In self-defence: hexokinase promotes voltage-dependent anion channel closure and prevents mitochondria-mediated apoptotic cell death

Abstract

In tumour cells, elevated levels of mitochondria-bound isoforms of hexokinase (HK-I and HK-II) result in the evasion of apoptosis, thereby allowing the cells to continue proliferating. The molecular mechanisms by which bound HK promotes cell survival are not yet fully understood. Our studies relying on the purified mitochondrial outer membrane protein VDAC (voltage-dependent anion channel), isolated mitochondria or cells in culture suggested that the anti-apoptotic activity of HK-I occurs via modulation of the mitochondrial phase of apoptosis. In the present paper, a direct interaction of HK-I with bilayer-reconstituted purified VDAC, inducing channel closure, is demonstrated for the first time. Moreover, HK-I prevented the Ca(2+)-dependent opening of the mitochondrial PTP (permeability transition pore) and release of the pro-apoptotic protein cytochrome c. The effects of HK-I on VDAC activity and PTP opening were prevented by the HK reaction product glucose 6-phosphate, a metabolic intermediate in most biosynthetic pathways. Furthermore, glucose 6-phosphate re-opened both the VDAC and the PTP closed by HK-I. The HK-I-mediated effects on VDAC and PTP were not observed using either yeast HK or HK-I lacking the N-terminal hydrophobic peptide responsible for binding to mitochondria, or in the presence of an antibody specific for the N-terminus of HK-I. Finally, HK-I overexpression in leukaemia-derived U-937 or vascular smooth muscle cells protected against staurosporine-induced apoptosis, with a decrease of up to 70% in cell death. These results offer insight into the mechanisms by which bound HK promotes tumour cell survival, and suggests that its overexpression not only ensures supplies of energy and phosphometabolites, but also reflects an anti-apoptotic defence mechanism.

The voltage-dependent anion channel-1 modulates apoptotic cell death

“…As observed with native VDAC1, E72Q-mVDAC1 is stable in a long-lived, fully opened state at À20 mV and showed different subconductance states at À40 mV (Figure 2a). Similar to the results obtained with rat liverpurified VDAC, 25 in all experiments performed in this study, at either À20 or À40 mV, addition of purified HK-I to recombinant native mVDAC1 induced channel closure to stable substates with a conductance reduced by 42.7574.8% at À20 mV and 5470.6% at À40 mV (mean7S.D., n ¼ 6 different experiments). Subsequent exposure to glucose-6-phosphate (G-6-P) reopened the HK-I-closed channel (Figure 2a).…”

“…25 Thus, the effect of HK-I on the channel activity of recombinant native and E72Q-mVDAC1 was considered next. The purity of the VDAC and HK-I preparations used is demonstrated in Figure 2b.…”

“…We have shown that HK-I and RuR directly interact with purified VDAC reconstituted into a PLB to induce channel closure. 7,10,12,25,31 Thus, it is probable that the effects of these reagents in preventing cell death are related to their interaction with VDAC. These reagents, however, are not specific for VDAC, and thus either the reagent or overexpression of HK-I could act via perturbation of cell physiology via other, VDAC-independent, mechanisms.…”

“…Recently, 25 we have demonstrated that HK-I induces VDAC closure, suppresses the release of cytochrome c and inhibits apoptotic cell death, thereby contributing to the survival advantage of tumor cells. The results presented here clearly demonstrate that a single mutation in VDAC molecule not only diminished HK-I binding to VDAC (Figures 1 and 2) but also prevented HK-I protection against cell death induced by VDAC overexpression ( Figure 5).…”

“…HK-I overexpression in leukemiaderived U-937 or vascular smooth muscle cells protects against staurosporine (STS)-induced apoptotic cell death. 25 HK-II has also been shown to inhibit Bax-induced cytochrome c release and apoptosis in HeLa cells. 24 In the present study, we have investigated the relationship between HK-I-and RuR-induced VDAC closure and protection against apoptotic cell death.…”