CCN5 activation by free or encapsulated EGCG is required to render triple-negative breast cancer cell viability and tumor progression

Abstract

Epigallocatechin-3-gallate (EGCG) has been considered an anticancer agent despite conflicting and discrepant bioavailability views. EGCG impairs the viability and self-renewal capacity of triple-negative breast cancer (TNBC) cells and makes them sensitive to estrogen via activating ER-α. Surprisingly, the mechanism of EGCG's action on TNBC cells remains unclear. CCN5/WISP-2 is a gatekeeper gene that regulates viability, ER-α, and stemness in TNBC and other types of cancers. This study aimed to investigate whether EGCG (free or encapsulated in nanoparticles) interacts with the CCN5 protein by emphasizing its bioavailability and enhancing its anticancer effect. We demonstrate that EGCG activates CCN5 to inhibit in vitro cell viability through apoptosis, the sphere-forming ability via reversing TNBC cells' stemness, and suppressing tumor growth in vivo. Moreover, we found EGCG-loaded nanoparticles to be functionally more active and superior in their tumor-suppressing ability than free-EGCG. Together, these studies identify EGCG (free or encapsulated) as a novel activator of CCN5 in TNBC cells and hold promise as a future therapeutic option for TNBC with upregulated CCN5 expression.

Keywords: CCN5; EGCG; FA-PEG-NPs; PCNA; TNBC; bioavailability; breast cancer; drug delivery; folic acid; nanoparticles.

FIGURE 1

Diagrammatic illustration of spatio‐selective activation and synthetic route of EGCG‐loaded tumor cell‐targeted nanoparticles. (A) The schematic illustration of the synthetic route toward the preparation of nanoparticles. (B) Plot and table of hydrodynamic diameters of EGCG containing nanoparticles. (C) Images of nanoparticles obtained using transmission electron microscopy (TEM). (D) Table showing the amount of drug in each nanoparticle formulation

FIGURE 2

EGCG reactivates CCN5 through transcriptional activation. (A‐C). Immunoblot analysis and quantification of CCN5 in lysates of untreated and different doses of EGCG‐treated MDA‐MB‐231, MCF‐7, and 4T1 TNBC cell lines. P‐value determined by Student's t test, data are mean ± SD when n = 3. (D) Immunoblot analysis and quantification of CCN5 in lysates of untreated and EGCG‐treated Panc‐1 pancreatic cancer cell line. P‐value determined by Student's t‐test, data are mean ± SD when n = 3. (E) Quantification of relative expression of CCN5 mRNA in EGCG‐treated MDA‐MB‐231 and 4T1 cell extracts using qRTPCR. P‐value determined by Student's t test, data are mean ± SD when n = 8. (F) Dose‐dependent induction of the CCN5 promoter constructs by EGCG in MDA‐MB‐231 and MCF‐7 cell lines. CCN5 promoter‐luciferase was performed as described under the Method section. P‐value determined by Student's t test, data are mean ± SD when n = 3

FIGURE 3

EGCG reduces cell viability via apoptosis. (A‐D) Dose‐dependent effect of EGCG on cell viability and defined the IC₅₀ in MCF‐7 and TNBC cell lines. P‐value determined by Student's t test, data are mean ± SD when n = 3. (E‐F) Detection and quantification of apoptotic cells using propidium iodide‐flow cytometry. The graph shows the mean ± SD of three independent experiments. (G) EGCG‐treated MDA‐MB‐231 and MCF‐7 cell lysates were analyzed by immunoblot to detect BAX and Bcl‐2 proteins. The graph shows the mean ± SD of three independent experiments. (H) Detection of cell viability in MDA‐MB‐231 cells treated with EGCG in the presence or absence of CCN5 neutralizing antibody

FIGURE 4

Effect of EGCG and human recombinant CCN5 (hrCCN5) protein on TNBC cell viability and the synergistic cytotoxic activity of combined EGCG and hrCCN5. (A‐B) The dose‐dependent synergistic cytotoxic activity of combined hrCCN5 and EGCG on TNBC cell lines. (C‐D) Combination treatment of hrCCN5 and EGCG on colony‐forming ability of TNBC cells reveals synergy. The graphs show the mean ± SD of three independent experiments

FIGURE 5

Suppression of mammosphere‐forming ability of TNBC cells by EGCG was enhanced by hrCCN5 protein and rescued by CCN5 antibody treatment. (A‐C) Effect of EGCG on the sphere‐forming ability of MDA‐MB‐231 cells. The graphs show the mean ± SD of five independent experiments. (D‐E) Immunoblot analysis and quantification of mesenchymal/stemness (left) and epithelial (right) protein markers in lysates of untreated and EGCG‐treated MDA‐MB‐231 cells. The graph shows the mean ±SD of five independent experiments. F‐G. Sphere‐forming ability of MDA‐MB‐231 and 4T1 cells were measured following treatment with EGCG alone or a combination of hrCCN5 protein or CCN5 antibody (CCN5^Ab). The graphs show the mean ± SD of five independent experiments

FIGURE 6

Differential effect of EGCG‐loaded structurally different nanoparticles on CCN5 reactivation in MDA‐MB‐231 cells. (A‐B) Confocal images of MDA‐MB‐231 cells showing cellular uptake of fluorescently labeled EGCG‐loaded NPs with the different chemical structures after 24 h of incubation. Detailed protocols are described in the Method section. a, nanoparticle without fluorescently tagged, b, PEG‐NPs, c, FA‐NPs‐PEG, and d, FA‐PEG‐NPs. (C‐D) Immunoblot analysis of CCN5 expression in the lysates of MDA‐MB‐231 cells treated with free‐ or encapsulated EGCG. β‐Actin was used as a loading control. The graph shows the mean ± SD of three independent experiments

FIGURE 7

EGCG‐loaded FA‐PEG‐NPs is equally effective as free‐EGCG in inhibiting colony‐ and sphere‐forming ability of TNBC cells. (A‐B) The colony‐forming ability of free‐EGCG and EGCG‐loaded nanoparticles treated TNBC cells as determined using anchorage‐dependent growth (ADG) assay. The graph shows the mean ± SD from triplicate measurements, ns, non‐significant. (C‐D) The sphere‐forming ability of free‐EGCG and EGCG‐loaded nanoparticles treated TNBC cells as determined using the mammosphere assay (C). The graph (D) shows the mean ± SD from triplicate measurements, ns, non‐significant

FIGURE 8

Inhibition of tumor growth in a xenograft model by EGCG correlates with CCN5 expression level in tumor tissue. (A‐C) Scheme of the EGCG treatment strategy (A). The figure illustrates the outcome of the effect of EGCG on the MDA‐MB‐231‐tumor xenograft mouse model (A). Quantification of tumor volume (B) and tumor mass (C) in the MDA‐MB‐231‐xenograft mouse model. Data represent error bar mean ± SD, n = 4 animal/group, ns = non‐significant. (D) IHC staining of PCNA and quantification of PCNA‐positive cells in EGCG‐treated and untreated MDA‐MB‐231‐xenograft tumor sections. Data represent error bar mean ± SD, n = 4 animal/group. (E) IHC staining of CCN5 in EGCG‐treated and untreated MDA‐MB‐231‐xenograft tumor sections. (F) Western blot analysis of CCN5 in lysates of untreated and EGCG‐treated MDA‐MB‐231‐tumor xenograft samples. Data represent error bar mean ± SD, n = 4 animals/group. P‐value determined by one‐way ANOVA and Student's t test, data represent error bar mean ± SD, n = 4 animals/group. (G) Quantification of tumor volume in free‐EGCG and EGCG‐loaded nanoparticles treated (14 days) tumor xenograft mouse model. Data represent error bar mean ± SD, n = 4 animal/group