Electric Fields Regulate In Vitro Surface Phosphatidylserine Exposure of Cancer Cells via a Calcium-Dependent Pathway

Abstract

Cancer is the second leading cause of death worldwide after heart disease. The current treatment options to fight cancer are limited, and there is a critical need for better treatment strategies. During the last several decades, several electric field (EF)-based approaches for anti-cancer therapies have been introduced, such as electroporation and tumor-treating fields; still, they are far from optimal due to their invasive nature, limited efficacy and significant side effects. In this study, we developed a non-contact EF stimulation system to investigate the in vitro effects of a novel EF modality on cancer biomarkers in normal (human astrocytes, human pancreatic ductal epithelial -HDPE-cells) and cancer cell lines (glioblastoma U87-GBM, human pancreatic cancer cfPac-1, and MiaPaCa-2). Our results demonstrate that this EF modality can successfully modulate an important cancer cell biomarker-cell surface phosphatidylserine (PS). Our results further suggest that moderate, but not low, amplitude EF induces p38 mitogen-activated protein kinase (MAPK), actin polymerization, and cell cycle arrest in cancer cell lines. Based on our results, we propose a mechanism for EF-mediated PS exposure in cancer cells, where the magnitude of induced EF on the cell surface can differentially regulate intracellular calcium (Ca²⁺) levels, thereby modulating surface PS exposure.

Keywords: calcium modulation; cancer biomarker; electric field; enhanced cancer therapy; p38 MAPK-actin pathway; surface phosphatidylserine exposure.

Figure 1

Electric field stimulation is safe and does not cause detrimental effects on cell growth, viability, and membrane integrity. (A) In vitro electric field stimulation setup. (B) There is no difference in the number of viable glioblastoma (U87-GBM) cells following EF stimulation and the medium control group. Sample size (n) = 5. Flow cytometry dot plots (C) and a bar graph (D) demonstrate that there is no difference in the percentage of PI-positive cells between EF-stimulated and control groups. Sample size (n) = 3.

Figure 2

Electric field stimulation induces PS exposure in cancer cells via caspase-independent mechanism. (A) Flow cytometric measurements of annexin V (FITC Fluorescence) demonstrate a significant decrease or no change in cell PS exposure in U87-GBM (top first panel) and cfpac-1 (top second panel) cancer cells following low amplitude EF (top panel) for 4–6 h, whereas moderate amplitude EF (bottom panels) stimulation significantly increased the PS exposure in U87-GBM (bottom first panel) and cfpac-1 (bottom second panel). This effect is absent in healthy astrocytes (top third, bottom third panels) and HPDE cells (top fourth, bottom fourth panels). The top and bottom fifth panels show representative histograms, with blue denoting Control and red indicating the EF-treated groups (6 h). Sample size (n) = 3 (B) Western blot analysis showed that 24-h EF stimulation did not change cleaved caspase 3 and 9 expressions in glioblastoma cells (U87-GBM) (left panel) or healthy cells (astrocytes) (right panel), with the levels in the stimulated group similar to the controls. Positive control: Cells were treated with 1 μM staurosporine for 4 h.

Figure 3

The electric field differentially regulates the intracellular Ca²⁺ levels in cancer cells. (A) Intracellular Ca²⁺ concentration (Fluo-3) fold changes of glioblastoma cells (U87-GBM) and healthy cells (astrocytes) cultured in normal medium (DMEM) following low and moderate amplitude EF stimulation for 6 h. The results show that low-amplitude EF stimulation decreases intracellular Ca²⁺ concentration, while moderate-amplitude EF stimulation increases intracellular Ca²⁺ concentration in cancer cells. Sample size (n) = 3 (B) Flow cytometric measurements of annexin V (FITC Fluorescence/10⁵ cells) on glioblastoma (U87-GBM) cells following low and moderate amplitude EF stimulation in the Ca²⁺-free medium. The results show that the EF-induced PS exposure in cancer cells, as shown in (A), is completely abolished in the absence of Ca²⁺, suggesting the critical role of Ca²⁺-dependent mechanisms in the EF-mediated change in PS exposure in cancer cells. Sample size (n) = 3.

Figure 4

Actin polymerization is involved in the regulation of EF-induced PS externalization in cancer cells. (A) Representative immunofluorescence images of glioblastoma cells (U87-GBM; per 10⁵ cells) (left panel) demonstrating that low-amplitude EF stimulation decreases actin polymerization, while moderate-amplitude EF stimulation increases actin polymerization levels in cancer cells. Negative control: culture medium; positive control: oxidative stress induction with 1.2 mM H₂O₂ for 20 min. (B) There is a trend toward a positive relationship between F-actin and PS exposure of cancer cells in both low and moderate amplitude EF stimulation groups (r² = 0.9085, p = 0.0121 vs. r² = 0.7615, p = 0.0535, low vs. moderate amplitude, respectively).

Figure 5

Electric field modulates the p38 MAPK activity in cancer cells. Western blot analysis showing the expression of p38 MAPK, phospho-p38 MAPK, and beta-actin of glioblastoma cells (U87-GBM) treated with DMEM, low and moderate amplitude EF for 24 h (top left panel). Western blot analysis of relative phospho-p38 MAPK protein levels between medium control, low and moderate amplitude groups (top right panel). Sample size (n) = 3. Western blot analysis showing the expression of p38 MAPK, phospho-p38 MAPK, P44/42 ERK1/2, p-AKT, and beta-actin of glioblastoma cells (U87-GBM) treated with DMEM (control) and low amplitude EF for indicated time periods (bottom left panel). Western blot analysis showing the expression of p38 MAPK, phospho-p38 MAPK, P44/42 MAPK, p-AKT, and beta-actin of glioblastoma cells (U87-GBM) treated with DMEM (control) or moderate amplitude EF for indicating time periods (bottom right panel).

Figure 6

Moderate-amplitude electric field stimulation causes cell cycle arrest in glioblastoma cancer cells. Following EF stimulation of glioblastoma cells (U87-GBM) for 24 h, there was a significant change in the cell cycle distribution pattern (left panel), with lower percentage of cells in G1 and higher percentage of cells in G2/M phases in the EF groups as compared to the respective percentages in the control groups. Western blot analyses demonstrate that 24 h stimulation with moderate amplitude EF results in a decreased protein expression of cyclin D1 in glioblastoma cells (U87-GBM) (right panel). Sample size (n) = 3.

Figure 7

(A) Possible mechanism of action of the electric field in cancer cells. Moderate amplitude EF activates the voltage-gated calcium channels on the cancer cell membrane, leading to cytosolic calcium increase within the cells. Increased cytosolic calcium inhibits flippase activity and further accumulates PS on the outer cell membrane. Next, higher cytosolic calcium induces actin polymerization and activates the p38 MAPK pathway in cancer cells; this activation blocks the expression of cyclin D. On the other hand, low amplitude EF inhibits the activation of calcium channels, which leads to a lower cytosolic calcium concentration within the cells. Decreased levels of cytosolic calcium results in the activation of the flippase enzymes, which leads to reduced PS localization on the outer cell membrane. Moreover, this process inhibits further activation of p38 MAPK and actin polymerization. (B) Potential therapeutic application of low amplitude electric field. Moderate amplitude EF increases the PS exposure of cancer cells, whereas low amplitude EF decreases their PS exposure. The cancer cells treated with the moderate amplitude EF will end up with higher PS exposure and become more sensitive to high-PS targeting treatments such as SapC-DOPS. Conversely, cancer cells treated with low amplitude EF will have lower PS surface exposure and, therefore, become more sensitive to radiation and chemotherapy. Thus, the combination of moderate amplitude EF treatment with SapC-DOPS or low amplitude EF treatment with chemo/radiation may lead to enhanced cancer cell death, the main goal of anti-cancer therapy.