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

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.