Voltage-gated Sodium Channel Activity Promotes Cysteine Cathepsin-dependent Invasiveness and Colony Growth of Human Cancer Cells

Abstract

Voltage-gated sodium channels (Na(V)) are functionally expressed in highly metastatic cancer cells derived from nonexcitable epithelial tissues (breast, prostate, lung, and cervix). MDA-MB-231 breast cancer cells express functional sodium channel complexes, consisting of Na(V)1.5 and associated auxiliary beta-subunits, that are responsible for a sustained inward sodium current at the membrane potential. Although these channels do not regulate cellular multiplication or migration, their inhibition by the specific blocker tetrodotoxin impairs both the extracellular gelatinolytic activity (monitored with DQ-gelatin) and cell invasiveness leading to the attenuation of colony growth and cell spreading in three-dimensional Matrigel-composed matrices. MDA-MB-231 cells express functional cysteine cathepsins, which we found play a predominant role ( approximately 65%) in cancer invasiveness. Matrigel invasion is significantly decreased in the presence of specific inhibitors of cathepsins B and S (CA-074 and Z-FL-COCHO, respectively), and co-application of tetrodotoxin does not further reduce cell invasion. This suggests that cathepsins B and S are involved in invasiveness and that their proteolytic activity partly depends on Na(V) function. Inhibiting Na(V) has no consequence for cathepsins at the transcription, translation, and secretion levels. However, Na(V) activity leads to an intracellular alkalinization and a perimembrane acidification favorable for the extracellular activity of these acidic proteases. We propose that Na(v) enhance the invasiveness of cancer cells by favoring the pH-dependent activity of cysteine cathepsins. This general mechanism could lead to the identification of new targets allowing the therapeutic prevention of metastases.

FIGURE 1.

Characterization of Na_V in breast cancer cells. A, reverse transcription-PCR experiments showing the mRNA expression of the Na_V1.5 α-subunit and of the β1, β2, and β4 subunits in MDA-MB-231 breast cancer cells. This figure is representative of four separate experiments. B, current-voltage relationship of the sodium current obtained from a holding potential of -100 mV (n = 80 cells). C, conductance (•) and availability (▪) voltage relationships obtained in the same cells as in B. D, effect of 30 μm TTX on a current elicited by a depolarization to -30 mV from a holding potential of -100mV.

FIGURE 2.

Involvement of the sodium influx through Na_V in invasion. A, effect of veratridine (50 μm) on a sodium current elicited by a depolarization to -5 mV from a holding potential of -100 mV. The inset emphasizes the veratridine-induced increase of the sustained current. B, effect of 30 μm TTX, alone or in combination with veratridine (Ver, 10 or 50 μm), on the MDA-MB-231 human breast cancer cells invasion. Statistically different: ^*, versus control condition; +, versus veratridine 10 and ×, versus veratridine 50 (n = 8 separate experiments). C, effect of external Na⁺ on MDA-MB-231 invasion. Panel a, effect of reducing external Na⁺ in culture medium, from 155 mm (Normo Na) to 45 mm (Low Na), on cell invasion (n = 4 separate experiments). Above each bar is shown the corresponding sodium current elicited from a holding potential of -100 to -5 mV in both external solutions (DMEM Normo Na and Low Na). Panel b, effect of TTX on cell invasion as a function of the extracellular culture medium: Normo Na or Low Na. Statistically different: ^*, versus control condition in absence of TTX; +, versus Normo Na condition (one symbol for p < 0.05; two symbols for p < 0.01, and three symbols for p < 0.001).

FIGURE 3.

Effect of transcriptional disruption of Na_V channels in MDA-MB-231 cells, using 20 nm siRNA against Na_V1. 5 (siNa_V1.5) versus control (siCTL). A, Na_V1.5 mRNA expression assessed by Q-PCR (n = 2 experiments). B, Na_V1.5 currents recorded at -5 mV from a holding potential of -100 mV in untransfected cells (CTL, n = 80) or in cells transfected with 20 nm control siRNA (siCTL, n = 10) or siRNA against Na_V1.5 (siNa_V1.5, n = 17). Representative currents elicited by a depolarization from -100 mV to -5 mV, recorded from non-transfected cells (CTL), siCTL, or siNa_V1.5 transfected cells, are shown on the right. C, Matrigel® invasion by cells transfected with siCTL or siNa_V1.5 and treated or not with 30 μm TTX, normalized to siCTL without TTX treatment (n = 4 separate experiments). The values are statistically different at p < 0.001 when compared with siCTL (A-C) or CTL (B).

FIGURE 4.

Effect of Na_V activity on MDA-MB-231 cell proliferation and colony growth. A, cell growth for 6 days on noncoated (squares) and Matrigel®-coated (circles) 24-well plates, in presence (gray) or not (black) of 30 μm TTX, expressed as 570 nm absorbance. B, cell sizes were assessed using the ImageJ 1.38I software, in control (CTL, n = 42) or TTX conditions (n = 42).

FIGURE 5.

Effect of Na_V activity on MDA-MB-231 colony growth. A, cancer cell colonies in a three-dimensional Matrigel® matrix in the presence or absence of 30 μm TTX. The pictures were taken with different objectives at the optical microscope: ×20 (top pictures) and ×40 (bottom pictures). The scale bars correspond to 100 μm. B, sizes of cancer cell colonies in control (CTL,n = 166) and TTX conditions (n = 274). C, number of cells escaping from colonies and invading the matrix were assessed in control (n = 2088) and TTX conditions (n = 245), using the ImageJ 1.38I software.

FIGURE 6.

Effect of Na_V activity on MDA-MB-231 cells pericellular gelatinolysis. A, confocal imaging of MDA-MB-231 cells grown for 24 h in a three-dimensional Matrigel® matrix containing DQ-Gelatin® in control conditions (CTL) or in presence of 30 μm TTX (TTX). The scale bars correspond to 20 μm. B, quantification of fluorescence intensity from DQ-gelatin cleavage in control conditions (CTL) or in presence of 30 μm TTX. ^**, p < 0.01 (n = 3).

FIGURE 7.

Proteolytic activities in MDA-MB-231 cells: involvement of cathepsins in the invasion process. A, enzymatic assays performed on total cell lysates (sodium-acetate buffer pH 5.5, 2 mm dithiothreitol, and 2 mm EDTA) in the presence of different fluorogenic substrates of Cat (see “Experimental Procedures”) (n = 4 separate experiments). B, effect of specific or broad spectrum (E-64) Cat inhibitors on cell invasiveness. All the Cat inhibitors were responsible for a significant (^***, p < 0.001) reduction of the cell invasiveness compared with the control condition (n = 8 separate experiments). C, effects of Cat inhibitors alone or in combination with 30 μm TTX on normalized Matrigel® invasion of MDA-MB-231 cancer cells (n = 8 separate experiments). D, effects of Cat inhibitors in presence or not of 3 μm TTX on Matrigel® invasion by H460 metastatic non-small cell lung cancer cells. Invasion through Matrigel® was normalized to the invasion in the absence of treatment (see “Experimental Procedures”). The effect of each inhibitor on invasion is compared with the same condition in presence of TTX. ^*, p < 0.05; ^***, p < 0.001.

FIGURE 8.

Regulation of cathepsins by Na_V. A, quantitative PCR showing the relative effect of the TTX treatment on the mRNA expression of Na_V1.5, Cat, and their physiological inhibitors. The results are expressed as percentages of control condition without TTX (n = 4 separate experiments). B, effect of TTX treatment on the membrane-associated Cat activity of intact cells. Cat titration was performed by using E-64. Alternatively, CA-074 was used to determine CatB concentration (see “Experimental Procedures”) (n = 6 separate experiments). C, effect of TTX treatment on the Cat activity measured in concentrated supernatants (Buffer: 0.1 m sodium acetate, pH 5.5, containing 2 mm dithiothreitol, and 2 mm EDTA). Enzymatic assays were performed using Z-FR-AMC as a substrate (n = 6 separate experiments). D, Western blots for the Cat B and S in total cell lysates or in concentrated supernatants (×100) from MDA-MB-231 grown for 24 h on Matrigel® in the presence or absence of 30 μm TTX. White and black arrowheads indicate proforms and mature Cat, respectively. These WB are representative of seven and four separate experiments for cathepsin B and S, respectively.

FIGURE 9.

Regulation of internal and perimembrane pH by Na_V activity. A, effect of TTX on intracellular pH (pH_i) measured using BCECF (n = 10 cells for TTX and n = 9 cells for control condition). B, effect of Na_V activity on the perimembrane pH (pH_m) measured using the outer membrane leaflet pH-sensitive probe DHPE. ^***, p < 0.001 (n = 8-10 cells).