Microwave-induced Apoptosis and Cytotoxicity of NK Cells through ERK1/2 Signaling
Objective To investigate microwave-induced morphological and functional injury of natural killer (NK) cells and uncover their mechanisms. Methods NK-92 cells were exposed to 10, 30, and 50 mW/cm2 microwaves for 5 min. Ultrastructural changes, cellular apoptosis and cell cycle regulation were detected at 1 h and 24 h after exposure. Cytotoxic activity was assayed at 1 h after exposure, while perforin and NKG2D expression were detected at 1 h, 6 h, and 12 h after exposure. To clarify the mechanisms, phosphorylated ERK (p-ERK) was detected at 1 h after exposure. Moreover, microwave-induced cellular apoptosis and cell cycle regulation were analyzed after blockade of ERK signaling by using U0126. Results Microwave-induced morphological and ultrastructural injury, dose-dependent apoptosis (P < 0.001) and cell cycle arrest (P < 0.001) were detected at 1 h after microwave exposure. Moreover, significant apoptosis was still detected at 24 h after 50 mW/cm2 microwave exposure (P < 0.01). In the 30 mW/cm2 microwave exposure model, microwaves impaired the cytotoxic activity of NK-92 cells at 1 h and down regulated perforin protein both at 1 h and 6 h after exposure (P < 0.05). Furthermore, p-ERK was down regulated at 1 h after exposure (P < 0.05), while ERK blockade significantly promoted microwave-induced apoptosis (P < 0.05) and downregulation of perforin (P < 0.01). Conclusion Microwave dose-dependently induced morphological and functional injury in NK-92 cells, possibly through ERK-mediated regulation of apoptosis and perforin expression.
注释:1) Authors' contributions: 2) CONFLICT OF INTEREST:
- Microwave /
- Natural killer cells /
- Cytotoxicity /
- Apoptosis /
- Cell cycle /
Figure 1. Microwave-induced morphological and functional injury of NK-92 cells. (A) NK-92 cells were exposed to 30 mW/cm2 microwaves, the ultrastructure of NK-92 cells was also observed at 1 h and 24 h after exposure by transmission electron microscopy (TEM). (B) NK-92 cells were exposed to 0 (Sham), 10, 30, and 50 mW/cm2 microwaves. One hour after exposure, NK-92 cells were collected and co-cultured with K562 cells at ratio of 10:1 for 4 h. Then, the lactate dehydrogenase (LDH) level in media was analyzed and the percentage of dead cells was calculated. **, P < 0.01 vs. Sham group.
Figure 2. Microwave-induced cellular apoptosis and cell cycle arrest. NK-92 cells were exposed to 0 (Sham), 10, 30, and 50 mW/cm2 microwaves. At 1 h and 24 h after exposure, cells were collected and labeled with Annexin-V-FITC and PI. Then, cellular apoptosis was analyzed by flow cytometry. The representative images after 30 mW/cm2 microwave exposure are shown in (A). And the quantification of apoptosis rates is presented in (B). To confirm the mechanisms of microwave-induced apoptosis, the activity of NK cells was detected at 0.5 h, 1 h, 6 h, and 12 h after microwave exposure by using the Caspase-3 activity kit according to the manufacturer's protocol (C). For analysis of the cell cycle, cells were collected at 1 and 24 h after exposure, and fixed in 70% ethanol at -20 ℃ for 24 h. Then, cells were processed for cell cycle analysis by using flow cytometry. The representative images after 30 mW/cm2 microwave exposure are shown in (D). The percentages of cells in G0/G1 phase, in G2/M phase, and in S phase are shown in (E), (F), and (G), respectively. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. sham group at the same time point after exposure.
Figure 3. Microwaves regulate the expression of perforin and NKG2D in NK-92 cells. NK-92 cells were exposed to 0 (Sham) and 30 mW/cm2 microwaves. Protein was extracted from cells collected at 1 h, 6 h, and 12 h after exposure. The protein expression of perforin and NKG2D was analyzed by western-blotting (A) and the corresponding results of quantification are shown in (B) and (C). *, P < 0.05, **, P < 0.01 vs. sham group at the same time points after exposure. Cells were collected at 1 h, 6 h, and 12 h after exposure, and total RNA was isolated by using TRIzol reagent. After cDNA was synthesized, the mRNA expression of perforin (D) and NKG2D (E), an activating receptor on NK cells, were measured by quantitative reverse transcription polymerase chain reaction (RT-PCR).
Figure 4. ERK signaling participates in microwave-induced injury of NK-92 cells. NK-92 cells were exposed to 0 (Sham) and 30 mW/cm2 microwaves. At 1 h, 6 h, and 12 h after exposure, cells were collected, and the expression of phosphorylated ERK (p-ERK) was detected by western-blotting. Then, p-ERK expression was normalized to total ERK (A). *, P < 0.05 vs. sham group at 1 h after exposure. To blocking ERK signaling, U0126 (a specific ERK inhibitor) was added to NK-92 cells 30 min before exposure at a concentration of 50 μmol/L. One hour after exposure to 0 (Sham) and 30 mW/cm2 microwaves, the level of p-ERK was confirmed by western-blotting and normalized to ERK (B), and the expression of perforin was also measured by western-blotting and normalized to GAPDH (C). The quantitative results of p-ERK and perforin are also shown. *, P < 0.05, **, P < 0.01 vs. sham group; ##, P < 0.01 vs. microwave exposure group (MW). Thirty minutes before microwave exposure, 50 μmol/L U0126 were added to NK-92 cells, using DMSO as a negative control. At 1 h, 6 h, 12 h, and 24 h after 0 and 30 mW/cm2 microwave exposure, cells were collected and used to analyze the cellular apoptosis and cell cycle by using flow cytometry. The apoptosis rates are shown in (D), and the percentage of cells in G0/G1 phase was calculated and displayed in (E). *, P < 0.05, **, P < 0.01, ***, P < 0.001 vs. sham group at the same time point; ##, P < 0.01, ###, P < 0.001 vs. microwave exposure group (MW) at the same time point.
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