Chloride Ion Modulates Cell Proliferation of Human Androgen-independent Prostatic Cancer Cell

In the present study, we investigated if the intracellular Cl(-) affects cell growth and cell cycle progression of androgen-independent prostate cancer PC3 cells. PC3 cells cultured in a medium containing 113 mM Cl(-) for 96 h grew up 9-fold in cell number, while PC3 cells cultured in an 8 mM-Cl(-)-containing culture medium showed complete arrest of cell growth even after culture for 96 h. Exposure of cells to the 8 mM-Cl(-) culture medium diminished phosphorylation levels of Rb and cdc2, which are respectively key accelerators of transition from G(1) to S phase and G(2) to M phase in cell cycle progression. Culturing cells in the 8 mM-Cl(-)-containing culture medium upregulated the protein expression level of p21 (a CDK inhibitor) inhibiting transition of G(1) to S phase, and diminished the incorporation of 5-ethynyl-2'-deoxyuridine (EdU; a thymidine analogue) into DNA. These results suggest that cells cultured in the low Cl(-) medium prolonged the duration of all phases of the cell cycle (G(1), S, and G(2)/M), thereby abolishing overall cell cycle progression. Effects of culturing cells in the low Cl(-) culture medium on cell cycle progression would be mediated via a change in the intracellular Cl(-) concentration ([Cl(-)](i)), since [Cl(-)](i) was decreased under a low Cl(-) culture medium. To clarify this possibility, we studied effects of furosemide and bumetanide, Na+/K+/2Cl(-) cotransporter (NKCC) inhibitors, on proliferation of PC3 cells. Furosemide and bumetanide decreased [Cl(-)](i) and cell growth of PC3 cells. These results suggest that a change in [Cl(-)](i) would play a critical role in this growth mechanism.