In this study, we tested synergistic chemopreventive effects of CUR and BBR in combination on ER-positive and triple-negative breast cancer cell lines. The data convincingly demonstrated that co-treatment of CUR and BBR markedly exerted a synergistic growth inhibitory effect on both cell lines through triggering apoptosis and autophagic cell death. The apoptosis involved ERK activation and caspase-dependent pathway, while the autophagic cell death involved JNK activation, Bcl-2 phosphorylation and the dissociation of Beclin1/Bcl-2 complex. Taken together, co-treatment of CUR and BBR markedly inhibits cell growth and might be a promising strategy for breast cancer chemoprevention.

CUR and BBR are renowned phytochemicals which possess distinct chemical structures and exhibit remarkable anticancer activities via different molecular mechanisms. However, their anticancer capability is compromised due to the limited intrinsic cytotoxic potential and nonspecific cytotoxicity when high dosage is applied. We speculate that combination treatment of CUR and BBR could target different molecules or pathways simultaneously and therefore exert synergistic cancer chemopreventive effects. In our experiments, CUR (2–10 μM) and BBR (3–50 μM) treatment alone resulted in a dose-dependent reduction of cell growth, accounting for 3–15% and 3–43% reduction in MCF-7 cells, respectively. Similar results were found in MDA-MB-231 cells, accounting for 3–34% and 13–36% reduction, respectively. These results were consistent with previous reports32,33. However, CUR and BBR co-treatment at the same range of concentrations caused growth inhibition of 15–62% and 20–82% in MCF-7 and MDA-MB-231 cells, respectively, which were significantly higher than the compounds treated alone. Besides, all CI values of these compounds in combination were less than one, indicating that these two compounds in combination markedly exerted synergistic growth inhibitory effects on MCF-7 and MDA-MB-231 cells.

Apoptosis, the major type of programmed cell death (PCD), is an active cell suicide process. It can be divided into two categories: caspsase-dependent and caspase-independent9. Among them, apoptosis in most cases is mediated in a caspase-dependent manner through death receptor (extrinsic) or mitochondrial (intrinsic) pathways. Mitochondria plays crucial roles in the intrinsic apoptosis. Bcl-2 family proteins, including anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins, which mainly regulate mitochondrial membrane potential accompanied by the release of mitochondrial proteins, such as cytochrome c34. The release of cytochrome c from mitochondria to cytosol activates caspase-9, the initiator of intrinsic pathway that binds to Apaf-1 and cytochrome c and then activates caspase-3 and subsequently leads to apoptosis. In this study, we found that CUR at 5 μM and BBR at 25 μM treatment alone slightly induced apoptosis (<20%) in both breast cancer cell lines, however, combination treatment synergistically augmented apoptosis induction to more than 40% in MCF-7 and MDA-MB-231 cells (Fig. 2A,B). Meanwhile, the activities of caspase-3 and -9 and accumulation of Sub-G1 phase were also markedly increased in both cell lines co-treated with these two compounds comparing to that of either agent alone (Fig. 2E–G). In addition, we observed that the co-treatment more greatly decreased the expression of Bcl-2 and increased expressions of Bax, cleaved caspase-3 and cleaved PARP comparing to either agent treated alone. Addition of the pan-caspase inhibitor Z-VAD, however, prominently diminished the effects of CUR and BBR co-treatment on the protein levels (Fig. 3D) and cell viability of both MCF-7 cells and MDA-MB-231 cells. These data suggested that the synergistic pro-apoptotic activities of the co-treatment might be through inducing the caspase-dependent mitochondrial pathway35, which is in consistent with previous reports when either compound used alone36,37.

Interestingly we found that combination treatment of CUR and BBR caused more ERK phosphorylation compared with groups of CUR and BBR treated alone in both cell lines. Pretreatment with ERK inhibitor markedly restrained phosphorylation of ERK, Bax/Bcl-2 ratio and the cytotoxicity induced by these two compounds. These results indicated that co-treatment of CUR and BBR induced apoptosis in the breast cancer cells via activation of ERK pathway. ERK plays complicated roles in cell proliferation and apoptosis. On the one hand, ERK activation has generally been found to prevent apoptotic cell death and induce cell proliferation through blockage of a critical component of the cell-intrinsic death machinery and enhancement of the expression of pro-survival genes through the Ras-Raf-MEK-ERK signaling pathway27. On the other hand, a number of studies indicated that activation of ERK could trigger apoptotic cell death depending upon the cell types and stimuli28. It was suggested that the intensity and duration of pro- versus anti-apoptotic signals transduced by ERK determined the cell fate38. ERK activation by sustained DNA damage could lead to p53 activation and subsequently induce both growth arrest and apoptosis. In addition, ERK-mediated cell apoptosis may be associated with suppression of nuclear translocation and cytosolic retention of activated ERK through its interaction with the pro-apoptotic proteins Bik, PEA-15 and DAPK and activation of these protein scaffolding38. The detailed mechanisms remain to be further investigated.

Autophagy is an evolutionarily conserved catabolic process which is primarily used for cellular adaptive response to deficiency in nutrient resources. Autophagy can also be broadly induced by multiple external stimuli such as ionizing radiation and cytotoxic chemicals, including anticancer reagents. It plays dual roles in cancers, i.e. pro-survival or pro-death of cancer cells, depending on types of cells and stimuli and stimulus concentration and duration of treatment39,40. Although autophagy is generally regarded as a cytoprotective process, prolonged stress and sustained autophagy could eventually lead to cell death41. Autophagic cell death (ACD) has been proposed as an additional mechanism for cell death in the absence of the apoptosis signaling pathways42. Here we found that CUR and BBR could significantly induce autophagy in both MCF-7 and MDA-MB-231 cells, which is in accordance with previous reports43,44. Interestingly, co-treatment of these two compounds much more potently induced autophagy in the breast cancer cells, as evidenced by the increased number and size of punctate structures in cells (Fig. 5), which are the feature of autophagic vacuole formation, higher levels of LC3-II turnover and p62 protein degradation, when compared to either agent treated alone. The turnover of LC3-II and its subcellular localization pattern were also observed using EGFP-LC3-expressing cells. Quantification analysis indicated that there were 36.15% and 12.48% of EGFP-LC3 puncta expressing in MDA-MB-231 and MCF-7 cells co-treated with CUR and BBR, respectively, comparing to 9.61% and 4.07% of positive cells without treatment. The differential responses to the autophagy induction by the co-treatment might be due to different background of the two breast cancer cell lines. Similar phenomenon was observed that MDA-MB-231 cells were more sensitive to rapamycin-induced autophagy than MCF-7 cells45. The presumed reason is that autophagy could be mediated by AKT/mTOR and/or JNK/Bcl-2/Beclin1 pathways, which are cell type-dependent46. Inhibition of autophagy by CQ and 3-MA, which were used to suppress autophagy at early and late stages, respectively, greatly reduced the cytotoxicity induced by combination treatment in both cell lines. Furthermore, inhibition of autophagy by CQ also markedly attenuated the increased Bax/Bcl-2 and LC3II/LC3I ratios and the decreased p62 expression. These results convincingly demonstrated that the enhanced cytotoxicity by co-treatment of CUR and BBR could be also mediated, at least partially, by inducing autophagic cell death in both ER-positive and ER-negative breast cancer cells.

In mammalian cells, anti-apoptosis protein Bcl-2 binds to autophagy protein Beclin1 and blocks the initiation of autophagosome formation in normal conditions47. JNK signaling pathway has been proved to modulate autophagy in response to cell stresses47. Increasing evidence shows that JNK activation facilitates the phosphorylation of Bcl-2 and dissociation of the Beclin1/Bcl-2 complex and the dissociated active Beclin1 induces autophagy47. Hence, Bcl-2 regulates both apoptosis and autophagy and Beclin1/Bcl-2 complex functions as a toggle switch in triggering autophagy48. In this study, our results demonstrated that co-treatment of CUR and BBR more strongly increased the protein levels of phosphorylated JNK and Beclin1 and decreased the phosphorylated Bcl-2, suggesting that the co-treatment activated JNK signaling pathway, leading to phosphorylation of Bcl-2 and dissociation of Beclin1 and consequently resulted in autophagic cell death in the two breast cancer cells. Inhibition of JNK by SP600125 significantly increased p-Bcl-2 and decreased Beclin1 levels and reduced the cytotoxicity of the co-treatment of CUR and BBR, further confirm our speculation. Numerous studies have suggested that phosphorylation of Bcl-2 could strengthen the anti-apoptotic activity of Bcl-2 and could inhibit autophagy49. Others reported that phosphorylation of Bcl-2 may lead to its ubiquitination and proteasomal degradation and disarm its anti-apoptotic function30,31. In the current study, we found that phosphorylated Bcl-2 induced by JNK activation was significantly reduced following the combination treatment of CUR and BBR. In contrast, inhibition of JNK markedly restored Bcl-2 phosphorylation. JNK can be activated by multiple stimuli or pathways, including oxidative stress or genotoxic agents, growth factors, inflammatory cytokines and ligands of G-protein-coupled receptors (GPCRs), among which, the stresses or genotoxic agents are the most powerful inducers of JNK50. We suppose that activation of JNK by co-treatment of CUR and BBR is through oxidative stress response and/or genotoxic pathways since CUR is a potent pro-oxidant14 and BBR could insert DNA and interrupt DNA replication17. NF-κB signaling system plays an important role in the regulation of autophagy51. It could inhibit autophagy through increasing the expression of autophagy repressors A20, Bcl-2 and NLRP receptors and suppressing autophagy inducers BNIP3, JNK and ROS. Both CUR and BBR are known to be NF-κB inhibitors52,53, which could explain, at least partially, the synergistic induction of autophagy in the breast cancer cells by the combination treatment.

In conclusion, the present study demonstrated that co-treatment of CUR and BBR exhibited synergistic anticancer effects on both ER-positive and ER-negative breast cancer MCF-7 and MDA-MB-231 cells via induction of apoptotic and autophagic cell death. The co-treatment-induced apoptosis was associated with the activation of ERK signaling pathway and the autophagic cell death was likely attributable to the activation of JNK, followed by phosphorylation of Bcl-2 and dissociation of Beclin1/Bcl-2 complex (Fig. 8). This study strongly suggests that curcumin and berberine in combination could be used for the prevention and treatment of breast cancers.