Eupolyphaga sinensis Walker Ethanol Extract Suppresses Cell Growth and Invasion in Human Breast Cancer Cells

Abstract

Aim of the study: To examine the antiproliferation and anti-invasion of Eupolyphaga sinensis Walker 70% ethanol extract (ESWE) on breast cancer and elucidate the underlying signaling mechanisms.

Methods: MTT and colony formation assays were used to investigate the effect of ESWE on proliferation of breast cancer cells in vitro. The xenograft mouse tumor model was used to determine the effect of ESWE on breast cancer in vivo. To investigate the underlying molecular mechanisms, we used western blotting to analyze the expression of ERK1/2, CXCR4, matrix metalloproteinase 2 (MMP2), and MMP9 pretreated with ESWE. The stromal cell-derived factor (SDF)-1α-induced migration and invasion potential of breast cancer cells were examined by wound-healing assays and Matrigel invasion chamber assays.

Results: ESWE effectively inhibited the proliferation of MDA-MB-435s and MDA-MB-231 cells and exhibited antitumor effects in an MDA-MB-231 xenograft mice model. Furthermore, ESWE suppressed the activity of ERK1/2, a key molecule of MAPK signaling. We also observed that ESWE treatment led to downregulation of CXCR4 expression as well as greatly reduced MMP2 and MMP9. ESWE affected CXCR4 expression partially through the modulation of autocrine vascular endothelial growth factor. However, suppression of CXCR4 expression was the result of downregulation of mRNA expression. Inhibition of CXCR4 expression by ESWE further correlated with the suppression of SDF-1α-induced migration and invasion in breast cancer cells.

Conclusion: ESWE exerted its antiproliferation and antiinvasion by regulating MAPK signaling and related metastasis factorsand thus could be a useful therapeutic candidate for breast cancer intervention.

Keywords: CXCR4; ERK1/2; Eupolyphaga sinensis Walker; MMP2; MMP9; breast cancer.

Figure 1.

Eupolyphaga sinensis Walker 70% ethanol extract (ESWE) suppressed breast cancer cell proliferation and colony formation. A. Effect on proliferation of MDA-MB-435s, MDA-MB-231, and MCF-10A cells by ESWE. Cells were treated with ESWE at a series of increasing concentrations (0.125, 0.25, 0.50, 0.75, 1.00, 1.50 mg/mL) for 48 hours. ESWE inhibited MDA-MB-435s and MDA-MB-231 cell growth in a dose-dependent manner and was considerably less active on MCF-10A cells. B. Effect on colony formation of MDA-MB-435s and MDA-MB-231 cells by ESWE. C. Quantification of (B); data are represented as the means ± standard error of the mean from 3 repeated experiments. D. Photograph of the representative tumor in each group.

Figure 2.

Eupolyphaga sinensis Walker 70% ethanol extract (ESWE) suppressed ERK1/2 activity in breast cancer cells. MDA-MB-435s (A) and MDA-MB-231 (B) cells were incubated with ESWE at different concentrations for 48 hours. Whole-cell extracts were prepared and analyzed by western blot analysis with antibodies against p-ERK1/2, ERK1/2, and GAPDH (glyceraldehyde 3-phosphate dehydrogenase). C. Quantification of (A) and (B). The results shown were representative of 3 independent experiments.^{a a}*P < .05, **P < .01 compared with untreated control cells.

Figure 3.

Eupolyphaga sinensis Walker 70% ethanol extract (ESWE) downregulated CXCR4 in breast cancer cells: MDA-MB-435s (A) and MDA-MB-231 (B) cells were incubated with ESWE at different concentrations for 48 hours. Whole-cell extracts were prepared and analyzed by western blot analysis with antibodies against CXCR4. The same blots were stripped and reprobed with GAPDH antibody to show equal protein loading. C. Quantification of (A) and (B). The results shown are representative of 3 independent experiments.^{a a}*P < .05, **P < .01 compared with untreated control cells.

Figure 4.

Effect of ESWE on VEGF expression in breast tumor cells: MDA-MB-435s (A) and MDA-MB-231 (B) cells were treated with the indicated concentrations of ESWE for 48 hours, after which western blotting for VEGF₁₆₅ was done as described above. The same blots were stripped and reprobed with GAPDH antibody to show equal protein loading. C. Quantification of (A) and (B). The results shown are representative of 3 independent experiments.^a Abbreviation: ESWE, Eupolyphaga sinensis Walker 70% ethanol extract; VEGF, vascular endothelial growth factor. ^a*P < .05, **P < .01 compared with untreated control cells.

Figure 5.

Eupolyphaga sinensis Walker 70% ethanol extract (ESWE) suppressed CXCR4 partial by proteasomal degradation: MDA-MB-435s (A) and MDA-MB-231 (B) cells were treated with indicated concentrations of chloroquine or MG132 for 1 hour, followed by treatment with ESWE for 48 hours. Whole-cell extracts were prepared and analyzed by western blot analysis using antibodies against CXCR4. The same blots were stripped and reprobed with GAPDH antibody to show equal protein loading. C. Quantification of (A) and (B). The results shown are representative of 3 independent experiments.

Figure 6.

Eupolyphaga sinensis Walker 70% ethanol extract (ESWE) suppressed CXCR4 mRNA level in breast tumor cells. MDA-MB-435s (A) and MDA-MB-231 (B) cells were treated with ESWE for the indicated concentrations for 48 hours, and then, real-time polymerase chain reaction was performed to measure the relative quantities of CXCR4 mRNA, with GAPDH as endogenous control for measurement of equal loading of RNA samples. The results shown are representative of 3 independent experiments.^{a a}*P < .05, **P < .01 compared with untreated control cells.

Figure 7.

ESWE inhibited migration and invasion of breast tumor cells: (A) and (B), Photographs of wounds of cells treated with ESWE. Wound-healing assay was performed for evaluating the inhibitory effect of ESWE on MDA-MB-435s (A) and MDA-MB-231 (B) cell migration. Confluent monolayers of cells were scarred and pretreated with ESWE for 48 hours before being exposed to 100 ng/mL SDF-1α for 24 hours. The average distance that the cells migrated into the wound surface was determined under an inverted microscope. The representative photographs show the same area at time 0 and after 48 hours of incubation. (C) and (D), Photographs of the cell invasion through the Matrigel-coated polycarbonate membrane stained by 0.2% crystal violet. MDA-MB-435s (C) and MDA-MB-231 (D) cells were seeded in the top chamber of the Matrigel. After pretreatment with or without ESWE for 48 hours, Millicell chambers were incubated with either the basal medium only or basal medium containing 100 ng/mL SDF-1α for 24 hours. After incubation, they were assessed for cell invasion as described in the Materials and Methods section. The representative photographs show the cell migration through the polycarbonate membrane stained by 0.2 % crystal violet. Abbreviation: ESWE, Eupolyphaga sinensis Walker 70% ethanol extract; SDF, stromal cell–derived factor.

Figure 8.

Effect of ESWE on expression of MMP-2 and MMP-9 in breast tumor cells: (A) and (B), western blot analysis of MMP-2 and MMP-9 protein expression in MDA-MB-435s cells (A) and MDA-MB-231 cells (B) after treatment with ESWE at indicated concentrations for 48 hours. C. Quantification of (A) and (B). The results shown are representative of 3 independent experiments. Data are expressed as mean ± standard error of the mean.^a Abbreviations: ESWE, Eupolyphaga sinensis Walker 70% ethanol extract; MMP, matrix metalloproteinase. ^a*P < .05, **P < .01 compared with untreated control cells.