Nitric oxide, a communicator between tumor cells and endothelial cells, mediates the anti-tumor effects of Marsdenia Tenacissima Extract (MTE)

Ethnopharmacological relevance: 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. 7'enacissima Extract) have been intensely studied. However, the roles of microenvironmental cells in mediating the anti-tumor actions of MTE remain to be defined. Aim of the study: 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. Materials and methods: 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. Results: 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 mu M) was introduced into the co-culture system, the NSCLC-inhibiting effects of MTE were significantly suppressed. By contrast, addition of L-NAME (300 mu M) 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. Conclusions: 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.