External use of Ruyanneixiao cream efficiently blocks precancerous mammary lesions by interfering with glycolysis induced by inhibition of hypoxia inducible factor-1α, hexokinase 2, phosphofructokinase, and pyruvate kinase M2 expression

OBJECTIVE:To investigate the effect of Ruyanneixiao cream(RYNX) on the expression of hypoxia inducible factor-1α(HIF-1α), hexokinase 2(HK2),phosphofructokinase(PFK), and pyruvate kinase M2(PKM2) mRNA and protein in MCF-10 AT cells and in an animal model of precancerous mammary lesions.METHODS:Following treatment of MCF-10 AT cells with RYNX, tamoxifen(TAM) and YC-1 for 48 h,HIF-1α, HK2, PFK, PKM2 mRNA and protein expression was analyzed.Fifty female SD rats were randomly divided into control, model, TAM, and highand low-dose RYNX groups, with 10 rats in each group.A precancerous mammary lesion model was established for all groups except the control group.High-and low-dose RYNX cream containing TAM was coated on the breasts of animals in the corresponding groups.The rat mammary tissue was removed in the 10 th week and HIF-1α, HK2, PFK,PKM2 mRNA and protein was analyzed.RESULTS:In vitro analyses demonstrated that, compared with the matrix group, HIF-1α, HK2, PFK,PKM2 mRNA and protein expression was significantly decreased in the RYNX group(P < 0.05).Compared with the YC-1 + RYNX group, HK2, PFK,and PKM2 protein expression was significantly reduced in the RYNX group.HIF-1α, HK2, PFK, and PKM2 protein expression was increased significantly in the model group(P < 0.05) compared with the control group, while HIF-1α, HK2, PFK, and PKM2 mRNA and protein expression was significantly decreased in both the high-and low-dose RYNX groups(P < 0.05), with the effect being greater in the high-dose group.CONCLUSION:RYNX can block precancerous breast lesions by decreasing the expression of HK2,PFK, and PKM2 mRNA and protein via inhibition of HIF-1α mRNA and protein overexpression in a dose-dependent manner.

乳岩内消霜外用对大鼠乳腺癌癌前病变组织VEGF、FGF2表达的影响

目的:观察乳岩内消霜外用对大鼠乳腺癌癌前病变模型乳腺组织中血管内皮生长因子(VEGF)和碱性成纤维细胞生长因子(FGF2)表达的影响,探寻乳岩内消霜治疗乳腺癌癌前病变的机制.方法:36只雌性未孕SD大鼠,随机分为空白对照组、疾病模型组、三苯氧胺组及乳岩内消霜质量分数分别为4 000、2 000、1 000 mg/g组.采用二甲基苯并蔥(DMBA)联合雌、孕激素诱导癌前病变大鼠模型,对乳房外用药物干预,第10周处死动物,进行组织病理学检测,Western blotting及免疫组化法检测乳腺组织中VEGF、FGF2的表达.结果:各药物干预组乳腺组织VEGF、FGF2的表达均明显低于疾病模型组(P<0.05);Western blotting检测乳岩内消霜质量分数4 000 mg/g组VEGF表达低于乳岩内消霜质量分数2 000和1 000 mg/g组(P<0.05);免疫组化检测乳岩内消霜质量分数4 000mg/g组FGF2表达低于乳岩内消霜质量分数1 000 mg/g组(P<0.05).结论:乳岩内消霜外用能够有效下调VEGF、FGF2在乳腺癌癌前病变大鼠乳腺组织中的表达,可能通过抑制血管生成因子表达而防治乳腺癌的有效机制,具有量效关系.

乳岩内消霜通过内源性caspase途径诱导乳腺癌前病变凋亡作用的机制研究

目的研究乳岩内消霜诱导乳腺癌前病变细胞凋亡的作用,探寻乳岩内消霜治疗乳腺癌前病变的机制。方法 MTT法观察不同浓度乳岩内消霜透皮液对乳腺癌前MCF-10AT细胞的生长抑制作用;流式细胞术检测MCF-10AT细胞凋亡率;Western blotting检测MCF-10AT细胞中bcl-2和bax蛋白;60只雌性未孕SD大鼠,采用二甲基苯并蔥(DMBA)联合雌、孕激素诱导癌前病变模型,同时使用霜剂涂抹大鼠乳房,第10周处死大鼠,进行组织病理学检测;Western blotting分别检测大鼠乳腺组织和MCF-10AT细胞cleaved caspase9、cleaved caspase3蛋白的表达。结果 MTT结果显示,乳岩内消霜透皮液对MCF-10AT细胞增殖有显著抑制作用;1%、2%和4%乳岩内消霜处理MCF-10AT细胞24 h,细胞凋亡率分别为(24.8±4.1)%、(43.2±5.3)%和(65.6±6.8)%;与空白对照组比较,乳岩内消霜和三苯氧胺干预MCF-10AT细胞后bcl-2表达下降,bax表达上升(P<0.05);与正常对照组比较,大鼠乳腺组织和MCF-10AT细胞中乳岩内消霜及三苯氧胺干预组cleaved caspase9、cleaved caspase3表达均明显上升(P<0.01)。结论乳岩内消霜有效抑制乳腺癌前病变发展,可能是通过内源性caspase途径诱导乳腺癌前病变细胞凋亡实现的。