柳氮磺吡啶拮抗电离辐射对乳腺癌转移促进作用的实验研究

Sulfasalazine antagonizes boosted metastasis of breast cancer induced by ionizing radiation

目的 评价柳氮磺吡啶(sulfasalazine, SSZ)对电离辐射(ionizing radiation, IR)诱导离体乳腺癌细胞上皮-间质转化(epithelial-to-mesenchymal transition, EMT)、迁移、侵袭等转移相关表型的抑制作用,探讨其作为治疗措施对在体局部IR促进乳腺癌肺转移的拮抗作用。方法 以体外培养两种乳腺癌细胞株(MCF-7、4T1)为研究对象,采用不同剂量(2、4、6、8、10 Gy)X射线照射后观察细胞形态改变,Western blot检测Vimentin、Snail等EMT相关标志物的表达变化。选取4 Gy X射线照射细胞,评价SSZ(0.5 mmol/L)对IR后细胞EMT、迁移、侵袭等转移相关表型的影响,并检测MMP2/9和xCT等蛋白的表达。在BALB/c雌鼠建立4T1细胞乳腺癌肺转移模型,实验分4组:Con组、SSZ组、IR组和IR+SSZ组。IR采用局部X射线(4 Gy)照射;SSZ采用腹腔给药8 mg/只,2次/d;IR+SSZ组采用局部X射线(4 Gy)照射后,以腹腔给药SSZ 8 mg/只,2次/d。荷瘤30 d后取材,计算原位瘤大小和肺转移结节数目,HE染色观察原位瘤和肺转移灶情况。结果 体外培养乳腺癌细胞在IR后出现明显的EMT形态改变,Vimentin、Snail等EMT相关蛋白表达上调,呈一定剂量相关性。SSZ能够抑制IR诱导的乳腺癌细胞EMT形态和标志蛋白改变,抑制IR诱导的乳腺癌细胞迁移和侵袭能力增强的表型(P<0.05),下调IR诱导的MMP2/9、xCT等蛋白增强表达。在体动物模型与离体细胞结果相对应,IR可以显著抑制原位瘤生长,与Con组比较,IR组[(1.24±0.32)cm^(3)]和IR+SSZ组[(0.83±0.54)cm^(3)]的小鼠原位瘤平均体积显著降低(P<0.05),且IR+SSZ组较IR组降低更显著(P<0.05);IR组小鼠肺转移结节数为[(19.00±8.31)个],较Con组[(8.67±7.88)个]显著增多(P<0.05),IR+SSZ组小鼠肺转移结节数[(6.10±7.78)个]较IR组显著降低(P<0.05),而单纯SSZ组小鼠肺转移结节数[(4.30±5.39)个]与Con组相比差异无统计学意义。HE染色结果显示,IR组肺转移灶数量增多,面积增加,边界欠清,有明显弥散的趋势;而IR+SSZ组肺转移灶面积较小,边界较清晰。结论 SSZ能有效抑制IR诱导乳腺癌细胞EMT、迁移、侵袭,并显著拮抗在体局部照射对乳腺癌肺转移的促进作用。

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 4 T1 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 4 T1 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 cm^(3)), the mean tumor volume was significantly smaller in the IR group(1.24±0.32 cm^(3)) and IR+SSZ group(0.83±0.54 cm^(3))(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.