Warburg revisited: regulation of mitochondrial metabolism by voltage-dependent anion channels in cancer cells

Abstract

The bioenergetics of cancer cells is characterized by a high rate of aerobic glycolysis and suppression of mitochondrial metabolism (Warburg phenomenon). Mitochondrial metabolism requires inward and outward flux of hydrophilic metabolites, including ATP, ADP and respiratory substrates, through voltage-dependent anion channels (VDACs) in the mitochondrial outer membrane. Although VDACs were once considered to be constitutively open, closure of the VDAC is emerging as an adjustable limiter (governator) of mitochondrial metabolism. Studies of VDACs reconstituted into planar lipid bilayers show that tubulin at nanomolar concentrations decreases VDAC conductance. In tumor cell lines, microtubule-destabilizing agents increase cytoplasmic free tubulin and decrease mitochondrial membrane potential (ΔΨ(m)), whereas microtubule stabilization increases ΔΨ(m). Tubulin-dependent suppression of ΔΨ(m) is further potentiated by protein kinase A activation and glycogen synthase kinase-3β inhibition. Knockdown of different VDAC isoforms, especially of the least abundant isoform, VDAC3, also decreases ΔΨ(m), cellular ATP, and NADH/NAD+, suggesting that VDAC1 and VDAC2 are most inhibited by free tubulin. The brake on mitochondrial metabolism imposed by the VDAC governator probably is released when spindles form and free tubulin decreases as cells enter mitosis, which better provides for the high ATP demands of chromosome separation and cytokinesis. In conclusion, tubulin-dependent closure of VDACs represents a new mechanism contributing to the suppression of mitochondrial metabolism in the Warburg phenomenon.

Fig. 1.

Scheme of the role of the voltage-dependent anion channel in tubulin-dependent suppression of mitochondrial metabolism. Respiratory substrates (e.g., fatty acids, pyruvate, and glutamine), ADP, and Pi move from the cytosol across the MOM into the intermembrane space (IMS) via the VDAC and across the MIM into the matrix via numerous individual transporters, including the adenine nucleotide transporter (ANT), the acylcarnitine transporter of the carnitine shuttle (CS), the pyruvate carrier (PC), the glutamine carrier (GC), and the phosphate transporter (PT). Respiratory substrates feed into the tricarboxylic acid (TCA) cycle, which generates mostly NADH. Transfer of reducing equivalents (electrons) from NADH to oxygen by complexes I to IV produces electrogenic proton translocation from the matrix into the IMS, generating a proton electrochemical gradient. Return of protons into the matrix drives ATP synthesis from ADP and Pi by the F₁F₀-ATP synthase (complex V). ATP then exchanges for ADP via ANT and subsequently moves through the VDAC into the cytosol. We propose that high free tubulin levels in proliferating cancer cells act to inhibit VDAC and cause global suppression of mitochondrial metabolism in the Warburg phenomenon. PKA through phosphorylation of VDAC sensitizes to inhibition by tubulin.

Fig. 2.

Effect of free tubulin and protein kinase A on mitochondrial membrane potential in HepG2 cells. A, nocodazole (Ncz; 10 μM), a microtubule-depolymerizing agent that increases free tubulin, decreased ΔΨ_m in HepG2 human hepatoma cells, as shown by decreased fluorescence (visualized in pseudocolor) of the ΔΨ_m indicator TMRM. Paclitaxel (Ptx; 10 μM), a microtubule stabilizer that decreases free tubulin, blocked depolarization induced by nocodazole and instead promoted hyperpolarization (increase of TMRM fluorescence). B, activation of PKA with dibutyryl-cAMP (db-cAMP, 2 mM) decreased ΔΨ_m, as shown by decreased TMRM fluorescence. Subsequent addition of H89 (1 μM), a PKA inhibitor, reversed the depolarizing effect of db-cAMP and promoted mitochondrial hyperpolarization. C, PKA inhibition with H89 hyperpolarized mitochondria and prevented tubulin-induced depolarization after colchicine (Col; 10 μM), another microtubule-destabilizing agent. Additions in B and C were approximately 20 min apart.

Fig. 3.

VDAC knockdown decreases ΔΨ_m. siRNA knockdowns were performed against each of the three VDAC isoforms in HepG2 cells. ΔΨ_m assessed by TMRM fluorescence decreased after knockdown of each isoform. Knockdown of VDAC3 produced the greatest decrease of ΔΨ_m.