Aerobic Exercise Improves Radiation Therapy Efficacy in Non-Small Cell Lung Cancer: Preclinical Study Using a Xenograft Mouse Model

Abstract

The "oxygen effect" improves radiation efficacy; thus, tumor cell oxygen concentration is a crucial factor for improving lung cancer treatment. In the current study, we aimed to identify aerobic exercise-induced changes in oxygen concentrations in non-small cell lung cancer (NSCLC) cells. To this end, an NSCLC xenograft mouse model was established using human A549 cells. Animals were subsequently subjected to aerobic exercise and radiation three times per week for 2 weeks. Aerobic exercise was performed at a speed of 8.0 m/m for 30 min, and the tumor was irradiated with 2 Gy of 6 MV X-rays (total radiation dose 12 Gy). Combined aerobic exercise and radiation reduced NSCLC cell growth. In addition, the positive effect of aerobic exercise on radiation efficacy through oxygenation of tumor cells was confirmed based on hypoxia-inducible factor-1 and carbonic anhydrase IX expression. Finally, whole-transcriptome analysis revealed the key factors that induce oxygenation in NSCLC cells when aerobic exercise was combined with radiation. Taken together, these results indicate that aerobic exercise improves the effectiveness of radiation in the treatment of NSCLC. This preclinical study provides a basis for the clinical application of aerobic exercise to patients with NSCLC undergoing radiation therapy.

Keywords: NSCLC; aerobic exercise; lung cancer; oxygenation; radiation therapy.

Figure 1

Aerobic exercise in combination with radiation reduced NSCLC growth. (A) Experimental procedure in a mouse xenograft model designed to investigate tumor growth in vivo. After subcutaneous injection of A549 cells, BALB/c athymic nude mice (n = 5 per group) were irradiated three times a week for 2 weeks and aerobic exercise was performed for 30 min three times per week; (B,C) Inhibitory effect on tumor volume in mouse xenograft models subjected to aerobic exercise and radiation; (D) H & E staining and Ki-67 immunohistochemical staining of mouse tumor sections showing inhibited cell proliferation upon radiation combined with aerobic exercise.; con: control, Ex: exercise, and IR: irradiation.

Figure 2

The “oxygen effect” was induced in tumor cells when aerobic exercise was combined with radiation. (A,B) HIF-α and CA IX immunohistochemical staining of tumor segments from mice treated with aerobic exercise and radiation exhibit increased oxygenation levels in tumor cells; (C) increased ROS levels in A549 cells in response to aerobic exercise and radiation confirmed by 4-hydroxynonenal and nitrotyrosine immunohistochemistry.

Figure 3

Combining aerobic exercise and radiation induces changes in various genes. (A) Through comparative combination analysis, DEGs that were upregulated or downregulated by more than 2-fold compared to the control group were identified. (B) Comparisons between the radiation-alone and combination groups: 215 DEGs were specifically altered by aerobic exercise. (C) Top 20 major enrichment biological processes in DEGs. The x-axis represents GeneRadio and False Discovery Rate (FDR) values. The y-axis shows various biological processes. (D) Chart showing the top 20 major enrichment cellular components of DEGs. The x-axis represents GeneRadio and FDR values. The y-axis shows the various cellular components. (E) Chart showing the top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathways of DEGs.

Figure 3

Combining aerobic exercise and radiation induces changes in various genes. (A) Through comparative combination analysis, DEGs that were upregulated or downregulated by more than 2-fold compared to the control group were identified. (B) Comparisons between the radiation-alone and combination groups: 215 DEGs were specifically altered by aerobic exercise. (C) Top 20 major enrichment biological processes in DEGs. The x-axis represents GeneRadio and False Discovery Rate (FDR) values. The y-axis shows various biological processes. (D) Chart showing the top 20 major enrichment cellular components of DEGs. The x-axis represents GeneRadio and FDR values. The y-axis shows the various cellular components. (E) Chart showing the top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathways of DEGs.

Figure 4

DEGs associated with oxygenation and ROS in the combination group compared to the radiation-alone group. (A) Overall heat-map of DEGs associated with oxygenation and ROS, and heat-map of specific genes; (B) KEGG analysis focusing on the ROS signaling pathway of DEGs altered in the combination group compared to the radiation-alone group.