Metformin-Induced Apoptosis Is Mediated Through Mitochondrial VDAC1

Abstract

Background: Besides diabetes mellitus, metformin has been identified as a potential therapeutic agent for treating various other conditions that include various cancers, cardiovascular diseases, neurodegenerative diseases, and aging. In cancer, metformin increased apoptotic cell death, while inhibiting it in neurodegenerative diseases. Thus, different modes of metformin action at the molecular level have been proposed. Methods: In this study, we present the mitochondria and the VDAC1 (voltage-dependent anion channel) as a potential target of metformin. Results: Metformin induces VDAC1 overexpression, its oligomerization, and subsequent apoptosis. Metformin analogs phenformin and buformin at much lower concentrations also induce VDAC1 overexpression, oligomerization, and cell death. We demonstrate the interaction of metformin with purified VDAC1, which inhibited its channel conduction in a voltage-dependent manner. Metformin bound to the synthetic VDAC1-N-terminal peptide and binding to this domain was also found by its molecular docking with VDAC1. Moreover, we demonstrated metformin binding to purified hexokinases (HK-I) with a 400-fold lower metformin concentration than that required for cell death induction. In cells, metformin induced HK-I detachment from the mitochondrial VDAC1. Lastly, metformin increased the expression of NLRP3 and ASC and induced their co-localization, suggesting inflammasome activation. Conclusions: The results suggest that VDAC1 is a target for metformin and its analogs, and this is associated with metformin's adverse effects on many diseases.

Keywords: VDAC1; apoptosis; hexokinase; metformin; mitochondria.

Figure 1

Metformin induces VDAC1 overexpression, oligomerization, and apoptotic cell death. SH-SY5Y (A–E) or U-87MG (F–I) cells were incubated with the indicated concentrations of metformin for 24 or 48 h (SH-SY5Y) or 48 h (U-87MG) and subsequently analyzed for VDAC1 expression levels (A,F) by immunoblotting using anti-VDAC1-specific antibodies. Immunoblotting β-Actin was used as a loading control. VDAC1 levels are presented below the blot in relative units (RUs). (B) SH-SY5Y cells were incubated with or without metformin (75 mM), harvested at the indicated times, and subjected to real-time quantitative PCR of VDAC1 mRNA as described in the Materials and Methods section. SH-SY5Y (C,D) or U-87MG (G,H) cells were treated as described above and analyzed for VDAC1 oligomerization by incubation protein samples (1 mg/mL) with the cross-linking reagent EGS (100 μM), followed by immunoblotting using anti-VDAC1 antibodies. The positions of VDAC1 monomers, dimers, trimers, tetramers, and higher oligomers are indicated. The level of VDAC1 dimers was analyzed using ImageJ software (version 1.54v) (D,H) and presented relative to its levels in control cells. Samples were also analyzed for cell death (E,I) by PI staining and flow cytometry. The results represent the means ± SE; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), or p < 0.0001 (****).

Figure 2

Metformin induces apoptosis. (A,B) SH-SY5Y cells were treated with the indicated concentrations of metformin for 24 or 48 h, followed by Annexin V–FITC and propidium iodide (PI) staining and flow cytometric analysis to assess apoptotic cell death. (A) Representative flow cytometry histograms are shown for control and 75 mM metformin-treated cells at 24 and 48 h. The percentages of live cells, Annexin V-positive cells, Annexin V and PI double-positive cells, and PI-only positive cells are shown. (B) Quantification of apoptotic populations from three independent experiments as shown in (A). (C,D) SH-SY5Y cells were cultured on 13 mm glass coverslips, treated with 75 mM metformin for 24 h, fixed, and subjected to IF staining using anti-cleaved caspase-3 antibodies. (C) Representative images are shown. (D) Quantification of cleaved caspase-3 staining intensity, performed using ImageJ software (version 1.54v). The data represent the mean ± SE from three independent experiments. ** p < 0.01; **** p < 0.0001.

Figure 3

Metformin induces the elevation of cellular [Ca²⁺] and ROS production. SH-SY5Y cells were incubated for 48 h with the indicated concentrations of metformin. Cells were harvested, and the intracellular calcium ([Ca²⁺]i) levels (A,B) or mitochondrial superoxide levels (C,D) were measured using Fluo-4-AM or MitoSOX Red^TM, respectively, and flow cytometry. Representative FACS histograms (A,C) and quantification (B,D) are presented. The results represent the means ± SEM (n = 3).

Figure 4

Buformin and phenformin induce VDAC1 overexpression, oligomerization, and apoptotic cell death. (A) Chemical structures of metformin and its analogs buformin (1-butylbiguanide) and phenformin. (B–E) SH-SY5Y cells were incubated for 48 h with the indicated concentrations of metformin, buformin, and phenformin. The cells were analyzed for VDAC1 expression levels (B) by immunoblotting using anti-VDAC1-specific antibodies, with β-actin used as a loading control. VDAC1 levels are shown below the blot in relative units (RUs). Additionally, VDAC1 oligomerization was assessed by incubating protein samples (1 mg/mL) with the cross-linking reagent EGS (100 μM), followed by immunoblotting using anti-VDAC1 antibodies. The positions of the VDAC1 monomers, dimers, trimers, tetramers, and higher oligomers are indicated (C). The level of VDAC1 dimers was quantified using ImageJ software (D) and presented relative to its levels in control cells. Samples were also analyzed for cell death (E) by PI staining and flow cytometry. The results represent the means ± SE; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), or p < 0.0001 (****), ns—non-significant.

Figure 5

Metformin binds and modulates VDAC1 channel conductance. (A) SDS-PAGE of rat liver purified VDAC1 stained with Coomassie blue. (B) Purified VDAC1 was reconstituted into a planar lipid bilayer (PLB) prepared from soybean asolectin [43], and single-channel currents through VDAC1, in response to a voltage step from 0 to +10 mV or −10 mV, were recorded before and 5 min after the addition of metformin. Then, the voltage was switched to 60 mV for 1 min and then back to +10 mV or −10 mV, and the current passing through the channels was measured. The dashed lines indicate the zero-level current. (C) Fluorescently labeled purified VDAC1 (●; 162 nM) or the FITC-labeled VDAC1-derived N-terminus peptide (○; 1–26 amino acids, 2.5 μM) was incubated for 30 min at 37 °C with the indicated concentrations of metformin, and then, micro-scale thermophoresis (MST) was performed. The results are presented as a % of the maximal bound fraction. (D) VDAC1 protein with the N-terminal domain within and outside of the pore. (E) Metformin (in red) docked near the N-terminal of the VDAC1, forming hydrogen bonds with threonine 6 and aspargine124 residues. (F) The VDAC1-metformin complex equilibrated structure with bilayer of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), with water molecules surrounding the top and bottom of the bilayer. Metformin (green color) during the start of molecular dynamic (MD) simulation, and after 5 ns, showing metformin out of VDAC1.

Figure 6

Metformin binds to HK-I and detaches it from the mitochondria. (A) Coomassie blue-stained recombinant HK-I was purified as described previously [75]. (B,C) Fluorescently labeled purified HK-I was incubated for 30 min at 37 °C with increasing concentrations of metformin (5 to 200 μM) (B) or with glucose-6-phosphate (0.6–60 mM) (C), and then micro-scale thermophoresis (MST) was performed. The results are presented as a % of the bound fraction. (D) SH-SY5Y cells were seeded on 13 mm glass coverslips, transfected to express HK-I-GFP (24 h, 1 μg, green staining), and incubated for 4 h with and without the indicated concentration of metformin. They were fixed, stained with DAPI (blue staining), and visualized under confocal microscopy. Enlarged areas in dashed squares are indicated as (a–c), with the arrows pointing to diffused HK-GFP. The results represent the means ± SEM (n = 3).

Figure 7

Metformin induces the activation of the NLRP3 inflammasome. SH-SY5Y cells were seeded on 13 mm glass coverslips and then incubated for 48 h with the indicated concentrations of metformin, followed by co-immuno-staining with anti-NLRP3 (red staining) and anti-ASC (green staining) antibodies. Cells then were stained with DAPI (blue staining) and images were visualized by confocal microscopy (A), and the staining intensity of NLRP3 (B), ASC (C), and their co-localization (D), as analyzed using Image J software (version 1.54v) is presented. The results represent the means ± SE; p < 0.01 (**).

Figure 8

Proposed model for metformin inducing VDAC1 overexpression, oligomerization, and apoptosis. Metformin entering the cell by the transporter OCP1 and hydrophobic buformin and phenformin directly crossing the cell membrane all enhancing VDAC1 expression levels with the overexpression of VDAC1 shifting the equilibrium towards the VDAC1 oligomeric state. This mediates the release of apoptogenic proteins and mitochondrial DNA (mtDNA), leading to apoptosis and inflammation. Metformin detaches HK-I from VDAC1, further enhancing VDAC1 oligomerization, which results in apoptosis and inflammation.