Abstract
The purpose of the present study was to clarify roles of cytosolic chloride ion (Cl−) in regulation of lysosomal acidification [intra-lysosomal pH (pHlys)] and autophagy function in human gastric cancer cell line (MKN28). The MKN28 cells cultured under a low Cl− condition elevated pHlys 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 G0/G1 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 pHlys and decreased [Cl−]lys associated with inhibition of cell proliferation via induction of G0/G1 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.
Highlights
Autophagy has been found to be a self-nutrient-providing system for cell survival under starvation conditions [1,2,3], and an important system for degradation of miss-folded or long-lived proteins, and superfluous or damaged organelle such as mitochondria [4,5,6].Starvation-caused poverty of nutrients induces autophagy for cell survival via autophagy-mediated recycling of nutrients contained in cells themselves [1]
MKN28 cells were seeded into 25 cm2 flasks at a density of 2.5 9 105 cells/flask and incubated for 24 hrs in RPMI1640 medium (SigmaAldrich) supplemented with 5% foetal bovine serum (FBS) in a humidified incubator at 37°C with 5% CO2 in air
Cells were cultured for 48 hrs under six different conditions: (1) Roswell Park Memorial Institute (RPMI) medium as control, (2) RPMI medium with a low ClÀ concentration, (3) RPMI medium with a low pH, (4) RPMI medium containing bafilomycin A1, (5) RPMI medium containing EIPA and (6) RPMI containing dimethyl sulfoxide (DMSO) as solvent control for bafilomycin A1 and EIPA
Summary
Autophagy has been found to be a self-nutrient-providing system for cell survival under starvation conditions [1,2,3], and an important system for degradation of miss-folded or long-lived proteins, and superfluous or damaged organelle such as mitochondria [4,5,6].Starvation-caused poverty of nutrients induces autophagy for cell survival via autophagy-mediated recycling of nutrients contained in cells themselves [1]. Autophagy has been found to be a self-nutrient-providing system for cell survival under starvation conditions [1,2,3], and an important system for degradation of miss-folded or long-lived proteins, and superfluous or damaged organelle such as mitochondria [4,5,6]. Cells mainly produce amino acids via autophagymediated process by digesting their own proteins [1]. It has been recently suggested that autophagy process functions even under conditions with rich nutrition [7], and that impairment or activation of autophagy closely relates to pathogenesis of diverse diseases including Parkinson disease [6], diabetes mellitus [8], inflammatory disease such as Crohn disease [9] and cancer [10]
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