Maackiain Modulates miR-374a/GADD45A Axis to Inhibit Triple-Negative Breast Cancer Initiation and Progression

Abstract

Breast cancer ranks as the leading cause of death in lethal malignancies among women worldwide, with a sharp increase of incidence since 2008. Triple negative breast cancer (TNBC) gives rise to the largest proportion in breast cancer-related deaths because of its aggressive growth and rapid metastasis. Hence, searching for promising targets and innovative approaches is indispensable for the TNBC treatment. Maackiain (MA), a natural compound with multiple biological activities, could be isolated from different Chinese herbs, such as Spatholobus suberectus and Sophora flavescens. It was the first time to report the anti-cancer effect of MA in TNBC. MA could suppress TNBC cell proliferation, foci formation, migration, and invasion. MA also exerted a significant inhibitory effect on tumor growth of TNBC. Furthermore, MA could induce apoptosis with an increase of GADD45α and a decrease of miR-374a. In contrast, overexpressing miR-374a would result in at least partly affecting the proapoptotic effect of MA and suppressing GADD45α stimulated by MA. These results reveal the anti-TNBC effect of MA in vitro and in vivo, providing evidence for its potential as a drug candidate utilized in TNBC therapy.

Keywords: EMT—epithelial to mesenchymal transformation; GADD45α; maackiain; miR-374a; triple negative breast cancer.

FIGURE 1

The chemical structure of MA constructed using ChemBioDraw.

FIGURE 2

MA attenuates TNBC cell growth. (A) Cell viability of TNBC cells after MA treatment. (B) Representative images and number of foci formation in TNBC cells after exposure to MA for 24 h. Compared with the control group (0 μM), *p < 0.05, **p < 0.01.

FIGURE 3

MA promotes TNBC cell apoptosis. (A) Representative images of flow cytometry and percentages of apoptotic cells in TNBC cells determined after MA treatment for 24 h. (B) Western blot analysis of Bax and Bcl-2 protein expression after 24 h MA treatment. (C) Real-time PCR analysis of BAX and BCL-2 mRNA expression after 24 h MA treatment. Compared with the control group (0 μM), **p < 0.01.

FIGURE 4

MA alleviates migration and invasion of TNBC cells. (A) Representative images of wound healing assay and healing percentage of TNBC cells after 24 h MA treatment. (B) Representative images of chamber invasion assay and number of invading cells after 24 h MA treatment. (C) Western blot analysis of EMT-related protein levels after 24 h MA intervention. (D) Real-time PCR analysis of E-cadherin after exposure to MA for 24 h. Compared with the control group (0 μM), *p < 0.05, **p < 0.01.

FIGURE 5

MA restrains TNBC tumor growth. (A) Collected tumor tissues after MA administration. (B) Tumor volumes in different groups during the experiment. (C) Tumor weights in different groups after MA administration. (D) Real-time PCR analysis of GADD45α mRNA levels in xenograft models at the end point. (E) Body weights of mice in different groups during the experiment. (F) Representative images of HE staining and IHC analysis of GADD45α after MA administration. Compared to the control group, **p < 0.01; compared to the low-dose group, ^## p < 0.01.

FIGURE 6

Overexpressed miR-374a is inhibited by MA in TNBC cells. (A) PCR array analysis of miRNAs variation in TNBC cells after 24 h MA intervention. (B) MiR-374a expression in different types of cancer tissues and the corresponding normal tissues. (C) MiR-374a expression in different types of breast cancer tissues and normal tissues. (D) Real-time PCR analysis of miR-374a expression in TNBC cells after 24 h MA intervention. (E) Relative luciferase activity determined through dual luciferase reporter assay in 293 T cells. Compared to the control group, **p < 0.01.

FIGURE 7

GADD45A is expressed at a low level in TNBC. (A) GADD45A expression in different types of cancer tissues. (B) GADD45A expression in different types of breast cancer tissues and normal tissues. (C) GADD45A is interacted with multiple genes. (D) KEGG annotation classification statistics of GADD45A-related genes.

FIGURE 8

MA regulates miR-374a/GADD45A axis. (A) Western blot analysis of GADD45α after 24 h MA treatment. (B) Real-time PCR analysis of GADD45α mRNA expression after 24 h MA treatment. (C) Real-time PCR analysis of miR-374a expression in different groups of TNBC cell (D) Real-time PCR analysis of GADD45A mRNA expression in different groups of TNBC cells. (E) Western blot analysis of GADD45α protein expression in different groups of TNBC cells. (F) Western blot analysis of Bax protein expression in different groups of TNBC cells. (G) Real-time PCR analysis of BAX in different groups of TNBC cells. Compared with the control group (0 μM), *p < 0.05, **p < 0.01.