Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Oct 13;23(1):240.
doi: 10.1186/s12935-023-03087-2.

EGCG inhibits the inflammation and senescence inducing properties of MDA-MB-231 triple-negative breast cancer (TNBC) cells-derived extracellular vesicles in human adipose-derived mesenchymal stem cells

Narjara Gonzalez Suarez  1 , Yuniel Fernandez-Marrero  2 , Mathieu P A Hébert  3 , Marie-Eve Roy  1 , Luc H Boudreau  3 , Borhane Annabi  4
Affiliations

EGCG inhibits the inflammation and senescence inducing properties of MDA-MB-231 triple-negative breast cancer (TNBC) cells-derived extracellular vesicles in human adipose-derived mesenchymal stem cells

Narjara Gonzalez Suarez et al. Cancer Cell Int. .

Abstract

Background: Triple-negative breast cancer (TNBC) cells' secretome can induce a pro-inflammatory phenotype in human adipose-derived mesenchymal stem cells (hADMSC). This can be prevented by the green tea polyphenol epigallocatechin-3-gallate (EGCG). The impact of EGCG on the paracrine regulation that the extracellular vesicles (EVs) specifically exert within the TNBC secretome remains unknown.

Methods: EVs were obtained from a TNBC-derived serum-starved MDA-MB-231 cell model treated or not with EGCG under normoxic or hypoxic (< 1% O2) culture conditions. RNA-Seq analysis was used to assess the EVs' genetic content. The modulation of inflammatory and senescence markers in hADMSC was evaluated by RT-qPCR using cDNA arrays and validated by immunoblotting. A protein profiler phospho-kinase array was used to explore signaling pathways.

Results: While hypoxic culture conditions did not significantly alter the genetic content of MDA-MB-231-secreted EVs, the addition of EGCG significantly modified EVs genetic material at low oxygen tension. Gene expression of cancer-associated adipocyte pro-inflammatory markers CXCL8, CCL2 and IL-1β was increased in hADMSC treated with EVs. Concomitantly, EVs isolated from MDA-MB-231 treated with EGCG (EGCG-EVs) downregulated CCL2 and IL-1β, while inducing higher expression of CXCL8 and IL-6 levels. EVs activated CHK-2, c-Jun, AKT and GSK-3β signaling pathways in hADMSC, whereas EGCG-EVs specifically reduced the latter two as well as the serum starvation-induced senescence markers p21 and β-galactosidase. Finally, the mitochondrial content within the TNBC cells-derived EVs was found reduced upon EGCG treatment.

Conclusion: This proof of concept study demonstrates that the chemopreventive properties of diet-derived polyphenols may efficiently target the paracrine regulation that TNBC cells could exert upon their surrounding adipose tissue microenvironment.

Keywords: Adipose-derived mesenchymal stem cells; EGCG; Extracellular vesicles; Inflammation; Senescence; Triple-negative breast cancer.

PubMed Disclaimer

Conflict of interest statement

The authors declare no potential conflicts of interest concerning the research, authorship, and/or publication of this article.

Figures

Fig. 1
Fig. 1
Characterization of the EVs isolated from the MDA-MB-231 cells conditioned media. Serum-starved triple-negative breast cancer-derived MDA-MB-231 cells were cultured for 48 h in the absence or presence of 30 µM EGCG. Conditioned media was next collected, concentrated, and extracellular vesicles (EVs) isolated as described in the Methods section. Dynamic light scattering particle size analysis of the A EVs and B EGCG-EVs distribution of the particles by number (upper panels) and by intensity (lower panels) of the refracted light. A representative experiment out of four is presented. C Immunoblotting of the exosomes enriched proteins CD9, CD63 and CD81, and of the negative marker BIP in MDA-MB-231 cell lysate and EVs lysate. D Gating strategy of the flow cytometry experiment performed to assess MemGlow-488 labelled EVs interaction with hADMSC. Merged histogram was obtained by the measurement by flow cytometry of the untreated cells (black lines) and the cells incubated with stained-EVs (red lines). A representative experiment out of two is presented. E Representative microscopy images of hADMSC incubated for two hours with the MDA-MB-231 cells-derived EVs labelled with MemGlow-488. A representative experiment out of three is presented (scale bar is 20 µm). F Relative cell migration rate of hADMSC treated with EVs (closed circle) or basal media (BM, open circle) in response to basal media supplemented with 1% FBS. Migration experiments were performed three times in quadruplicate. Diameter in nanometers (d.nm), polydispersity index (Pdl), intensity weighted mean hydrodynamic size of the particles (Z-Average). Statistically significant differences were determined by the non-parametric comparison test Mann–Whitney, * p < 0.05
Fig. 2
Fig. 2
Modulation of the EVs cargo by EGCG. The extracellular vesicles (EVs) were isolated from serum-starved MDA-MB-231 cells treated or not with 30 μM EGCG for 48 h. A Volcano plot for the expression profile of differentially expressed genes (DEG) with an adjusted p-value < 0.05 was selected as the threshold. B KEGG pathways enrichment analysis resulted from the comparison of DEG (EGCG-EVs vs EVs) with absolute fold change (FC) > 2, and adjusted p-value < 0.05. C Network graph showing the enriched pathways and their respective genes. Enriched terms are coloured in beige, while upregulated and downregulated genes are coloured in red and green, respectively
Fig. 3
Fig. 3
Signalling cascades triggered by the EVs. The hADMSC were incubated for one hour in basal media (BM), EVs, or EGCG-EVs using a Cell:EVs ratio of 1:0,5 (#:#). Cells were next lysed as described in the Methods section for Western blotting analysis. A phospho-kinase array was used to detect pathways activation states. A Immunoblotting results (1, p38a; 2, STAT5a/b; 3, p53; 4, Chk-2; 5, c-Jun; 6, Akt 1/2/3; 7, GSK-3b), and B Densitometric analysis of the highlighted immunoreactive spots performed using the ImageJ software. C Validation of the phosphorylated and total states of GSK-3β (Ser9) and AKT (Ser473) by immunoblotting. A representative experiment out of two is presented. D Ratios of the phosphorylated/total forms of AKT and GSK-3β resulted from the densitometric analysis performed with ImageJ. Mitogen-activated protein kinases (p38); signal transducer and activator of transcription 5A/B (STAT5a/b); Checkpoint kinase-2 (Chk-2); c-Jun N-terminal kinases JNK (c-Jun); protein kinase B signaling pathway (AKT); glycogen synthase kinase-3 (GSK-3); tumor protein 53 (p53)
Fig. 4
Fig. 4
Induction of a pro-inflammatory molecular signature by the MDA-MB-231 cells-derived EVs. The hADMSC were incubated for 24 h in Basal Media (BM, Control), EVs (white bars) or EGCG-EVs (black bars) with a Cell: EVs ratio of 1:0,5. Next, total RNA was isolated, and cDNA was synthesized. Gene expression levels were determined by qPCR using a Human Inflammatory Cytokine and Receptors RT2-Profiler gene array kit. Densitometric analysis was performed using the ImageJ software. A The fold change (FC) expression of genes related to the cancer-associated adipocyte (CAA) phenotype. Validation of the arrays results for CAA genes was performed in two independent experiments. B Immunoblotting of interleukin-6 (IL-6) and tubulin (10 µg protein/well). Immunoblotting is representative of three experiments. C FC of selected genes from the array to highlight the modulatory effect of the EGCG-EVs
Fig. 5
Fig. 5
EGCG-EVs rescue hADMSC from serum-starvation-induced senescence. hADMSC were incubated for 24 h in complete media (CM), serum-deprived basal media (BM), EVs or EGCG-EVs at a ratio Cell:EVs of 1:0.5. hADMSC were collected for protein and total RNA as described in the Methods section. A Immunoblotting detection of the senescence biomarker p21 and of the loading control tubulin from control hADMSC lysates, treated with CM, BM, or the respective EVs. Immunoblotting is representative of three independent experiments. B Confocal microscopy of hADMSC treated for 48 h at a Cell: EVs ratio of 1:2. The nucleus was stained with DAPI (red), and the expression of the senescence-associated β-galactosidase (β-gal) marker is coloured in green. One out of three experiments is presented. C Histograms showing the percent of positive β-gal cells obtained upon 48 h of treatment. D Gene expression of other senescence markers modulated in hADMSC by EGCG-EVs (black bars) compared with the expression level of the genes in cells incubated with EVs (white bars), using as cut-off a log2 FC ≥ 2 and quantified by qPCR using the Human Senescence RT2-Profiler gene array kit. The percent of positive β-gal cells/field was calculated using the following equation: (number of positive cells /total of cells)*100. The Kruskall-Wallis test determined statistically significant differences, showing a * p < 0.05
Fig. 6
Fig. 6
EGCG reduces the mitochondrial content within the EVs. Serum-starved MDA-MB-231 cells were cultured for 48 h in the presence or absence of 30 µM EGCG. EVs were isolated, stained with anti-CD44-FITC and MitoTracker Deep Red (MTR), and analyzed by flow cytometry. Four independent experiments were spaced in time from cell passage 3 to 8. The dots represent the mean of the counting, and results from the same experimental day were connected with a line in graphs A-C. Paired t-test was performed; **P < 0.01. A The total number of CD44+/MTR− microparticles (MPs) detected in EVs or EGCG-EVs. B Quantification of the mitochondria-containing particles (CD44+/MTR+, mitoMPs) in the EVs or EGCG-EVs. Four independent experiments were performed. C Citrate synthase activity was measured in particles isolated from the conditioned media as described in the Methods section. D Dot plot resulting from the flow cytometry analysis detecting the presence of mitochondria delivered by the EVs in hADMSC after incubation with basal media (BM, negative control), mitoTracker-labelled EVs (MTR-EVs), or mitoTracker-labelled ECGC-EVs (MTR-EGCG-EVs). E Mean of the fluorescence intensity of the EVs or EGCG-EVs -delivered mitochondria within hADMSC. F The percent of hADMSC positive for the presence of mitochondria delivered by the EVs or EGCG-EVs
Fig. 7
Fig. 7
Transcriptomic analysis of the influence of low oxygen tension and EGCG in loading the MDA-MB-231-derived EVs. A Principal Component Analysis (PCA) of the top 500 differential expressed genes (DEG) identified in the samples. B Venn diagram showing the number of DEG detected with a log2 FC ≥|2| in the EVs obtained by adding 30 µM EGCG at different oxygen tensions. The analysis was performed by comparing the EGCG-EVs obtained in hypoxia (EV_HE, C3) vs EGCG-EVs obtained in normoxia (EV_NE, C2). Genes with a significant or non-significant value were coloured in red and grey respectively. C) Robust k-means clustering visualized as a heatmap of the individual samples and their DEG with a log2 FC ≥|2| and p-adjusted value < 0.05. D Gene ontology (GO)-SLIM PANTHER analysis showing the biological process that involves genes downregulated by HE (cluster 1, heat map). E List of all biological processes downregulated by HE. F GO-SLIM PANTHER analysis of the downregulated genes in cluster 1 of the heatmap, showing their protein class. Fisher’s exact test and the false discovery rate (FDR) correction were used during the GO-SLIM analysis
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Tkach M, Thery C. Communication by extracellular vesicles: where we are and where we need to go. Cell. 2016;164(6):1226–1232. - PubMed
    1. Stein K, Chiang HL. Exocytosis and endocytosis of small vesicles across the plasma membrane in saccharomyces cerevisiae. Membranes (Basel) 2014;4(3):608–629. - PMC - PubMed
    1. Taylor J, Bebawy M. Proteins regulating microvesicle biogenesis and multidrug resistance in cancer. Proteomics. 2019;19(1–2):e1800165. - PubMed
    1. Becker A, et al. Extracellular vesicles in cancer: cell-to-cell mediators of metastasis. Cancer Cell. 2016;30(6):836–848. - PMC - PubMed
    1. Mause SF, et al. Platelet microparticles: a transcellular delivery system for RANTES promoting monocyte recruitment on endothelium. Arterioscler Thromb Vasc Biol. 2005;25(7):1512–1518. - PubMed

Grants and funding

-