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It has been reported that cancer stem cells (CSCs) may play a crucial role in the development, recurrence and metastasis of breast cancer. Targeting signaling pathways in CSCs is considered to be a promising strategy for the treatment of cancer. Here, we investigated the role of the A2B adenosine receptor (A2BAR) and its associated signaling pathways in governing the proliferation and viability of breast cancer cell line derived CSCs. CSCs were isolated from the breast cancer cell lines MCF-7 and MDA-MB-231 using a mammosphere assay. The effect of the A2BAR agonist BAY606583 on cell proliferation was evaluated using XTT and mammosphere formation assays, respectively. Apoptosis was assessed using Annexin-V staining and cell cycle analyses were performed using flow cytometry. The expression levels of Bax, Bcl-2, cyclin-D1, CDK-4 and (phosphorylated) ERK1/2 were assessed using Western blotting. Our data revealed that the breast cancer cell line derived mammospheres were enriched for CSCs. We also found that A2BAR stimulation with its agonist BAY606583 inhibited mammosphere formation and CSC viability. In addition, we found that the application of BAY606583 led to CSC cell cycle arrest and apoptosis through the cyclin-D1/Cdk-4 and Bax/Bcl-2 pathways, respectively. Notably, we found that BAY606583 significantly down-regulated ERK1/2 phosphorylation in the breast cancer cell line derived CSCs. From our results we conclude that A2BAR induces breast CSC cell cycle arrest and apoptosis through downregulation of the ERK1/2 cascade. As such, A2BAR may be considered as a novel target for the treatment of breast cancer.

Induction of apoptosis in cancer cells can be a promising treatment method in cancer therapy. Naturally derived products had drawn growing attention as agent in cancer therapy. The main target of anticancer drugs may be distinct, but eventually, they lead to identical cell death pathway, which is apoptosis. Here, we indicated that britannin, a sesquiterpene lactone isolated from Asteraceae family, has antiproliferative activity on the MCF-7 and MDA-MB-468 human breast cancer cells. Annexin V/propidium iodide (PI) staining, Hoechst 33258 staining, and caspase-3/9 activity assay confirmed that britannin is able to induce apoptosis in MCF-7 and MDA-MB-468 cells. The Western blot analysis showed that the expression of Bcl-2 was noticeably decreased in response to britannin treatment, while the expression of Bax protein was increased, which were positively correlated with elevated expression of p53. Moreover, britannin also increased reactive oxygen species (ROS) generation which in turn triggered the loss of mitochondrial transmembrane potential (ΔΨm) and the subsequent release of cytochrome c from mitochondria into cytosol. Taken together, these results suggest that britannin inhibits growth of MCF-7 and MDA-MB-468 breast cancer cells through the activation of the mitochondrial apoptotic pathway and may potentially serve as an agent for breast cancer therapy.