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 ␤-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 (ϳ65%) 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.Breast cancer is the most common female cancer and the primary cause of death in women by cancer worldwide (1).Deaths occur primarily after the development of metastases. The invasive potential of malignant cells is mainly linked to their capacity to degrade basement membranes and extracellular matrices by various proteases. Studies have mostly focused on metalloproteases, including matrix metalloproteinases and the closely related ADAMs (a disintegrin and metalloproteinase) and ADAMTs (a disintegrin and metalloproteinase with thrombospondin motifs) (2), that are key factors in growth, invasion, and angiogenesis, and to a lesser extent on aspartyl and serine proteases. Pharmaceutical inhibitors of matrix metalloproteinases have been developed, but the results from clinical trials with these drugs have so far been disappointing (3,4

Studies using inhibitors that reportedly discriminate between cathepsin B and related lysosomal cysteine proteinases have implicated the enzyme in a wide range of physiological and pathological processes. The most popular substance to selectively inhibit cathepsin B in vivo is CA-074Me, the methyl ester of the E-64 derivative CA-074. However, we now have found that CA-074Me inactivates both cathepsin B and cathepsin L within murine fibroblasts. In contrast, exposure of these cells to the parental compound CA-074 leads to the selective inhibition of endogenous cathepsin B, while intracellular cathepsin L remains unaffected. These results indicate that CA-074 rather than CA-074Me should be used to specifically inactivate cathepsin B within living cells.

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