Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Integration of Chinese and Western Medicine, Peking University, Cancer Hospital and Institute, Beijing, 100142, PR China.
Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Integration of Chinese and Western Medicine, Peking University, Cancer Hospital and Institute, Beijing, 100142, PR China.
J Ethnopharmacol. 2022 Nov 15;298:115607. doi: 10.1016/j.jep.2022.115607. Epub 2022 Aug 13.
Marsdenia tenacissima (Roxb.) Wight et Arn. is a traditional Chinese herbal medicine, and its water-soluble ingredient Marsdenia tenacissima extract (MTE), was widely used for cancer treatment. The multi-pharmacological efficacies and mechanisms of MTE in directly inhibiting tumor cells have been extensively studied. However, the anti-tumor effects of MTE in the tumor-associated macrophages (TAMs) microenvironment remain unclear.
To uncover the role of hepatoma-derived growth factor (HDGF) in the interaction between TAMs and non-small cell lung cancer (NSCLC) cells. To evaluate the anti-tumor effects of MTE on the vicious crosstalk between TAMs and NSCLC by targeting HDGF.
HDGF-overexpression PC-9 and H292 NSCLC cell lines were constructed and verified. RNA-sequencing (RNA-seq) was performed in HDGF-overexpression PC-9 cells to probe the differential expression of genes. THP-1-derived macrophages were characterized using specific markers after stimulation with phorbol-12-myristate 13-acetate (PMA) and rhIL-4 or rhHDGF. The role of HDGF both in NSCLC cells and TAMs was determined using approaches like Western blot, qRT-PCR, ELISA, and flow cytometry. The interaction between tumor cells and TAMs were assessed by indirect co-culture H1975, PC-9 cells with M2 type macrophages. The effects of MTE on anti-tumor and macrophage polarization were evaluated in vitro and in vivo.
RNA-seq results identified IL-4 as a critical response to HDGF in NSCLC. HDGF induced macrophages polarizing toward M2 type, and promoted NSCLC cells proliferation, migration and invasion in vitro. On the one hand, HDGF dose-dependently promoted IL-4 expression in NSCLC cells. On the other hand, HDGF induced M2 macrophage polarization through the IL-4/JAK1/STAT3 signaling pathway. MTE treatment significantly decreased the expression and secretion of HDGF in NSCLC cells. Meanwhile, MTE treatment led to M2 macrophage repolarization, as evidenced by decreased expression of M2 markers and increased levels of M1 markers. Importantly, MTE treatment significantly suppressed tumor development in C57BL/6 mice bearing Lewis lung cancer (LLC) cells in vivo, accompanied by decreased plasma HDGF levels, reduced M2 macrophages infiltration and increased M1 macrophages proportion in mice tumor tissues.
HDGF upregulated IL-4 expression in NSCLC cells, and promoted M2 polarization by the IL-4/JAK1/STAT3 signaling pathway in macrophages. MTE disturbed the interaction between NSCLC and TAMs in vitro, and inhibited tumor growth in vivo, at least in part, by suppressing HDGF. Therefore, our present study revealed a novel anti-tumor mechanism of MTE through inhibiting HDGF expression and enhancing macrophage polarization from M2 to M1 phenotype.
密蒙花（Roxb.）Wight et Arn. 是一种传统的中药，其水溶性成分密蒙花提取物（MTE）被广泛用于癌症治疗。MTE 对肿瘤细胞的直接抑制的多药效学作用及其机制已得到广泛研究。然而，MTE 在肿瘤相关巨噬细胞（TAMs）微环境中的抗肿瘤作用尚不清楚。
揭示肝癌衍生生长因子（HDGF）在 TAMs 与非小细胞肺癌（NSCLC）细胞相互作用中的作用。评估 MTE 通过靶向 HDGF 对 TAMs 和 NSCLC 之间恶性串扰的抗肿瘤作用。
构建并验证了 HDGF 过表达 PC-9 和 H292 NSCLC 细胞系。在 HDGF 过表达的 PC-9 细胞中进行 RNA 测序（RNA-seq），以探测基因的差异表达。用佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）和 rhIL-4 或 rhHDGF 刺激后，用特异性标志物对 THP-1 衍生的巨噬细胞进行特征描述。通过 Western blot、qRT-PCR、ELISA 和流式细胞术等方法确定 HDGF 在 NSCLC 细胞和 TAMs 中的作用。通过间接共培养 H1975、PC-9 细胞与 M2 型巨噬细胞来评估肿瘤细胞与 TAMs 之间的相互作用。在体外和体内评估 MTE 对抗肿瘤和巨噬细胞极化的影响。
RNA-seq 结果表明，IL-4 是 NSCLC 中对 HDGF 的关键反应。HDGF 诱导巨噬细胞向 M2 型极化，并促进 NSCLC 细胞在体外的增殖、迁移和侵袭。一方面，HDGF 剂量依赖性地促进 NSCLC 细胞中 IL-4 的表达。另一方面，HDGF 通过 IL-4/JAK1/STAT3 信号通路诱导 M2 巨噬细胞极化。MTE 处理可显著降低 NSCLC 细胞中 HDGF 的表达和分泌。同时，MTE 处理导致 M2 巨噬细胞再极化，表现为 M2 标志物的表达降低和 M1 标志物的水平增加。重要的是，MTE 处理可显著抑制 C57BL/6 小鼠体内 Lewis 肺癌（LLC）细胞的肿瘤发展，体内血浆 HDGF 水平降低，肿瘤组织中 M2 巨噬细胞浸润减少，M1 巨噬细胞比例增加。
HDGF 在 NSCLC 细胞中上调 IL-4 的表达，并通过 IL-4/JAK1/STAT3 信号通路促进巨噬细胞的 M2 极化。MTE 在体外干扰 NSCLC 和 TAMs 之间的相互作用，并通过抑制 HDGF 在体内抑制肿瘤生长，至少部分是通过抑制 HDGF。因此，我们的研究揭示了 MTE 通过抑制 HDGF 表达和增强巨噬细胞从 M2 向 M1 表型的极化来发挥抗肿瘤作用的新机制。
