Screening of Anti-tumor Active Sites of Smilax China L. and Its Regulation and Mechanism on Tumor-associated Macrophages

OBJECTIVE  To screen the anti-tumor activity of different polar fractions of the alcoholic extract of Smilax china L. and explore its regulatory mechanisms on tumor-associated macrophages.

METHODS  A subcutaneous melanoma transplantation model was established in mice to evaluate the anti-tumor effects of different polar fractions of Smilax china L., based on tumor volume and weight. The findings were further validated in mouse mammary tumor virus polyoma middle T antigen(MMTV-PyMT) breast cancer mice, and the effects of the active fractions on the tumor immune microenvironment were analyzed using flow cytometry. An M2 polarization model was constructed in vitro by stimulating primary mouse peritoneal macrophages with IL-4, and the M1/M2 ratio was measured by flow cytometry. The main signaling pathways responsible for the anti-breast cancer effects of Smilax china L. were identified using network pharmacology, and validated via Western blotting.

RESULTS  In vivo experiments showed that, compared to the B16-F10 model group, the tumor weight of mice treated with the ethanol extract and ethyl acetate fraction of Smilax china L. was significantly reduced(P<0.01 or P<0.05), while the n-butanol and water fractions exhibited no anti-tumor effects. These findings were confirmed in MMTV-PyMT mice, where the ethanol and ethyl acetate fractions significantly reduced the infiltration of M2 tumor-associated macrophages(CD86⁻CD206⁺) compared to the MMTV-PyMT model group(P<0.05). In vitro experiments showed that, compared with the model group, the ethanol extract(100 µg·mL⁻¹) and ethyl acetate fraction(12.5, 25, 50 µg·mL⁻¹) significantly inhibited IL-4-induced M2 polarization(P<0.001). Network pharmacology results suggested that the phosphatidylinositol-3-kinase/protein kinase B(PI3K/Akt) signaling pathway might be a key mechanism for the anti-breast cancer effects of Smilax china L.. Western blotting analysis revealed that, compared with the model group, the relative expression levels of P-PI3K/PI3K protein in the 50, 25 µg·mL⁻¹ ethyl acetate groups were significantly reduced(P<0.001). Similarly, the expression levels of P-Akt/Akt protein were also significantly decreased(P<0.01 or P<0.001).

CONCLUSION  The ethyl acetate fraction of Smilax china L. is the active anti-tumor fraction, and its anti-tumor effect is related to the inhibition of M2 polarization in tumor-associated macrophages, potentially via suppression of the PI3K/Akt signaling pathway.