Particular sensitivity to calcium channel blockers of the fast inward voltage-dependent sodium current involved in the invasive properties of a metastastic breast cancer cell line

Abstract

1. A voltage-dependent sodium current has been described in the highly invasive breast cancer cell line MDA-MB-231. Its activity is associated with the invasive properties of the cells. The aim of our study was to test whether this current (I(Na)) is sensitive to three representative calcium channel blockers: verapamil, diltiazem and nifedipine. I(Na) was studied in patch-clamp conditions. 2. I(Na) was sensitive to verapamil (IC(50)=37.6+/-2.5 microM) and diltiazem (53.2+/-3.6 microM), while it was weakly sensitive to nifedipine. 3. The tetrodotoxin (TTX) concentration, which fully blocks I(Na) (30 microM), did not affect cell proliferation. Diltiazem and verapamil, at concentrations that do not fully block I(Na), strongly reduced cell proliferation, suggesting, regarding proliferation, that these molecules act on targets distinct from sodium channels. These targets are probably not other ionic channels, since the current measured at the end of a 500 ms long pulse in the voltage range between -60 and +40 mV was unaffected by verapamil and diltiazem. 4. We conclude that the sodium channel expressed in MDA-MB-231 cells is sensitive to several calcium channel blockers. The present study also underlines the danger of concluding to the possible involvement of membrane channel proteins in any phenomenon on the sole basis of pharmacology, and without an electrophysiological confirmation.

Figure 1

Representative effect of nifedipine on I_Na. I_Na was elicited by a 30 ms long depolarisation to −5 mV from a holding potential of −100 mV every 500 ms. I_Na amplitude was measured during each depolarisation and plotted as a function of time. Nifedipine was externally applied to the cell at the times and concentrations indicated at the top of the graph. The same results were obtained with three other MDA-MB-231 cells.

Figure 2

Effect of diltiazem on I_Na. (a) Representative example of the effect of three different diltiazem concentrations on the amplitude of I_Na, elicited by a 30 ms long depolarisation to −5 mV from a holding potential of −100 mV every 500 ms. I_Na amplitude was measured during each depolarisation and plotted as a function of time. Diltiazem was externally applied to the cell at the times and concentrations indicated at the top of the graph. (b) Dose–response curve of diltiazem inhibition of I_Na. Numbers above each point give the number of cells used at the given diltiazem concentration. The logistic fit gives an IC₅₀ of 53.2±3.6 μM and a Hill coefficient of 0.91±0.06.

Figure 3

Effect of verapamil on I_Na. (a) Representative example of the effect of three different verapamil concentrations on the amplitude of I_Na, elicited by a 30 ms long depolarisation to −5 mV from a holding potential of −100 mV every 500 ms. I_Na amplitude was measured during each depolarisation and plotted as a function of time. Verapamil was externally applied to the cell at the times and concentrations indicated at the top of the graph. (b) Dose–response curve of verapamil inhibition of I_Na. Numbers above each point indicate the number of cells used at the given verapamil concentration. The logistic fit gives an IC₅₀ of 37.6±2.5 μM and a Hill coefficient of 1.05±0.08.

Figure 4

Relative effects of I_Na blockers on proliferation of MDA-MB-231 cells. The concentrations of the blockers were 30 μM TTX, 100 μM diltiazem and 100 μM verapamil. Results are normalised as explained in Methods section. ^*P<0.05 when comparing blocker vs control. Above each bar is shown a representative normalised I_Na obtained with the same drug concentration as the respective proliferation test.

Figure 5

Effect of diltiazem and verapamil on end-pulse current (Iep). (a) Mean current–voltage relationship obtained from five cells before (PSS) and after application of 100 μM diltiazem. (b) Mean current–voltage relationship obtained from eight cells before (PSS) and after application of 100 μM verapamil. Cells were depolarised from −60 to +40 mV for 500 ms by 10-mV increments from a holding potential of −100 mV.