Cytosolic chloride ion is a key factor in lysosomal acidification and function of autophagy in human gastric cancer cell.
Cytosolic chloride ion is a key factor in lysosomal acidification and function of autophagy in human gastric cancer cell.
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DOI：
10.1111/jcmm.12257
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发表时间：
2014-06
影响因子：
5.3
通讯作者：
Marunaka Y
中科院分区：
文献类型：
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作者：
Hosogi S;Kusuzaki K;Inui T;Wang X;Marunaka Y
本研究的目的是阐明胞浆氯离子（Cl−）在调节人胃癌细胞系（MKN 28）溶酶体酸化[溶酶体内pH（pHlys）]和自噬功能中的作用。在低Cl−条件下培养的MKN 28细胞通过降低胞浆Cl −浓度（[Cl−]c）而升高了pH lys并降低了溶酶体内Cl−浓度（[Cl−]lys），显示参与自噬功能的LC 3 II和p62的异常蓄积（自噬功能障碍），伴随着通过G 0/G1停滞抑制细胞增殖而没有诱导凋亡。我们还研究了直接修饰H+转运对溶酶体酸化和自噬的影响。应用巴弗洛霉素A1（一种V型H+-ATP酶抑制剂）或乙基异丙基阿米洛利[EIPA;一种Na+/H+交换器（NHE）抑制剂]可通过诱导G 0/G1期阻滞（类似于低Cl−条件下的培养）升高pHlys并降低[Cl−]lys，从而抑制细胞增殖。然而，与低Cl−条件不同，与低Cl −条件相比，化合物巴弗洛霉素A1或EIPA的应用诱导了与半胱天冬酶3和9增加相关的细胞凋亡，而[Cl−]c没有大幅降低。这些观察结果表明，[Cl−]c降低主要通过溶酶体内酸化障碍诱导的溶酶体功能障碍引起自噬功能障碍，而不引起凋亡。这是第一项研究表明，胞质Cl−是溶酶体酸化和自噬的关键因素。
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.