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. 2022 Jan 27;15(2):154.
doi: 10.3390/ph15020154.

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  1   2 , Hairul-Islam Mohamed Ibrahim  3   4 , Emad A Ahmed  3   5 , Promise Madu Emeka  1 , Ibrahim A Alhaider  1   6
Affiliations

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 et al. Pharmaceuticals (Basel). .

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.

Keywords: A549; COX-2 inhibitor; Trigonella hamosa L.; lung cancer; miRNA; migration; orientin.

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Conflict of interest statement

The authors declare that there are no conflict of interest.

Figures

Figure 1
Figure 1
The cell viability assay (CCK-8 kit) of TME against MDA-MB-231, A549 and HCT-116. Values represent the mean ± SD of triple experiments. * p < 0.05, treated groups were compared with the DMSO group.
Figure 2
Figure 2
HPLC chromatograms of SubFr.DMF1-3-4 (A), HPLC chromatogram of collected pure OT (B).
Figure 3
Figure 3
Effect of orientin on different cancer cell lines. Cytotoxicity of OT in different concentrations from 6.25 μM to 100 μM. The cell lines used for CCK-8 are MDA-MB-231, A549 and HCT-116 cell lines. IC50 concentration of OT was determined, found to be 25 μM, and this was chosen for further analysis (A). Cellular interaction of OT and COX-2 in different tested cell lines (MDA-MB-231, A549 and HCT-116 cell lines) using immunoprecipitation (B). OT 25 μM for different periods was used to measure A549 cell viability (C). mRNA expression of COX-2 with OT treatment was studied using real-time PCR (D). The effect of OT on protein expression of COX-2 in A549 cells (E,F). OT abrogated wound healing (migration) effect of A549 and it was compared with 0 h and 24 h time points (G,H). Invasion–Matrigel analysis was used to test OT in A549 invading capacity using gelatin-coated insert well method (I,J). A549 cells were stimulated with TGF-β (10 μg/mL) with supplemented OT 25 μM for 4 h in cell culture incubator. After incubation, stimulated cells were blocked with actinomycin D (5 μg/mL) (Act-D). Cells were harvested after 6 h and COX-2 mRNA stability expression was analyzed using RT-PCR. The estimation of COX-2 expression was relative compared with and without OT treatment with TGF-β induction (K). Values represent the mean ± SD of triplicate experiments. * p < 0.05, OT-treated groups compared with the DMSO group.
Figure 4
Figure 4
In silico docking of ligand (OT) and COX-2 protein binding analysis. Computational binding of OT (CID: 5281675) and human COX-2 (PDB-ID-5kir) using AutoDock software. Structure of OT with carbon arrangements (A). The 3D structure of OT in PDB format (B). The 3D structure of human COX-2 from PDB (C). Binding of OT with human COX-2 protein (D). Binding pocket of OT with COX-2 protein (E).
Figure 5
Figure 5
The effect of COX-2 inhibitor CLX on A549 cancer cell lines. The 3D structure of celecoxib using pymol software tool (A). The 3D structure of COX-2 derived from protein data bank PDB-RSCB (B). In silico binding of CLX with COX-2 protein and three prominent bindings in threonine and tyrosine amino acids (C). Binding pocket of CLX with COX-2 protein using AutoDock software (D). COX-2 mRNA expression level in CLX-treated A549 lung cancer cell lines (E). COX-2 protein expression of CLX-treated A549 cancer cell lines (F). Cytotoxic effect of CLX against OT in A549 cell lines (OT concentration was from 6.5 μM to 25 μM) (G). CLX-challenged OT treatment inhibits mRNA and protein expression level, tested groups included control, CLX, CLX + OT (25 μM) and OT (25 μM) alone (H–J). Values represent the mean ± SD of triplicate experiments. * p < 0.05, compared with the DMSO group.
Figure 6
Figure 6
The effect of OT on apoptotic modifications in A549 lung cancer cell lines. OT concentration of 25 μM was used in CLX-challenged A549 cells and cells were harvested after 24 h for microscopic and flow cytometry examination. AO/EB dual staining of CLX-challenged (5 μM) OT-treated (25 μM) A549 cell lines for detection of early and late apoptosis (A). DAPI staining of CLX-challenged OT treatment in A549 cell lines for detection of nuclear DNA damage (B). CLX-challenged A549 cells were treated with OT (25 μM) for 24 h, stained by annexin-V-FITC and PI and analyzed by flow cytometry gates (C). Each column represents the mean ± SEM of triplicate experiments. * p < 0.05, treated groups compared with the DMSO group.
Figure 7
Figure 7
Silencing model of COX-2 abrogates the cancer conditions in A549 cancer cell lines. COX-2-coding miRNA targets such as miR-146a, miR-26b expression levels were compared with U6 control miRNA for DMSO, OT with three time points (12 h, 24 h and 48 h) (A). The inhibition of A549 cell proliferation with OT (25 μM) was mitigated with Si-COX-2 after 24 h treatment. The cells were transfected with Si-COX-2 or SiNS by nucleofector. Cell proliferation was quantified colorimetrically at 450 nm using CCK-8 kit (B). The response of COX-2 mRNA and protein expression in Si-COX-2-transfected cells treated with OT (25 μM) (C–E). The expression of miR-146a and miR-26b in silenced model of Si-COX-2 with treatment of OT (25 μM) (F). Downstream of COX-2-mediated prostaglandin-2 (PGE-2) level was quantified in OT-treated (25 μM) A549 cells. Briefly, Si-COX-2-transfected cells were treated with OT and whole cell lysate was prepared with 0.1 M phosphate buffer. This cell lysate was used to quantify PGE-2 level and expressed in pg/mL using ELISA method (G). PGE-2 protein level was quantified in OT-treated A549 cells using Western blot (H,I). Data are shown as mean ± SD from representative experiment studied in triplicate. * p < 0.05, OT treatment groups were compared with the DMSO group.
Figure 8
Figure 8
COX-2 abrogates tumorigenic properties. Depletion of COX-2 abrogated the inhibitory effects of OT (25 μM) on migration of A549 lung cancer cells (A,B). Si-COX-2 mitigated the inhibitory effects of OT (25 μM) on invasion of A549 cells. Invasiveness of cancer cells was studied using Boyden chamber insert well model. The invaded cells were stained with Giemsa and counted in 4 different microscopic fields (C,D). The DNA damage and formation linear strand was excited and showed yellow-orange fluorescence. Si-COX-2 reduced the inhibitory effects of OT (25 μM) on DNA damage of A549 cells (E,F). Si-COX-2-transfected A549 cells were treated with OT (25 μM) for 24 h, nuclear stained by annexin-V-FITC and PI and analyzed by flow cytometry gates (G). COX-2 does not obstruct OT-induced apoptosis. Data are shown as mean ± SD from representative experiment studied in triplicate. * p < 0.05, treated groups were compared with the DMSO group.
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