Theaflavin-3-gallate, a natural antagonist for Hsp90: In-silico and in-vitro approach

https://doi.org/10.1016/j.cbi.2021.109774Get rights and content

Highlights

  • HSP90 key modulators for pre-metastatic niche.

  • Theaflavin-3-gallate inhibited cell migration through MMP signalling pathway.

  • Theaflavin-3-gallate has potential effect on the HSP90 marker.

Abstract

Poor prognosis and metastasis have been recognized as the major cause of breast cancer related deaths worldwide. Recent experimental evidence has shown that Hsp90, the prime chaperone, is overexpressed in many cancers and is responsible if reducing the 5-year survival rate of cancer patients. Therefore, targeted inhibition of Hsp90 may be a new and effective way to target cancer as well as enhancing therapeutic outcomes. In the present study, screening and simulation of potential natural compounds result in the identification of theaflavin-3-gallate as a promising inhibitory compound of Hsp90. Further in-vitro validation of the cytotoxic effect of theaflavin-3-gallate in human breast carcinoma cell line MCF7 and normal cell line MCF10A revealed that theaflavin-3-gallate significantly inhibited the cell proliferation of MCF7 cells whereas no cytotoxic effect was observed on MCF10A cells. We also found that theaflavin-3-gallate significantly induced programmed cell death by arresting cells in the G2/M phase of the cell cycle. A significant decrease in cell migration and colony formation by theaflavin-3-gallate treatment was also observed in MCF7 cells. Furthermore, theaflavin-3-gallate significantly downregulated the mRNA expression patterns of the HSP90, MMP9, VEGFA, and SPP1 genes. Collectively, our results demonstrated theaflavin-3-gallate as a potential natural Hsp90 inhibitor that can be used to enhance the therapeutic efficacy of existing breast cancer therapies and improve overall survival of breast cancer patients.

Introduction

Breast cancer metastasis is a terminal event and the leading cause of mortality in cancer patients; however, the mechanism is poorly understood [1]. In spite of progress in diagnosis and therapeutic modalities using newer technologies metastasis continue to be the major cause of cancer-related death until today [2]. Endocrine therapy, surgery, and chemotherapy are the main treatment approaches for breast cancer, and breast cancer that is identified early on always has an improved prognosis after surgery [3]. Owing to a lack of ample evidence on the mechanism that triggers the metastatic process in breast cancer, partial achievement has been made in the prevention and inhibition of metastatic events using target-specific inhibitors. Heat Shock Proteins (HSP) are among key targets of interest to many researchers who are putting effort to develop specific antagonists due to their potential role in tumorigenesis and metastasis [4].

Mammalian HSPs have been classified mainly in four families according to their molecular weight “Hsp90, Hsp70, Hsp60, and small HSP, Hsp90, a family of an important chaperone, were first identified in a complex form with steroid hormone receptors and oncoprotein viral Src kinase and it has been shown that HSPs were significantly associated with cancers [5,6]. In detail, heat shock protein (HSP) 90 has been found to play critical roles in protein folding, intracellular disposition, and proteolytic turnover of key regulators of cell growth and survival. Furthermore, many studies revealed that Hsp90 can regulate the conformation, stability, and function of various oncogenic proteins to participate in the proliferation, apoptosis, and metastasis of tumor cells [7]. Moreover, breast cancer is a sign that Hsp90 inhibitors should be discovered for a diversity of reasons such as inhibition of Hsp90 has also been known to modulate angiogenesis of breast cancer xenografts [8]. Beside, recent study also suggested that in breast cancer patient mRNA level of HSP90 was significantly upregulated in primary as well as bone metastases [9]. Since Hsp90 was recognized as a potential cancer target, numerous inhibitors targeting cytoplasmic Hsp90 have been established as anticancer agents but, none have shown satisfactory effectiveness to be approved by the FDA [10,11]. Therefore, it is the need of the hour to identify a potent lead compound as an hsp90 antagonist and evaluate its efficiency at the molecular & cellular levels. Interlinking biological experiments with computational modeling and investigation may lead to a decrease in numeral in vitro experiments, enhanced validation evaluations, and improved sensitivity.

Thus, this study aims to screen potential lead compounds targeting the Hsp90 protein using molecular docking and molecular dynamics simulation approach. 95 natural compounds were screened as potential lead compounds against the Hsp90 protein through various parameters such as binding energy and pattern. The therapeutic potential of the possible lead compound was further analyzed using an MTT assay, apoptotic, migration, colony formation, and cell cycle assay. The potent lead compound against Hsp90 may come up as a novel anti-metastatic agent in the future for breast cancer.

Section snippets

Retrieval of protein structures and its preparations

The Hsp90 protein target in complex with 4-(1,3-dihydro-2h-isoindol-2-ylcarbonyl)-6-(1-methylethyl) benzene-1,3-diol (PDB entry: 2xab, chain A) was retrieved from Protein Data Bank (PDB) [12]. The retrieved structures were pre-processed using Protein Preparation Wizard of Schrödinger suite (academic license) (Schrödinger Suite 2014). Furthermore, restrained minimization was carried out to obtain a geometrically-stable protein conformation. A curated ligand library consisting of 95 natural

Molecular docking of Hsp90 target

We performed molecular docking of Hsp90 target using a curated collection of 95 natural molecules with proven anti-cancer activity. Extensive docking calculations were carried out using the XP module of Schrödinger suite (academic license) with thorough examination of optimal intermolecular contacts with the target. The binding site of 2,4-dihydroxy benzamide molecule (co-crystal ligand of PDB entry: 2xab) was defined as the docking site which encompassed all amino acids with 8 Å distance to

Discussion

The heat shock protein (Hsp90) belongs to a family of molecular chaperones, involved in regulating biological and functional cell processes such as cell growth, invasion, migration, and recurrence that are responsible for the progression of different malignancies [[31], [32]]. Numerous studies have emphasized the importance of Hsp90 in the last two decades; however, there was a great difference in these reports, as all these studies fail to take into consideration that Hsp90 is a group of

Author contribution

Conceptualization, Methodology, Validation, Writing - Original Draft, Software, Writing - Review & Editing, Data analysis, Software, Software, Visualization, Supervision, Investigation, Funding, Review & Editing.

Author statement

All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Furthermore, each author certifies that this material or similar material has not been and will not be submitted to or published in any other publication before its appearance in the chemico-biological interaction.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

Kinjal Bhadresha is thankful to Indian Council of Medical Research (ICMR; New Delhi) for the financial assistance in the form of Senior Research fellowship (ortho/2018/NCD-I). The authors thank the Department of Botany, Bioinformatics and Climate Change Impacts Management, Gujarat University for providing the supercomputing facility support to carry out this study.

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