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

The study provides evidence that non-contact electric field (EF) stimulation can differentially modulate surface phosphatidylserine (PS) exposure in cancer cells through a calcium-dependent pathway, involving actin polymerization and p38 MAPK activation. These findings open new avenues for enhancing targeted cancer therapies by manipulating PS exposure using EF stimulation.

EXPERIMENT: The key components of the EF stimulation setup included a parallel plate capacitor with two plates measuring 135 mm × 128 mm, spaced 26 mm apart. A voltage source (Pasco, model SF-9586, Roseville, CA, USA) was used to generate the electric fields. Cells were seeded in a petri dish filled with cell culture media, which was placed between the capacitor plates to ensure exposure to the electric fields without direct contact with the electrodes. Two different EF amplitudes, 7.5 V/mm (low amplitude) and 15 V/mm (moderate amplitude), were selected based on previous theoretical and experimental studies to ensure safety and efficacy. The study utilized several cell lines, including glioblastoma (U87-GBM), human pancreatic cancer (cfPac-1 and MiaPaCa-2), human astrocytes, and human pancreatic ductal epithelial (HPDE) cells. Flow cytometry was employed to measure PS exposure, intracellular calcium levels, and membrane leakage, while immunofluorescence staining was used to visualize actin polymerization and p38 MAPK activation. Western blot analysis quantified protein expression levels of key markers such as cleaved caspase 3, cleaved caspase 9, p38 MAPK, and cyclin D1. Statistical analysis was performed using one or two-factor ANOVA with Bonferroni post-hoc comparisons, and a p-value of <0.05 was considered statistically significant.

Key Findings:

PS Exposure Modulation:

• Moderate amplitude EF stimulation significantly increased PS exposure on cancer cell surfaces.
• Low amplitude EF stimulation decreased PS exposure.
• This modulation was specific to cancer cells and was not observed in normal cell lines.

Calcium-Dependent Mechanism:

• EF-induced PS exposure is regulated by intracellular calcium levels.
• Moderate amplitude EF increases cytosolic calcium, while low amplitude decreases it.
• The increase in PS exposure under moderate EF is mediated by inhibition of flippase activity due to increased intracellular calcium.

Actin Polymerization and p38 MAPK Activation:

• Moderate amplitude EF stimulation led to increased actin polymerization and p38 MAPK activation.
• Low amplitude EF had the opposite effect, decreasing actin polymerization and inhibiting p38 MAPK activation.

Cell Cycle Arrest:

• Moderate amplitude EF stimulation caused cell cycle arrest in the G2/M phase in cancer cells.
• This arrest was accompanied by decreased cyclin D1 expression.

Clinical Implications:

1. Non-Invasive Modulation of Cancer Biomarkers: The ability to modulate PS exposure using non-contact EF stimulation provides a non-invasive means to alter key cancer biomarkers. This could be particularly valuable for targeting cancer cells without causing harm to normal cells.
2. Personalized Cancer Treatment: By adjusting the EF amplitude, it may be possible to tailor the treatment to the specific PS expression levels of a patient’s cancer cells. This approach could enhance the efficacy of treatments by making them more personalized and targeted.
3. Enhanced Targeted Therapies: The study suggests that cancer cells with higher PS exposure are more susceptible to PS-targeting treatments like SapC-DOPS nanovesicles. Conversely, cancer cells with lower PS exposure are more sensitive to chemotherapy and radiation. By modulating PS exposure, EF stimulation could be used to sensitize cancer cells to specific treatments, enhancing their efficacy.
4. Potential for Combination Therapies: Combining moderate amplitude EF treatment with SapC-DOPS or low amplitude EF treatment with chemotherapy/radiation could lead to enhanced cancer cell death. This approach could be particularly effective in treating cancer by leveraging the strengths of different therapeutic modalities.
5. Reduction of Side Effects: Non-contact EF stimulation offers a non-invasive method for cancer treatment, potentially reducing the side effects associated with current invasive therapies. This could improve patient quality of life and make treatments more tolerable.
6. Mechanistic Insights for Future Therapies: The findings on the calcium-dependent pathway and the role of actin polymerization and p38 MAPK in EF-induced PS exposure provide mechanistic insights that could lead to the development of new cancer therapies. Understanding these mechanisms could help identify new therapeutic targets and improve treatment strategies.
7. Safe and Effective Treatment Modality: The study demonstrates that non-contact EF stimulation is safe and does not cause detrimental effects on cell growth, viability, or membrane integrity. This supports the potential of EF stimulation as an effective and safe treatment modality for cancer.