Carbon Ion Therapy Inhibits Esophageal Squamous Cell Carcinoma Metastasis by Upregulating STAT3 Through the JAK2/STAT3 Signaling Pathway

Abstract

Radiation therapy is an important component of the comprehensive treatment of esophageal cancer. However, conventional radiation resistance is one of the main reasons for treatment failure. The superiority of heavy ion radiation in physics and biology has been increasingly highlighted in radiation therapy research. The Janus Kinase 2/Signal Transducer and Activator of Transcription 3 (JAK2/STAT3) pathway plays an important role in the occurrence, development and metastasis of esophageal squamous cell carcinoma (ESCC) and is related to the development of resistance to ionizing radiation in ESCC. Therefore, the aim of the present study was to investigate the relationship between carbon ion inhibition of the proliferation and metastasis of esophageal carcinoma cells and the JAK2/STAT3 signaling pathway. The results demonstrated that carbon ion beams significantly reduced cell viability and stimulated apoptosis in human ESCC cells in a dose-dependent manner. In addition, carbon ion beams induced G2/M phase cell cycle arrest in ESCC cells and inhibited tumor metastasis in a dose-dependent manner. Additionally, poorly differentiated KYSE150 cells were more sensitive to the same carbon ion beam dose than moderately differentiated ECA109 cells. Carbon ion beam exposure regulated the relative expression of metastasis-related molecules at the transcriptional and translational levels in ESCC cells. Carbon ion beams also regulated CDH1 and MMP2 downstream of the STAT3 pathway and inhibited ESCC cell metastasis, which activated the STAT3 signaling pathway. This study confirmed the inhibition of cell proliferation and the metastatic effect of carbon ion beam therapy in ESCC cells.

Keywords: JAK2/STAT3 pathway; apoptosis; carbon ion beam; esophageal squamous cell carcinoma; metastasis.

Figure 1

Carbon ion beam therapy inhibits ESCC cell proliferation. (A) Clonogenic survival curves of ECA109 and KYSE150 carcinoma cell lines after carbon ion therapy. (B,C) Colony formation of ECA109 and KYSE150 cells. (D,E) Carbon ions inhibit the proliferation of ECA109 and KYSE150 cells.

Figure 2

Carbon ion-induced apoptosis. (A,B) Apoptosis of ECA109 and KYSE150 cells 24 h after irradiation. (C) Carbon ion irradiation promotes apoptosis of ECA109 and KYSE150 cells. (*,#p < 0.05, ##p < 0.01).

Figure 3

Changes in cell cycle arrest 24 h after irradiation of ECA109 and KYSE150 cells with carbon ion rays. (A,B) Cell cycle distribution of ECA109 and KYSE150 cells 24 h after irradiation. (C,D) Inhibition of cell cycle arrest in ECA109 and KYSE150 cells by carbon ions. *p < 0.05, **p < 0.01.

Figure 4

The effect of carbon ion beam irradiation on ECA109 and KYSE150 cell migration as shown by a wound-healing assay. (A,B) Scratch area of ECA109 and KYSE150 cells 24 h after irradiation. (C) Inhibition of ECA109 and KYSE150 cell migration by carbon ions (*,#p < 0.05, **,##p < 0.01).

Figure 5

The effect of carbon ion beam irradiation on ECA109 and KYSE150 cell invasiveness by Transwell assay. (A) Number of invasive ECA109 cells after irradiation. (B) Number of invasive KYSE150 cells after irradiation. (C) Inhibition of ECA109 and KYSE150 cell invasion by carbon ions (*,#p < 0.05, **,##p < 0.01).

Figure 6

Effect of carbon ion irradiation on the transcription level of STAT3-related molecules in ECA109 and KYSE150 cells. (A) The relative expression of genes related to carbon ion regulation at 24 h after carbon ion irradiation was measured by qRT-PCR in ECA109 cell. (B) The relative expression of genes related to carbon ion regulation at 24 h after carbon ion irradiation was measured by qRT-PCR in KYSE150 cell. (*,^#,^Δ,^⋆, p < 0.05).

Figure 7

Carbon ion beams inhibit ECA109 and KYSE150 cell migration via regulation of JAK2/STAT3 signaling. (A,C) Western blotting analysis of protein expression changes in ECA109 and KYSE150 cells 24 h after irradiation. (B,D) Effect of carbon ion irradiation on relative protein expression in ECA109 and KYSE150 cells (*,^#,^Δ, ^⋆, p < 0.05).