Normothermic Microwave Irradiation Induces Death of HL-60 Cells through Heat-Independent Apoptosis

Abstract

Microwaves have been used in various cancer therapies to generate heat and increase tumor cell temperature; however, their use is limited by their side-effects in normal cells and the acquisition of heat resistance. We previously developed a microwave irradiation method that kills cultured cancer cells, including a human promyelomonocytic leukemia (HL-60) cell line, by maintaining a cellular temperature of 37 °C during treatment. In the present study, we investigated the mechanisms underlying HL-60 cell death during this treatment. The microwave-irradiated HL-60 cells appear to undergo caspase-independent apoptosis, whereby DNA fragmentation was induced by mitochondrial dysfunction-related expression of apoptosis-inducing factor (AIF). Caspase-dependent apoptosis was also interrupted by the loss of apoptotic protease-activating factor 1 (Apaf-1) and caspase 9. Moreover, these cells did not exhibit a heat-stress response, as shown by the lack of heat shock protein 70 (HSP70) upregulation. Alternatively, in HL-60 cells heated at 42.5 °C, HSP70 expression was upregulated and a pathway resembling death receptor-induced apoptosis was activated while mitochondrial function was maintained. Collectively, these results suggest that the cell death pathway activated by our 37 °C microwave irradiation method differs from that induced during other heating methods and support the use of normothermic microwave irradiation in clinical cancer treatments.

Figure 1

Cell death and cell cycle changes. Microwave irradiation (“MW”) was applied for 1 h with the temperature of the cultured cells maintained at 37 °C, and the temperature inside the applicator set at 12 °C. Negative-control cells were incubated at 37 °C in a CO₂ incubator without being subjected to microwave irradiation. Cells exposed to thermal treatment (42.5 °C) were incubated at an applicator temperature of 42.5 °C without receiving microwave irradiation. (A) Annexin-V/PI staining. Cells were regarded as follows: stained with neither Annexin V nor PI, live cells; stained only with Annexin V, early apoptotic cells; and stained with both Annexin V and PI, late apoptotic or necrotic cells. The corresponding histograms are shown in Fig. S1. (B) Cell cycle analysis through PI staining after 24-h incubation. Data are expressed as the means ± SD of 4 independent experiments. *P < 0.05 and **P < 0.01 versus the negative control.

Figure 2

Microwave irradiation induces mitochondrial damage without a heat-stress response. After microwave irradiation or thermal treatment followed by a 3-h incubation period, cells were examined for (A) Bcl-2 and Bax expression; (B) mitochondrial membrane potential, by staining with JC-1; and (C) HSP70 expression, measured using ELISA. The uncropped western blot images are shown in Fig. S3. Data are expressed as the means ± SD of 4 independent experiments. *P < 0.05 and **P < 0.01 versus the negative control.

Figure 3

Apoptosome activity potential of cells. Cells were exposed to microwave irradiation or thermal treatment, incubated for 3 h, and then analyzed for (A) apoptosome activity potential, based on western blotting (see Fig. S2 for COX IV results); (B) DNA fragmentation, by TUNEL staining; and (C) caspase 3/7 activity, measured using ELISA. The uncropped western blot images are shown in Fig. S3. Data are expressed as the means ± SD of 4 independent experiments. *P < 0.05 and **P < 0.01 versus the negative control.

Figure 4

Death receptor-induced cell death and endoplasmic reticulum stress. (A) Caspase 8 activity measured using ELISA, and RIP 1 and 3 expression examined through western blotting. (B) Caspase 12 activity measured using ELISA, and CHOP and ATF-4 expression analyzed through western blotting. The uncropped western blot images are shown in Fig. S3. Data are expressed as the means ± SD of 3 independent experiments for the caspase 12 assay, and 4 independent experiments for all other assays. *P < 0.05 and **P < 0.01 versus the negative control.

Figure 5

Assay of cellular autophagy. (A) LC3-I and -II expression after microwave irradiation or thermal treatment followed by a 3-h incubation period. (B) Trypan blue and (C) WST-8 assays of cells treated without or with chloroquine diphosphate. The uncropped western blot images are shown in Fig. S3. Data are expressed as the means ± SD of 4 independent experiments. *P < 0.05 and **P < 0.01 versus the negative control.

Figure 6

Schematic diagram of the cell death pathways induced by microwave irradiation and thermal treatment.

Figure 7

Transition of temperature and output under microwave irradiation. Temperatures (left axis) of the culture medium (blue lines) and applicator (green lines) are shown. Outputs of the incident and reflected waves are shown using red and pink lines, respectively. All parameters are shown for 0–30 min of microwave irradiation with the applicator temperature set at 12 °C. The lower graph shows an expansion of the initial transition period before the medium temperature was maintained constant at 37 °C, which is illustrated in the full period of microwave irradiation shown in the upper graph.