Volume-sensitive chloride channels are involved in cisplatin treatment of osteosarcoma

Abstract

Chemotherapy is the most common therapeutic strategy used to treat osteosarcoma. The present study aimed to investigate the effects of functionally activated chloride channels on cisplatin‑induced apoptosis of MG‑63 human osteosarcoma cells. An MTT assay and flow cytometry were used to detect proliferation and apoptosis of the cells, respectively. Live cell imaging was used to detect volume changes in response to treatment with cisplatin and/or chloride channel blockers. The effects of these treatments on chloride currents were also assayed using the patch‑clamp technique. The results of the present study indicate that chloride channel blockers may suppress cisplatin‑induced apoptosis. The MG‑63 cells cultured with cisplatin demonstrated an apoptotic volume decrease, as well as suppression of cell proliferation; which were reversed by co‑treatment with chloride channel blockers. These results suggest that cisplatin may activate chloride channels, and that channel activation is an early signal in the pathways that lead to cisplatin‑induced apoptosis and inhibition of proliferation in MG‑63 cells. In conclusion, these results indicate that chloride channels have an important role in cisplatin treatment of osteosarcoma.

Figure 1

Chloride channel blockers decrease the rate of cisplatin-induced apoptosis in MG-63 human osteosarcoma cells. The cells were treated with cisplatin (CDDP; 2 μg/ml) in Dulbecco’s modified Eagle’s medium alone, or in combination with chloride channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB; 100 μmol/l) or tamoxifen (20 μmol/l) for 72 h. The data represent the mean percentage of inhibition of apoptosis ± standard error of six experiments. ^**P<0.01, vs. the control.

Figure 2

Chloride channel blockers increase proliferation of cisplatin-treated MG-63 human osteosarcoma cells. The cells were cultured in 96-well culture plates (at a density of 2,500 cells/well) in control medium overnight (~16–18 h), and were then incubated in control medium (Ctrl) or medium containing cisplatin (CDDP; 2 μg/ml) alone, or in combination with 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB; 100 μM) or tamoxifen (20 μM) for 36 or 72 h. The relative cell numbers were detected by an MTT assay and the optical density (OD) of the cells was measured. The figure shows the comparison of cell growth to standardized OD values at different time points in MG-63 cells. The data represent the mean ± standard error of three experiments. ^**P<0.01, vs. the control.

Figure 3

Chloride channel blocker tamoxifen prevents cisplatin-induced decreases in cell volume of MG-63 human osteosarcoma cells. (A) Time-dependent changes in MG-63 cell volume following incubation in isotonic bath solutions, with or without cisplatin (CDDP; 2 μg/ml) and tamoxifen (20 μM) treatment. (B) Final cell volumes following treatment for 360 min with different bath solutions. Cell volume analysis was standardized to that of the control group. The data represent the mean ± standard error of 23–32 imaged cells. ^**P<0.01, vs. the control.

Figure 4

Cisplatin-induced chloride currents are inhibited by chloride channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and tamoxifen in MG-63 human osteosarcoma cells. (A) Typical time course of the chloride current activated by cisplatin (2 μg/ml) in isoosmotic bath solution (CDDP) alone or in combination with chloride channel blockers (B) NPPB (100 μM) and (C) tamoxifen (20 μM). (D) Typical current traces recorded in the control CDDP bath solutions. Typical timecourse of inhibition of cisplatin-induced chloride currents by the chloride channel blockers (E) NPPB (100 μM) and (F) tamoxifen (20 μM). (G) Current-voltage relationships recorded in the control isotonic bath solution (Control) and in the cisplatin isoosmotic bath solution (CDDP) (mean ± standard error, n=15). (H) Chloride currents of control (Ctrl), cisplatin isoosmotic (CDDP) and cisplatin isoosmotic solutions containing NPPB or tamoxifen (mean ± standard error of 5–15 cells; ^**P<0.01 vs. CDDP) at 80 mV. Cells were initially held at 0 mV, and voltage was then stepped to 0, ±40 and ±80 mV, repeatedly.

Figure 5

Anion permeability of the cisplatin-activated chloride channel. Anion selectivity of the cisplatin-activated chloride channel was determined by replacing the 70 mM NaCl of the isotonic solution with equimolar Na (X), where X represents the substituted anion I⁻, Br⁻ or gluconate. The anion permeability, relative to that of Cl⁻, was calculated from the shift in reverse potential. The order of anion permeability was Cl⁻=I⁻> Br⁻>gluconate acid ion. The data represent the mean ± standard error of six experiments. ^*P<0.05 and ^**P<0.01, vs. Cl⁻.