Author

Yuxi Liu

Year of Award

8-28-2020

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

School of Chinese Medicine

Principal Supervisor

Yu, Zhiling

Keywords

Melanoma ; Melanoma ; Medicine, Chinese

Language

English

Abstract

Huai-Hua-Jin-Yin-Jiu, a traditional Chinese medicine (TCM) formula (SL) comprising Sophorae Flos and Lonicerae Japonicae Flos, was used for treating melanoma in ancient China. Our group has previously shown that an ethanolic extract of SL (SLE) possesses anti-melanoma effects, and that inhibiting STAT3 signaling contributes to the action mechanisms. STAT3 activation promotes the formation of an immunosuppressive microenvironment of melanoma. Regulation of the miR-let- 7/CCR7 pathway is a strategy for treating metastatic melanoma. Chrysoeriol is a flavonoid identified in SLE. The compound has anti-tumor properties, but there is no report about its effects on melanoma. The first aim of this study is to determine whether SLE inhibits melanoma progression by reprogramming tumor microenvironment; the second aim is to explore the involvement of miR-let-7a/f- CCR7 signaling in the anti-metastatic effects of SLE; and the third aim is to elucidate the anti-melanoma mode and mechanisms of action of chrysoeriol. Results showed that SLE suppresses melanoma growth and angiogenesis in mice. SLE inhibits STAT3 signaling, and alters immune cell compositions and molecules involved in STAT3 signaling in melanoma tissues. Cell co-culture experiments showed that SLE inhibits STAT3 signaling in melanoma cells and splenic lymphocytes. Over-activation of STAT3 in melanoma cells diminishes SLE's effects in altering compositions of immune cells and STAT3 signaling molecules in the co- culture system. Small RNA sequencing showed that SLE upregulates miR-let-7a/f levels in B16F10 melanomas. RT-qPCR analyses confirms these results. SLE elevates miR-let- 7a/f levels, and inhibits CCR7 (a target gene of miR-let-7a/f) signaling in melanoma cells. In a lung metastasis mouse model, SLE inhibits melanoma metastasis, elevates miR-let-7a/f levels, and suppresses CCR7 signaling. Knockdown of miR-let-7a/f diminishes the effects of SLE on CCR7 signaling and melanoma cell invasion; and overexpression of CCR7 lessens the effects of SLE on melanoma cell invasion. Chrysoeriol shows in vitro and in vivo anti-melanoma effects. Molecular dynamics and microscale thermophoresis assays demonstrate that chrysoeriol directly binds to Src. Chrysoeriol suppresses the Src/STAT3 pathway in melanoma cells and tissues. Chrysoeriol's anti-proliferative effects are diminished by STAT3 over- activation in melanoma cells. Chrysoeriol also has inhibitory effects in vemurafenib- resistant melanoma cell and animal models. RNA-seq analyses shows that syndecan-3 mRNA level is lower in vemurafenib-resistant cells than in vemurafenib-sensitive cells, and chrysoeriol reverses this vemurafenib resistance-associated downregulation. RT-qPCR and Western blotting analyses confirmed the results. In conclusion, we have demonstrated that reprograming immune microenvironment, partially mediated by inhibiting STAT3 signaling, contributes to the anti-melanoma mechanisms of SLE. Regulation of the miR-let-7a/f-CCR7 pathway is another mechanism underlying the anti-melanoma effects of SLE. Chrysoeriol is identified to be one of the active components responsible for the anti- melanoma effects of SLE. Inhibiting the Src/STAT3 pathway contributes to the anti- melanoma mechanisms of chrysoeriol. Chrysoeriol is able to overcome vemurafenib resistance in melanoma, and upregulation of syndecan-3 is involved in the action mechanisms. This study provides further pharmacological and chemical justifications for the use of the formula SL in treating melanoma, and suggests that SLE and SLE- derived compounds have the potential to be developed as modern alternative and/or complimentary agents for melanoma management

Comments

Principal supervisor: Prof. Yu Zhiling ; Thesis submitted to the School of Chinese Medicine

Bibliography

Includes bibliographical references (pages 151-168)

Available for download on Wednesday, November 09, 2022



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