Phytomedicine ( IF 6.7 ) Pub Date : 2023-06-16 , DOI: 10.1016/j.phymed.2023.154928 Han Chen 1 , Xue Han 2 , Yue Zhang 2 , Ke Wang 2 , Da Liu 2 , Zhiqiang Hu 3 , Jing Wang 4
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Background
Triple-negative breast cancer (TNBC) has a poor prognosis because of its high degree of malignancy and the lack of effective treatment options. Cancer-associated fibroblasts (CAFs) comprise the most abundant stromal cells in the tumor microenvironment (TME), leading to functional impairments and facilitating tumor metastasis. Excessive TNF-α further promotes cross-talk between different cells in TME. Therefore, there is an urgent need to develop more effective therapies and potential drugs that target the key factors that promote TNBC metastasis.
Purpose
The study aimed to evaluate the efficacy of Bruceine D, an active compound derived from the Chinese herb Brucea javanica, in inhibiting metastasis and elucidate the underlying mechanism of action in TNBC.
Methods
In vitro, the clonogenic and the Transwell assays were used to assess the effects of Bruceine D on the proliferation, migration and invasion abilities of co-cultured CAFs and MDA-MB-231 (4T1) cells under TNF-α stimulation. TNF-α, IL-6, CXCL12, TGF-β1, and MMP9 levels in the supernatant of co-cultured cells were determined using ELISA. Western blotting was utilized to detect the expression levels of proteins related to the Notch1-Jagged1/NF-κB(p65) pathway. In vivo, the anti-tumor growth and anti-metastatic effectiveness of Bruceine D was evaluated by determining tumor weight, number of metastatic lesions, and pathological changes in the tumor and lung/liver tissues. The inhibitory effect of Bruceine D on α-SMA+ CAFs activation and CAF-medicated extracellular matrix remodeling was accessed using immunohistochemistry, immunofluorescence, and Masson and Sirius Red staining. The expression levels of Notch1, Jagged1 and p-NF-κB(p65) proteins in the primary tumors were measured by immunohistochemistry and western blotting.
Results
In vitro, Bruceine D significantly inhibited the migration and invasion of co-cultured CAFs and MDA-MB-231 (4T1) cells under TNF-α stimulation, reduced the expression of tumor-promoting and matrix-remodeling cytokines secreted by CAFs, and hindered the mutual activation of Notch1-Jagged1 and NF-κB(p65). In vivo, Bruceine D significantly suppressed tumor growth and the formation of lung and liver metastases by decreasing TNF-α stimulated α-SMA+ CAFs activation, collagen fibers, MMPs production, and inhibited Notch1-Jagged1/NF-κB(p65) signaling in TNBC-bearing mice.
Conclusion
Bruceine D effectively weakened the "tumor-CAF-inflammation" network by inhibiting the mutual activation of Notch1-Jagged1 and NF-κB(p65) and thereby suppressed TNBC metastasis. This study first explored that Bruceine D disrupted the cross-talk between CAFs and tumor cells under TNF-α stimulation to inhibit the metastasis of TNBC, and highlighted the potential of Bruceine D as therapeutic agent for suppressing tumor metastasis.
中文翻译：
Bruceine D 通过抑制 Notch1-Jagged1/NF-κB(p65) 信号传导抑制 TNF-α 刺激下 CAF 促进的 TNBC 转移
背景
三阴性乳腺癌（TNBC）因其恶性程度高且缺乏有效的治疗方案而预后较差。癌症相关成纤维细胞（CAF）包含肿瘤微环境（TME）中最丰富的基质细胞，导致功能损伤并促进肿瘤转移。过量的TNF-α进一步促进TME中不同细胞之间的串扰。因此，迫切需要针对促进TNBC转移的关键因素开发更有效的疗法和潜在药物。
目的
该研究旨在评估布鲁斯碱 D（一种从中草药鸦胆子中提取的活性化合物）抑制转移的功效，并阐明 TNBC 的潜在作用机制。
方法
在体外，通过克隆形成和Transwell实验评估Bruceine D对TNF-α刺激下共培养的CAF和MDA-MB-231 (4T1)细胞的增殖、迁移和侵袭能力的影响。使用 ELISA 测定共培养细胞上清液中的 TNF-α、IL-6、CXCL12、TGF-β1 和 MMP9 水平。 Western blotting检测Notch1-Jagged1/NF-κB(p65)通路相关蛋白的表达水平。在体内，通过测定肿瘤重量、转移病灶数量以及肿瘤和肺/肝组织的病理变化来评估布鲁斯碱D的抗肿瘤生长和抗转移功效。使用免疫组织化学、免疫荧光以及 Masson 和 Sirius Red 染色来评估 Bruceine D 对 α-SMA + CAF 激活和 CAF 治疗的细胞外基质重塑的抑制作用。采用免疫组化和免疫印迹法检测原发肿瘤中Notch1、Jagged1和p-NF-κB(p65)蛋白的表达水平。
结果
在体外，Bruceine D 显着抑制共培养的 CAF 和 MDA-MB-231 (4T1) 细胞在 TNF-α 刺激下的迁移和侵袭，减少 CAF 分泌的促肿瘤和基质重塑细胞因子的表达，并阻碍Notch1-Jagged1 和 NF-κB(p65) 的相互激活。在体内，Bruceine D 通过减少 TNF-α 刺激的 α-SMA + CAF 活化、胶原纤维、MMP 产生，并抑制 Notch1-Jagged1/NF-κB(p65) 信号传导，显着抑制肿瘤生长以及肺和肝转移的形成。携带 TNBC 的小鼠。
结论
Bruceine D 通过抑制 Notch1-Jagged1 和 NF-κB(p65) 的相互激活，有效削弱“肿瘤-CAF-炎症”网络，从而抑制 TNBC 转移。本研究首次探讨了Bruceine D在TNF-α刺激下破坏CAF与肿瘤细胞之间的串扰来抑制TNBC的转移，并强调了Bruceine D作为抑制肿瘤转移的治疗剂的潜力。
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