Several lines of evidence supported critical roles for Src and COX-2 signaling during breast tumorigenesis. Accordingly, there was a growing interest in studying Src and COX-2 pathways via their co-targeting by Dasatinib and Celecoxib in MDA-MB-231 TNBC cell line. To the best of our knowledge, this study is the first to assess the possible antitumor effects of Dasatinib/Celecoxib combination in MDA-MB-231 TNBC cell line.
The up-regulatory effect of Dasatinib on c-Src gene expression level could be a reflex mechanism resulting from the inhibition of c-Src on the protein level in our study. Previous studies documented an increase in the level of total Src upon treatment with c-Src inhibitors in Malignant Mesothelioma (MSTO-211H, NCI-H28, and NCI-H2052) (16, 17). Furthermore, this is consistent with what was reported in various tumor cell lines following treatment with other Src inhibitors (18, 19).
It was documented that Dasatinib exert an effect on Src/FAK pathway (20) and this was proved by several studies in which Dasatinib inhibited growth, migration, and invasion of non-small cell lung cancer, and head and neck squamous cell carcinoma (HNSCC) cell lines (21, 22). The molecular mechanisms suggested for Dasatinib were Src inhibition and epidermal growth factor receptor and estrogen receptor α down-regulation (23). In colorectal cancer cell lines, overexpression of epidermal growth factor receptor was correlated with Src activation. Src enables epidermal growth factor receptor to evade degradation by inactivation of Cbl, a kinase responsible for the ubiquitination and degradation of ligand-activated receptors (24). It was reported that Dasatinib decreased phosphorylation of c-Src in burkitt's esophageal cells (25) and MDA-MB-468 cells (26).
Our results depicted that Dasatinib significantly decreased FAK protein levels compared to the control group suggesting that it has the potential to control cell adhesion, migration, and invasion. Previous studies on different BC cell lines showed that Dasatinib strongly inhibited FAK phosphorylation at the activating site Y576 (27). This effect was also evident in multiple studies conducted on hepatocellular carcinoma (HCC) cell lines suggesting that Dasatinib may interplay with other molecules to block FAK phosphorylation, and therefore suppresses motility and invasion (28). Not only studies on HCC cell lines, but also studies on nasopharyngeal carcinoma cell lines showed that FAK is downstream of Src (29). In addition, cell migration requires FAK activity, whereas FAK activation requires Src activity, suggesting a reciprocal catalytic activation mechanism of FAK and Src (30).
Consistent with mesenchymal-like tumor characteristics, MDA-MB 231 cells showed a high level of basal AKT activity. In our study, Dasatinib suppressed the phosphorylation of AKT at serine 473. The potentiality of Dasatinib in nasopharyngeal carcinoma treatment was investigated and AKT phosphorylation was found to be reduced by Dasatinib in CNE2 cells (27).
In the present study, Dasatinib significantly reduced cyclin D1 protein levels indicating that Dasatinib has the capacity to cause cell cycle G1-S arrest. It was reported earlier that Dasatinib decreased proliferation in lung, and head and neck cancer cells (22, 31), malignant pleural mesothelioma (32), melanoma cells (33), HCT-116 colorectal cancer cells (34), nasopharyngeal carcinoma cells (35), neuroblastoma cells (36), myxoid liposarcoma (37), papillary thyroid carcinoma cells (20), breast cancer cells (26), ovarian cancer cells (38, 39), HCC cells (40), acute myeloid leukemia cells (41), and acute myeloid leukemia Kasumi-1 cells (42).
The significant increase in caspase-3 by Dasatinib in our study could be linked to the inhibition of both Src and FAK. Supporting our findings, Dasatinib promoted apoptosis in pancreatic cancer cells (44), HNSCC cells (e.g., Ca9-22, HSC3, and SCC-25 cells) (45), BC cells (46), laryngeal cancer cell line (Hep-2) (47), chronic lymphoid leukemia cells (48), neuroblastoma cells (36), nasopharyngeal carcinoma cells (35), SKOv3 and HEY ovarian cancer cells (38), and Kasumi-1 cells (42).
In the current study, Dasatinib markedly decreased VEGF protein level. Supporting our finding, a previous study conducted on chronic myeloid leukemia cells inferred that Dasatinib reduced the phosphorylation of e-Proline-Rich Homeodomain which regulates myeloid survival via direct transcriptional repression of various genes encoding VEGF signaling pathway components (49). Another study depicted that Src family kinases affect tumor angiogenesis, where results in advanced non-small cell lung cancer patients suggested that levels of pro-angiogenic factors including VEGF are decreased by Dasatinib (50).
Our data also inferred that Dasatinib up-regulated COX-2 gene although it was expected that Dasatinib would down-regulate COX-2 expression. This could be the consequence of the observed reflex upregulation of src in our study which requires further investigation and evidence.
As for Celecoxib, it down-regulated COX-2 gene expression in our study and this was confirmed by multiple studies (53, 54). Herein, the effect of Celecoxib on PGE2 protein level was concordant with the results of another study conducted on MCF-7 BC cell line in which the PGE2 level gradually decreased in a dose-dependent manner (53). It was suggested that Celecoxib decreased PGE2 synthesis via Wnt pathway and conversion of arachidonic acid to bioactive prostanoids (21).
Celecoxib decreased FAK protein level herein and this is consistent with what was reported earlier in HCC cells (55), non-small cell lung cancer cells (56–57), and acute myeloid leukemia cells (58). Furthermore, Celecoxib reduced AKT protein level making our results corroborating with a previous study in which Celecoxib significantly decreased the phosphorylation of AKT in MDA-MB-231 cells but not in MDA-MB-468 cells, suggesting that the mechanism of apoptosis induction in MDA-MB-231 cells was in part dependent upon decreased AKT phosphorylation where AKT acts as a critical signaling component in cell survival by enhancing the downstream apoptotic proteins (22).
The present data revealed a significant reduction in Cyclin D1 protein levels by Celecoxib. Consistent with the current finding, Celecoxib markedly suppressed tumor growth in a number of animal models of colon, skin, lung, bladder, and breast cancers (59). In a spontaneous metastatic BC mouse model, Celecoxib reduced tumor growth via proliferation and angiogenesis inhibition, Bax up-regulation, and AKT and Bcl-2 down-regulation (60). Furthermore, Celecoxib decreased cyclin D1 expression in both HN30 and HN31 HNSCC lines (61) and in mouse colon carcinoma cell line (62). Celecoxib suppressed growth and promoted cell-cycle arrest at the G0/G1 phase in nasopharyngeal cell lines (63), BC cell lines (64), murine mammary tumor cell lines (65), human pancreatic cancer cell lines (66), and human ovarian cancer cell lines (67). Celecoxib inhibited cell cycle progression via the G1-S transition in SKOV-3 cells in-vivo (68). Celecoxib inhibited proliferation via the PGE2 pathway in BC, HCC (69), and human cholangiocarcinoma cell lines (70). Celecoxib reduced cyclin D1 in U373 and T98G human glioblastoma cells by regulating NF-κB target genes expression and inhibited proliferation in GBM cells at least partly by suppressing NF-κB activation (71).
Our results were in agreement with several studies in which caspase-3 was increased significantly by Celecoxib in a concentration-dependent manner in both MDA-MB-231 and SK-BR-3 BC cells. This supports the notion that Celecoxib can enhance caspase 3-dependent pathways in BC cells (72). It was found that the blockade of caspase activation is enough to suppress apoptosis (22) due to decreased AKT phosphorylation and increased Bax expression (73). It was reported that Celecoxib prevented colon tumorigenesis by promoting apoptosis via both COX-dependent and COX-independent mechanisms (59). It induced apoptosis in cervical cancer cells via Fas-ligand independent FADD activation in a cell type-specific manner (74) and via an apoptosome-dependent pathway, independent of death receptor pathways in lymphoma (74). In BJMC3879 mammary adenocarcinoma cells, it enhanced apoptosis via the activation of the intrinsic mitochondrial pathway. Furthermore, Celecoxib induced apoptosis in human glioblastoma cells at least partly by suppressing NF-κB activation (71). Celecoxib also induces a p53-independent apoptotic response which may be highly relevant in treating human neoplasms (75) and promoted apoptosis in lung cancer cells which seems to be dose-dependent (76).
Our results were in line with previous studies that reported that VEGF is reduced by Celecoxib (77, 78). It was reported that COX-2 overexpression in tumor cells influences angiogenesis through the production of COX-2 derived eicosanoids, which enhance endothelial cell migration and angiogenesis by elevating VEGF expression and stimulating the proliferation of endothelial cells (79, 80). Inhibition of COX-2 activity by Celecoxib reduces all these effects and leads to inhibition of angiogenesis and reduction of tumor growth (81, 82). A previous study found that VEGF was reduced by Celecoxib in a dose-dependent manner in MDA-MB-231 cells suggesting that COX-2/PGE2 pathway might play a pivotal role in channel formation and angiogenesis in part by enhancing proangiogenic proteins such as VEGF (22). The involvement of COX-2 inhibition in the antiangiogenic effect of Celecoxib was seen in a rat cornea model (83, 84). Prostaglandins binding to its receptor might enhance VEGF expression via hypoxia-inducible factor 1 alpha explaining at least in part, the cross-talk between VEGF and COX-2/PGs pathways (85). COX-2 independent mechanisms contributing to the antiangiogenic effects of Celecoxib was also described in rat hepatoma cells (86), human umbilical vein endothelial cells (87), ovarian SKOV-3 carcinoma xenografts (88), human colon carcinoma cells in nude mice (83), and human BC cells (89).
Taken all together, our results refer to the potential points of crosstalk between the two signaling pathways c-Src and COX-2 with their downstream molecular targets that are involved in BC. Furthermore, up to our knowledge, this is the first study that offers supporting evidence of the beneficial antitumor effects of combining Dasatinib and Celecoxib in MDA-MB-231 cells. Further preclinical and clinical investigational studies are highly recommended to explore the proposed favorable antitumor effects of combining Dasatinib and Celecoxib, not only in BC, but also in other different types of cancer.