoblotting and band intensities were quantified

Figure 7

α-Solanine inhibited Akt/mTOR signaling. (a) A549 cells were analyzed for Akt/mTOR activity by western blot assay using pathway-specific antibodies after incubation with or without α-solanine for 24 h. (b) Densitometric analysis on band intensity of corresponding proteins relative to loading-control. ^#P<0.05; *P<0.005. A549 cells were treated with α-solanine in presence or absence of 10 μM Akt1/2 inhibitor (c) and 10 nM rapamycin (d). Cell lysates were analyzed by immunoblotting with corresponding antibodies

Figure 8

Proposed mechanism of α-solanine-induced cell death