Tumor-Treating Fields Induce RAW264.7 Macrophage Activation Via NK-?B/MAPK Signaling Pathways

Park, Jeong-In; Song, Kyung-Hee; Jung, Seung-Youn; Ahn, Jiyeon; Hwang, Sang-Gu; Kim, Joon; Kim, Eun Ho; Song, Jie-Young

Macrophages are crucial for host defense against infections and in inflammatory processes through the release of molecules, such as NO, TNF-a, and IL-6. 26 On the basis of our results, the study showed that TTFs increase macrophage activation in RAW 264.7 cells. Furthermore, TTFs significantly enhanced the production of NO in RAW 264.7 cells in vitro. After its release from macrophages, NO acts as an intracellular messenger to mediate the nonspecific immune response 27 and has also been suggested to be a critical factor in the immune response. 28 Nitric oxide is a gaseous, free radical and acts as a signaling molecule in biological systems, recruiting leukocytes to affected tissues. 29, 30 Inducible NOS is the inducible enzyme for NO production and is responsible for increased levels of NO. Accumulating evidence indicates that intracellular ROS also serves as a second messenger in inflammatory signal transduction by modulating the release of other inflammatory mediators and stimulating MAPK activity. [31] [32] [33] In our result, we observed that the stimulation of RAW 264.7 cells by TTFs increases iNOS expression as well as the production of NO and ROS, although the increase was much lower than that observed in LPS-treated cells. Therefore, TTFs may have immune mediating/modulating effects, including activation of macrophages and mediating their biological functions such as tumoricidal activity through NO-dependent pathways. 34 Once activated, macrophages release abundant cytokines that act as signals to control homeostasis via regulating cell differentiation, proliferation, apoptosis, and immune responses. 35 Figure 3 . Upregulation of inflammatory cytokines in TTFs-treated RAW 264.7 cells. A, Cells were treated with TTFs (0.9 V/cm) or LPS (1 ng/mL) for 24 hours, and mRNA levels of IL-1b and TNF-a were evaluated by qRT-PCR. B, RAW 264.7 cells were treated with TTFs (0.9 V/cm) for 24 hours, and the cells were cocultured with 4T1 cells at the indicated ratios (4T1:TTFs-RAW 264.7). 4T1 cells, untreated-or TTFs-treated RAW 264.7 cells were designated as 4T1, Ct, and TTF, respectively. Co-cultivations were performed for 48 hours, and the levels of proinflammatory cytokines (IL-1b, TNF-a, and IL-6) were determined by ELISA. **P < .01, ***P < .001 compared with the TTF group. C, RAW 264.7 cells were treated with TTFs (0.9 V/cm) or LPS (1 ng/mL) for 24 hours, and the culture medium (CM) of RAW 264.7 cells was used to treat 4T1 cells for 24 and 48 hours. The untreated RAW 264.7 cells were designated as Ct. The percentage of cell viability was determined by the MTT assay and calculated relative to that of untreated 4T1 cells (none). Data represent the mean (standard deviation) of triplicate samples. **P < .01, ***P < .001 compared with the 24 hours none group. yy P < .01 compared with the 48 hours none group. ELISA indicates enzyme-linked immunoassay; IL, interleukin; LPS, lipopolysaccharide; mRNA, messenger RNA; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; TNF, tumor necrosis factor; TTFs, tumor-treating fields; qRT-PCR, quantitative reverse transcription polymerase chain reaction.