Isoliquiritigenin Derivative Regulates miR-374a/BAX Axis to Suppress Triple-Negative Breast Cancer Tumorigenesis and Development

Abstract

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that accounts for the largest proportion of breast cancer-related deaths. Thus, it is imperative to search for novel drug candidates with potent anti-TNBC effects. Recent studies suggest that isoliquiritigenin (ISL) can significantly suppress the growth, migration, and invasion of breast cancer cells. We previously synthesized ISL derivatives and found that 3',4',5',4″-tetramethoxychalcone (TMC) inhibits TNBC cell proliferation to a greater degree than ISL. The present study aimed to investigate the mechanisms underlying the anti-TNBC effects of TMC in vitro and in vivo. We show that TMC significantly inhibits the proliferative, migratory, and invasive abilities of MDA-MB-231 and BT549 cells. TMC induces apoptosis through the upregulation of Bax and downregulation of Bcl-2. PCR arrays demonstrate a significant decrease in miR-374a expression in TNBC cells after 24-h TMC treatment. MiR-374a is overexpressed in TNBC cells and has oncogenic properties. Real-time PCR analysis confirmed that TMC inhibits miR-374a in a dose-dependent manner, and luciferase assays confirmed that BAX is targeted by miR-374a. Further, we show that TMC increases Bax protein and mRNA levels by inhibiting miR-374a. TMC also attenuates TNBC tumor volumes and weights in vivo. These results demonstrate that TMC inhibits TNBC cell proliferation, foci formation, migration, invasion, and tumorigenesis, suggesting its potential to serve as a novel drug for treating TNBC through miR-374a repression.

Keywords: 3′,4′,5′,4″-tetramethoxychalcone; Bax; apoptosis; miR-374a; triple-negative breast cancer.

Figure 1

The chemical structure of tetramethoxychalcone (TMC) constructed using ChemBioDraw.

Figure 2

TMC inhibits triple-negative breast cancer (TNBC) cell proliferation. (A) Cell viability of MDA-MB-231 and BT549 cells after exposure to TMC for 24 and 48 h. (B) Representative images and rate of foci formation in MDA-MB-231 and BT549 cells after 24-h treatment. (C) Number of foci in MDA-MB-231 and BT549 after TMC 24-h treatment.

Figure 3

TMC suppresses TNBC cell migration and invasion. (A) Representative images and percentages of wound healing in MDA-MB-231 cells after exposure to TMC for 24 h. (B) Representative images and percentages of wound healing in BT549 cells after exposure to TMC for 24 h. (C) Representative images of chamber invasion assays in MDA-MB-231 and BT549 cells. (D) Percentages of invading cells in MDA-MB-231 and BT549 cells after exposure to TMC for 24 h. Compared with the control group (0 μM), *p < 0.05,**p < 0.01.

Figure 4

TMC increases the percentage of apoptotic cells in TNBC. (A) Representative images of apoptosis in MDA-MB-231 and BT549 cells determined by flow cytometry analysis after exposure to TMC for 24 h. (B) Percentages of apoptotic cells in MDA-MB-231 and BT549 cells after exposure to TMC for 24 h. (C) Western blot analysis of Bax and Bcl-2 after exposure to TMC for 24 h. (D) Real-time PCR analysis of BAX expression after exposure to TMC for 24 h. Compared with the control group (0 μM), *p < 0.05,**p < 0.01.

Figure 5

TMC represses TNBC tumorigenesis. (A) Tumor tissues collected at the end point. (B) Mice weights measured during the experiment. (C) Tumor volumes measured during the experiment. (D) Tumor weights at the end point. (E) Western blot analysis of Bax and Bcl-2 after TMC administration. (F) Real-time PCR analysis of BAX and BCL2 after TMC administration. Compared with the control group (0 μM), **p < 0.01.

Figure 6

TMC attenuates upregulation of miR-374a in TNBC. (A) PCR array analysis of varied miRNA expression in MDA-MB-231 and BT549 cells after exposure to TMC for 24 h. (B) Real-time PCR analysis of miR-374a in MCF-10A cells and various types of breast cancers. (C) Real-time PCR analysis of miR-374a in MDA-MB-231 and BT549 cells after exposure to TMC for 24 h. Compared with the control group (0 μM), *p < 0.05, **p < 0.01.

Figure 7

TMC promotes Bax protein and mRNA expression by decreasing miR-374a. (A) Dual luciferase reporter assays confirmed the direct binding of miR-374a to the BAX 3′UTR in 293T cells. (B) Real-time PCR analysis of miR-200c after exposure to TMC for 24 h following transfection of the miR-374a mimic or miRNA negative control. (C) BAX expression in breast cancer tissues and normal breast tissues according to data from the Oncomine database. (D) Western blot analysis of Bax after exposure to TMC for 24 h and miR-374a mimic interference. (E) Real-time PCR analysis of BAX after exposure to TMC for 24 h and miR-374a mimic interference. Compared with the control group (0 μM), *p < 0.05, **p < 0.01.