Berberine Inhibits Breast Cancer Stem Cell Development and Decreases Inflammation: Involvement of miRNAs and IL-6

Abstract

Background: Breast cancer (BC) is a health concern worldwide and is often accompanied by depressive symptoms in patients. In BC, elevated interleukin-6 (IL-6) levels contribute to an inflammatory signature linked to cancer stem cell (CSC) stemness and depressive behaviors. Bioactive food components, such as berberine (BBR), have preventative effects against BC by targeting CSCs.

Objectives: This study aimed to investigate the effects of BBR on breast CSC proliferation, on levels of specific micro (mi)RNAs and IL-6 in vitro and in vivo, and in alleviating depressive-like behaviors in mice with BC.

Methods: Mammosphere formation assays were conducted by treating murine 4T1 and human MDA-MB-231 BC cell lines with BBR. qPCR analysis of miRNAs miR-let-7c and miR-34a-5p was performed on 4T1 CSCs exposed to BBR. BBR was administered orally to female BALB/c, followed by injection with mammary carcinoma cells to induce BC. Behavioral tests were conducted to assess depressive-like behaviors. Tumor tissues were collected for ex vivo mammosphere assays, miRNA expression analysis, and IL-6 detection by ELISA. Serum was also collected for IL-6 analysis.

Results: BBR treatment inhibited mammosphere formation and proliferation of CSCs derived from 4T1 and MDA-MB-231 cell lines. Quantification of mammosphere formation showed a significant decrease in both cell lines at 75 μM BBR (4T1: P < 0.001; MDA-MB-231: P < 0.0001). BBR upregulated the expression of miRNAs miR-let-7c and miR-34a in both cell lines, with miR-34a showing a significant increase (P < 0.001) and let-7c showing a significant increase (P < 0.05) in expression. In vivo, oral administration of BBR reduced mammosphere formation in breast tumor tissues (P < 0.0001) and elevated expression of miR-145 and miR-34a, with both showing significant upregulation (P < 0.0001), indicating its potential tumor-suppressive effects. BBR treatment resulted in a significant decrease in serum IL-6 levels (P < 0.05), suggesting anti-inflammatory properties, while the IL-6 in tumor tissue did not show significant changes (P > 0.05). However, no significant differences were observed in depressive-like behaviors between control and treatment groups.

Conclusions: BBR may have the potential to be used as an "Epi-Natural Compound" to prevent cancer by reducing inflammation and affecting epigenetics.

Keywords: berberine; breast cancer; cancer stem cells; epigenetics; interleukin-6; microRNAs.

FIGURE 1

CSC growth and proliferation from both cell lines. (A) Untreated 4T1 CSCs (10× magnification). (B) 4T1 CSCs treated with 75 μM BBR (10× magnification). (C) Quantification of 4T1 mammospheres in different BBR concentrations. (D) Untreated MDA-MB-231 CSCs (10× magnification). (E) MDA-MB-231 CSCs treated with 75 μM BBR (10× magnification). (F) Quantification of MDA-MB-231 mammospheres in different BBR concentrations. Statistical significance was determined using a 1-way ANOVA and Tukey multiple comparisons test. ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001. ANOVA, analysis of variance; BBR, berberine; CSC, cancer stem cell.

FIGURE 2

Relative expression of miRNAs in 4T1 cell culture in vitro. (A) miR-34a relative expression. (B) miR-let-7c relative expression. Results presented as mean ± SEM and statistically evaluated with a simple t test (∗P < 0.05; ∗∗∗P < 0.001).

FIGURE 3

Ex vivo mammospheres of the breast tumors both groups. (A) Microscope image of mammospheres (10× magnification) from untreated mice consuming 1% sucrose water injected with the 4T1 cell line. (B) Mammospheres of BBR-treated mice consuming BBR solution injected with the 4T1 cell line. (C) Ex vivo mammosphere quantification of the breast tumors in the control and treatment groups; results presented as mean ± SEM. Statistical significance was determined using a simple t test.

FIGURE 4

Relative expression of miRNAs in tumor tissues. (A) miR-145 relative expression in breast cancer tumor tissues. (B) miR-34a relative expression in breast cancer tumor tissues. Results presented as mean ± SEM and statistically evaluated with a simple t test (∗∗∗∗P < 0.0001).

FIGURE 5

IL-6 levels measured with ELISA. (A) IL-6 levels in BALB-c mouse breast tumor. (B) IL-6 levels in BALB-c mouse plasma pools (pg/mL). The graphs show the mean ± SEM; statistical significance was determined using t test analysis (∗P < 0.05; ns, no significance).

FIGURE 6

Depressive-like behavior tests in female mice exposed to either 1% sucrose water or berberine in 1% sucrose water treatment at week 11 of age. (A) The duration of inactivity in seconds (sec) during the forced swim test (FST). (B) The duration below the threshold in the tail suspension test (TST). Data are presented as mean (± SEM); no significant difference between groups; n = 12 for each group.