Anthocyanidins Inhibit Growth and Chemosensitize Triple-Negative Breast Cancer via the NF-κB Signaling Pathway

Abstract

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Due to the lack of drug-targetable receptors, chemotherapy is the only systemic treatment option. Although chemotherapeutic drugs respond initially in TNBC, many patients relapse and have a poor prognosis. Poor survival after metastatic relapse is largely attributed to the development of resistance to chemotherapeutic drugs. In this study, we show that bilberry-derived anthocyanidins (Anthos) can inhibit the growth and metastasis of TNBC and chemosensitize paclitaxel (PAC)-resistant TNBC cells by modulating the NF-κB signaling pathway, as well as metastatic and angiogenic mediators. Anthos administered orally significantly decreased MDA-MB-231 orthoxenograft tumor volume and led to lower rates of lymph node and lung metastasis, compared to control. Treatment of PAC-resistant MDA-MB-231Tx cells with Anthos and PAC in combination lowered the IC₅₀ of PAC by nearly 20-fold. The combination treatment also significantly (p < 0.01) decreased the tumor volume in MDA-MB-231Tx orthoxenografts, compared to control. In contrast, Anthos and PAC alone were ineffective against MDA-MB-231Tx tumors. Our approach of using Anthos to inhibit the growth and metastasis of breast cancers, as well as to chemosensitize PAC-resistant TNBC, provides a highly promising and effective strategy for the management of TNBC.

Keywords: anthocyanidins (Anthos); breast cancer; chemosensitization; drug resistance; metastasis; paclitaxel.

Figure 1

Effect of Anthos on cell viability, cell-cycle progression, apoptosis, and cell-cycle regulatory proteins: (a) BC cells were treated with a native mixture of Anthos (0–400 μM) isolated from bilberry extract for 72 h and MTT assay was performed. Data denote the mean of three experiments (SD < 10%); (b,c) MDA-MB-231, MDA-MB-436, and HCC1937 cells were treated with Anthos (0–200 μM), and cell-cycle arrest (b) and apoptosis (c) were determined after staining cells with propidium iodide by flow cytometry; (d,e) BC cells were treated with Anthos (0–200 μM) for 48 h and cell lysates were analyzed by Western blot for indicated proteins. Equal loading was confirmed by β-actin. Densitometry data are presented in Figures S2 and S3.

Figure 2

Effect of Anthos on NF-κB activation, nuclear translocation, and related protein molecules in TNBC: (a) BC cells were treated with Anthos (0–50 μM) in the presence or absence of TNFα for 48 h and nuclear extracts were analyzed by EMSA; (b) Cells were treated with Anthos (0–200 μM) for 48 h, and nuclear and cytosol lysates were probed for NF-κB and IκBα proteins by Western blot. Equal loading was confirmed by tata binding protein (TBP) and β-actin, respectively; (c) Effect of bilberry Anthos on NF-κB activation. Breast cancer cells were treated with Anthos (100 and 200 μM) in presence or absence of TNFα for 48 h, and nuclear extracts were analyzed by ELISA: (d) Whole-cell lysates were prepared and immune-precipitated with anti-IKKβ antibody. The immuno-complex kinase assay was performed by ELISA using a CycLex^® IKKα and β Kinase Assay/Inhibitor Screening Kit; (e) Whole-cell lysates were probed for IκBα and P65 levels. Equal loading confirmed by β-actin. Statistical analysis was carried out using student’s t-test; * p < 0.05; ** p < 0.01; *** p < 0.001. Densitometry data are presented in Figures S5–S7.

Figure 3

Effect of Anthos on cell migration, invasion, and EMT pathway proteins: (a) BC cells seeded in 2-Well Culture-Insert in a 24-well plate (Ibidi^®, Munich, Germany), resulting in a 1 mm wide wound. The inserts were removed after overnight incubation and cells were treated with Anthos (0–200 μM). Migration was assessed after 24 h and 48 h of Anthos treatment, represented as the percentage reduction in wound area; (b,c) Cells seeded in matrigel-coated transwell inserts. The bottom chamber was treated with Anthos (0–200 μM) in the absence (b) and presence (c) of TGFβ. Invasive cells at the bottom of the trans-well insert were visualized after staining with 0.2% toluidine blue; (d) Cells were treated with Anthos (0–200 μM) for 48 h and whole-cell lysates were probed for EMT pathway proteins. Equal loading confirmed by β-actin. Densitometry data from panel D are presented in Figure S9.

Figure 4

Effect of Anthos on tumor growth inhibition and lymph node metastasis: (a) NOD-Scid mice were inoculated (Ventral right, under second inguinal nipple) with MDA-MB-231 BC cells (2.5 × 10⁶ cells) to produce an orthotopic tumor. When the tumor grew to ~120 mm³, animals were treated (oral gavage, three times a week) with Anthos (30 mg/kg or 60 mg/kg b.wt.). Control groups received PBS; (b) At euthanasia, lymph nodes were scored for size (as 0, 1+, 2+, or 3+). Data represent average ± SE (n = 9–10). Data represent average ± SE (n = 9–10). Statistical analysis was carried out using student’s t-test; * p < 0.05; ** p < 0.005.

Figure 5

Anti-proliferative and synergistic activity of Anthos against parental and paclitaxel (PAC)-resistant BC cells and effects on resistant gene markers: (a,b) Drug-sensitive (MDA-MB-231) and drug-resistant (MDA-MB-231Tx) cells were treated with Anthos and PAC, alone and in combination, at various concentrations. Anti-proliferative activity was determined by MTT assay. IC₅₀ values, of individual and combination treatments, and combination index were calculated using the Calcusyn software; (c) Expression levels of resistant gene markers (MRP1, MDR1, BRCP, and MVP) were determined using RT-PCR. The bar graph depicts the fold increase in mRNA expression of resistance proteins in MDA-MB-231Tx cells compared to parental MDA-MB- 231 cells; (d) MDA-MB-231Tx were treated with indicated doses of Anthos (A; μM) and PAC (P; nM). C1 and C2 indicate combination treatment with PAC:Anthos at 1:1250; C1 = PAC 80 nM + Anthos 100 μM while C2 = PAC 160 nM + Anthos 200 μM. Significant differences between groups are indicated by a and a**; ** p < 0.01, *** p < 0.001.

Figure 6

Effect of Anthos on NF-κB activation, NF-κB pathway proteins, and tumor growth inhibition of PAC-resistant BC cells: (a) MDA-MB-231Tx cells were treated with Anthos and PAC, alone and in combination, for 48 h and nuclear extracts were analyzed by EMSA; (b) BC cells were treated with Anthos (100 and 200 μM) in presence or absence of TNF-α for 48 h and nuclear extracts were analyzed by ELISA; (c) Whole-cell lysates were prepared and immune-precipitated with anti-IKKβ antibody. The immuno-complex kinase assay was performed by ELISA using CycLex^® IKKα and β Kinase Assay/Inhibitor Screening Kit; (d) BC cells were treated with Anthos (0–200 μM) for 48 h. Whole-cell lysates were probed for IκBα and P65 levels. Equal loading confirmed by β-actin; (e) Effect of Anthos on MDA-MB-231Tx tumor growth inhibition. NOD-Scid mice were inoculated with MDA-MB-231Tx BC cells (2.5 × 10⁶ cells) under the nipple to produce orthotopic tumors. When tumor xenografts grew to ~120 mm³, animals were treated with Anthos (60 mg/kg b. wt.; 3 times/week; oral gavage), PAC (4 mg/kg, b. wt; once weekly; i.p.), or both. Control groups were treated with PBS. Data represent average ± SE (n = 10). Statistical analysis was c using student’s t-test; * p < 0.05; ** p < 0.01, *** p < 0.001. Densitometry data from panels A and D are presented in Figure S12.