Distinct Modulatory Effects of Fever-Range Hyperthermia on the Response of Breast Cancer Cells and Macrophages to Mistletoe (Viscum album L.) Extract

Abstract

Heat utility as a critical component of fever is often ignored, although the symptom is observed in many medical conditions. Mistletoe extract (ME) is an adjunctive medication prescribed to cancer patients. The increase in body temperature is frequently observed in patients following ME administration. Nevertheless, the impact of this fever on the effectiveness of therapy is unknown. Therefore, we aimed to investigate the effect of fever-range temperatures on ME-treated breast cancer cells and macrophages. The cells were simultaneously stimulated with ME and subjected to fever-range hyperthermia (FRH; 39 °C or 41 °C). After co-treatment, the cell viability, generation of reactive oxygen species (ROS), cell cycle distribution, and production of pro-inflammatory factors (interleukin (IL)-1β, IL-6, and cyclooxygenase (COX)-2) were evaluated. The results showed that the exposure of ME-treated breast cancer cells to FRH at 39 °C resulted in a slight decrease in their viability, whereas FRH of 41 °C enhanced this effect. Only FRH of 41 °C induced minor changes in ROS level in ME-treated breast cancer cell lines. In ME-treated macrophages, FRH stimulated cell proliferation. The cell cycle distribution analysis showed a difference between cells cultured at 39 °C and 41 °C in all examined cell lines. Moreover, hyperthermia at 41 °C completely inhibited the ME-induced increase in IL-1β and IL-6 expression in MCF-7 breast cancer cells, whereas this effect was not observed in 4T1 breast cancer cells. In contrast, in ME-treated macrophages, FRH of 41 °C strongly up-regulated expression of the pro-inflammatory factors. We conclude that fever is an important component of ME therapy that differentially affects cancer and immune cells.

Keywords: cell cycle distribution; cytokines; fever; hyperthermia; inflammation; mistletoe extract; reactive oxygen species.

Figure 1

Effect of heat exposure on the viability, as measured using the MTT test (A), ROS production, as measured using carboxy–H₂DCFDA staining followed by flow cytometry analysis (B), and cell cycle distribution, as measured using propidium iodide staining followed by flow cytometry analysis (C,D), of ME-treated MCF-7 cells. Data are presented as the mean ± S.E.M. of three independent experiments. Asterisks indicate statistical significance in comparison to untreated control cells, and slashes indicate significance in comparison to ME-treated cells cultured at 37 °C (*** or ### p < 0.001, ** or ## p < 0.01, * or # p < 0.05). ME indicates mistletoe extract.

Figure 2

Effect of heat exposure on the viability, measured using MTT test (A), ROS production, measured using carboxy-H₂DCFDA staining followed by flow cytometry analysis (B), and cell cycle distribution, measured using propidium iodide staining followed by flow cytometry analysis (C,D) of ME-treated 4T1 cells. Data are presented as the mean ± S.E.M. of three independent experiments. Asterisks indicate statistical significance in comparison to untreated control cells, and slashes indicate significance in comparison to ME-treated cells cultured at 37 °C (*** or ### p < 0.001, ** or ## p < 0.01, * or # p < 0.05). ME indicates mistletoe extract.

Figure 3

Effect of heat exposure on the viability, measured using MTT test (A), ROS production, measured using carboxy-H₂DCFDA staining followed by flow cytometry analysis (B), and cell cycle distribution, measured using propidium iodide staining followed by flow cytometry analysis (C,D) of ME-treated RAW 264.7 cells. Data are presented as the mean ± S.E.M. of three independent experiments. Asterisks indicate statistical significance in comparison to untreated control cells and slashes indicate significance in comparison to ME-treated cells cultured at 37 °C (*** or ### p < 0.001, ## p < 0.01, * or # p < 0.05). ME indicates mistletoe extract.

Figure 4

Effect of heat exposure on ME-induced mRNA expression of (A,B) IL-1β, (C,D) IL-6 in MCF-7 cells (A,C), and 4T1 cells (B,D), respectively. mRNA expression was determined by quantitative real time-PCR. Data are presented as the mean ± S.E.M. of three independent experiments. Asterisks indicate statistical significance in comparison to untreated control cells, and slashes indicate significance in comparison to ME+/37 °C (*** or ### p < 0.001, ** or ## p < 0.01, * or # p < 0.05). ME indicates mistletoe extract.

Figure 5

Effect of heat exposure on ME-induced mRNA expression in RAW 264.7 cells. (A) IL-1β, (B) IL-6, and (C) COX-2 mRNA expression was determined by quantitative real-time PCR. Data are presented as the mean ± S.E.M. of three independent experiments. Asterisks indicate statistical significance in comparison to untreated control cells and slashes indicate significance in comparison to ME-treated cells cultured at 37 °C (*** or ### p < 0.001, ** or ## p < 0.01, * or # p < 0.05). ME indicates mistletoe extract.