An ethanolic extract of a two-herb formula Huai-Hua-Jin-Yin-Jiu reprograms melanoma immune microenvironment

Background: A traditional Chinese medicine formula Huai-Hua-Jin-Yin-Jiu comprising Sophorae Flos and Lonicerae Japonicae Flos (SL for short) was used for treating melanoma in ancient China. We have previously shown that an ethanolic extract of SL (SLE) possesses anti-melanoma effects and suppresses STAT3 signaling in vitro and in vivo. STAT3 has been linked to the development of melanoma immunosuppressive microenvironment. In this work, we investigated whether SLE inhibits melanoma growth by reprogramming the tumor microenvironment in mouse and co-culture cell models.

Results: In B16F10 melanoma-bearing mice, we found that intragastric administration of SLE (1.2 g/kg) dramatically inhibited tumor growth and angiogenesis, decreased tumor cell proliferation, and induced tumor cell apoptosis. These observations were associated with the downregulation of protein levels of phospho-STAT3 (Tyr 705) and STAT3-targeted genes involved in tumor growth, angiogenesis, and immune evasion. We also observed decreased immune suppressive cytokines and increased Th, Tc and dendritic cells in B16F10 melanomas of the SLE-treated mice. In a co-culture system composed of B16F10 cells and mouse primary splenic lymphocytes, we found that SLE not only inhibited the proliferation of and STAT3 activation in B16F10 cells, but also increased the percentages of Th, Tc and dendritic cells. In the co-culture setting, SLE downregulated mRNA levels of STAT3-targeted genes in the splenic lymphocytes as well. Further, it was found that over-activation of STAT3 in B16 melanoma cells diminished SLE’s effects for increasing the numbers of Th, Tc, dendritic and NK cells, decreasing the numbers of MDSCs and Tregs, and lowering the levels of immune suppressive cytokines in a co-culture system consisting of B16STAT3C cells (stable cells harboring a constitutively active STAT3 variant STAT3C) and primary splenic lymphocytes.

Conclusions: Our findings indicate that reprograming immune microenvironment, partially mediated by inhibiting STAT3 signaling, contributes to the anti-melanoma mechanisms of SLE. This study provides further pharmacological groundwork for developing SLE as a modern agent for melanoma prevention/treatment, and provide further justifications for the traditional use the formula SL in treating melanoma. Moreover, this study supports the notion that reprograming immunosuppressive microenvironment is a viable anti-melanoma strategy.