N-((1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-methylene-3-oxo-olean-12-en-28-amide Induces Apoptosis in Human Breast Cancer Cells by Stimulating Oxidative Stress and Inhibiting the Notch-Akt Signaling Pathway

Abstract

Breast cancer is of the leading causes of cancer-related deaths and the most frequently diagnosed cancer among females worldwide. Despite advancements in breast cancer therapy, the disease eventually progresses in most patients because of de novo or secondary resistance. Thus, discovering novel drugs with high effectiveness and low toxicity for systemic therapy is essential. In this study, we investigated whether a new oleanolic derivative N-((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-methylene-3-oxo-olean-12-en-28-amide (ZQL-4c) exhibits potential anticancer effects against breast cancer. We determined that ZQL-4c strongly inhibited cell proliferation and invasion and induced G2/M phase arrest and apoptosis in breast cancer cells. We then found that ZQL-4c induced the production of reactive oxygen species (ROS). We then found that ZQL-4c significantly inhibited Notch-AKT signaling pathways that are related to oxidative stress. Taken together, this study is the first to show that ZQL-4c can significantly suppress the growth and invasion of breast cancer by blocking Notch-Akt signaling pathways, which are mainly regulated by ROS-mediated oxidative stress. Thus, ZQL-4c might be considered a novel and potential anticancer drug for breast cancer treatment.

Figure 1

Synthesis of the OA derivative N-((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-methylene-3-oxo-olean-12-en-28-amide (ZQL-4c.

Figure 2

ZQL-4c significantly inhibited the growth of breast cancer cells. (a)–(c) MCF-7, MDA-MB-231, and SK-BR-3 cells were treated with various concentrations of ZQL-4c for increasing times. Line graphs showed the percentage of viable MCF-7, MDA-MB-231, and SK-BR-3 cells after treatment with 0, 0.4, 0.8, 1.0, 2.0, 4.0, and 8.0 μmol/L ZQL-4c for 24 and 48 h, as determined by the CCK8 assay. Data are expressed as the mean ± standard error of the mean of three independent experiments performed in triplicate (n = 3; ^∗P < 0.05). (d) Histogram showing the IC₅₀ values (24 h and 48 h) of ZQL-4c in MCF-7, MDA-MB-231, and SK-BR-3 cells.

Figure 3

ZQL-4c induces G2/M phase arrest in breast cancer cells. MCF-7, MDA-MB-231, and SK-BR-3 cells were treated with 0, 0.4, 0.8, and 1.6 μmol/L ZQL-4c for 24 h. (a) Cells were fixed and stained with PI and analyzed by flow cytometry. (b) The percentage of cells in the G2/M phase is shown in the histogram. Data are expressed as the mean ± standard error of the mean of three independent experiments performed in triplicate (n = 3; ^∗P < 0.05). (c) The protein levels of cyclin B1, cyclin D1, p21, and p27 were detected by western blotting. Actin was used as a loading control.

Figure 4

ZQL-4c induces apoptosis in breast cancer cells. MCF-7, MDA-MB-231, and SK-BR-3 cells were treated with 0, 0.4, 0.8, and 1.6 μmol/L ZQL-4c for 24 h. (a) Light microscopy showed apoptosis-related changes induced by ZQL-4c. (b) Cells were stained with annexin V/PI and analyzed by flow cytometry. (c) The percentage of apoptotic cells is shown in the histogram. Data are expressed as the mean ± standard mean of the error of three independent experiments performed in triplicate (n = 3; ^∗P < 0.05). (d) The protein levels of cleaved caspase-3,-7,-9, p53, Bax, and Bcl-2 were detected by western blotting. Actin was used as a loading control.

Figure 5

ZQL-4c inhibits cell migration and invasion in breast cancer cells. MCF-7, MDA-MB-231, and SK-BR-3 cells were treated with 0, 0.4, 0.8, and 1.6 μmol/L ZQL-4c for 24 h. (a) A pipette tip was used to scratch the cultured cells and the width of the scratch was measured. Untreated cancer cultures of each cell line were used as negative controls. (b) The wound surface area was quantified by Image J software. Data are expressed as the mean ± standard error of the mean of three independent experiments performed in triplicate (n = 3; ^∗P < 0.05).

Figure 6

ZQL-4c induces reactive oxygen species production in breast cancer cells. MCF-7, MDA-MB-231, and SK-BR-3 cells were treated with 0, 0.4, 0.8, and 1.6 μmol/L ZQL-4c for 24 h. Cells were loaded with 10 μmol/L DCFH-DA for 20 min. The nuclei were stained with Hoechst 33342. The level of ROS production was detected by fluorescence microscopy (a) and flow cytometry (b).

Figure 7

ZQL-4c suppresses Notch-Akt signaling pathways in breast cancer cells. MCF-7, MDA-MB-231, and SK-BR-3 cells were treated with 0, 0.4, 0.8, and 1.6 μmol/L ZQL-4c for 24 h. (a)–(c) The protein levels of Notch1, Notch2, mTOR, phospho-mTOR, AKT, phospho-AKT, JAK2, phospho-JAK2, STAT3, and phospho-STAT3 were detected by western blotting. Actin was used as a loading control.

Figure 8

Proposed model for ZQL-4c-induced cell death in breast cancer cells.