Bioelectricity and cell growth of normal and tumor mammary epithelial cells
Figure 1a compares the resting membrane potential (Em) in HMEC, MCF7, and MDA-MB-231 cells. MCF7 cells are 30.4 mV more depolarized in comparison to HMEC cells (Em_MCF7 = −36.5 ± 5.4 mV, Em_HMEC = −66.9 ± 4.4 mV, n = 8, p < 0.005). MDA-MB-231 cells are 27.3 mV more positive compared to HMEC (Em_MDA-MB-231 = −39.5 ± 5.4 mV, Em_HMEC = −66.9 ± 4.4 mV, n = 8, p < 0.001). HMEC at days 1 and 5 are shown in Fig. 1b and c, respectively. Compared to day 1, cells grew 1.47 ± 0.16-fold in HMEC (n = 4, p < 0.05), 10.33 ± 2.19-fold in MCF7 (n = 4, p < 0.05), and 19.93 ± 3.83-fold in MDA-MB-231 (n = 4, p < 0.05) (Fig. 1d).
Stimulation of breast cancer cell growth by membrane depolarization
The normal potassium concentration in standard cell culture medium (DMEM) is 5 mM. Altering potassium concentration in DMEM changes membrane potential, which affected growth of MDA-MB-231 cells (Fig. 2). We started cell culture with approximately the same number of cells on day 1 under different potassium concentrations (Fig. 2a, 5 mM, Fig. 2c, 50 mM). After 5 days, cells grew significantly more in culture containing 50 mM K+ ions (Fig. 2d) than in culture containing 5 mM K+ (Fig. 2c). On average, the growth rate was increased by 1.89 ± 0.07-fold in 50 mM K+ medium than in 5 mM K+ medium (growth rate was normalized to 5 mM K+, n = 3, p < 0.01) after 5 days of culture (Fig. 2e).
Voltage-gated potassium channels control the plasma membrane potential and blocking these channels induces membrane depolarization [20]. We used a non-selective potassium channel blocker, TEA, to study the effect of depolarization on growth of MCF7 cells. In the absence of TEA, cells grew 225 % after 4 days in culture (Fig. 3a, b) (n = 128.2 ± 12 at day 0, n = 416.7 ± 22 at day 4, p < 0.01, n = 3). In the presence of 1 mM TEA, cells grew 4.78 ± 0.50 fold at day 4 compared to day 0 (normalized) (Fig. 3c–e, n = 3, p < 0.01). These results indicate that changes in membrane potential have significant effects on growth rate of breast cancer cells.
Calcium as a key modulator in breast cancer cell growth
To understand how depolarized membranes can stimulate growth of breast cancer cells, we studied the effect of calcium ions on cell proliferation. After 5 days, MCF7 cells barely grew in the absence of Ca2+ ions in the culture medium (Fig. 4b) compared with cells growing to nearly 100 % confluence in medium that contains 2 mM Ca2+ ions (Fig. 4a). The average cell population growth in the absence and presence of Ca2+ ions is shown in 4C for MCF7 and in 4D for MDA-MB-231, respectively. Cell numbers at day 1 were normalized (Fig. 4c, d). In normal DMEM containing Ca2+, MCF7 cells grew 2.5 ± 0.07-fold at day 2, 4.56 ± 0.13 at day 3, 9.55 ± 0.28-fold at day 4, 20.78 ± 0.60-fold at day 5, and 34.83 ± 1.01-fold at day 6; and MDA-MB-231 cells grew 2.03 ± 0.06-fold at day 2, 3.87 ± 0.11-fold at day 3, 7.66 ± 0.22-fold at day 4, 13.71 ± 0.40-fold at day 6, and 19.93 ± 0.58-fold at day 6. In the absence of external Ca2+, cells barely grew (Fig. 4c, dark bars) or grew in a significantly slower rate compared to that with Ca2+ (Fig. 4d, dark bars). These results indicate the essential requirement of external Ca2+ ions during growth of breast cancer cells.
External Ca2+ ions can be blocked from entering the cell specifically through voltage-gated calcium channels [11] with verapamil. Figure 5a shows MDA-MB-231 cells grown in normal medium without verapamil. After 1 day of incubation with 10 μM verapamil, cell number dramatically decreased (Fig. 5b). Figure 5c shows the growth of MDA-MB-231 cells at day 2 and 3 under different concentrations of verapamil. Compared to the absence of verapamil (0 μM), verapamil inhibited cell growth by 74 % at 10 μM and 92 % at 20 μM, respectively (n = 3, p < 0.01). Figure 5d shows inhibited growth of MCF7 cells by verapamil. At day 3, verapamil inhibited cell growth by 49 % at 10 μM and 85 % at 20 μM, respectively (n = 3, p < 0.05).
Mechanism of verapamil-induced death in breast cancer cells
One common cellular mechanism associated with calcium in cell death is the caspase signaling pathway [15]. Among the many caspases in breast cancer cells, we focused on caspase-3 not only because it is the principle caspase in caspase family [21], but also since it is present in MDA-MB-231 but not in MCF7 cells [17].
Figure 6 shows the representative western blots for caspase levels in MCF7 and MDA-MB-231 cells. The levels of protein expression of caspase-3 were increased by verapamil in a concentration-dependent manner in MDA-MB-231, whereas no caspase-3 protein expression was detected in MCF7 cells (Fig. 6a). In comparison to the absence of verapamil, α-actin normalized caspase-3 levels are increased by 28-fold in 10 μM verapamil (0 μM = 0.026 ± 0.007, 10 μM = 0.72 ± 0.19, n = 3, p < 0.05) and 45-fold in 20 μM verapamil (0 μM = 0.026 ± 0.007, 20 μM = 1.16 ± 0.04, n = 3, p < 0.001) (Fig. 6b). In MCF7 cells the caspase-9 levels are increased after verapamil treatment (Fig. 6c). In comparison to the absence of verapamil, α-actin normalized caspase-9 levels are increased by 1.9-fold in 10 μM verapamil (0 μM = 0.42 ± 0.12, 10 μM = 0.79 ± 0.20, n = 3), but insignificantly (p > 0.05), and 2.6-fold in 20 μM verapamil (0 μM = 0.42 ± 0.12, 20 μM = 1.11 ± 0.19, n = 3, p < 0.05) (Fig. 6d).