Ethanol Extracts of Dietary Herb, Alpinia nantoensis, Exhibit Anticancer Potential in Human Breast Cancer Cells

Abstract

Recent advances in mammography screening, chemotherapy, and adjuvant treatment modalities have improved the survival rate of women with breast cancer. Nevertheless, the breast tumor with metastatic progression is still life-threatening. Indeed, combination therapy with Ras-ERK and PI3K inhibitors is clinically effective in malignant breast cancer treatment. Constituents from genus Alpinia plants have been implicated as potent anticancer agents in terms of their efficacy of inhibiting tumor cell metastasis. In this study, we tested the effects of ethanol extracts of Alpinia nantoensis (rhizome, stem, and leaf extracts) in cultured human breast cancer cells and particularly focused on the Ras-ERK and PI3K/AKT pathways. We found that the rhizome and leaf extracts from A nantoensis inhibited cell migration, invasion, and sphere formation in MCF-7 and MDA-MB-231 cells. The potency was extended with the inhibition of serum-induced PI3K/AKT and Ras-ERK activation and epidermal growth factor (EGF)-mediated EGFR activation in MDA-MB-231 cells. These results indicate that extracts of A nantoensis could inhibit signal transduction at least involved in EGFR as well as the PI3K/AKT and Ras-ERK pathways, which are crucial players of tumor cell migration and invasion. Our study strongly supports that the extracts of A nantoensis could be a novel botanical drug lead for the development of an antimetastatic agent for the treatment of human malignant breast cancer.

Keywords: EGFR; PI3K/AKT; Ras-ERK; breast cancer; metastasis.

Figure 1.

Effect of Alpinia nantoensis extracts on cell survival and proliferation in cancer cell lines. Cell viability was measured by MTT assay after 48 hours of treatment with increasing concentrations of ANR, ANS, or ANL in MDA-MB-231 (A) and MCF-7 (B) cells. The survival cells were measured by MTT assay at 0 to 96 hours after exposure to 2.5 to 20 µg/mL A nantoensis extracts was used to test the cell doubling in MDA-MB-231 (C) and MCF-7 (D) cells. The insoluble formazan was dissolved by DMSO at the end of the experiment, and the percentage of viable cells to the DMSO group was calculated from the absorbance measured at 550 nm. All results are presented as mean ± standard error from 3 independent experiments, n = 3.

Figure 2.

Effect of Alpinia nantoensis extracts on cell migration in MDA-MB-231 cells. The ability of cell migration was determined using the wound healing assay and immunoblots for MDA-MB-231 cells. (A) The wounds are 400 µm wide created by a Culture-Insert, and the wound distances were measured on the microscope images at 0, 8, 16, and 24 hours after cells were incubated with or without 10 µg/mL of extracts. These results were shown by representative photomicrographs. (B) Bar graph illustrating the quantitative analysis of wound distances at the different extracts following various exposure times. Data represent the mean ± standard error of 3 independent experiments, *P < .05 compared with the DMSO group. (C) Immunoblots results showed the effect of extracts on the E-cadherin phosphorylation following exposure to 10 µg/mL of extracts for 24 hours.

Figure 3.

Effect of Alpinia nantoensis extracts on cell migration and cell invasion in MDA-MB-231 cells. The ability of cell migration was determined using the Transwell inserts assay, invasion chamber assay, and Griess assay for MDA-MB-231 cells. (A) 5 × 10⁴ cells were plated into noncoding Boyden chambers together with tested extracts for 16 hours. (C) 2.5 × 10³ of MDA-MB-231 cells were seeded into the invasion chamber together with extracts for 24 hours. The migratory and invasive cells were stained with 1% w/v crystal violate solution and become visible under an inverted microscope. Representative photomicrographs are shown, and the bar chart for mean ± standard error of mean was calculated from 3 independent experiments (B and D). **P < .01 compared with DMSO group. ***P < .001 compared with the DMSO group. MMP expression and activity assessment of Alpinia nantoensis extracts by gelatin zymography in conditioned medium (E) and cell lysates (F).

Figure 4.

Mammosphere formation of MDA-MB-231 cells treated with Alpinia nantoensis extracts at different concentration. (A) Photomicrographs of the mammospheres following treatment with Alpinia nantoensis extracts. (B) The number and the size of mammospheres formed per dish were enumerated, and 100 spheres were picked up for each group to analyze. The counts represented are mean ± standard error of mean from 3 independent experiments.

Figure 5.

Effect of Alpinia nantoensis extracts on the PI3K/AKT and Ras/ERK signaling in MDA-MB-231 cells. Immunoblots results showed the effect of extracts on the AKT and ERK activation by detecting phosphorylation of AKT Ser473 and ERK Tyr204 following exposure to extracts at 10 µg/mL for 24 hours in MDA-MB-231 (A) and MCF-7 (B) cells. Immunoblots results showed the effect of extracts on serum-induced AKT and ERK activations by detecting phosphorylation of AKT Ser473 and ERK Tyr204 following pretreatment to extracts at 10 µg/mL for 30 minutes, then stimulated with 20% FBS (fetal bovine serum) for 30 minutes in MDA-MB-231 (C) and MCF-7 (D) cells. Treatment of a PI3K inhibitor (Wortmannin, 1 µM) was served as a control. Immunoblots results showed the effect of extracts on EGF-induced EGFR and AKT activations by detecting phosphorylation of EGFR Tyr1068 and AKT Ser473 following pretreatment to extracts at 10 µg/mL for 30 minutes before stimulated with EGF for 30 minutes in MDA-MB-231 (E) and MCF-7 (F) cells.