Department of Integration of Chinese and Western Medicine, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education, Beijing), Peking University Cancer Hospital & Institute, Beijing, 100142, PR China.
Department of Gastroenterology, Peking University International Hospital, Beijing, 102206, PR China.
J Ethnopharmacol. 2020 Mar 25;250:112524. doi: 10.1016/j.jep.2019.112524. Epub 2019 Dec 26.
Marsdenia tenacissima (Roxb.) Wight & Arn is a well-known traditional Chinese medicine for treating cancer. The anti-tumor effects of the water soluble component of M. tenacissima (MTE, M. Tenacissima Extract) have been intensely studied. However, the roles of microenvironmental cells in mediating the anti-tumor actions of MTE remain to be defined.
To determine the roles of nitric oxide (NO) released by endothelial cells (ECs), an important component of tumor microenvironment, in regulating the anti-cancer effects of MTE, and to explore the underlying mechanisms.
Co-culture system of ECs and A549 non-small cell lung cancer (NSCLC) cells was established for determining the interactions of ECs and lung cancer cells. Nitro-L-arginine methyl ester hydrochloride (L-NAME) was used to inhibit the production of NO. Cell viability was examined using cell counting kit 8 and 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. NO assay and Western blot were used to determine the involved signaling pathway. Primary lung microenvironmental cells (PLMCs) were cultured to examine the roles of NO released from the lung microenvironment in regulating the anti-cancer effects of MTE. A subcutaneous xenograft model was established to determine the involvement of NO in effects of MTE against NSCLCs in vivo.
In the co-culture system of ECs and A549 NSCLC cells, MTE (30 mg/mL) treatment reduced viability of lung cancer cells. However, when L-NAME (a nitric oxide synthase (NOS) inhibitor, 300 μM) was introduced into the co-culture system, the NSCLC-inhibiting effects of MTE were significantly suppressed. By contrast, addition of L-NAME (300 μM) did not affect the anti-cancer efficiency of MTE when ECs were not present. Mechanistically, MTE enhanced endothelial production of NO via stimulating PKA-endothelial nitric oxide synthase (eNOS) signaling. Elevated levels of NO inhibited proliferation and promoted apoptosis of the A549 NSCLC cells. Importantly, PKA-eNOS-NO signaling was effective in mediating the anti-cancer effects of MTE, when lung cancer cells were co-cultured with PLMCs. Finally, oral administration of MTE to the subcutaneous xenograft mice significantly suppressed tumor growth, while elevated NO productions. Plasma NO was also revealed to be negatively correlated with the tumor weight.
ECs significantly contributed to anti-cancer effects of MTE by elevating production of NO, in a PKA-dependent manner. The present study revealed a novel anti-cancer mechanism of MTE through regulating the function of ECs, an important component of tumor microenvironment.
密花藤（Roxb.）Wight & Arn 是一种众所周知的传统中药，用于治疗癌症。密花藤水溶性成分（MTE，密花藤提取物）的抗肿瘤作用已得到深入研究。然而，微环境细胞在调节 MTE 抗肿瘤作用中的作用仍有待确定。
确定内皮细胞（ECs）释放的一氧化氮（NO）在调节 MTE 抗癌作用中的作用，探讨其潜在机制。
建立 ECs 和 A549 非小细胞肺癌（NSCLC）细胞共培养体系，确定 ECs 和肺癌细胞之间的相互作用。使用硝基-L-精氨酸甲酯盐酸盐（L-NAME）抑制 NO 的产生。使用细胞计数试剂盒 8 和 3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物（MTT）测定细胞活力。使用 NO 测定和 Western blot 测定来确定涉及的信号通路。培养原代肺微环境细胞（PLMCs），以研究肺微环境中释放的 NO 调节 MTE 抗癌作用的作用。建立皮下异种移植模型，以确定 NO 在 MTE 对 NSCLC 体内作用中的参与。
在 ECs 和 A549 NSCLC 细胞的共培养体系中，MTE（30mg/mL）处理降低了肺癌细胞的活力。然而，当将一氧化氮合酶（NOS）抑制剂 L-NAME（300μM）引入共培养体系时，MTE 对 NSCLC 的抑制作用明显受到抑制。相比之下，当不存在 ECs 时，加入 L-NAME（300μM）不会影响 MTE 的抗癌效率。机制上，MTE 通过刺激蛋白激酶 A-内皮型一氧化氮合酶（eNOS）信号来增强内皮细胞产生 NO。NO 水平升高抑制 A549 NSCLC 细胞的增殖并促进其凋亡。重要的是，当肺癌细胞与 PLMCs 共培养时，PKA-eNOS-NO 信号在介导 MTE 的抗癌作用方面是有效的。最后，口服 MTE 给药显著抑制皮下异种移植小鼠的肿瘤生长，同时升高 NO 产生。还发现血浆 NO 与肿瘤重量呈负相关。
ECs 通过以 PKA 依赖性方式升高 NO 的产生，对 MTE 的抗癌作用有重要贡献。本研究通过调节肿瘤微环境重要组成部分 ECs 的功能，揭示了 MTE 的一种新的抗癌机制。
