植物医学1区: 鸦胆子苦素D抑制Notch1-Jagged1/NF-κB信号抑制TNF-α刺激下CAF促进的TNBC转移

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Bruceine D suppresses CAF-promoted TNBC metastasis under TNF-α stimulation by inhibiting Notch1-Jagged1/NF-κB(p65) signaling

Abstract

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.

鸦胆子苦素D通过抑制Notch1-Jagged1/NF-κB(p65)信号抑制TNF-α刺激下CAF促进的TNBC转移

摘要

背景：三阴性乳腺癌（TNBC）由于恶性程度高且缺乏有效的治疗方案，预后较差。肿瘤相关成纤维细胞（CAFs）是肿瘤微环境（TME）中最丰富的基质细胞，可导致功能障碍并促进肿瘤转移。过量的TNF-α进一步促进了肿瘤微环境中不同细胞之间的交叉对话。因此，亟需针对促进TNBC转移的关键因素开发更有效的疗法和潜在药物。

目的：本研究旨在评估从中草药苦参子中提取的活性化合物鸦胆子苦素D抑制TNBC转移的疗效，并阐明其在TNBC中的作用机制。

方法: 在体外，采用克隆形成试验和Transwell试验评估布鸦胆子苦素D在TNF-α刺激下对共培养的CAFs和MDA-MB-231（4T1）细胞的增殖、迁移和侵袭能力的影响。用酶联免疫吸附法测定共培养细胞上清液中TNF-α、IL-6、CXCL12、TGF-β1和MMP9的水平。用免疫印迹法检测与Notch1-Jagged1/NF-κB(p65) 通路相关的蛋白质的表达水平。在体内，通过测定肿瘤重量、转移灶数量以及肿瘤和肺/肝组织的病理变化来评估鸦胆子苦素D的抗肿瘤生长和抗转移效果。免疫组化、免疫荧光、Masson和Sirius Red染色法检测了鸦胆子苦素D对α-SMA+ CAFs活化和CAF介导的细胞外基质重塑的抑制作用。免疫组化和免疫印迹法检测了原发性肿瘤中Notch1、Jagged1和p-NF-κB(p65)蛋白的表达水平。

结果: 在体外，鸦胆子苦素D能明显抑制CAFs和MDA-MB-231（4T1）细胞在TNF-α刺激下的迁移和侵袭，降低CAFs分泌的促肿瘤细胞因子和基质重塑细胞因子的表达，阻碍Notch1-Jagged1和NF-κB(p65)的相互激活。在体内，鸦胆子苦素D通过减少TNF-α刺激的α-SMA+ CAFs的活化、胶原纤维和MMPs的产生，以及抑制Notch1-Jagged1/NF-κB(p65)信号传导，明显抑制了TNBC小鼠的肿瘤生长以及肺和肝转移灶的形成。

结论:鸦胆子苦素D通过抑制Notch1-Jagged1和NF-κB(p65)的相互激活，有效削弱了"肿瘤-CAF-炎症"网络，从而抑制了TNBC的转移。该研究首次发现了在TNF-α刺激下，鸦胆子苦素D能破坏CAFs与肿瘤细胞之间的交叉对话，从而抑制TNBC的转移，凸显了鸦胆子苦素D作为抑制肿瘤转移的治疗药物的潜力。

1. Bruceine D单独或由CAFs促进抑制MDA-MB-231细胞、4T1细胞和CAFs的增殖和集落形成能力。

2.在TNF-α刺激下，Bruceine D抑制CAF促进的MDA-MB-231细胞迁移和侵袭能力。

3.Bruceine D对小鼠原发性肿瘤生长的抑制作用。

4.Bruceine D抑制小鼠肿瘤细胞的肺和肝转移。

5.在TNF-α诱导的炎症微环境中，Bruceine D通过减少胶原纤维的形成和MMPs的产生来减少α-SMA+CAF的激活并重塑ECM。

6.Bruceine D抑制小鼠原发性肿瘤中Notch1、Jagged1和p-NF-κB（p65）蛋白的表达。

总之，我们的体外数据表明，TNF-α通过增强Notch1-Jagged1和NF-κB（p65）的相互调节，以及通过促进TNF-α、IL-6、CXCL12、TGF-β1和MMP9的产生，加速了CAF促进两种TNBC细胞系的迁移和侵袭。我们的体内结果证实了肿瘤CAF炎症网络的存在，这是TNBC侵袭性的重要驱动因素。体外和体内研究结果表明，Bruceine D有可能通过降低Notch1、Jagged1、NF-κB（p65）和TNF-α的表达来重塑ECM，重新教育CAFs和肿瘤细胞的恶性行为。基于Bruceine D的研究数据，本研究为控制TNBC攻击提供了一种新的治疗策略，并强调Bruceine D是干预TME的候选药物。

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