The hEag1 K+ Channel Inhibitor Astemizole Stimulates Ca2+ Deposition in SaOS-2 and MG-63 Osteosarcoma Cultures

Abstract

The hEag1 (Kv10.1) K⁺ channel is normally found in the brain, but it is ectopically expressed in tumor cells, including osteosarcoma. Based on the pivotal role of ion channels in osteogenesis, we tested whether pharmacological modulation of hEag1 may affect osteogenic differentiation of osteosarcoma cell lines. Using molecular biology (RT-PCR), electrophysiology (patch-clamp) and pharmacology (astemizole sensitivity, IC₅₀ = 0.135 μM) we demonstrated that SaOS-2 osteosarcoma cells also express hEag1 channels. SaOS-2 cells also express to KCa1.1 K⁺ channels as shown by mRNA expression and paxilline sensitivity of the current. The inhibition of hEag1 (2 μM astemizole) or KCa1.1 (1 mM TEA) alone did not induce Ca²⁺ deposition in SaOS-2 cultures, however, these inhibitors, at identical concentrations, increased Ca²⁺ deposition evoked by the classical or pathological (inorganic phosphate, Pi) induction pathway without causing cytotoxicity, as reported by three completer assays (LDH release, MTT assay and SRB protein assay). We observed a similar effect of astemizole on Ca²⁺ deposition in MG-63 osteosarcoma cultures as well. We propose that the increase in the osteogenic stimuli-induced mineral matrix formation of osteosarcoma cell lines by inhibiting hEag1 may be a useful tool to drive terminal differentiation of osteosarcoma.

Keywords: Ca2+ deposition; Kv10.1; SaOS-2 osteosarcoma cells; hEag1 potassium channel.

Figure 1

hEag1 and KCa1.1 currents in SaOS-2 cells (A) Activation kinetics of the current from different holding potentials. Whole-cell currents were recorded using two different holding potentials (Vh): −60 mV (black) and −120 mV (red). The depolarizing test pulses to +50 mV were 500 ms-long. (B) Inhibition of hEag1 currents by the open channel blocker astemizole. Astemizole (20 nM, green; 2 µM, blue) was administered in whole-cell configuration during 3000-ms-long test pulses to +50 mV from a holding potential of −60 mV, the inhibition was reversible (control: black; wash-out: brown) (C) Astemizole concentration–response curve. Six different doses of astemizole were tested as indicated. The remaining current fractions (RCF) were determined as I/I₀, where I is the from the current at equilibrium block measured at the end of the 3000-ms-long depolarizing pulses, and I₀ is the current in the absence of astemizole (see panel (B) for the demonstration of equilibrium block). (D) Paxilline-sensitive KCa1.1 current. Whole-cell current measurements were carried out using a 4.5 µM free Ca²⁺-containing intracellular solution and +100 mV test pulses (300 ms duration) to maximally activate the KCa1.1 K⁺ current. Traces were recorded in the absence (control, black), and in the presence of 1 µm paxilline (magenta) and following the wash-out (wash-out, green). The paxilline-sensitive current fraction was 17% in this cell.

Figure 2

Lack of hErg1 currents in SaOS-2 cells (A) hErg1 whole-cell current was recorded using hErg1 solutions (see Methods for details) in HEK 293 cells stably expressing hErg1 (Kv11.1) gene. The measurements were carried out using a +20 mV test pulse (1250 ms-long) and followed by a 2000-ms-long test pulse to −40 mV. The holding potential was −80 mV. These measurements were repeated in hEag1(Kv10.1)-transfected HEK cells (B) and SaOS-2 cells (C) using the same experimental conditions and voltage protocol.

Figure 3

Ca²⁺ deposition induced by the classical pathway of osteogenesis in the presence of TEA in SaOS-2 cells. Ca²⁺ deposits were stained using alizarin red S (see Materials and Methods). Alizarin red S-stained cultures were photographed with a microscope equipped with a digital camera. The photos are presented according to the time of the examination by the rows and the type of treatment by the columns. Column I.: non-differentiated cell cultures (Ctrl, no differentiation cocktail) on Day 3 (row (A)), Day 4 (row (B)) and on Day 5 (row (C)). Column II.: differentiated cells (Diff, classical induction pathway cocktail) after the osteogenic induction in the same order as above. Column III.: differentiated cells in the presence of TEA (1 mM TEA, classical induction pathway cocktail+1 mM TEA) in the same order as above. The hydroxyapatite containing bone nodules are recognizable with the red color.

Figure 4

Classical pathway-induced Ca²⁺ deposition in SaOS-2 cultures in the presence and absence of TEA and the hEag1 inhibitor astemizole. TEA (1 mM) and astemizole (2 µM) were added to the cell cultures at the beginning of osteogenic induction. The Ca²⁺ deposits were detected using alizarin red staining and quantification was achieved by dissolving alizarin red–calcium complexes in CPC and measuring absorbance (see Materials and Methods). Normalized Ca²⁺ deposition was calculated as A/A_PBS where A is the absorbance of a given sample and A_PBS is the average absorbance of differentiation-induced cells in the presence of PBS (vehicle control). Labels indicate SaOS-2 cell cultures treated with ctrl: no differentiation cocktail; diff+PBS: induced by the classical pathway of osteogenesis and added PBS as vehicle control for TEA; diff+1 mM TEA: induced by the classical pathway and treated with 1 mM TEA; diff+DMSO: induced by the classical pathway and added DMSO as vehicle control for astemizole; diff+2 µM astemizole: induced by classical pathway of osteogenesis and treated with 2 µM astemizole. Data were obtained on Day 3 (A) and Day 4 (B). Data are presented as mean ±SEM (numbers in the bars indicate the number of data points and in parentheses the number of independent experiments) and analyzed using the one-way RM ANOVA statistical test, * p < 0.05.

Figure 5

Pathological pathway (Pi)-induced Ca²⁺ deposition in SaOS-2 cultures in the presence and absence of TEA and the hEag1 inhibitor astemizole. Mineralization of SaOS-2 cells was induced via the pathological pathway using inorganic phosphate (Pi). Alizarin red–calcium complexes were determined using alizarin red assay on Day 3 (A) and Day 4 (B) (See Figure 4 and Methods for details). Normalized Ca²⁺ deposition was calculated as A/A_PBS where A is the absorbance of a given sample and A_PBS is the average absorbance of differentiation-induced cells in the presence of PBS (vehicle control). TEA and astemizole were added to differentiation-induced cultures in 1 mM and 2 µM concentrations, respectively, at the beginning of osteogenic induction. Labels indicate SaOS-2 cell cultures treated with ctrl: no differentiation induction by Pi; diff+PBS: differentiation induced by Pi and added PBS as vehicle control for TEA; diff+1 mM TEA: differentiation induced by Pi and treated with 1 mM TEA; diff+DMSO: differentiation induced by Pi and added DMSO as vehicle control for astemizole; diff+2 µM astemizole: differentiation induced by Pi and treated with 2 µM astemizole. Data are presented as mean ±SEM (numbers in the bars indicate the number of data points and in parentheses the number of independent experiments) and analyzed using the one-way RM ANOVA statistical test, * p < 0.05.

Figure 6

Delayed application of ion channel blockers to mineralization-induced SaOS-2 cells. Osteogenic differentiation of SaOS-2 cells was induced by the classical pathway and the ion channel blockers in the indicated concentrations were added 24 h after the induction. Alizarin red–calcium complexes were determined using alizarin red assay on Day 4 (A) and Day 5 (B) (See Figure 4 and Methods for details). Normalized Ca²⁺ deposition was calculated as A/A_PBS where A is the absorbance of a given sample and A_PBS is the average absorbance of differentiation-induced cells in the presence of PBS (vehicle control). Labels indicate SaOS-2 cell cultures treated with ctrl: no differentiation cocktail; diff+PBS: induced by the classical pathway of osteogenesis and added PBS as vehicle control for TEA; diff+1 mM TEA: induced by the classical pathway and treated with 1 mM TEA; diff+DMSO: induced by the classical pathway and added DMSO as vehicle control for astemizole; diff+2 µM astemizole: induced by classical pathway of osteogenesis and treated with 2 µM astemizole. Data are presented as mean ±SEM (numbers in the bars indicate the number of data points and in parentheses the number of independent experiments) and analyzed using One Way RM ANOVA statistical test.

Figure 7

K⁺ channel blockers do not increase cytotoxicity. Normalized absorbance was calculated as [A/A_PBS] × 100 (%), where A and A_PBS are the absorbances of a given sample and that of SaOS-2 cells in the presence of PBS control, respectively. Astemizole and TEA were added to the cell culture medium in the indicated concentrations. Absorbance determinations were carried out on samples harvested on Day 3 (gray columns) and Day 4 (hatched columns). Bar heights and error bars indicate mean ± SEM (n > 9). Data were analyzed using the one-way ANOVA statistical test, * p < 0.05. (A) LDH release assay. Labels indicate SaOS-2 cell cultures treated with ctrl+PBS: added PBS as vehicle control for TEA; ctrl+DMSO: added DMSO as vehicle control for astemizole; ctrl+1 mM TEA: treated with 1 mM TEA; ctrl+2 µM astemizole: treated with 2 µM astemizole; diff+DMSO: mineralization induced by the classical pathway and added DMSO as vehicle control for astemizole. (B) MTT assay. The same set of experiments as in panel A was repeated using MTT assay, treatments and variables are the same as above. Similarly, PBS treatment was used as vehicle control, results were normalized to SaOS-2 cells treated with PBS control. (C) SRB assay. The same set of experiments as in panels A and B were repeated using SRB assay, treatments and variables are the same as above. PBS treatment was used as vehicle control, results were normalized to SaOS-2 cells treated with PBS control.