Orientin, a Bio-Flavonoid from Trigonella hamosa L., Regulates COX-2/PGE-2 in A549 Cell Lines via miR-26b and miR-146a.

Hany Ezzat Khalil,Hairul-Islam Mohamed Ibrahim,Emad A Ahmed,Promise Madu Emeka,Ibrahim A Alhaider

doi:10.3390/ph15020154

Abstract

Cancer is a severe health condition and considered one of the major healthcare issues and is in need of innovative strategy for a cure. The current study aimed to investigate the chemical profile of Trigonella hamosa L. and a potential molecular approach to explain its regulation in cancer progression through an inflammatory mediator (COX-2) in A549 non-small lung cancer cell lines via in silico, mechanistic and molecular aspects. T. hamosa was extracted and then subjected to a CCK-8 cell viability assay in different cancer cell lines including MDA-MB-231, A549 and HCT-116. Total extract was subjected to several chromatographic techniques to yield orientin (OT); the structure was elucidated by inspection of NMR spectroscopic data. To achieve anticancer effects of OT, a cell viability assay using a CCK-8 kit, immunoprecipitation by Western blot, cell migration using a wound healing assay, cell invasion using a Matrigel-Transwell assay, apoptosis by AO/EB dual staining, flow cytometric analysis and DAPI staining, a silenced COX-2 model to determine PGE-2 production and real-time PCR and Western blot of BCL-2, CYP-1A1, iNOS and COX-2 markers were carried out. The results demonstrated that OT decreased the cell proliferation and controlled cell migration and invasive properties. OT destabilized the COX-2 mRNA and downregulated its expression in A549 cell lines. Virtual binding showed interaction (binding energy −10.43) between OT and COX-2 protein compared to the selective COX-2 inhibitor celecoxib (CLX) (binding energy −9.4). The OT-CLX combination showed a superior anticancer effect. The synergistic effect of OT-CLX combination was noticed in controlling the migration and invasion of A549 cell lines. OT-CLX downregulated the expression of BCL-2, iNOS and COX-2 and activated the proapoptotic gene CYP-1A1. OT mitigated the COX-2 expression via upregulation of miR-26b and miR-146a. Interestingly, COX-2-silenced transfected A549 cells exhibited reduced expression of miR-26b and miR-146a. The findings confirmed the direct interaction of OT with COX-2 protein. PGE-2 expression was quantified in both naïve and COX-2-silenced A549 cells. OT downregulated the release of PGE-2 in both tested conditions. These results confirmed the regulatory effect of OT on A549 cell growth in a COX-2-dependent manner. OT activated apoptosis via activation of CYP-1A1 expression in an independent manner. These results revealed that the OT-CLX combination could serve as a potential synergistic treatment for effective inflammatory-mediated anticancer strategies.

Highlights

In spite of the recent approaches in the field of synthetized pharmaceutical candidates, plants and plant-derived products remain the pivotal reservoir of bioactive, eco-friendly and unique molecules which play crucial therapeutic roles
(5 μM) and OT (25 μM), which was significant compared to cells treated with OT alone (Figure 5H,J). These results revealed that OT (25 μM) treatment alone regulates positive function against COX-2, inducible nitric oxide synthase (iNOS) and BCL-2 markers, but it has a synergistic function with
The results suggested that OT is able to reduce the COX-2 mRNA and this leads to a reduction in its bioavailability, diminishing functional mRNA for translation and the subsequent release of PGE-2

Summary

Introduction

In spite of the recent approaches in the field of synthetized pharmaceutical candidates, plants and plant-derived products remain the pivotal reservoir of bioactive, eco-friendly and unique molecules which play crucial therapeutic roles. Many plant-derived natural products have been reported as anticancer agents. Almost 50% of the anticancer drugs under clinical trials are from natural sources or their derivatives [1]. The genus Trigonella belongs to the family Fabaceae. This genus comprises annual or perennial herbs, often with fragrant, trifoliate compound leaves. Six species from this genus are found in Saudi flora and especially in Eastern Province [6]

Methods

Discussion

Conclusion