背景:珠毒配方(ZDF)由雷公藤甲素组成,华蟾素和紫杉醇,这是雷公藤的活性成分。F,干蟾蜍皮和红豆杉wallichianavar。chinensis(Pilg)Florin,分别。现代药理研究表明,雷公藤甲素,华蟾素,紫杉醇是众所周知的天然化合物,通过干扰DNA合成发挥抗肿瘤作用,诱导肿瘤细胞凋亡,抑制微管蛋白的动态平衡。然而,三种化合物抑制三阴性乳腺癌(TNBC)转移的机制尚不清楚.
目的:本研究的目的是研究ZDF对TNBC转移的抑制作用,并阐明其潜在机制。
方法:雷公藤甲素(TPL)的细胞活力,华蟾素(CBF),使用CCK-8测定评估MDA-MB-231细胞上的紫杉醇(PTX)。采用Chou-Talalay方法体外测定三种药物对MDA-MB-231细胞的药物相互作用。MDA-MB-231细胞被鉴定为迁移,通过实施划痕试验在体外的侵袭和粘附,transwell测定和粘附测定,分别。免疫荧光法检测细胞骨架蛋白F-肌动蛋白的形成。ELISA法检测细胞上清液中MMP-2和MMP-9的表达。采用Westernblot和RT-qPCR方法研究与RhoA/ROCK和CDC42/MRCK双信号通路相关的蛋白表达。在小鼠4T1TNBC模型中研究了ZDF的体内抗肿瘤功效及其初步机制。
结果:结果表明ZDF能显著降低MDA-MB-231细胞的活力,实际相容性实验点的组合指数(CI)值均小于1,表现出良好的协同相容性关系。发现ZDF降低RhoA/ROCK和CDC42/MRCK双信号通路,它们负责MDA-MB-231细胞的迁移,入侵,和附着力。此外,细胞骨架相关蛋白的表现明显减少。此外,RhoA的表达水平,CDC42、ROCK2和MRCKβmRNA和蛋白表达下调。ZDF显著降低波形蛋白的表达,细胞角蛋白8,Arp2和N-WASP,并抑制肌动蛋白聚合和肌动球蛋白收缩。此外,高剂量ZDF组MMP-2和MMP-9水平分别下降30%和26%,分别。ZDF显著降低肿瘤组织中ROCK2和MRCKβ的肿瘤体积和蛋白质表达,而不会引起小鼠身体质量的任何可察觉的改变,与BDP5290治疗组相比,下降更为明显。
结论:目前的研究表明,ZDF通过RhoA/ROCK和CDC42/MRCK的双重信号通路调节细胞骨架蛋白,对TNBC转移具有良好的抑制作用。此外,结果表明,ZDF在乳腺癌动物模型中具有显著的抗肿瘤和抗转移特性。
BACKGROUND: Zhuidu Formula (ZDF) is composed of triptolide, cinobufagin and paclitaxel, which are the active ingredients of Tripterygium wilfordii Hook. F, dried toad skin and Taxus wallichiana var. chinensis (Pilg) Florin, respectively. Modern pharmacological studies show that triptolide, cinobufagin, and paclitaxel are well-known natural compounds that exert anti-tumor effects by interfering with DNA synthesis, inducing tumor cell apoptosis, and inhibiting the dynamic balance of the tubulin. However, the mechanism by which the three compounds inhibit triple-negative breast cancer (TNBC) metastasis is unknown.
OBJECTIVE: The objective of this investigation was to examine the inhibitory essences of ZDF on the metastasis of TNBC and elucidate its potential mechanism.
METHODS: Cell viability of triptolide (TPL), cinobufagin (CBF), and paclitaxel (PTX) on MDA-MB-231 cells was assessed employing a CCK-8 assay. The drug interactions of the three drugs on MDA-MB-231 cells were determined in vitro utilizing the Chou-Talalay method. MDA-MB-231 cells were identified for migration, invasion and adhesion in vitro through the implementation of the scratch assay, transwell assay and adhesion assay, respectively. The formation of cytoskeleton protein F-actin was detected by immunofluorescence assay. The expressions of MMP-2 and MMP-9 in the supernatant of the cells were determined by ELISA analysis. The Western blot and RT-qPCR were employed to explore the protein expressions associated with the dual signaling pathways of RhoA/ROCK and CDC42/MRCK. The anti-tumor efficacy of ZDF in vivo and its preliminary mechanism were investigated in the mouse 4T1 TNBC model.
RESULTS: The results demonstrated that ZDF could significantly reduce the viability of the MDA-MB-231 cell, and the combination index (CI) values of actual compatibility experimental points were all less than 1, demonstrating a favorable synergistic compatibility relationship. It was found that ZDF reduces RhoA/ROCK and CDC42/MRCK dual signaling pathways, which are responsible for MDA-MB-231cell migration, invasion, and adhesion. Additionally, there has been a significant reduction in the manifestation of cytoskeleton-related proteins. Furthermore, the expression levels of RhoA, CDC42, ROCK2, and MRCKβ mRNA and protein were down-regulated. ZDF significantly decreased the protein expressions of vimentin, cytokeratin-8, Arp2 and N-WASP, and inhibited actin polymerization and actomyosin contraction. Furthermore, MMP-2 and MMP-9 levels in the high-dose ZDF group were decreased by 30% and 26%, respectively. ZDF significantly reduced the tumor volume and protein expressions of ROCK2 and MRCKβ in tumor tissues without eliciting any perceptible alterations in the physical mass of the mice, and the reduction was more pronounced than that of the BDP5290 treated group.
CONCLUSIONS: The current investigation demonstrates that ZDF exhibits a proficient inhibitory impact on TNBC metastasis by regulating cytoskeletal proteins through the dual signaling pathways of RhoA/ROCK and CDC42/MRCK. Furthermore, the findings indicate that ZDF has significant anti-tumorigenic and anti-metastatic characteristics in breast cancer animal models.