O-145 Green Tea catechins EGCG and pro-drug of EGCG (Pro-EGCG) inhibit endometriosis through targeting molecules regulating macrophages and B cells
Authors: Hung S W , Gaetani M , Tan Z Y R , Zhang R Z , Zubarev R A , Wang C C 
Published: August 2021

Abstract

Study question
What are the therapeutic targets and mechanisms of green tea EGCG and Pro-EGCG in treating endometriosis?

Summary answer
EGCG and Pro-EGCG have unique molecular targets to regulate interactions of B cells, macrophages and endometriotic cells and limit the growth and development of endometriosis.

What is known already
Current treatments of endometriosis are mainly hormonal suppression and surgical ablation or removal. Our previous studies showed EGCG significantly inhibits development of experimental endometriosis in mice. Pro-EGCG is more effective than EGCG in term of anti-endometriosis, anti-angiogenesis and anti-oxidation (Wang, et. al., 2013; Xu, et al., 2011). Dysfunctional immunological activities of macrophages and B cells were found in women with endometriosis. The molecular targets, underlying mechanism and differential therapeutic efficacy of EGCG and Pro-EGCG, as well as their anti-inflammatory activities are still not known.

Study design, size, duration
Multiplexed Proteome Integral Stability Alteration (PISA) assay (Gaetani et al.,2019), followed by MS/MS was applied to identify the molecular targets of EGCG and Pro-EGCG in endometriotic cells. Pharmacological studies of EGCG and Pro-EGCG on endometriotic cell line and endometriosis models in mice were performed to characterise their anti-endometriosis and anti-inflammatory effects. Gene silencing and over-expression experiments were conducted to confirm the immunoregulatory mechanisms.

Participants/materials, setting, methods
Endometriotic (Hs832(C)T) cell lines in culture and lysate were treated for chemical proteomics analysis. SiRNA and overexpression vectors were transfected to the cells in vitro and lesions in vivo. Hs832(C).T, monocytic cells (THP-1) and control B cell (Raji null) lines were used for co-culture assays to study the interaction between endometriotic and immune cells in vitro. Endometriosis mice model was established for immunostaining and microarray analysis of lesions to characterise the molecular pathways in vivo.

Main results and the role of chance
MTDH and PXK were the strongest and most differential targets of EGCG and Pro-EGCG in both cells lysate and cell culture of Hs832(C).T, respectively. Gene silencing and overexpression of the protein targets in vitro and in vivo significantly altered expressions of downstream proteins, including BLK and EGF after PXK, and MYC and AKT after MTDH, as well as endometriosis-related genes such as VEGFC and MMP9. Co-culture assays of Hs832(C).T with Raji null or THP-1 induced macrophages showed that expressions of PXK, MTDH, downstream targets, and immune-related genes were significantly increased after incubation of recombinant proteins, but were significantly decreased after EGCG and Pro-EGCG treatment. M1 and M2 macrophages, as well as B cells were significantly reduced after the treatments in vitro and in vivo. Double immunofluorescent staining of lesions showed that CD68, CD163 or CD20 co-expressed with MTDH, PXK and downstream targets, and numbers of the co-expressed cells were significantly reduced after treatments in vivo. Microarray experiment further identified the upstream and downstream genes of MTDH or PXK contributing to the growth and development of endometriosis.

Limitations, reasons for caution
Results of this pharmacological and mechanistic study require clinical samples to validate the anti-endometriosis effects of EGCG and Pro-EGCG. Effects of other potential pharmaceuticals targeting the macrophages and B cells on endometriosis are needed.

Wider implications of the findings
The findings provide pharmacological and mechanistic data for future development of EGCG and Pro-EGCG as new treatment for endometriosis. This study shows that macrophage and B cell could be potential therapeutic targets for treatment of endometriosis, which opens up new horizon for the novel immunotherapy for endometriosis.

Trial registration number
NA