Sulfasalazine antagonizes boosted metastasis of breast cancer induced by ionizing radiation

Abstract

Objective To evaluate the inhibitory effects of sulfasalazine (SSZ) on ionizing radiation (IR) induced epithelial-to-mesenchymal transition (EMT), migration, invasion, and other metastasis-related phenotypes in breast cancer cells in vitro, and to investigate its antagonistic effects as a therapeutic measure against breast cancer lung metastasis promoted by in vivo local IR. Methods Cultured breast cancer cell lines MCF-7 and 4T1 were used to study the morphological changes of cells after different doses of X-ray irradiation (2, 4, 6, 8 and 10 Gy) and the expression changes of EMT-related markers such as Vimentin and Snail were detected by Western blotting. A cell model with 4 Gy of X-ray irradiation was selected to evaluate the effect of SSZ (0.5 mmol/L) on metastasis-related phenotypes such as EMT, migration, and invasion of cells after IR, and on the protein expression of MMP2/9 and xCT after IR. After xenograft model of 4T1 cell breast cancer lung metastasis was established in BALB/c female mice, the mice were divided into control group, SSZ group (intraperitoneal injection of 8 mg SSZ, 2 times per day), IR group (X-ray 4 Gy irradiation) and IR+SSZ group (X-ray 4 Gy irradiation, intraperitoneal injection of 8 mg SSZ, 2 times per day). The tumor masses were harvested in 30 d after tumor loading, and the size of in situ tumor and the number of lung metastatic nodes were measured and calculated, and HE staining was used to observed in situ tumor and lung metastases. Results Cultured breast cancer cells showed significant EMT morphological alterations after IR, and the expression of Vimentin and Snail was upregulated in a dose-related manner. SSZ inhibited IR-induced changes in EMT morphology and marker proteins, suppressed IR-induced phenotypes of enhanced migration and invasion of breast cancer cells (P < 0.05), and down-regulated IR-induced enhanced expression of MMP2/9, xCT, and other proteins. In vivo animal model corresponded to the ex vivo cellular results that IR significantly inhibited in situ tumor growth. Compared with the control mice (1.70±0.54 cm3), the mean tumor volume was significantly smaller in the IR group (1.24±0.32 cm3) and IR+SSZ group (0.83± 0.54 cm3) (P < 0.05), and the reduction was more significant in the latter than in the former group (P < 0.05). The number of lung metastases in the IR group (19.00±8.31) was significantly higher than that in the Con group (8.67±7.88) (P < 0.05), and the number in the IR+SSZ group (6.10±7.78) was significantly lower than the IR group (P < 0.05), while the number in the SSZ group (4.30±5.39) was not statistically different from the Con group. HE pathology showed that the number of metastases in the IR group was increased, the area was enlarged, the border was not clear, and the metastases had a tendency to diffuse. In contrast, the lung metastases in the IR+SSZ group had clearer borders and a weaker tendency to invade outward. Conclusion SSZ effectively inhibits IR-induced EMT, migration, and invasion of breast cancer cells and significantly antagonizes the lung metastasis-promoting effects of in vivo local irradiation of breast cancer.

Keywords

• breast cancer
• metastasis
• ionizing radiation
• epithelial-mesenchymal transition
• sulfasalazine