P-306 How ProEGCG achieves superior anti-angiogenic efficacy to prevent and treat endometriosis?
Authors: Hung S.W , Li Y , Zhouyurong T , Zhang T , Wang C.C 
Published: June 2022

Abstract

Study question
What are the therapeutic targets and molecular mechanisms that bring superior pharmacological effects of ProEGCG against angiogenesis in endometriosis?

Summary answer
ProEGCG binds to PXK and downregulates PXK-mediated EGF expression in inhibiting angiogenesis via HIF-1α pathway during the growth and development of endometriosis.

What is known already
Current endometriosis treatment shows great effects in managing the symptoms, but associated with significant recurrence risk and hormonal side effects. Previously, we demonstrated the greater efficacies and potency of prodrug of green tea epigallocatechin gallate (ProEGCG) over EGCG to inhibit angiogenesis and oxidative stress in experimental endometriosis mouse models ( Xu, et al., 2011; Wang, et. al., 2013; Hung, et. al. 2021). However, the therapeutic targets and molecular mechanism to bring the superior anti-angiogenic effects of ProEGCG are still not known.

Study design, size, duration
Efficiency of ProEGCG as preventive or therapeutic medication were compared with that of EGCG in endometriosis mice model Therapeutic targets were studied by Proteome Integral Stability Alteration and proteomic analysis (Gaetani et al.,2019) using endometriotic cells, and further validated by computational simulation, target kinetic analysis and immunostaining. Anti-angiogenic mechanism were confirmed in endometriotic cell lines and mouse models.

Participants/materials, setting, methods
Endometriotic (Hs832(C)T) cell line was treated with EGCG or ProEGCG to identify the binding target with chemical proteomics analysis. The stability of ProEGCG-protein complexes was assessed by molecular docking analysis and bio-layer interferometry. Gene and protein expressions of the binding targets and downstream angiogenesis pathway were assessed by qRT-PCR, ELISA and immunostaining to characterise the molecular pathways in vitro and in vivo.

Main results and the role of chance
ProEGCG significantly reduced the endometriotic lesions in both preventive and treatment regimens with no recurrence risk after discontinuation in mice. MTDH and PXK were identified as differential protein targets of EGCG and ProEGCG respectively in Hs832(C)T cells via proteomics analysis. Molecular docking and kinetic studies validated stabilities of the drug-target complexes. Protein expressions of MTDH and PXK were significantly higher in various kinds of human endometriotic tissues compared with normal endometrium, which were found significantly downregulated in mouse endometriotic tissues after treatment of EGCG and ProEGCG, respectively. Angiogenesis was significantly inhibited by ProEGCG and EGCG in endometriosis in mice and downstream angiogenic factors HIF-1α and VEGF were downregulated in both mRNA and protein levels. However EGCG bound to MTDH and inhibited AKT-mediated angiogenesis via AKT/PI3K signalling pathway, while ProEGCG bound to PXK and inhibited EGF-mediated angiogenesis via more profound EGF/AKT1/HIF-1α/VEGF signalling pathways. It indicated distinct and specific potency of ProEGCG as an anti-proliferation and anti-angiogenesis agent in inhibiting the endometriosis progression.

Limitations, reasons for caution
The crosstalk of binding targets-induced therapeutic effects in endometriosis and their corresponding effects on angiogenesis are still unclear. Clinical studies are also needed to confirm the efficacy and mechanisms in humans.

Wider implications of the findings
Novel drug discovery identifies an ideal pharmaceutical for clinical use in endometriosis, which alleviates the symptoms, cures the disease and prevents the recurrence. Molecular targets and mechanisms as preclinical research priorities will optimize pharmacological and physiochemical properties of new drugs, as well as maximize specificity and efficacy for endometriosis treatment.

Trial registration number
NA