Cytosolic chloride ion is a key factor in lysosomal acidification and function of autophagy in human gastric cancer cell

Abstract

The purpose of the present study was to clarify roles of cytosolic chloride ion (Cl ⁻) in regulation of lysosomal acidification [intra-lysosomal pH (pH _lys)] and autophagy function in human gastric cancer cell line (MKN28). The MKN28 cells cultured under a low Cl ⁻ condition elevated pH _lys and reduced the intra-lysosomal Cl ⁻ concentration ([Cl ⁻] _lys) via reduction of cytosolic Cl ⁻ concentration ([Cl ⁻] _c), showing abnormal accumulation of LC3II and p62 participating in autophagy function (dysfunction of autophagy) accompanied by inhibition of cell proliferation via G ₀/G ₁ arrest without induction of apoptosis. We also studied effects of direct modification of H ⁺ transport on lysosomal acidification and autophagy. Application of bafilomycin A1 (an inhibitor of V-type H ⁺-ATPase) or ethyl isopropyl amiloride [EIPA; an inhibitor of Na ⁺/H ⁺ exchanger (NHE)] elevated pH _lys and decreased [Cl ⁻] _lys associated with inhibition of cell proliferation via induction of G ₀/G ₁ arrest similar to the culture under a low Cl ⁻ condition. However, unlike low Cl ⁻ condition, application of the compound, bafilomycin A1 or EIPA, induced apoptosis associated with increases in caspase 3 and 9 without large reduction in [Cl ⁻] _c compared with low Cl ⁻ condition. These observations suggest that the lowered [Cl ⁻] _c primarily causes dysfunction of autophagy without apoptosis via dysfunction of lysosome induced by disturbance of intra-lysosomal acidification. This is the first study showing that cytosolic Cl ⁻ is a key factor of lysosome acidification and autophagy.