Baicalein Suppresses Stem Cell-Like Characteristics in Radio- and Chemoresistant MDA-MB-231 Human Breast Cancer Cells through Up-Regulation of IFIT2

Abstract

Resistance to both chemotherapy and radiation therapy is frequent in triple-negative breast cancer (TNBC) patients. We established treatment-resistant TNBC MDA-MB-231/IR cells by irradiating the parental MDA-MB-231 cells 25 times with 2 Gy irradiation and investigated the molecular mechanisms of acquired resistance. The resistant MDA-MB-231/IR cells were enhanced in migration, invasion, and stem cell-like characteristics. Pathway analysis by the Database for Annotation, Visualization and Integrated Discovery revealed that the NF-κB pathway, TNF signaling pathway, and Toll-like receptor pathway were enriched in MDA-MB-231/IR cells. Among 77 differentially expressed genes revealed by transcriptome analysis, 12 genes involved in drug and radiation resistance, including interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), were identified. We found that baicalein effectively reversed the expression of IFIT2, which is reported to be associated with metastasis, recurrence, and poor prognosis in TNBC patients. Baicalein sensitized radio- and chemoresistant cells and induced apoptosis, while suppressing stem cell-like characteristics, such as mammosphere formation, side population, expression of Oct3/4 and ABCG2, and CD44^highCD24^low population in MDA-MB-231/IR cells. These findings improve our understanding of the genes implicated in radio- and chemoresistance in breast cancer, and indicate that baicalein can serve as a sensitizer that overcomes treatment resistance.

Keywords: IFIT2; baicalein; cancer stem cell; chemoresistance; radioresistance; triple-negative breast cancer.

Figure 1

Comparison of parental MDA-MB-231 cells with radio- and chemoresistant MDA-MB-231/IR cells. (A) Representative images of the clonogenic assay and (B) the surviving fraction of MDA-MB-231 and MDA-MB-231/IR cells after five days of irradiation. MTT assay of MDA-MB-231 and MDA-MB-231/IR cells after (C) Adriamycin and (D) cisplatin treatment for 24 h. Asterisks (*) indicate significant differences at p < 0.05.

Figure 2

Stem cell-like characteristics of MDA-MB-231/IR cells. (A) Morphologies of MDA-MB-231 cells and MDA-MB-231/IR cells. (B,C) Migration and (D,E) invasion were analyzed on same number of MDA-MB-231 cells and MDA-MB-231/IR cells for 24 h. (F,G) Western blot assay for detection of EMT markers expressed in MDA-MB-231 cells and MDA-MB-231/IR cells. GAPDH was used as a control; band intensities were quantified using ImageJ. (H,I) Mammosphere formation over seven days, (J,K) expression of cell surface markers, and (L,M) side population (SP) were detected by FACS analyses. (N,O) Western blot assay for stem cell markers on MDA-MB-231 cells and MDA-MB-231/IR cells. Asterisks (*) indicate significant differences at p < 0.05.

Figure 3

Analyses of differentially expressed genes (DEGs) in MDA-MB-231/IR cells compared to parental MDA-MB-231 cells. (A) Heatmap of DEG expression. (B) Pathway analysis and (C) biological, (D) cellular, and (E) molecular gene ontology (GO) analyses of DEGs in MDA-MB-231/IR cells.

Figure 4

Baicalein treatment reversed levels of IFIT2, which is known to be associated with metastasis and recurrence. (A) MTT assay of baicalein on MDA-MB-231/IR cells for 6, 12, and 24 h. (B) Expression levels of DEGs involved in resistance in MDA-MB-231/IR cells. (C) IFIT2 gene expression after baicalein treatment for 24 h. (D) IFIT2 expression in primary tumor, metastatic, and solid normal tissue of breast cancer patients based on Xena browser. (E) Relapse-free survival (RFS) plot of TNBC patients analyzed by IFIT2 expression with the Kaplan–Meier plotter. Asterisks (*) indicate significant differences at p < 0.05.

Figure 5

Effects of baicalein on characteristics of MDA-MB-231/IR cells. (A,B) Migration assay and (C,D) Western blot analysis of EMT proteins in MDA-MB-231/IR cells were performed after baicalein treatment for 24 h. GAPDH was used as a control; band intensities were quantified using ImageJ. Effects of baicalein on stem cell-like characteristics were determined by (E,F) invasion assay for 24 h and (G,H) mammosphere formation assay for one week. (I,J) Western blot assay for stem cell markers in MDA-MB-231 cells after baicalein treatment for 24 h. (K,L) The percentage of expression of cell surface markers and (M,N) side population (SP) on MDA-MB-231/IR cells were detected by FACS after baicalein treatment for 24 h. Asterisks (*) indicate significant differences at p < 0.05.

Figure 6

Baicalein induced apoptosis and sensitized resistant MDA-MB-231/IR cells. (A,B) Expression levels of IFIT2 (left-hand y-axis) and apoptosis marker proteins (right-hand y-axis) after baicalein treatment for 24 h. (C) Cell cycle analysis, (D,E) annexin V/PI staining, and (F,G) JC-1 staining were performed to observe induction of apoptosis by baicalein. (H,I) Baicalein sensitized MDA-MB-231/IR cells to irradiation. (J,K) Baicalein sensitized MDA-MB-231/IR cells to Adriamycin (0, 12.5, 25, 50, and 100 nM) and cisplatin (0, 10, 20, 30, and 40 μM) treatment (▬: Adriamycin or cisplatin alone, ▬: Adriamycin or cisplatin with baicalein 10 μM, ▬: Adriamycin or cisplatin with baicalein 20 μM). Asterisks (*) indicate significant differences at p < 0.05.