ASIC1a Inhibitor mambalgin-2 Suppresses the Growth of Leukemia Cells by Cell Cycle Arrest.

Although tyrosine kinase inhibitors have brought significant success in the treatment of chronic myelogenous leukemia, the search for novel molecular targets for the treatment of this disease remains relevant. Earlier, expression of acid-sensing ion channels, ASIC1a, was demonstrated in the chronic myelogenous leukemia K562 cells. Three-finger toxins from the black mamba (Dendroaspis polylepis) venom, mambalgins, have been shown to efficiently inhibit homo- and heteromeric channels containing the ASIC1a subunit; however, their use as possible antitumor agents had not been examined. In this work, using the patch-clamp technique, we detected, for the first time, an activation of ASIC1a channels in the leukemia K562 cells in response to an extracellular pH decrease. Recombinant mambalgin-2 was shown to inhibit ASIC1a activity and suppress the proliferation of the K562 cells with a half-maximal effective concentration (EC50) ~ 0.2 µM. Maximum mambalgin-2 inhibitory effect is achieved after 72 h of incubation with cells and when the pH of the cell medium reaches ~ 6.6. In the K562 cells, mambalgin-2 caused arrest of the cell cycle in the G1 phase and reduced the phosphorylation of G1 cell cycle phase regulators: cyclin D1 and cyclin-dependent kinase CDK4, without affecting the activity of CDK6 kinase. Thus, recombinant mambalgin-2 can be considered a prototype of a new type of drugs for the treatment of chronic myelogenous leukemia.

[Molecular and functional identification of sodium channels in K562 cells].

Modulations of ion channel activity underlie rapid changes in membrane transport of cations in various non-excitable cells. Previously, in smooth muscle cells, macrophages, lymphocytes, carcinoma and leukemia cell lines, non-voltage-gated sodium (NVGS) channels have been found. The activity of NVGS channels was shown to be critically dependent on the organization of actin cytoskeleton. The molecular identity of NVGS channels remains unclear. The present work is focused on molecular and functional identification of NVGS channels in human myeloid leukemia K562 cells. Degenerin/epithelial Na+ channels (DEG/ENaC) could be considered as a possible molecular correlates. Using RT-PCR, expression of alpha-, beta-, gamma-hENaC subunits in K562 cells was detected. Various modes of the patch-clamp method were employed to examine functional properties of sodium channels, specifically, to test the effect of amiloride on single channel and integral currents. Biophysical characteristics of NVSG channels were close to ENaC; the channels have unitary conductance 12 pS (145 mM Na+) and were impermeable for divalent cations (Ca2+ and Mg2+). We found that amiloride did not inhibit NVGS channels. Importantly, no amiloride-blockable sodium current was detected in the plasma membrane of K562 cells. Taken together, our observations suggest that amiloride-insensitive sodium channels in K562 cells belong to the ENaC family.

[Functional properties of sodium channels in cholesterol-depleted K562 cells].

The level of cellular cholesterol is known to determine functional compartmentalization of membrane lipids into ordered microdomains (rafts). Lipid rafts are assumed to play an essential role in the interactions between cell membrane and cortical cytoskeleton. As we have shown earlier, the activity of non-voltage-gated sodium channels in K562 human leukaemia cells is critically dependent on actin cytoskeleton organization. In the present paper, functional properties of sodium channels in K562 cells were examined after cholesterol-depleting treatment using methyl-beta-cyclodextrin (MbCD), selective acceptor of sterols. Single currents through sodium channels were recorded in cell-attached and inside-out mode experiments with the use of patch clamp technique. After incubation with MbCD (2.5 or 5.0 mM), an activation of sodium channels in response to cytochalasin B or D was observed in membrane fragments as well as in native cells. Characteristics of the channels in cholesterol-depleted K562 cells were similar to those in control; unitary conductance was 12 pS. Inside-out experiments with the use of globular actin have indicated that filament assembly on cytoplasmic membrane side causes an inactivation of sodium channels. These data imply that there is no association of sodium channels with cholesterol-rich membrane microdomains in K562 cells. Possible mechanisms underlying an interplay between plasma membrane and cortical cytoskeleton are discussed.