纳米雄黄的抗小鼠原位乳腺癌作用及其机制

目的: 研究纳米雄黄体内体外对小鼠乳腺癌细胞(4T1)的增殖抑制作用及其机制。方法: 以萤火虫荧光素酶(Luciferase, Luc)基因标记小鼠4T1乳腺癌(4T1-Luc)细胞,应用噻唑蓝(MTT)比色法和生物发光法(Bioluminescent method, BLM)检测细胞增殖活性;细胞形态学及Annexin V/PI 双标记法观察细胞凋亡。4T1-Luc细胞接种雌性BALB/c小鼠乳腺脂肪垫制作原位乳腺癌模型,纳米雄黄(4和 8 mg·kg^-1·d^-1)灌胃治疗 20 d,小动物活体成像系统连续动态观察小鼠乳腺肿瘤生长变化,治疗末期处死动物、剥离肿瘤块称重,并制片HE染色和CD₃₄标记观测肿瘤组织内细胞核分裂像、新生血管形成及坏死改变。结果: MTT法和BLM法检测显示 1.56~50 μg/mL 纳米雄黄体外显著抑制 4T1-Luc 细胞的增殖(P<0.05);形态学观察和Annexin V/PI染色显示细胞呈现典型的凋亡改变。体内纳米雄黄呈时间和剂量依赖性地抑制小鼠4T1-Luc原位乳腺癌的生长(P<0.05);肿瘤组织制片观察,经纳米雄黄治疗后肿瘤组织内细胞核分裂像和微小血管显著减少(P<0.01),肿瘤组织内部呈显著的坏死改变。结论: 纳米雄黄体外抑制小鼠乳腺癌4T1细胞增殖和诱导细胞凋亡,并主要通过减低原位肿瘤组织内新生血管的形成而导致肿瘤组织坏死而发挥抗乳腺癌作用。

The anti-tumor effects and mechanism of Realgar nanoparticles on orthotopic breast cancer in mice

AIM: To study the anti-proliferating action of realgar nanoparticles (nano-realgar) on murine orthotopic transplanted breast carcinoma (4T1) in vitro and in vivo.METHODS: The firefly luciferase gene was transferred into murine 4T1 breast cancer cells with a lentiviral vector (4T1-Luc cells). Both MTT colorimetric assay and bioluminescence (BLM) assay was used to detect the cellular cell proliferation, the morphological observation and Annexin V and propidium iodide (Annexin V/PI) double-labeling were employed to assess the cell apoptosis in vitro. The 4T1-Luc cells were orthotopically implanted into the mammary fat pad of female BALB/c mice to establish the orthotopic transplanted model of mouse breast cancer, the tumor-bearing mice were treated with 4 mg·kg^-1·d^-1 and 8 mg·kg^-1·d^-1 nano-realgar, by gavage once a day, for 20 days, respectively, the optical in vivo imaging system was used to continuously and dynamically observe the growth, and at the end of the treatment the animals were sacrificed, and the tumor tissue was removed and weighed. The tumor tissue sections were prepared and stained with HE stain for examination of karyokinesis and necrosis in tumors, and the tumor angiogenesis was observed by CD₃₄ immunocytochemistry.RESULTS: Both MTT and BLM assays showed that 1.56-50 μg/mL nano-realgar significantly inhibited the proliferation of 4T1-Luc cells (P<0.05), and the morphological observation and Annexin V/PI staining displayed the typical apoptotic characteristics in the cells. Nano-realgar significantly inhibited the growth of the mouse 4T1 orthotopic breast cancer in vivo at a time- and dose-dependent manner (P<0.05). The pathological and immunocytochemistry examination showed that after nano-realgar administration, the numbers of mitotic cells and microvessels in tumor tissue markedly reduced, and the extensive necrosis region could be observed at the center of the tumor tissue.CONCLUSION: Realgar nanoparticles significantly inhibits the proliferation of murine 4T1 breast cancer cells and induces to apoptosis in vitro, and plays the anti-tumor action on orthotopic transplanted breast cancer in mice via repressing neovascularization to impel necrosis of the tumor.