Abstract：
BACKGROUND: Triple-negative breast carcinomas (TNBCs) are a breast carcinoma with the most aggressive form, which is demonstrated as enhanced invasion and recurrence. Britannin is extracted mainly from the traditional Chinese herb Inula japonica Thunb, and few studies have focused on its effect on TNBC. Moreover, there is still no report concerning the role of Britannin in degrading the transcripts of Zinc finger E-box-binding homeobox 1 (ZEB1) proteins.
OBJECTIVE: To explore the potential effect of Britannin on invasion and stemness of TNBCs and its underlying mechanism.
METHODS: Cellular activity was measured using MTT, and cell cycle was measured using flow cytometry (FCM). The effect of Britannin on the migrating and invading abilities of MDA-MB-231 and 4T1 cells were measured using the wound healing and transwell assays. The sizes and number of breast carcinoma cells were measured by tumor formation assay and in vitro limiting-dilution assay. CD44 expression in tumor spheroids was tested by immunofluorescence assay. Nextly, the expressions of epithelial-mesenchymal transition (EMT) markers and ZEB1 protein expressional level were detected by western blot . ZEB1 mRNA expressional level was analyzed using RT-qPCR. Drug affinity-responsive target stability (DARTS) method was used to detect the binding activity between Britannin and ZEB1. Co-immunoprecipitation (Co-IP) analysis was applied to test the ubiquitination of ZEB1. The mouse models for experimental lung metastasis of 4T1 cells were established to detect the anti-metastasis effect of Britannin in vivo, and the expressional levels of EMT markers in lung metastases were detected by immunohistochemistry.
RESULTS: Britannin could inhibit cell growth and G2/M arrest in TNBC cells. Britannin could inhibit the migrating and invading ability without inducing severe apoptosis of MDA-MB-231 and 4T1 cells. Meanwhile, Britannin reduced the size and number of spheroids formed in these two cells, and decreased the expressional level of stem cells biomarker CD44 in tumor spheroids. Mechanism research showed that Britannin specifically bound to ZEB1 and induced its ubiquitination in MDA-MB-231 cells. Afterwards, Britannin disturbed protein stability and promoted ZEB1 protein degradation. Importantly, Britannin could not inhibit cell invasion and spheroid formation after ZEB1 expression was knocked down. Finally, Britannin inhibition of 4T1 cell metastasis was confirmed through establishing mouse models for the experimental lung metastasis. It was proved that both Britannin and paclitaxel could decrease the lung metastases, and Britannin could also down-regulate the protein expressional levels of ZEB1, MMP9 and CD44.
CONCLUSIONS: This study reveals that Britannin suppresses the invasion and metastasis of TNBC cells through degrading ZEB1, which suggests that Britannin can be used to prevent tumor metastasis and recurrence via degrading ZEB1proteins.
OBJECTIVE: To explore the potential effect of Britannin on invasion and stemness of TNBCs and its underlying mechanism.
METHODS: Cellular activity was measured using MTT, and cell cycle was measured using flow cytometry (FCM). The effect of Britannin on the migrating and invading abilities of MDA-MB-231 and 4T1 cells were measured using the wound healing and transwell assays. The sizes and number of breast carcinoma cells were measured by tumor formation assay and in vitro limiting-dilution assay. CD44 expression in tumor spheroids was tested by immunofluorescence assay. Nextly, the expressions of epithelial-mesenchymal transition (EMT) markers and ZEB1 protein expressional level were detected by western blot . ZEB1 mRNA expressional level was analyzed using RT-qPCR. Drug affinity-responsive target stability (DARTS) method was used to detect the binding activity between Britannin and ZEB1. Co-immunoprecipitation (Co-IP) analysis was applied to test the ubiquitination of ZEB1. The mouse models for experimental lung metastasis of 4T1 cells were established to detect the anti-metastasis effect of Britannin in vivo, and the expressional levels of EMT markers in lung metastases were detected by immunohistochemistry.
RESULTS: Britannin could inhibit cell growth and G2/M arrest in TNBC cells. Britannin could inhibit the migrating and invading ability without inducing severe apoptosis of MDA-MB-231 and 4T1 cells. Meanwhile, Britannin reduced the size and number of spheroids formed in these two cells, and decreased the expressional level of stem cells biomarker CD44 in tumor spheroids. Mechanism research showed that Britannin specifically bound to ZEB1 and induced its ubiquitination in MDA-MB-231 cells. Afterwards, Britannin disturbed protein stability and promoted ZEB1 protein degradation. Importantly, Britannin could not inhibit cell invasion and spheroid formation after ZEB1 expression was knocked down. Finally, Britannin inhibition of 4T1 cell metastasis was confirmed through establishing mouse models for the experimental lung metastasis. It was proved that both Britannin and paclitaxel could decrease the lung metastases, and Britannin could also down-regulate the protein expressional levels of ZEB1, MMP9 and CD44.
CONCLUSIONS: This study reveals that Britannin suppresses the invasion and metastasis of TNBC cells through degrading ZEB1, which suggests that Britannin can be used to prevent tumor metastasis and recurrence via degrading ZEB1proteins.
摘要：
背景：三阴性乳腺癌（TNBC）是一种侵袭性最强的乳腺癌，这被证明是增强的侵袭和复发。大蒜素主要从传统中草药InulajaponicaThunb中提取，很少有研究关注其对TNBC的影响。此外，仍然没有关于不列颠宁在降解锌指E盒结合同源异型盒1（ZEB1）蛋白转录物中的作用的报道。
目的：探讨Britannin对TNBC侵袭和干性的潜在影响及其潜在机制。
方法：使用MTT测量细胞活性，使用流式细胞术(FCM)测量细胞周期。使用伤口愈合和transwell测定法测量了Britannin对MDA-MB-231和4T1细胞的迁移和入侵能力的影响。通过肿瘤形成测定和体外有限稀释测定来测量乳腺癌细胞的大小和数量。通过免疫荧光法检测肿瘤球体中CD44的表达。Nextly,Westernblot检测上皮间质转化（EMT）标志物的表达和ZEB1蛋白的表达水平。使用RT-qPCR分析ZEB1mRNA表达水平。使用药物亲和响应靶标稳定性（DARTS）方法检测Britannin与ZEB1之间的结合活性。应用免疫共沉淀(Co-IP)分析来测试ZEB1的泛素化。建立小鼠实验性4T1细胞肺转移模型，检测Britannin在体内的抗转移作用。免疫组织化学检测肺转移灶中EMT标志物的表达水平。
结果：Britannin可抑制TNBC细胞的生长和G2/M期阻滞。Britannin可以抑制MDA-MB-231和4T1细胞的迁移和侵袭能力，而不会引起严重的凋亡。同时,Britannin减少了这两个细胞中形成的球体的大小和数量，并降低肿瘤球体中干细胞生物标志物CD44的表达水平。机制研究表明,在MDA-MB-231细胞中,Britannin与ZEB1特异性结合并诱导其泛素化。之后,Britannin搅扰了卵白的稳固性，增进了ZEB1卵白的降解。重要的是,在敲除ZEB1表达后，Britannin不能抑制细胞侵袭和球体形成。最后,通过建立实验性肺转移的小鼠模型证实了Britannin对4T1细胞转移的抑制作用。研究证明，不列颠宁宁和紫杉醇均能减少肺转移，和Britannin也可以下调ZEB1，MMP9和CD44的蛋白质表达水平。
结论：本研究揭示了大英霉素通过降解ZEB1抑制TNBC细胞的侵袭和转移，提示大英霉素可通过降解ZEB1蛋白预防肿瘤转移和复发。
目的：探讨Britannin对TNBC侵袭和干性的潜在影响及其潜在机制。
方法：使用MTT测量细胞活性，使用流式细胞术(FCM)测量细胞周期。使用伤口愈合和transwell测定法测量了Britannin对MDA-MB-231和4T1细胞的迁移和入侵能力的影响。通过肿瘤形成测定和体外有限稀释测定来测量乳腺癌细胞的大小和数量。通过免疫荧光法检测肿瘤球体中CD44的表达。Nextly,Westernblot检测上皮间质转化（EMT）标志物的表达和ZEB1蛋白的表达水平。使用RT-qPCR分析ZEB1mRNA表达水平。使用药物亲和响应靶标稳定性（DARTS）方法检测Britannin与ZEB1之间的结合活性。应用免疫共沉淀(Co-IP)分析来测试ZEB1的泛素化。建立小鼠实验性4T1细胞肺转移模型，检测Britannin在体内的抗转移作用。免疫组织化学检测肺转移灶中EMT标志物的表达水平。
结果：Britannin可抑制TNBC细胞的生长和G2/M期阻滞。Britannin可以抑制MDA-MB-231和4T1细胞的迁移和侵袭能力，而不会引起严重的凋亡。同时,Britannin减少了这两个细胞中形成的球体的大小和数量，并降低肿瘤球体中干细胞生物标志物CD44的表达水平。机制研究表明,在MDA-MB-231细胞中,Britannin与ZEB1特异性结合并诱导其泛素化。之后,Britannin搅扰了卵白的稳固性，增进了ZEB1卵白的降解。重要的是,在敲除ZEB1表达后，Britannin不能抑制细胞侵袭和球体形成。最后,通过建立实验性肺转移的小鼠模型证实了Britannin对4T1细胞转移的抑制作用。研究证明，不列颠宁宁和紫杉醇均能减少肺转移，和Britannin也可以下调ZEB1，MMP9和CD44的蛋白质表达水平。
结论：本研究揭示了大英霉素通过降解ZEB1抑制TNBC细胞的侵袭和转移，提示大英霉素可通过降解ZEB1蛋白预防肿瘤转移和复发。
