Betulin Attenuates TGF-beta 1-and PGE(2)-Mediated Inhibition of NK Cell Activity to Suppress Tumor Progression and Metastasis in Mice

YY Transforming growth factor (TGF)-beta 1 and prostaglandin E2 (PGE2) are humoral factors critically involved in the induction of immunosuppression in the microenvironment of various types of tumors, in-cluding melanoma. In this study, we identified a natural compound that attenuated TGF-beta 1-and PGE2- induced immunosuppression and examined its effect on B16 melanoma growth in mice. By screening 502 natural compounds for attenuating activity against TGF-beta 1-or PGE2-induced suppression of cytolysis in poly(I:C)-stimulated murine splenocytes, we found that betulin was the most potent compound. Betulin also reduced TGF-beta 1-and PGE2-induced downregulation of perforin and granzyme B mRNA expression and cell surface expression of NKG2D and CD69 in natural killer (NK) cells. Cell depletion and coculture experi-ments showed that NK cells, dendritic cells, B cells, and T cells were necessary for the attenuating effects of betulin. Structure-activity relationship analysis revealed that two hydroxyl groups at positions C3 and C28 of betulin, their cis-configuration, and methyl group at C30 played crucial roles in its attenuating activity. In a subcutaneous implantation model of B16 melanoma in mice, intratumor administration of betulin and LY2157299, a TGF-beta 1 type I receptor kinase inhibitor, significantly retarded the growth of B16 melanoma. Notably, betulin increased significantly the number of CD69 positive NK cells in tumor sites at early stages of post-tumor cell injection. Our data suggest that betulin inhibits the growth of B16 melanoma by enhancing NK cell activity through attenuating the immunosuppressive tumor microenvironment.