<i>Withania</i><i> somnifera</i> Root Extract Inhibits Mammary Cancer Metastasis and Epithelial to Mesenchymal Transition

Though clinicians can predict which patients are at risk for developing metastases, traditional therapies often prove ineffective and metastatic disease is the primary cause of cancer patient death; therefore, there is a need to develop anti-metastatic therapies that can be administered over long durations to specifically inhibit the motility of cancer cells. Withaniasomnifera root extracts (WRE) have anti-proliferative activity and the active component, Withaferin A, inhibits the pro-metastatic protein, vimentin. Vimentin is an intermediate filament protein and is part of the epithelial to mesenchymal transition (EMT) program to promote metastasis. Here, we determined whether WRE standardized to Withaferin A (sWRE) possesses anti-metastatic activity and whether it inhibits cancer motility via inhibition of vimentin and the EMT program. Several formulations of sWRE were created to enrich for Withaferin A and a stock solution of sWRE in EtOH could recover over 90% of the Withaferin A found in the original extract powder. This sWRE formulation inhibited breast cancer cell motility and invasion at concentrations less than 1µM while having negligible cytotoxicity at this dose. sWRE treatment disrupted vimentin morphology in cell lines, confirming its vimentin inhibitory activity. To determine if sWRE inhibited EMT, TGF-β was used to induce EMT in MCF10A human mammary epithelial cells. In this case, sWRE prevented EMT induction and inhibited 3-D spheroid invasion. These studies were taken into a human xenograft and mouse mammary carcinoma model. In both models, sWRE and Withaferin A showed dose-dependent inhibition of tumor growth and metastatic lung nodule formation with minimal systemic toxicity. Taken together, these data support the hypothesis that low concentrations of sWRE inhibit cancer metastasis potentially through EMT inhibition. Moreover, these doses of sWRE have nearly no toxicity in normal mouse organs, suggesting the potential for clinical use of orally administered WRE capsules.尽管临床医生能够预测哪些患者存在发生转移（metastases）的风险，但传统疗法往往收效甚微，而转移性疾病仍是癌症患者死亡的首要原因；因此，亟需开发可长期给药、能够特异性抑制癌细胞运动能力的抗转移疗法。睡茄（Withaniasomnifera）根提取物（WRE）具有抗增殖活性，其活性成分睡茄内酯A（Withaferin A）可抑制促转移波形蛋白（vimentin）。波形蛋白是一种中间丝蛋白，参与上皮间质转化（epithelial to mesenchymal transition, EMT）程序以促进转移。本研究旨在评估以睡茄内酯A为标准品标准化的睡茄根提取物（sWRE）是否具有抗转移活性，以及其是否通过抑制波形蛋白与上皮间质转化程序来抑制癌细胞运动能力。我们制备了多种富集睡茄内酯A的sWRE制剂，其乙醇储备液可回收原提取物粉末中90%以上的睡茄内酯A。该sWRE制剂在低于1µM的浓度下即可抑制乳腺癌细胞的运动与侵袭能力，且在此剂量下细胞毒性可忽略不计。sWRE处理可破坏细胞系中波形蛋白的形态，证实了其抑制波形蛋白的活性。为验证sWRE是否抑制上皮间质转化，我们使用转化生长因子-β（TGF-β）诱导MCF10A人乳腺上皮细胞发生上皮间质转化。在此模型中，sWRE可阻止上皮间质转化的诱导，并抑制三维球体侵袭。我们将这些研究拓展至人源异种移植（human xenograft）与小鼠乳腺癌模型。在两种模型中，sWRE与睡茄内酯A均呈现剂量依赖性的肿瘤生长抑制与肺部转移结节形成抑制，且全身性毒性极低。综上，这些数据支持如下假说：低浓度sWRE可能通过抑制上皮间质转化来抑制癌症转移。此外，该剂量的sWRE对正常小鼠器官几乎无毒性，提示口服给药的睡茄根提取物胶囊具备临床应用潜力。