CDKs are serine/threonine kinases that control cell cycle progression by forming a complex with their respective cyclin partners (43,44). Altered expression of CDKs, overexpression of cyclins and loss of expression of CDK inhibitors are frequently observed in malignant cells (44). Dysregulated CDK activity provides cancer cells with a selective growth advantage. In this way, dysregulated overexpression of cyclin D1 triggers progression of aggressive breast cancer (45). Previous studies in mammary epithelial carcinoma cells suggest that curcumin inhibits cell cycle progression by blocking the association of cyclin D1 with CDK4, thus reducing cyclin D1 activity (46,47). In MCF-7 breast cancer cells, curcumin reduces cell proliferation by arresting cells in G₁ phase. The drug achieves this arrest by stimulating the proteosomal degradation of cyclin E and upregulating CDK inhibitors, p53, p21 and p27; the addition of specific proteosomal inhibitors suppresses these effects of curcumin (48). Cyclin E is a nuclear protein that serves an important role in G₁/S progression by interacting with its catalytic partner, CDK2, and by interacting with the retinoblastoma (Rb) protein (49,50). It appears likely that the anti-proliferative effects of curcumin are due to proteasome-mediated downregulation of cyclin E and upregulation of CDK inhibitors (51).

The anti-proliferative effects of curcumin appear to be selective to cells overexpressing CDK 2. In mammary carcinoma cells, curcumin induces p53-dependent apoptosis and causes G₂ phase arrest. However, in normal human mammary cells, curcumin causes G₀ cell cycle arrest by blocking the association between CDK 4 and CDK 6, and inhibiting the phosphorylation of Rb (52). In this way, curcumin also prevents the initiation of p53-mediated apoptosis, which occurs only in cells arrested in G₂ phase (52).

The p53 protein is one of the most important tumor suppressor proteins, regulating a wide range of cellular processes, including cell proliferation, DNA damage and apoptosis (53). It is encoded by the tumor protein p53 gene, which is frequently mutated in numerous types of human cancer (53), leading to loss of cell proliferative control, DNA check points and DNA repair mechanisms. As a result, cancer cells become immortal. Restoring the function of p53 is an attractive therapeutic strategy in cancer therapy (54).

Curcumin induces apoptosis in breast cancer cells via p53-dependent and -independent pathways. For instance, curcumin arrests the cell cycle and induces p53-dependent apoptosis in MCF-7 breast cancer cells (55). Notably, curcumin exerts no anti-proliferative effects on MDAH041 cells lacking p53 or on TR9-7 cells that express p53 at low levels; rather, its effects are most notable in p53-expressing TR9-7 and MCF-7 cells. Expression of the pro-apoptotic protein apoptosis regulator Bax (Bax) is also higher in curcumin-treated MCF-7 cells. These results suggest that curcumin exerts its anti-proliferative effects via p53-dependent and p53-independent pathways (55,56).

Ras is a small transmembrane protein belonging to the large GTPase family of enzymes that hydrolyze guanosine triphosphate in order to transduce signals inside the cell (57). Mammalian cells have three Ras proteins (K-, H- and N-Ras), each of which serves a different function (57). Blocking oncogenic Ras signaling is an attractive strategy in cancer therapy.

Curcumin has been extensively studied for its effects on oncogenic Ras signaling pathways. In MCF-10A human breast epithelial cells transformed using H-Ras, curcumin induces reactive oxygen species production, which downregulates activity of matrix metalloproteinase (MMP)-2 and Bcl-2 and upregulates the activity of Bax and caspase-3 (58).

Potentially acting through a similar mechanism, curcumin arrests Ras-transfected HAG-1 human adenocarcinoma cells in G₂/M phase by inducing expression of extracellular signal-regulated kinase 1/2 and Bax, and reducing expression of Bcl-xL. These results suggest that curcumin may be a potent therapy against Ras-overexpressing cancer (59). Preclinical studies in animals, and ultimately clinical trials, are required to clarify the therapeutic effect of curcumin in Ras-induced cancer.

PI3Ks are a family of lipid kinases that phosphorylate inositol phospholipids and generate the secondary messenger phosphatidylinositol-3,4,5-trisphosphate in the plasma membrane (60). PI3K interacts with Akt to trigger the latter's translocation inside the cytoplasm. Activated Akt interacts with a number of substrates to perform numerous functions in cell survival, cell cycle progression and cell growth (60). Constitutive expression of PI3K and Akt, in addition to silencing of phosphatase and tensin homolog and glycogen synthase kinase 3β (GSK3β), are frequently observed in a number of human malignancies. Therefore, PI3K/Akt-mediated signaling is an attractive target in cancer chemotherapy (61,62).

Cancer cells survive for a prolonged time by activating survival pathways involving PI3K, Akt and mTOR, in addition to anti-apoptotic pathways involving Bcl-2. Targeting survival and apoptosis pathways is likely to be essential for controlling highly metastatic breast cancer. Curcumin on its own weakly stimulates apoptosis in breast cancer cells; however, combining it with the PI3K-specific inhibitor LY294002 stimulates apoptosis more strongly (63,64). The authors of these previous studies hypothesized that the PI3K obstruction overcomes the oncogenic expression of Bcl-2. Further studies are required to verify whether curcumin may inhibit PI3K/Akt/mTOR signaling in breast cancer cells and identify the mechanism(s) involved.

Wnts are a family of secreted glycoproteins that regulate multiple signaling pathways through β-catenin-dependent and -independent mechanisms (65–67). Wnts serve a crucial role in development, survival and metabolism. Inappropriate regulation and hyperactivation of Wnt/β-catenin signaling have been implicated in numerous human malignancies. Overexpression of β-catenin leads to constitutive activation of cell proliferation (68), and tumor cells downregulate the tumor suppressor GSK3β, which limits the activity of β-catenin by triggering its ubiquitin-mediated proteosomal degradation. Therefore, targeting the Wnt/β-catenin signaling pathway is an attractive approach in cancer therapy (69,70).

In MCF-7 and MDA-MB-231 cells, curcumin arrests the cell cycle in G₂/M cells by modulating Wnt/β-catenin signaling. In these cells, curcumin upregulates GSK3β and causes loss of nuclear β-catenin. Loss of nuclear β-catenin results in a loss of its downstream target cyclin D1 (71). This suggests that, at least in MCF-7 and MDA-MB-231 cells, the antitumor effects of curcumin are due to abrogation of Wnt/β-catenin signaling (71).

NF-κB is a family of transcription factors that are involved in the immune response and inflammation. Gene expression profiling studies suggest that the NF-κB pathway is a key regulator in triple-negative breast cancer (TNBC), with activation of NF-κB signaling strongly implicated in the pathogenesis of specific TNBCs (72–74). Cytoplasmic NF-κB is bound to a group of inhibitory proteins known as inhibitors of NF-κB (IκB); accumulation of non-phosphorylated IκB prohibits the translocation of NF-κB from cytoplasm to nucleus, resulting in inactivation of NF-κB and its downstream targets (74). NF-κB promotes the transcription of numerous key regulators of cancer invasion and progression, including cytokines, chemokines, cell adhesion molecules and inducible pro-inflammatory enzymes (74). In addition, NF-κB has been postulated to be a useful marker of the epithelial-mesenchymal transition (EMT) and invasiveness in breast cancer (19).

A number of previous studies suggest that curcumin inhibits NF-κB expression and therefore additional downstream signaling pathways, ultimately leading to the silencing of inflammatory cytokines, including chemokine (C-X-C motif) ligand (CXCL)1 and CXCL2 (19); and to alterations in the expression of MMP-9, urokinase plasminogen activator (uPA), uPA receptor, intercellular adhesion molecule 1 and chemokine receptor 4 (3,72,75). In this manner, curcumin is likely to inhibit the growth and invasion of breast cancer, in part, by downregulating NF-κB signaling pathways.

Curcumin may modulate the expression of NF-κB target genes (76,77), which include Bcl-2, ornithine decarboxylase (ODC) and c-myc, which are associated with apoptosis or cell survival (78). For example, ODC is the rate-limiting enzyme in polyamine biosynthesis and curcumin has been demonstrated to suppress ODC activity and inhibit cell proliferation (79). Activation of the NF-κB/Bcl-2 pathway is associated with drug resistance in cancer cells (80).

Accumulating evidence suggests that targeting NF-κB to inhibit cell growth and reverse EMT may be a novel therapeutic strategy in breast cancer.

Angiogenesis is the normal physiological mechanism by which novel blood vessels are formed from pre-existing blood vessels. It occurs during embryogenesis, menstruation and wound healing (81). Angiogenesis in tumors is crucial for cancer progression. Tumor cells procure nutrients for their uncontrolled growth through tumor angiogenesis (81). Tumor cells constitutively produce pro-angiogenic factors, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor, which curcumin may inhibit in order to modulate tumor angiogenesis (81,82).

Curcumin inhibits angiogenesis and growth of breast cancer tumors implanted into nude mice. These effects are associated with downregulated expression of a number of VEGF isomers, including VEGF-A, VEGF-C and VEGF receptor 2, in addition to decreased microvessel density (83). These results are in agreement with other previous studies demonstrating that suppression of VEGF function inhibits breast tumor growth (83,84). In nude mice, which were implanted with MDA-MB-231 tumors and treated with osteopontin (OPN; additionally termed secreted phosphoprotein 1) to stimulate angiogenesis, curcumin blocked NF-κB/cyclic AMP-dependent transcription factor ATF-4 binding and prevented OPN-induced upregulation of VEGF (85). This suggests that curcumin acts as a potent anti-angiogenic agent in regulating OPN-induced tumor angiogenesis in breast cancer.