Elaeagnus angustifolia Plant Extract Inhibits Epithelial-Mesenchymal Transition and Induces Apoptosis via HER2 Inactivation and JNK Pathway in HER2-Positive Breast Cancer Cells

Abstract

Elaeagnus angustifolia (EA) is a medicinal plant used for treating several human diseases in the Middle East. Meanwhile, the outcome of EA extract on HER2-positive breast cancer remains nascent. Thus, we herein investigated the effects of the aqueous EA extract obtained from the flowers of EA on two HER2-positive breast cancer cell lines, SKBR3 and ZR75-1. Our data revealed that EA extract inhibits cell proliferation and deregulates cell-cycle progression of these two cancer cell lines. EA extract also prevents the progression of epithelial-mesenchymal transition (EMT), an important event for cancer invasion and metastasis; this is accompanied by upregulations of E-cadherin and β-catenin, in addition to downregulations of vimentin and fascin, which are major markers of EMT. Thus, EA extract causes a drastic decrease in cell invasion ability of SKBR3 and ZR75-1 cancer cells. Additionally, we found that EA extract inhibits colony formation of both cell lines in comparison with their matched control. The molecular pathway analysis of HER2 and JNK1/2/3 of EA extract exposed cells revealed that it can block HER2 and JNK1/2/3 activities, which could be the major molecular pathway behind these events. Our findings implicate that EA extract may possess chemo-preventive effects against HER2-positive breast cancer via HER2 inactivation and specifically JNK1/2/3 signaling pathways.

Keywords: EMT; Elaeagnus angustifolia; apoptosis; breast cancer; chemoprevention.

Figure 1

(a,b) The effects of different concentrations of Elaeagnus angustifolia (EA) plant extract on cell proliferation of HER2-positive breast cancer cell lines SKBR3 (a) and ZR75-1 (b) at 48 h. Data indicate an inverse relation between concentrations of EA extract and cell proliferation in both SKBR3 and ZR75-1 cell lines. Data are expressed as percent of growth ± SEM.

Figure 2

(a,b) Flow cytometry data analysis of SKBR3 and ZR75 cells after EA-treatment. Data demonstrate an increase in G₀/G₁ phase with simultaneous reduction in S and G₂/M phases in both cell lines. Meanwhile, there is a significant increase in cell apoptosis (Sub/G₁ phase) of SKBR3 cells treated with EA, and a small increase in cell apoptosis of treated ZR75 cells.

Figure 3

(a,b) Induction of apoptosis by EA extract in SKBR3 (a, b) and ZR75 (c, d) cells, as determined by Annexin V-FITC and 7-AAD apoptosis assay.

Figure 4

(a,b) EA plant extract induces morphological changes in HER2-positive cell lines, SKBR3 and ZR75-1. (a) We observe that treatment for 48 h with 100 and 200 μL/mL of EA extract induces epithelial transition and the formation of a monolayer of cells in both cell lines, in comparison with untreated (control) cells which display a round phenotype and form multilayers; arrows indicate epithelial morphology with clear cell-cell adhesion. (b) At three days of treatment of SKBR3, ZR75-1, and HNME-E6/E7 cell lines with 200 μL/mL of EA plant extract, the two cancer cell lines start detaching from the surface of the tissue culture dish, indicating cell death; this observation was not noted in the HNME-E6/E7 cells (images a and b at ×20 magnification).

Figure 5

(a,b): The effects of EA flower extract on cell invasion of human HER2-positive breast cancer cells. EA extract inhibits cell invasion ability of SKBR3 (a) and ZR75-1 (b) cell lines by approximately 70% in comparison with their matched control cells (unexposed) (p < 0.05). Boyden chambers were used to assess cell-invasion ability of SKBR3 and ZR75-1 cell lines. Cancer cells treated for 24 h with 100 and 200 µL/mL EA plant extract showed a significant inhibition of cell invasion in both cell lines, when compared with their matched control (p < 0.05). Data are quantified by normalizing the number of invasive cells by their total number.

Figure 6

(a,b) Effect of EA flower extract on colony formation, in soft agar, in human HER2-positive cancer cell lines, SKBR3 (a) and ZR75-1 (b). EA extract inhibits colony formation of SKBR3 and ZR75-1, in comparison with their matched control cells (images of figure a,b at ×10 magnification). Colony formation in soft agar is a solid indicator of tumor formation in vivo. The colonies were counted manually and expressed as percentage of treatment relative to the control (mean ± SEM).

Figure 7

(a,b) Protein expression and molecular mechanisms of EA inhibitory actions in SKBR3 cell line. This plant extract induces an overexpression of E-cadherin, β-catenin, and downregulation of vimentin and fascin, while upregulating pro-apoptotic markers (Bax and Caspase-3), in comparison with their control and inhibiting anti-apoptotic markers (Bcl-2). Furthermore, EA plant extract inhibits the phosphorylation of ErbB2 and β-catenin, as well as the expression of JNK1/2/3. β-actin was used as a control for the proteins amount in this assay. Cells were treated with 100 and 200 μL/mL of EA extract for 48 h, as explained in the materials and methods and the results sections. (a) Blot image and (b) quantification of bands.

Figure 8

(a,b) Protein expression and molecular mechanisms of EA inhibitory actions in ZR75 cell line. This plant extract induces an overexpression of E-cadherin, β-catenin, and downregulation of vimentin and fascin; in addition, pro-apoptotic markers Bax and Caspase-3 are upregulated in comparison with their control, while anti-apoptotic marker Bcl-2 is inhibited. Furthermore, EA plant extract inhibits the phosphorylation of ErbB2 and β-catenin, as well as JNK1/2/3 expression. β-actin served as a control in this assay. Cells were treated with 100 and 200 μL/mL of EA extract for 48 h, as explained in the materials and Methods section. (a) Blot image and (b) quantification of bands.