Background

Triple negative breast cancer (TNBC) has the worst prognosis of the any breast cancer subtype, and the efficient therapeutical treatment is extremely limited. Antenoron filiforme (Thunb.) Roberty & Vautier (AF) is a Traditional Chinese Medicine (TCM), which is well-known for a diverse array of pharmacological activities, including but not limited to anti-inflammatory, antioxidant and anti-tumors properties. Clinically, AF is commonly prescribed for the treatment of gynecological diseases.

Purpose

Since TNBC is one of the worst gynecological diseases, the objective of this research is to study the anti-TNBC function of the ethyl acetate extract (EAE) of AF (AF-EAE) and disclose its mechanism of action.

Materials and methods

With the aim of elucidating the underlying molecular mechanism and possible chemical basis of AF-EAE in the treatment of TNBC, a comprehensive approach combining system pharmacology and transcriptomic analysis, functional experimental validation, and computational modeling was implemented. Firstly, the potential therapeutic targets of AF-EAE treating TNBC were analyzed by systemic pharmacology and transcriptome sequencing. Subsequently, cell viability assays, cell cycle assays, and transplantation tumor assays were employed to detect the inhibitory effect of AF-EAE on TNBC. Apart from that, the western blot and RT-qPCR assays were adopted to verify its mechanism of action. Finally, the potential chemical basis of anti-TNBC function of AF-EAE was screened through molecular docking and validated by molecular dynamics.

Results

This study analyzed the differentially expressed genes after AF-EAE treatment by RNA-sequencing (RNA-seq). It was found that most of the genes were abundant in the gene set termed “cell cycle”. Besides, AF-EAE could suppress the proliferation of TNBC cells in vitro and in vivo by inhibiting the function of Skp2 protein. AF-EAE could also lead to the accumulation of p21 and a decrease of CDK6/CCND1 protein, thereby stalling the cycle of cell in the G1/S stage. Notably, clinical data survival analysis clearly demonstrated that Skp2 overexpression has been negatively correlated with survival rates in breast cancer (BC) patients. Further, as suggested by molecular docking and molecular dynamics, the quercetin and its analogues of AF-EAE might bind to Skp2 protein.

Conclusion

In summary, AF-EAE inhibits the growth of TNBC in vitro and in vivo through targeting Skp2/p21 signaling pathway. While providing a novel potential drug for treating TNBC, this study might establish a method to delve into the action mechanism of TCM.

中文翻译：

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丝状触角乙酸乙酯提取物通过抑制Skp2/p21信号轴抑制三阴性乳腺癌细胞增殖

背景

三阴性乳腺癌（TNBC）是所有乳腺癌亚型中预后最差的一种，有效的治疗方法极为有限。Antenonon filiforme (Thunb.) Roberty & Vautier (AF) 是一种传统中药 (TCM)，以其多种药理活性而闻名，包括但不限于抗炎、抗氧化和抗肿瘤特性。临床上，房颤常用于治疗妇科疾病。

目的

由于 TNBC 是最严重的妇科疾病之一，本研究的目的是研究 AF 的乙酸乙酯提取物 (EAE) (AF-EAE) 的抗 TNBC 功能，并揭示其作用机制。

材料和方法

为了阐明 AF-EAE 治疗 TNBC 的潜在分子机制和可能的化学基础，实施了一种结合系统药理学和转录组学分析、功能实验验证和计算模型的综合方法。首先，通过系统药理学和转录组测序分析了AF-EAE治疗TNBC的潜在治疗靶点。随后，采用细胞活力测定、细胞周期测定和移植肿瘤测定来检测AF-EAE对TNBC的抑制作用。除此之外，还采用了蛋白质印迹和RT-qPCR检测来验证其作用机制。最后，通过分子对接筛选AF-EAE抗TNBC功能的潜在化学基础，并通过分子动力学进行验证。

结果

本研究通过 RNA 测序 (RNA-seq) 分析了 AF-EAE 处理后的差异表达基因。发现大多数基因在称为“细胞周期”的基因组中是丰富的。此外，AF-EAE可通过抑制Skp2蛋白的功能，在体内外抑制TNBC细胞的增殖。AF-EAE 还可能导致 p21 的积累和 CDK6/CCND1 蛋白的减少，从而使细胞周期停滞在 G1/S 期。值得注意的是，临床数据生存分析清楚地表明，Skp2 过表达与乳腺癌 (BC) 患者的生存率呈负相关。此外，正如分子对接和分子动力学所表明的，槲皮素及其 AF-EAE 类似物可能与 Skp2 蛋白结合。

结论

总之，AF-EAE通过靶向Skp2/p21信号通路在体外和体内抑制TNBC的生长。在为治疗 TNBC 提供一种新的潜在药物的同时，本研究可能建立一种深入研究中药作用机制的方法。