Proton ATPase are involved in metastasis in human breast cancer cell
Cancer cells live in a more acidic environment than the normal tissue, but the intracellular pH (pHin) of tumors is more alkaline than in non-tumor cells. This indicates that cancer cells maintain a larger transmembrane pH gradient than normal cells. Our previous studies suggested that vacuolar type H'^-ATPases (V-H''ATPases) that normally reside in acidic organelles may be also expressed at the plasma membrane (pmVATPase) of highly metastatic human tumors. We hypothesize that pmV-ATPase plays a role in maintaining an alkaline intracellular environment favorable for growth, while maintaining an acidic extracellular environment favorable for invasion. Human breast cancer cell lines with distinct metastatic potential (MCF-7, MB-231, MB468 and MB- 435s) were used as experimental models. Immunocytochemical experiments showed that pmV-ATPases are located at the leading edge of metastatic human breast cancer cells. We employed spectral imaging and line scanning confocal microscopy (LSCM) to monitor the pHfn of discrete cellular regions, as well as fluorescence spectroscopy to determine proton fluxes in these cancer cell lines. These approaches are complementary in that they offer unsurpassed spatial, temporal and spectral resolution. Our data show that the pHin is more alkaline at the leading than at the lagging edge in the more invasive cells. The magnitude ofthe pHin gradient is larger in the highly than in the lowly metastatic cells. The proton fluxes are faster in the highly than in the lowly metastatic cells. Pharmacological and ion substitution experiments indicated that pmV-H"^ATPase expression was more elevated in the highly than in lowly metastatic cells. Invasion assays of these cancer cells lines show that bafilomycin Ai inhibits the invasive behavior. These novel observations suggest that the magnitude ofthe pH gradients is determined by pmVH^ ATPase, and pmV-H^ ATPase is involved in the invasion ofthe cancer cells.