Fig 1.
Electrical stimulation device and experimental setup used in this study.
(A) Schematic representation of the electrical stimulation device with the chamber and two incorporated electrodes. (B) Experimental design of co-culture with NK cells and target tumor cells to analyze the effect of electrical stimulation on the cytolytic activity of NK cells.
Fig 2.
Effects of electrical stimulation on NK cell viability.
(A) CCK-8 cell viability assays were conducted after exposure to electrical stimulation with constant direct current (DC) or a biphasic (BP) square waveform (10 Hz frequency, column with slashed line) for 1 h (n = 3). (B) The relative mRNA expression ratio of the apoptosis-related genes BAX/BCL-2 was evaluated using RT-qPCR after 1 h of constant DC electrical stimulation (n = 3). Results are presented with average and error bars. Student’s t-test was used to determine the statistical significance of the results (*, P < 0.05; **, P < 0.01 and ***, P < 0.001).
Table 1.
Primer sequences of target genes.
Fig 3.
Cytotoxicity of NK cells against tumor cells using LDH cytotoxicity assays.
(A) LDH cytotoxicity data of MDA-MB-231, actin response-positive cell line, and MCF-7, actin response-negative cell line treated for 24 h with electrically stimulated or non-stimulated KHYG-1 cells (n = 3). (B) Live/dead cytotoxicity imaging of MCF-7 cells co-cultured with stimulated or unstimulated KHYG-1 cells for 6 h. Results are presented with average and error bars. Student’s t-test was used to determine the statistical significance of the results (*, P < 0.05; **, P < 0.01 and ***, P < 0.001).
Fig 4.
Fold changes in gene expression levels related to cytotoxic activity and granzyme B production in NK cells after electrical stimulation.
(A) Relative mRNA expression levels of degranulation markers (PRF1, GZMB) and cytokines (IFNG, TNFA) in NK cells were analyzed and normalized to the housekeeping gene GAPDH and non-stimulated control (n = 3). (B) Granzyme B levels were detected in intracellular protein and extracellular levels were released into the culture media after incubation for 4 and 8 h by sandwich ELISA (n = 3). Results are presented with average and error bars. Student’s t-test was used to determine the statistical significance of the results (*, P < 0.05; **, P < 0.01 and ***, P < 0.001).
Fig 5.
Intracellular Ca2+ was measured by Fluo-4 fluorescence and relative gene expression levels involved in the Ca2+-NFAT pathway in response to electrical stimulation.
(A) Quantitative analysis of calcium with the Fluo-4 NW assay and fluorescence imaging of Fluo-4-stained cells was performed independently to detect Ca2+ influx triggered by constant electrical stimulation (n = 3). (B) Gene expression levels of calcium signaling markers were analyzed using RT-qPCR after 1 h of stimulation (n = 3). Results are presented with average and error bars. Student’s t-test was used to determine the statistical significance of the results (*, P < 0.05; **, P < 0.01 and ***, P < 0.001).
Fig 6.
Effects of electrical stimulation-induced increase in calcium levels on gene expression and NFAT1 dephosphorylation with the calcium chelator BAPTA-AM.
(A) RT-qPCR was performed to analyze GZMB expression levels and calcineurin-NFAT signaling proteins (CALM, CALN, and NFAT1) after Ca2+ chelation. KHYG-1 cells were cultured with DMSO (control) or a calcium chelator (5 μM BAPTA-AM), with or without electrical stimulation (n = 3). (B) Western blot and densitometric analyses were conducted under the same conditions (1.0 V/cm) to determine the dephosphorylation of NFAT1, which could represent the transcriptional activation in the Ca2+-dependent pathway. Quantification of dephosphorylated NFAT1 was calculated as the NFAT1/p-NFAT1 ratio relative to the control (n = 3). Results are presented with average and error bars. Student’s t-test was used to determine the statistical significance of the results (*, P < 0.05; **, P < 0.01 and ***, P < 0.001).
Fig 7.
Calcineurin-NFAT signaling pathway.
Calcium-mediated signaling is involved in the activation of diverse subsets of gene transcription, including GZMB. Target gene expression can change with the activation of transcriptional factors in response to electrical stimulation, thus providing a link between intracellular Ca2+ and gene expression in immune effector functions. ER, endoplasmic reticulum; IP3R, inositol 1,4,5-trisphosphate receptor; CaM, calmodulin; CN, calcineurin; NFAT, nuclear factor of activated T cells; GZMB, granzyme B.