Microwave hyperthermia promotes caspase‑3-dependent apoptosis and induces G2/M checkpoint arrest via the ATM pathway in non‑small cell lung cancer cells

Abstract

Post-operative microwave (MW) hyperthermia has been applied as an important adjuvant therapy to enhance the efficacy of traditional cancer treatment. A better understanding of the molecular mechanisms of MW hyperthermia may provide guided and further information on clinical hyperthermia treatment. In this study, we examined the effects of MW hyperthermia on non‑small cell lung carcinoma (NSCLC) cells in vitro, as well as the underlying mechanisms. In order to mimic clinical treatment, we developed special MW heating equipment for this study. Various NSCLC cells (H460, PC-9 and H1975) were exposed to hyperthermia treatment using a water bath or MW heating system. The results revealed that MW hyperthermia significantly inhibited cell growth compared with the water bath heating system. Furthermore, MW hyperthermia increased the production of reactive oxygen species (ROS), decreased the levels of mitochondrial membrane potential (MMP) and induced caspase‑3 dependent apoptosis. It also induced G2/M phase arrest through the upregulation of the expression of phosphorylated (p‑) ataxia telangiectasia mutated (ATM), p‑checkpoint kinase 2 (Chk2) and p21, and the downregulation of the expression of cdc25c, cyclin B1 and cdc2. On the whole, the findings of this study indicate that the exposure of NSCLC cells to MW hyperthermia promotes caspase‑3 dependent apoptosis and induces G2/M cell cycle arrest via the ATM pathway. This preclinical study may help to provide laboratory-based evidence for MW hyperthermia treatment in clinical practice.

Figure 1

Schematic principles of the microwave hyperthermia device. (A) Photographic appearance of the microwave hyperthermia device: The novel irradiation system consisted of a (B) 433 MHz microwave generator and computer control system with MW radiator and (C) a temperature sensor. (D) Schematic representation of the MW hyperthermia device. Petri dishes or plates were positioned under the radiator and exposed to microwave radiation at 433 MHz. (E) Changes in temperature and output of microwave. Blue line indicates the temperature of the cultured cells measured using thermometer probe 1 under the Petri dishes. Green line indicates the temperature of the surrounding circulating water measured by thermometer probe 2. The red line indicates the output of the incident wave. (F) Photographic appearance of the water bath system.

Figure 2

Microwave (MW) hyperthermia inhibits growth in different cell lines. (A-D) NSCLC cells (1×10⁴/well) were seeded in a 96-well plate and were exposed to the water bath or MW hyperthermia for different periods of time. Following incubation for 6, 12 or 24 h, cell viability was determined by CCK-8 assay. (E) Primary astrocytes (1×10⁴/well) and human normal lung epithelial cells, BEAS-2B cells (1×10⁴/well) were seeded in a 96-well plate and exposed to MW hyperthermia for 60 or 90 min. Following incubation for 24 h, cell viability was determined by CCK-8 assay. Data are expressed as the means ± SEM of 3 independent experiments. ^*P<0.05 vs. the control group, ^**P<0.01 vs. the control group, ^***P<0.01 vs. the control group.

Figure 3

Microwave (MW) hyperthermia promotes apoptosis. H460, PC-9 and H1975 cells were exposed to a water bath or MW hyperthermia at 43°C for 90 min, then allowed to recover at 37°C until 24 h. (A, C and E) Representative images of flow cytometry results. (B, D and F) Quantitative analyses of Annexin-FITC positive H460, PC-9 and H1975 cells are shown. (G-I) Quantitative analyses of Annexin-FITC positive H460, PC-9 and H1975 cells subjected to different treatments are shown. ^*P<0.05 vs. the control group, ^**P<0.01 vs. the control group.

Figure 4

Microwave (MW) hyperthermia triggers cell death through caspase-3-mediated apoptosis. H460, PC-9 and H1975 cells were treated with or without Ac-DEVD-CHO for 3 h prior to MW hyperthermia (43°C for 90 min), then allowed to recover at 37°C until 24 h. (A) Cell viability was determined by CCK-8 assay. Data are expressed as the means ± SEM of 3 independent experiments. ^*P<0.05 vs. the control group, ^**P<0.01 vs. the control group. (B) Quantitative analyses of Annexin-FITC positive H460, PC-9 and H1975 cells are shown. ^*P<0.05 vs. the control group, ^**P<0.01 vs. the control group. (C) Caspase-3 activity following treatment was analyzed. Data are expressed as the means ± SEM of 3 independent experiments. ^**P<0.01 vs. the control group, ^***P<0.001 vs. the control group; ^#P<0.05 vs. MW treatment group, ^###P<0.001 vs. MW treatment group.

Figure 5

Microwave (MW) hyperthermia treatment increases reactive oxygen species (ROS) levels. Cells were seeded onto 6-well plate at 5×10⁵/well and then exposed to the water bath or MW radiation at 43°C for 90 min. Following incubation for 6 h, cells were pre-loaded with DCFH-DA and then mounted under a fluorescence microscope. (A-C) Representative photographs from DCF fluorescence staining in the H460, PC-9 and H1975 cells, respectively. Scale bars, 200 µm. (D) Quantitative analyses of the mean fluorescence intensity. ^*P<0.05 vs. the control group in H460 cells, ^***P<0.001 vs. the control group in PC-9 cells, ^**P<0.01 vs. the control group in H1975 cells.

Figure 6

Microwave (MW) hyperthermia treatment alters mitochondrial membrane potential (MMP). Cells were seeded onto 6-well plate at 2×10⁵/well and then exposed to a water bath or MW radiation at 43°C for 90 min. Following incubation for 6 h, MMP was examined by JC-1 staining with a confocal microscope. (A-C) Representative photographs from DCF fluorescence staining in the H460, PC-9 and H1975 cells, respectively. Scale bars, 50 µm. (D) Quantitative analyses of relative fluorescence are shown. ^***P<0.001 compared to each control group.

Figure 7

Microwave (MW) hyperthermia induces G₂/M arrest. Cells were seeded onto 6-well plate at 1×10⁶/well and then exposed to a water bath or MW at 43°C for 90 min. The cells were cultivated for 24 h, cell cycle distribution were analyzed by flow cytometry. (A-C) The cell cycle distributions following treatment of H460, PC-9 and H1975 cells. (D) Western blot analysis of ATM, p-ATM, p-Chk2, p21, cdc25c, cyclin B1 and cdc2 in NSCLCs cells. β-actin expression was included as an internal control. Values were the means of triplicate analyses.