Dasatinib/Celecoxib combination: A new hope in triple negative breast cancer treatment

Dasatinib/Celecoxib combination: A new hope in triple negative breast cancer treatment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dasatinib/Celecoxib combination: A new hope in triple negative breast cancer treatment Nermine Aly Moussa, Mahira Mohamed, Medhat Haroun, Maged Helmy Wasfy This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-904306/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 5 You are reading this latest preprint version Abstract Despite the tremendous efforts to implement new paradigms for breast cancer, the disease still remains a major challenge worldwide. Genetic deregulation is evident in all breast cancer subtypes and comprises a multitude of mutated genes and deregulated signaling cascades. In this sense, co-targeting Src and COX-2 signaling cascades have attracted fervent interest. This work explored the probable anti-carcinogenic effects of Dasatinib as a Src inhibitor, Celecoxib as a selective COX-2 inhibitor, and their combination in MDA-MB-231 triple-negative breast cancer cell line. Drug growth inhibition 50 (GI50) was determined using the MTT assay and the obtained results were analyzed using CompuSyn 3.0.1 software. MDA-MB-231 cells were divided into four treatment groups including a positive control, Dasatinib-treated, Celecoxib-treated, and combination-treated groups. Standard sandwich ELISA was used for the determination of the protein levels of c-Src, Bcl-2, p-AKT, FAK, PGE2, VEGF, and cyclin D1. Active caspase-3 was determined colorimetrically and the expression of COX-2 and c-Src genes was quantitatively determined via quantitative real-time polymerase chain reaction. The GI50 for Dasatinib was 0.05699 µM while that for Celecoxib was 69.0976 µM. Dasatinib up-regulated c-Src gene while Celecoxib and Dasatinib/Celecoxib combination down-regulated such expression level. COX-2 gene was down-regulated by Celecoxib while it was up-regulated by both Dasatinib and Dasatinib/Celecoxib combination. On one hand, Dasatinib, Celecoxib, and their combination significantly reduced the protein levels of c-Src, Bcl-2, p-AKT, FAK, PGE2, VEGF, and cyclin D1. On the other hand, they elevated active caspase-3. To sum up, Dasatinib/Celecoxib combination increased the capability for apoptosis and suppressed proliferation, angiogenesis, migration, and invasion suggesting a strong cross-talk between Src signaling cascade and COX-2/PGE2 via the intermediate PI3K/AKT/mTOR pathway. Further in-vitro and in-vivo studies are warranted to verify the present findings. Cancer Biology Oncology Triple-negative breast cancer Src signaling cascade COX-2/PGE2 pathway PI3K/AKT/ mTOR pathway Dasatinib Celecoxib Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Breast cancer (BC) is the most frequently diagnosed cancer and the leading cause of cancer death among females according to GLOBOCAN 2018 ( 1 ) . As a multifaceted disease, BC comprises an array of molecular subtypes characterized by differences in molecular signatures, responses to therapies, and prognoses ( 2 ) . Triple-negative breast cancer (TNBC), accounting for almost 15-20% of all BCs, is the most aggressive form that lacks estrogen, progesterone, and human epidermal growth factor receptors expression ( 3 ) . Improved understanding of the pivotal role that signaling pathways have in the establishment and maintenance of the tumorigenic state will be crucial for the development of new chemotherapeutic agents. Small-molecule inhibitors, either as single agents or in combination therapy, provide a foundation for exploiting these pathways as probable targets in BC treatment specifically the most aggressive TNBC subtype ( 4 ) . Novel approaches for better disease management aimed at co-targeting more than one of these pathways. Herein, we investigated Src and cyclooxygenase-2 pathways are plausible targets involved in BC pathogenesis. Src is a versatile target at the nexus of a multitude of signaling cascades. Src acts as an intermediate between growth factor receptor binding and downstream signaling which is vital for various cellular processes, including survival, proliferation, differentiation, invasion, and metastasis ( 5 , 6 ) . Dasatinib, an inhibitor of Src/Abl family kinases, is approved by the FDA for the treatment of imatinib-resistant chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia ( 7 , 8 ) . Dasatinib inhibits tumor growth in a number of solid tumors and several mechanisms underlie such suppression including G1 arrest of the cell cycle, induction of apoptosis, and inhibition of cell migration/invasion/metastasis ( 9 ) . Cyclooxygenase-2 (COX-2) is another target overexpressed in BC and is a fundamental step in BC pathogenesis acting via prostaglandin-dependent and independent mechanisms. Epidemiological studies suggest that non-steroidal anti-inflammatory drugs offer a moderate degree of benefit against BC. Nevertheless, further work is warranted to better understand how this enzyme system can be employed for therapeutic benefit. Celecoxib, as a selective COX-2 inhibitor, is believed to have potential anticancer effects in a wide variety of cancer types including colorectal, breast, and lung cancers such as suppression of cell growth, promotion of apoptotic cell death, immunoregulation, modulation of tumor microenvironment, and antiangiogenic effect. Meanwhile, COX-2-independent pathways also contribute to the anticancer effects of Celecoxib ( 10 ) . Taken together, this study was undertaken to investigate the antitumor effects of Dasatinib as a Src inhibitor, Celecoxib as a selective COX-2 inhibitor as well as their combination in MDA-MB-231 TNBC cell line. Materials And Methods Drugs Dasatinib and Celecoxib (Selleckchem, TX, USA) were prepared at the concentration of 10 mM in dimethyl sulphoxide and then were stored at -20°C. Cell lines The current study used MDA-MB-231 cell line obtained from the American Type Culture Collection (ATCC ® HTB-26™). It is an epithelial BC cell line taken from the pleural effusion of a 51-year-old Caucasian female suffering from metastatic mammary adenocarcinoma. Cell cultures MDA-MB-231 cells were kept as a monolayer culture in T-25 flasks in Dulbecco's Modified Eagle's Medium (Lonza Biowhitaker™, B-4800 Verviers, Belgium) supplemented with 10% (v/v) fetal bovine serum (Sigma-Aldrich Co., Germany) and 1% penicillin-streptomycin (Lonza Biowhitaker™, B-4800 Verviers, Belgium) at 37°C with 5% CO 2 . Cells were passaged when they reached 80% confluence. Growth inhibition assay MTT assay was used to determine cell viability (11) . Briefly, MDA-MB-231 cells were seeded in 96-well plates, treated with six different concentrations of the tested drugs. The six different concentrations for Dasatinib were 0.04 µM, 0.02 µM, 0.01 µM, 0.005 µM, 0.003 µM and 0.001 µM while those for Celecoxib were 100 µM, 50 µM, 25 µM,.5 µM, 6.25 µM, and 3.125 µM. MTT (10 µl) was added after 72 hours then incubation was carried out at 37°C for 4 hours and finally the absorbance was measured at 570 nm. The GI50 was assessed for Dasatinib and Celecoxib utilizing CompuSyn 3.0.1 software. Determination of the combination and dose reduction indices The combination index (CI) was assessed as described earlier (12) to determine whether there is synergism, antagonism, or additive effect between Dasatinib and Celecoxib, where CI lower than 1 indicates synergism, =1 indicates additive effect and greater than 1 indicates antagonism. Additionally, the dose reduction index (DRI) was determined using the CompuSyn software as described earlier (12) . Experimental design Three replicas of MDA-MB-231 cells received either dimethyl sulphoxide as a vehicle, Dasatinib (0.05699µM), Celecoxib (69.0976µM), or Dasatinib (0.05699µM)/Celecoxib (69.0976µM) combination. The regulatory aspects regarding the use of cell lines were followed in all the experiments. Biochemical analyses Protein levels of v-akt murine thymoma viral oncogene homolog 1 (p-AKT), c-Src, Focal adhesion kinase (FAK), Bcl-2, prostaglandin E2 (PGE2), cyclin-D1, and vascular endothelial growth factor (VEGF1) were determined using the following ELISA kits: Human p-AKT (Ser473) ELISA kit (RayBiotech, USA) (Cat#: PEL-AKT-S473-T), Human c-Src kinase ELISA kit (LifeSpan, Bioscience, USA) (Cat#: LS-F11230), Human FAK ELISA kit (LifeSpan, Bioscience, USA) (Cat#: MBS2515396, 96T), Bcl-2 ELISA kit (Sigma-Aldrich, USA) (Cat#: CS0520), PGE2 ELISA kit (Sigma-Aldrich, USA) (Cat#: MBS721434), cyclin-D1 ELISA kit (USCN Life Science and Technology Co.) (Cat#: E0585h) and VEGF based ELISA assay kit (Cusabio, USA) (Product Code CSB-E11718h), respectively according to the manufacturer’s instructions. Determination of capase-3 activity Caspase 3 activity, expressed as μmol p-nitroaniline/min/ml, was assessed using Caspase-3 colorimetric kit (Sigma Aldrich, USA) (Product Code CASP-3-C) according to the manufacturer’s instructions. Gene expression analysis of c-Src and Cox-2 genes using quantitative real-time polymerase chain reaction Cox-2 and c-Src gene expression levels were determined using step one real-time polymerase chain reaction (PCR) system (Applied Biosystem, USA). First, total messenger RNA was isolated using the Easy-RED TM total RNA extraction kit (Intron Biotechnology, South Korea) (Product Code 17063) according to the instructions of the manufacturer. Second, quantification and purity checking were performed using the NanoDrop 2000 spectrophotometer (Thermo Fischer Scientific, USA). Quantitative real-time PCR reactions were performed using the SensiFast™ SYBR ® No-ROX one-step kit (Bioline Co., USA) (Product Code BIO-72001). Finally, the relative expression of Cox-2 and c-Src genes was assessed against glyceraldehyde 3-phosphate dehydrogenase ( GAPDH) as a housekeeping gene. The sequences of the forward and reverse primers for c-Src gene were: forward: 5'- GGACAGTGGCGGATTCTACATC-3' and reverse: 5'- AGCTGCTGCAGGCTGTTGA-3'; for Cox-2, forward, 5′CTGTTGCGGAGAAAGGAGTC-3′; reverse, 5′-TCAAACAAGCTTTTACAGGTGA-3′, whereas those for GAPDH gene were: forward: 5'- TGCACCACCAACTGCTTAGC-3' and reverse: 5'- GGCATGGACTGTGGTCATGAG-3' (13,14,15) . To confirm the amplification of c-Src , Cox-2 , and GAPDH genes, primer sequences were blasted against NCBI/Primer Blast. The analyses were carried out as triplicates. The relative expression of the aforementioned genes against GAPDH depended on the ∆∆ comparative threshold method. Statistical analysis of the data Data were presented as mean ± standard error of the mean. Results were analyzed using one-way analysis of variance test followed by Tukey post-hoc test. The statistical analyses were executed by Graph Pad Prism Software (version 3.0). The level of significance was fixed at p < 0.05. Results Determination of GI50 for Dasatinib and Celecoxib in MDA-MB-231 cells The GI50 was 0.05699 µM for Dasatinib and 69.0976 µM for Celecoxib as demonstrated in (Figure 1 a and 1 b), respectively. Determination Of The Combination And Dose Reduction Indices Based on the MTT assay results shown in (Figure 2 a and 2 b) and the statistical analyses using Compusyn software, Dasatinib /Celecoxib combination showed a strong synergistic effect as evidenced from the combination index (CI=0.98621 µM). Likewise, the dose reduction index revealed that Celecoxib decreased the dose of Dasatinib by approximately 3.8 folds which could decrease its undesired adverse effect as a monotherapy. In addition, Dasatinib decreased the dose of Celecoxib by approximately 1.4 folds. Effect of Dasatinib, Celecoxib and their combination on c-Src gene expression and protein levels in MDA MB-231 cell lysates after 72 hours of treatment Data presented in (Figure 3 a) inferred that Dasatinib up-regulated c-Src gene expression; however, Celecoxib and the combination down-regulated such expression compared to its expression in positive control cells. Results shown in (Figure 3 b) revealed that c-Src protein levels were significantly reduced by 57%, 46%, and 76% when compared with the control group in Dasatinib-treated, Celecoxib-treated, and combination-treated cells, respectively (p < 0.001). Effect of Dasatinib, Celecoxib and their combination on COX-2 gene expression level, and PGE2 protein level (Pg/mg total protein) in MDA-MB-231 cell lysates after 72 hours of treatment As shown in (Figure 4 a), Dasatinib and Dasatinib/Celecoxib combination up-regulated COX-2 gene expression; however, Celecoxib down-regulated such expression compared to its expression in positive control cells. The results shown in (Figure 4 b) revealed that PGE2 protein levels were significantly reduced by 56%, 45%, and 73% when compared with the positive control group in Dasatinib-treated, Celecoxib-treated, and combination-treated cells, respectively (p < 0.001). Likewise, the combination decreased PGE2 levels significantly compared with single treatments with either Dasatinib or Celecoxib (p < 0.05 and p < 0.001; respectively). Effect of Dasatinib, Celecoxib and their combination on FAK protein level (ng/mg total protein), p-AKT protein level (Units/mg total protein), and cyclin-D1 protein level (Units/mg total protein) in MDA-MB-231 cell lysates after 72 hours of treatment Our findings herein (Figure 5 a) inferred that FAK protein levels were significantly reduced by about 59%, 50 %, and 74% in Dasatinib-treated, Celecoxib-treated, and combination-treated cells, respectively (p < 0.001). Moreover, Dasatinib/Celecoxib combination significantly reduced FAK protein levels when compared with single treatments with either Dasatinib or Celecoxib (p < 0.001). The results presented in (Figure 5 b) revealed that p-AKT protein levels were significantly decreased by approximately 64%, 54%, and 77% compared with the positive control group in Dasatinib-treated, Celecoxib-treated, and combination-treated cells, respectively ( p < 0.001). Furthermore, the combination reduced p-AKT protein levels significantly compared with single treatments with either Dasatinib or Celecoxib (p < 0.01 and p < 0.001; respectively). The findings depicted in (Figure 5 c) demonstrated that cyclin D1 protein levels were significantly reduced by approximately 66%, 56%, and 76% when compared with the positive control group in Dasatinib-treated, Celecoxib-treated, and combination-treated cells, respectively (p < 0.001). Effect of Dasatinib, Celecoxib and their combination on Bcl-2 protein level (Units/mg total protein), active caspase-3 (ng/mg total protein), and VEGF protein level (Pg/mg total protein) in MDA-MB 231 cell lysates after 72 hours of treatment The data presented in (Figure 6 a) showed that Dasatinib, Celecoxib, and their combination reduced Bcl-2 protein levels by 45%, 37%, and 79% compared with the positive control group (p < 0.001). Our results herein (Figure 6 b) showed that caspase-3 protein levels were significantly increased by approximately 196%, 152%, and 538% compared with the positive control group in Dasatinib-treated, Celecoxib-treated, and combination-treated cells respectively (p < 0.001). Additionally, Dasatinib/Celecoxib combination significantly elevated caspase-3 levels compared with single treatments with either Dasatinib or Celecoxib (p < 0.001).As presented in (Figure 6 c), Dasatinib, Celecoxib, and their combination reduced VEGF protein levels significantly by about 68%, 63%, and 82% compared with the positive control group (p < 0.001). Discussion 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. Declarations Ethical approval: Not applicable Consent to participate: Not applicable Consent to publish: We declare that no part of the work referred to in here has been published before. Author ’ s Contributions: N.M, M.M, M.W.H contributed to the research idea. N.M, M.M, M.W.H conceived and designed the experiments. N.M, M.M, M.W.H conducted the experiments. N.M, M.M, M.W.H, M.H contributed to data analysis and presentation. N.M wrote the manuscript. N.M, M.M, M.W.H, M.H revised the manuscript and declared that the data were generated in-ho u se and that no paper m ill was u sed. Funding: The authors declare that this research was conducted in the absence of any commercial or financial funding. 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Molecular Cancer 9(1):257 Le X-F, Mao W, Lu Z et al (2010) Dasatinib Induces Autophagic Cell Death in Human Ovarian Cancer Cancer 116(21):4980–4990 Konecny GE, Glas R, Dering J et al (2009) Activity of the multikinase inhibitor dasatinib against ovarian cancer cells. British Journal of Cancer 101(10):1699–1708 Chang AY, Wang M (2013) Molecular mechanisms of action and potential biomarkers of growth inhibition of dasatinib (BMS-354825) on hepatocellular carcinoma cells. BMC Cancer 13(1):267 Guerrouahen BS, Futami M, Vaklavas C et al (2010) Dasatinib inhibits the growth of molecularly heterogeneous myeloid leukemias. Clinical Cancer Research 16(4):1149–1158 Mpakou VE, Kontsioti F, Papageorgiou S et al (2013) Dasatinib inhibits proliferation and induces apoptosis in the KASUMI-1 cell line bearing the t(8;21)(q22;q22) and the N822K c-kit mutation. Leuk Res 37(2):175–182 Sun Q, Wang Y, Desgrosellier JS (2019) Combined Bcl-2/Src inhibition synergize to deplete stem-like breast cancer cells. 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Int J Cancer 118(10):2381–2389 Dai ZJ, Ma XB, Kang HF et al (2012) Antitumor activity of the selective cyclooxygenase-2 inhibitor, celecoxib, on breast cancer in Vitro and in Vivo. Cancer Cell Int 12:1–8 Friedrich M, Reichert K, Woeste A et al (2018) Effects of Combined Treatment with Vitamin D and COX2 Inhibitors on Breast Cancer Cell Lines. Anticancer Res 38(2):1201–7 Xiao-Ming B, Wei Z, Ning-Bo L, Hui J (2009) Focal adhesion kinase: Important to prostaglandin E-2-mediated adhesion, migration and invasion in hepatocellular carcinoma cells. Oncol Rep 21(1):129–136 Angfang C, Minghui C, Daolong Z (2017) Curcumol potentiates celecoxib-induced growth inhibition and apoptosis in human non-small cell lung cancer. Oncotarget 8:115526–115545 Chen M-H, Hua Z-C (2014) P0089 Combination of curcumol with celecoxib synergistically enhances their anti-migration effects via FAK/ERK/PI3K/AKT pathways in human non-small-lung cancer cells. 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J Pharmacol Exp Ther 298(3):976–985 Denkert C, Furstenberg A, Daniel PT et al (2003) Induction of G0/G1 cell cycle arrest in ovarian carcinoma cells by the anti-inflammatory drug NS-398, but not by COX-2-specific RNA interference. Oncogene 22(54):8653–8661 Masamha CP, Benbrook DM (2009) Cyclin D1 degradation is sufficient to induce G1 cell cycle arrest despite constitutive expression of cyclin E2 in ovarian cancer cells. Cancer Research 69(16):6565–6572 Brandão RD, Veeck J, Van de Vijver KK et al (2013) A randomised controlled phase II trial of pre-operative celecoxib treatment reveals anti-tumour transcriptional response in primary breast cancer. Breast Cancer Research 15(2):R29 Wu GS, Zou SQ, Liu ZR et al (2003) Celecoxib inhibits proliferation and induces apoptosis via prostaglandin E2 pathway in human cholangiocarcinoma cell lines. 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Anticancer Research 26(6B):4245–4254 Sun SY, Schroeder CP, Yue P et al (2005) Enhanced growth inhibition and apoptosis induction in NSCLC cell lines by combination of celecoxib and 4HPR at clinically relevant concentrations. Cancer Biol Ther 4(4):407–413 Kim Y-Y, Lee E-J, Kim Y-K et al (2010) Anti-cancer effects of celecoxib in head and neck carcinoma. Molecules and Cells 29(2):185–194 Rodriguez DA, Tapia JC, Fernandez JG et al (2009) Caveolin-1–mediated Suppression of Cyclooxygenase-2 via a β-catenin-Tcf/Lef–dependent Transcriptional Mechanism Reduced Prostaglandin E2 Production and Survivin Expression. Mol Biol Cell 20(8):2297–2310 Li G, Yang T, Yan J (2002) Cyclooxygenase-2 increased the angiogenic and metastatic potential of tumor cells. Biochemical and Biophysical Research Communications . ; 299 (5):886-90 Liu XH, Kirschenbaum A, Yao S et al (1999) Upregulation of vascular endothelial growth factor by cobalt chloride-simulated hypoxia is mediated by persistent induction of cyclooxygenase-2 in a metastatic human prostate cancer cell line. Clin Exp Metas 17(8):687–694 Gately S, Li WW (2004) Multiple roles of COX-2 in tumor angiogenesis: a target for antiangiogenic therapy. Seminars in Oncology 31:2–11 Grosch S, Maier TJ, Schiffmann S, Geisslinger G (2006) Cyclooxygenase-2 (COX-2)-independent anticarcinogenic effects of selective COX-2 inhibitors. J Natl Cancer Inst 98(11):736–747 Masferrer JL, Leahy KM, Koki AT et al (2000) Antiangiogenic and antitumor activities of cyclooxygenase-2 inhibitors. Cancer Res 60(5):1306–1311 Leahy KM, Ornberg RL, Wang Y et al (2002) Cyclooxygenase-2 inhibition by celecoxib reduces proliferation and induces apoptosis in angiogenic endothelial cells in vivo. Cancer Research 62(3):625–631 Konturek PC, Konturek SJ, Brzozowski T (2006) Gastric cancer and Helicobacter pylori infection. Journal of Physiology and Pharmacolog 57:51–65 Ostrowski J, Wocial T, Skurzak H, Bartni W (2003) Do altering in ornithine decarboxylase activity and gene expression contribute to antiproliferative properties of COX inhibitors? Br J Cancer 88(7):1143–1151 Lin HP, Kulp SK, Tseng PH et al (2004) Growth inhibitory effects of celecoxib in human umbilical vein endothelial cells are mediated through G1 arrest via multiple signaling mechanisms. Molecular Cancer Therapeutics 3(12):1671–1680 Li W, Jiang HR, Xu XL et al (2010) Cyclin D1 expression and the inhibitory effect of celecoxib on ovarian tumor growth in vivo. Int J Mol Sci 11(10):3999–4013 Chang S-H, Liu CH, Conway R et al (2004) Role of prostaglandin E2-dependent angiogenic switch in cyclooxygenase 2-induced breast cancer progression. Proceedings of the National Academy of Sciences of the United States of America 101(2):591–596 Supplementary Files 1MTTandCIFig12.pzm 2ParametersandPCRFig345.pzm 3ELISAParameters.pzm Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Major Revisions Needed 31 Dec, 2021 Reviews received at journal 22 Nov, 2021 Reviewers invited by journal 10 Nov, 2021 Editor assigned by journal 09 Nov, 2021 First submitted to journal 13 Sep, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-904306","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":62518280,"identity":"565d1ee1-a3dc-419a-b31e-8428d6b02174","order_by":0,"name":"Nermine Aly Moussa","email":"data:image/png;base64,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","orcid":"","institution":"Institute of Graduate Studies and Research","correspondingAuthor":true,"prefix":"","firstName":"Nermine","middleName":"Aly","lastName":"Moussa","suffix":""},{"id":62518281,"identity":"dac35892-8baa-4d16-87f4-248010027cd5","order_by":1,"name":"Mahira Mohamed","email":"","orcid":"","institution":"Institute of Graduate Studies and Research","correspondingAuthor":false,"prefix":"","firstName":"Mahira","middleName":"","lastName":"Mohamed","suffix":""},{"id":62518282,"identity":"a4fa5242-85b8-4783-bcf1-c2db95f5caf0","order_by":2,"name":"Medhat Haroun","email":"","orcid":"","institution":"Institute of Graduate Studies and Research","correspondingAuthor":false,"prefix":"","firstName":"Medhat","middleName":"","lastName":"Haroun","suffix":""},{"id":62518283,"identity":"e161bee5-eb07-4aee-a78d-8853890674db","order_by":3,"name":"Maged Helmy Wasfy","email":"","orcid":"","institution":"Damanhour University Faculty of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Maged","middleName":"Helmy","lastName":"Wasfy","suffix":""}],"badges":[],"createdAt":"2021-09-14 14:16:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-904306/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-904306/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":15484092,"identity":"59f261b1-86bf-42bc-89df-b627fb21c35d","added_by":"auto","created_at":"2021-11-12 15:47:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":16099,"visible":true,"origin":"","legend":"Viability of MDA-MB-231 cells treated with (a) Dasatinib and (b) Celecoxib. MTT was conducted to determine the cell viability. Data points represent mean ± standard error of the mean, each performed in triplicate. ","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-904306/v1/957e0d3a76b6be171ec66969.png"},{"id":15484096,"identity":"118c4f80-09dc-403a-b1ec-e77683ca6d76","added_by":"auto","created_at":"2021-11-12 15:47:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":45633,"visible":true,"origin":"","legend":"Viability of MDA-MB-231 cells treated with (a) Dasatinib/Celecoxib. (b) Dose reduction index for Dasatinib/Celecoxib combination. Data points represent mean ± standard error of the mean, each performed in triplicate. ","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-904306/v1/835a269c9c3f0a9cb0a79d51.png"},{"id":15484766,"identity":"496884ec-c1c3-40b7-b3a5-245c7d8d3ffd","added_by":"auto","created_at":"2021-11-12 15:50:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":33949,"visible":true,"origin":"","legend":"Effect of Dasatinib (0.05699 μM), Celecoxib (69.0976 μM) and their combination on (a) c-Src gene expression level, and (b) c-Src protein level (ng/mg total protein) in MDA-MB 231 cell lysates after 72 hours of treatment. Data presented as Mean ± standard error of the mean. * p \u003c 0.05 versus Control; † p \u003c 0.05 versus Dasatinib; ‡ p \u003c 0.05 versus Celecoxib; # p \u003c 0.05 versus Dasatinib+Celecoxib combination. ","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-904306/v1/791dd91806c20c154edede80.png"},{"id":15484093,"identity":"f388a70b-c058-4e0d-9523-ef64fd7e5e02","added_by":"auto","created_at":"2021-11-12 15:47:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":34886,"visible":true,"origin":"","legend":"Effect of Dasatinib (0.05699 μM), Celecoxib (69.0976 μM) and their combination on (a) COX-2 gene expression level, and (b) PGE2 protein level (Pg/mg total protein) in MDA-MB 231 cell lysates after 72 hours of treatment. Data presented as Mean ± standard error of the mean. * p \u003c 0.05 versus Control; † p \u003c 0.05 versus Dasatinib; ‡ p \u003c 0.05 versus Celecoxib; # p \u003c 0.05 versus Dasatinib+Celecoxib combination. ","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-904306/v1/c87488e114f7b58b8de73e9e.png"},{"id":15484765,"identity":"ba6d2efd-976a-4e01-b5bc-70569b5007ca","added_by":"auto","created_at":"2021-11-12 15:50:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":25676,"visible":true,"origin":"","legend":"Effect of Dasatinib (0.05699 μM), Celecoxib (69.0976 μM) and their combination on (a) FAK, (b) p-AKT, and (c) cyclin D1 protein levels in MDA-MB 231 cell lysates after 72 hours of treatment. Data presented as Mean ± standard error of the mean. * p \u003c 0.05 versus Control; † p \u003c 0.05 versus Dasatinib; ‡ p \u003c 0.05 versus Celecoxib; # p \u003c 0.05 versus Dasatinib+Celecoxib combination. ","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-904306/v1/d6a345223748a52acd282a60.png"},{"id":15484094,"identity":"ea348a8b-2a53-4f7d-8629-e442e0f0a70f","added_by":"auto","created_at":"2021-11-12 15:47:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":24931,"visible":true,"origin":"","legend":"Effect of Dasatinib (0.05699 μM), Celecoxib (69.0976 μM) and their combination on (a) Bcl-2, (b) caspase-3, and (c) VEGF protein levels in MDA-MB 231 cell lysates after 72 hours of treatment. Data presented as Mean ± standard error of the mean. * p \u003c 0.05 versus Control; † p \u003c 0.05 versus Dasatinib; ‡ p \u003c 0.05 versus Celecoxib; # p \u003c 0.05 versus Dasatinib+Celecoxib combination. ","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-904306/v1/80c9e79f5800af1d011a089d.png"},{"id":15484792,"identity":"d0de275d-b80c-4496-8ad7-8c8fca12c985","added_by":"auto","created_at":"2021-11-12 15:50:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":675056,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-904306/v1/d8b631db-e311-479a-9dcb-f9cac1a947a3.pdf"},{"id":15484097,"identity":"b6e95a8f-4559-42fc-aa53-440452d469f6","added_by":"auto","created_at":"2021-11-12 15:47:11","extension":"pzm","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":707545,"visible":true,"origin":"","legend":"","description":"","filename":"1MTTandCIFig12.pzm","url":"https://assets-eu.researchsquare.com/files/rs-904306/v1/0448c8476f42dcc8c0ff027d.pzm"},{"id":15484100,"identity":"5257c4fc-38e3-4a38-9088-0c98f4d48db5","added_by":"auto","created_at":"2021-11-12 15:47:11","extension":"pzm","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":661293,"visible":true,"origin":"","legend":"","description":"","filename":"2ParametersandPCRFig345.pzm","url":"https://assets-eu.researchsquare.com/files/rs-904306/v1/317225190d3010d7e323b9ea.pzm"},{"id":15484099,"identity":"095059a1-a9f6-4d6c-ad2e-d2a87248c224","added_by":"auto","created_at":"2021-11-12 15:47:11","extension":"pzm","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":542630,"visible":true,"origin":"","legend":"","description":"","filename":"3ELISAParameters.pzm","url":"https://assets-eu.researchsquare.com/files/rs-904306/v1/f487e444493c3358bfd5eef1.pzm"}],"financialInterests":"","formattedTitle":"\u003cp\u003eDasatinib/Celecoxib combination: A new hope in triple negative breast cancer treatment\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer (BC) is the most frequently diagnosed cancer and the leading cause of cancer death among females according to GLOBOCAN 2018 \u003csup\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003c/sup\u003e. As a multifaceted disease, BC comprises an array of molecular subtypes characterized by differences in molecular signatures, responses to therapies, and prognoses \u003csup\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/sup\u003e. Triple-negative breast cancer (TNBC), accounting for almost 15-20% of all BCs, is the most aggressive form that lacks estrogen, progesterone, and human epidermal growth factor receptors expression \u003csup\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eImproved understanding of the pivotal role that signaling pathways have in the establishment and maintenance of the tumorigenic state will be crucial for the development of new chemotherapeutic agents. Small-molecule inhibitors, either as single agents or in combination therapy, provide a foundation for exploiting these pathways as probable targets in BC treatment specifically the most aggressive TNBC subtype \u003csup\u003e(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u003c/sup\u003e. Novel approaches for better disease management aimed at co-targeting more than one of these pathways. Herein, we investigated Src and cyclooxygenase-2 pathways are plausible targets involved in BC pathogenesis.\u003c/p\u003e \u003cp\u003eSrc is a versatile target at the nexus of a multitude of signaling cascades. Src acts as an intermediate between growth factor receptor binding and downstream signaling which is vital for various cellular processes, including survival, proliferation, differentiation, invasion, and metastasis \u003csup\u003e(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u003c/sup\u003e. Dasatinib, an inhibitor of Src/Abl family kinases, is approved by the FDA for the treatment of imatinib-resistant chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia \u003csup\u003e(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e)\u003c/sup\u003e. Dasatinib inhibits tumor growth in a number of solid tumors and several mechanisms underlie such suppression including G1 arrest of the cell cycle, induction of apoptosis, and inhibition of cell migration/invasion/metastasis \u003csup\u003e(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCyclooxygenase-2\u003c/em\u003e (COX-2) is another target overexpressed in BC and is a fundamental step in BC pathogenesis acting via prostaglandin-dependent and independent mechanisms. Epidemiological studies suggest that non-steroidal anti-inflammatory drugs offer a moderate degree of benefit against BC. Nevertheless, further work is warranted to better understand how this enzyme system can be employed for therapeutic benefit. Celecoxib, as a selective COX-2 inhibitor, is believed to have potential anticancer effects in a wide variety of cancer types including colorectal, breast, and lung cancers such as suppression of cell growth, promotion of apoptotic cell death, immunoregulation, modulation of tumor microenvironment, and antiangiogenic effect. Meanwhile, COX-2-independent pathways also contribute to the anticancer effects of Celecoxib \u003csup\u003e(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTaken together, this study was undertaken to investigate the antitumor effects of Dasatinib as a Src inhibitor, Celecoxib as a selective COX-2 inhibitor as well as their combination in MDA-MB-231 TNBC cell line.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eDrugs\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDasatinib\u0026nbsp;and\u0026nbsp;Celecoxib\u0026nbsp;(Selleckchem, TX, USA) were prepared at the concentration of 10 mM in dimethyl sulphoxide and then were stored at -20\u0026deg;C.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe current study used MDA-MB-231 cell line obtained from the American Type Culture Collection (ATCC\u003csup\u003e\u0026reg;\u003c/sup\u003e HTB-26\u0026trade;). \u0026nbsp;It is an epithelial BC cell line taken from the pleural effusion of a 51-year-old Caucasian female suffering from metastatic mammary adenocarcinoma.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell cultures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMDA-MB-231 cells were kept as a monolayer culture in T-25 flasks in Dulbecco\u0026apos;s Modified Eagle\u0026apos;s Medium (Lonza Biowhitaker\u0026trade;, B-4800 Verviers, Belgium) \u0026nbsp;supplemented with 10% (v/v) fetal bovine serum (Sigma-Aldrich Co., Germany) and 1% penicillin-streptomycin (Lonza Biowhitaker\u0026trade;, B-4800 Verviers, Belgium) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were passaged when they reached 80% confluence.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrowth inhibition assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMTT assay was used to determine cell viability\u0026nbsp;\u003csup\u003e(11)\u003c/sup\u003e. Briefly, MDA-MB-231 cells were seeded in 96-well plates, treated with six different concentrations of the tested drugs.\u0026nbsp;The six different concentrations for Dasatinib were 0.04 \u0026micro;M, 0.02 \u0026micro;M, 0.01 \u0026micro;M, 0.005 \u0026micro;M, 0.003 \u0026micro;M and 0.001 \u0026micro;M while those for Celecoxib were 100 \u0026micro;M, 50 \u0026micro;M, 25 \u0026micro;M,.5 \u0026micro;M, 6.25 \u0026micro;M, and 3.125 \u0026micro;M.\u0026nbsp;MTT (10 \u0026micro;l)\u0026nbsp;was added after 72 hours then incubation was carried out at 37\u0026deg;C for 4 hours and finally the absorbance was measured at 570 nm. The\u0026nbsp;GI50 was assessed for\u0026nbsp;Dasatinib\u0026nbsp;and\u0026nbsp;Celecoxib\u0026nbsp;utilizing\u0026nbsp;CompuSyn 3.0.1 software.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of the combination and dose reduction indices\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe combination index (CI) was assessed as described earlier \u003csup\u003e(12)\u003c/sup\u003e to determine whether there is synergism, antagonism, or additive effect between Dasatinib and Celecoxib, where CI lower than 1 indicates synergism, =1 indicates additive effect and greater than 1 indicates antagonism. Additionally, the dose reduction index (DRI) was determined using the CompuSyn software as described earlier \u003csup\u003e(12)\u003c/sup\u003e.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental design\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree replicas of MDA-MB-231 cells received either\u0026nbsp;dimethyl sulphoxide\u0026nbsp;as a vehicle, Dasatinib (0.05699\u0026micro;M), Celecoxib (69.0976\u0026micro;M), or Dasatinib (0.05699\u0026micro;M)/Celecoxib (69.0976\u0026micro;M) combination. The regulatory aspects regarding the use of cell lines were followed in all the experiments.\u0026nbsp;\u003cstrong\u003e\u003cins cite=\"mailto:Abd%20El-%20Halim%20EL-%20Kishky%20[2]\" datetime=\"2018-02-21T21:22\"\u003e\u0026nbsp;\u003c/ins\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical analyses\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein levels of\u0026nbsp;v-akt murine thymoma viral oncogene homolog 1 (p-AKT),\u0026nbsp;c-Src,\u0026nbsp;Focal adhesion kinase (FAK),\u0026nbsp;Bcl-2, prostaglandin E2 (PGE2), cyclin-D1, and vascular endothelial growth factor (VEGF1) were determined using the following\u0026nbsp;ELISA kits:\u0026nbsp;Human p-AKT (Ser473) ELISA kit (RayBiotech, USA) (Cat#: PEL-AKT-S473-T),\u0026nbsp;Human c-Src kinase ELISA kit (LifeSpan, Bioscience, USA) (Cat#: LS-F11230), Human FAK ELISA kit (LifeSpan, Bioscience, USA) (Cat#: MBS2515396, 96T),\u0026nbsp;Bcl-2 ELISA kit (Sigma-Aldrich, USA) (Cat#: CS0520), PGE2 ELISA kit (Sigma-Aldrich, USA) (Cat#: MBS721434),\u0026nbsp;cyclin-D1 ELISA kit\u0026nbsp;(USCN Life Science and Technology Co.) (Cat#: \u0026nbsp; E0585h)\u0026nbsp;and VEGF based ELISA assay kit (Cusabio, USA) (Product Code\u0026nbsp;CSB-E11718h), respectively\u0026nbsp;according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of capase-3 activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCaspase 3 activity,\u0026nbsp;expressed as \u0026mu;mol p-nitroaniline/min/ml,\u0026nbsp;was assessed using\u0026nbsp;Caspase-3 colorimetric kit (Sigma Aldrich, USA) (Product Code CASP-3-C) according to the manufacturer\u0026rsquo;s instructions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene expression analysis of \u003cem\u003ec-Src\u003c/em\u003e and \u003cem\u003eCox-2\u003c/em\u003e genes\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eusing quantitative real-time polymerase chain reaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCox-2\u003c/em\u003e and \u003cem\u003ec-Src\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003egene\u0026nbsp;expression levels were determined\u0026nbsp;using step one real-time polymerase chain reaction (PCR) system (Applied Biosystem, USA).\u0026nbsp;First, total messenger RNA was isolated using the\u0026nbsp;Easy-RED\u003csup\u003eTM\u003c/sup\u003e total RNA extraction kit (Intron Biotechnology, South Korea) (Product Code 17063) according to the instructions of the manufacturer. Second, quantification and purity checking were performed using the NanoDrop 2000 spectrophotometer (Thermo Fischer Scientific, USA). Quantitative real-time PCR reactions were performed using the SensiFast\u0026trade; SYBR\u003csup\u003e\u0026reg;\u003c/sup\u003e No-ROX one-step kit (Bioline Co., USA) (Product Code BIO-72001). \u0026nbsp; Finally, the relative expression of \u003cem\u003eCox-2\u003c/em\u003e and \u003cem\u003ec-Src\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003egenes was assessed against glyceraldehyde 3-phosphate dehydrogenase (\u003cem\u003eGAPDH)\u003c/em\u003e as a housekeeping gene. The sequences of the forward and reverse primers for \u003cem\u003ec-Src\u003c/em\u003e gene were: forward: 5\u0026apos;- GGACAGTGGCGGATTCTACATC-3\u0026apos; and reverse: \u0026nbsp;5\u0026apos;- AGCTGCTGCAGGCTGTTGA-3\u0026apos;; for \u003cem\u003eCox-2,\u003c/em\u003e forward, 5\u0026prime;CTGTTGCGGAGAAAGGAGTC-3\u0026prime;; reverse, 5\u0026prime;-TCAAACAAGCTTTTACAGGTGA-3\u0026prime;, whereas those for \u003cem\u003eGAPDH\u003c/em\u003e gene were: forward: 5\u0026apos;- TGCACCACCAACTGCTTAGC-3\u0026apos; and reverse: 5\u0026apos;- GGCATGGACTGTGGTCATGAG-3\u0026apos; \u003csup\u003e(13,14,15)\u003c/sup\u003e. To confirm the amplification of \u003cem\u003ec-Src\u003c/em\u003e, \u003cem\u003eCox-2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003eand \u003cem\u003eGAPDH\u003c/em\u003e genes, primer sequences were blasted against NCBI/Primer Blast. The analyses were carried out as triplicates.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe relative expression of the\u0026nbsp;aforementioned\u003cem\u003e\u0026nbsp;\u003c/em\u003egenes against \u003cem\u003eGAPDH\u003c/em\u003e depended on the ∆∆ comparative threshold method.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis of the data\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were presented as mean \u0026plusmn; standard error of the mean. Results were analyzed using one-way analysis of variance test followed by Tukey post-hoc test. The statistical analyses were executed by Graph Pad Prism Software (version 3.0). The level of significance was fixed at p \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of GI50 for Dasatinib and Celecoxib in MDA-MB-231 cells\u003c/h2\u003e \u003cp\u003eThe GI50 was 0.05699 \u0026micro;M for Dasatinib and 69.0976 \u0026micro;M for Celecoxib as demonstrated in (Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch2\u003eDetermination Of The Combination And Dose Reduction Indices\u003c/h2\u003e\n\u003cp\u003eBased on the MTT assay results shown in (Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) and the statistical analyses using Compusyn software, Dasatinib /Celecoxib combination showed a strong synergistic effect as evidenced from the combination index (CI=0.98621 \u0026micro;M). Likewise, the dose reduction index revealed that Celecoxib decreased the dose of Dasatinib by approximately 3.8 folds which could decrease its undesired adverse effect as a monotherapy. In addition, Dasatinib decreased the dose of Celecoxib by approximately 1.4 folds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of Dasatinib, Celecoxib and their combination on\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ec-Src\u003c/span\u003e \u003cb\u003egene expression and protein levels in MDA MB-231 cell lysates after 72 hours of treatment\u003c/b\u003e\u003c/p\u003e \u003cp\u003eData presented in (Figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) inferred that Dasatinib up-regulated \u003cem\u003ec-Src\u003c/em\u003e gene expression; however, Celecoxib and the combination down-regulated such expression compared to its expression in positive control cells. Results shown in (Figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) revealed that c-Src protein levels were significantly reduced by 57%, 46%, and 76% when compared with the control group in Dasatinib-treated, Celecoxib-treated, and combination-treated cells, respectively (p \u0026lt; 0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of Dasatinib, Celecoxib and their combination on\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eCOX-2\u003c/span\u003e \u003cb\u003egene expression level, and PGE2 protein level (Pg/mg total protein) in MDA-MB-231 cell lysates after 72 hours of treatment\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs shown in (Figure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), Dasatinib and Dasatinib/Celecoxib combination up-regulated \u003cem\u003eCOX-2\u003c/em\u003e gene expression; however, Celecoxib down-regulated such expression compared to its expression in positive control cells. The results shown in (Figure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) revealed that PGE2 protein levels were significantly reduced by 56%, 45%, and 73% when compared with the positive control group in Dasatinib-treated, Celecoxib-treated, and combination-treated cells, respectively (p \u0026lt; 0.001). Likewise, the combination decreased PGE2 levels significantly compared with single treatments with either Dasatinib or Celecoxib (p \u0026lt; 0.05 and p \u0026lt; 0.001; respectively).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of Dasatinib, Celecoxib and their combination on FAK protein level (ng/mg total protein), p-AKT protein level (Units/mg total protein), and cyclin-D1 protein level (Units/mg total protein) in MDA-MB-231 cell lysates after 72 hours of treatment\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur findings herein (Figure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) inferred that FAK protein levels were significantly reduced by about 59%, 50 %, and 74% in Dasatinib-treated, Celecoxib-treated, and combination-treated cells, respectively (p \u0026lt; 0.001). Moreover, Dasatinib/Celecoxib combination significantly reduced FAK protein levels when compared with single treatments with either Dasatinib or Celecoxib (p \u0026lt; 0.001). The results presented in (Figure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) revealed that p-AKT protein levels were significantly decreased by approximately 64%, 54%, and 77% compared with the positive control group in Dasatinib-treated, Celecoxib-treated, and combination-treated cells, respectively (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). Furthermore, the combination reduced p-AKT protein levels significantly compared with single treatments with either Dasatinib or Celecoxib (p \u0026lt; 0.01 and p \u0026lt; 0.001; respectively). The findings depicted in (Figure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) demonstrated that cyclin D1 protein levels were significantly reduced by approximately 66%, 56%, and 76% when compared with the positive control group in Dasatinib-treated, Celecoxib-treated, and combination-treated cells, respectively (p \u0026lt; 0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of Dasatinib, Celecoxib and their combination on Bcl-2 protein level (Units/mg total protein), active caspase-3 (ng/mg total protein), and VEGF protein level (Pg/mg total protein) in MDA-MB 231 cell lysates after 72 hours of treatment\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe data presented in (Figure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) showed that Dasatinib, Celecoxib, and their combination reduced Bcl-2 protein levels by 45%, 37%, and 79% compared with the positive control group (p \u0026lt; 0.001). Our results herein (Figure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) showed that caspase-3 protein levels were significantly increased by approximately 196%, 152%, and 538% compared with the positive control group in Dasatinib-treated, Celecoxib-treated, and combination-treated cells respectively (p \u0026lt; 0.001). Additionally, Dasatinib/Celecoxib combination significantly elevated caspase-3 levels compared with single treatments with either Dasatinib or Celecoxib (p \u0026lt; 0.001).As presented in (Figure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec), Dasatinib, Celecoxib, and their combination reduced VEGF protein levels significantly by about 68%, 63%, and 82% compared with the positive control group (p \u0026lt; 0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSeveral 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.\u003c/p\u003e \u003cp\u003eThe up-regulatory effect of Dasatinib on \u003cem\u003ec-Src\u003c/em\u003e gene expression level could be a reflex mechanism resulting from the inhibition of \u003cem\u003ec-Src\u003c/em\u003e 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) \u003csup\u003e(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e)\u003c/sup\u003e. Furthermore, this is consistent with what was reported in various tumor cell lines following treatment with other Src inhibitors \u003csup\u003e(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt was documented that Dasatinib exert an effect on Src/FAK pathway \u003csup\u003e(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e)\u003c/sup\u003e 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 \u003csup\u003e(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)\u003c/sup\u003e. The molecular mechanisms suggested for Dasatinib were Src inhibition and epidermal growth factor receptor and estrogen receptor α down-regulation \u003csup\u003e(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e)\u003c/sup\u003e. 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 \u003csup\u003e(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e)\u003c/sup\u003e. It was reported that Dasatinib decreased phosphorylation of c-Src in burkitt's esophageal cells \u003csup\u003e(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e)\u003c/sup\u003e and MDA-MB-468 cells \u003csup\u003e(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur 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 \u003csup\u003e(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e)\u003c/sup\u003e. 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 \u003csup\u003e(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e)\u003c/sup\u003e. Not only studies on HCC cell lines, but also studies on nasopharyngeal carcinoma cell lines showed that FAK is downstream of Src \u003csup\u003e(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e)\u003c/sup\u003e. In addition, cell migration requires FAK activity, whereas FAK activation requires Src activity, suggesting a reciprocal catalytic activation mechanism of FAK and Src \u003csup\u003e(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eConsistent 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 \u003csup\u003e(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn 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 \u003csup\u003e(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e)\u003c/sup\u003e, malignant pleural mesothelioma \u003csup\u003e(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e)\u003c/sup\u003e, melanoma cells \u003csup\u003e(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e)\u003c/sup\u003e, HCT-116 colorectal cancer cells \u003csup\u003e(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e)\u003c/sup\u003e, nasopharyngeal carcinoma cells \u003csup\u003e(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e)\u003c/sup\u003e, neuroblastoma cells \u003csup\u003e(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e)\u003c/sup\u003e, myxoid liposarcoma \u003csup\u003e(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e)\u003c/sup\u003e, papillary thyroid carcinoma cells \u003csup\u003e(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e)\u003c/sup\u003e, breast cancer cells \u003csup\u003e(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e)\u003c/sup\u003e, ovarian cancer cells \u003csup\u003e(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e)\u003c/sup\u003e, HCC cells \u003csup\u003e(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e)\u003c/sup\u003e, acute myeloid leukemia cells \u003csup\u003e(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e)\u003c/sup\u003e, and acute myeloid leukemia Kasumi-1 cells \u003csup\u003e(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe 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 \u003csup\u003e(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e)\u003c/sup\u003e, HNSCC cells (e.g., Ca9-22, HSC3, and SCC-25 cells) \u003csup\u003e(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e)\u003c/sup\u003e, BC cells \u003csup\u003e(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e)\u003c/sup\u003e, laryngeal cancer cell line (Hep-2) \u003csup\u003e(\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e)\u003c/sup\u003e, chronic lymphoid leukemia cells \u003csup\u003e(\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e)\u003c/sup\u003e, neuroblastoma cells \u003csup\u003e(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e)\u003c/sup\u003e, nasopharyngeal carcinoma cells \u003csup\u003e\u003cb\u003e(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e)\u003c/b\u003e\u003c/sup\u003e, SKOv3 and HEY ovarian cancer cells \u003csup\u003e(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e)\u003c/sup\u003e, and Kasumi-1 cells \u003csup\u003e(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn 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 \u003csup\u003e(\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e)\u003c/sup\u003e. 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 \u003csup\u003e(\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur data also inferred that Dasatinib up-regulated \u003cem\u003eCOX-2\u003c/em\u003e gene although it was expected that Dasatinib would down-regulate \u003cem\u003eCOX-2\u003c/em\u003e expression. This could be the consequence of the observed reflex upregulation of src in our study which requires further investigation and evidence.\u003c/p\u003e \u003cp\u003eAs for Celecoxib, it down-regulated \u003cem\u003eCOX-2\u003c/em\u003e gene expression in our study and this was confirmed by multiple studies \u003csup\u003e(\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e)\u003c/sup\u003e. 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 \u003csup\u003e(\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e)\u003c/sup\u003e. It was suggested that Celecoxib decreased PGE2 synthesis via Wnt pathway and conversion of arachidonic acid to bioactive prostanoids \u003csup\u003e(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCelecoxib decreased FAK protein level herein and this is consistent with what was reported earlier in HCC cells \u003csup\u003e(\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e)\u003c/sup\u003e, non-small cell lung cancer cells \u003csup\u003e(\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e)\u003c/sup\u003e, and acute myeloid leukemia cells \u003csup\u003e(\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e)\u003c/sup\u003e. 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 \u003csup\u003e(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe 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 \u003csup\u003e(\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e)\u003c/sup\u003e. 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 \u003csup\u003e(\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e)\u003c/sup\u003e. Furthermore, Celecoxib decreased cyclin D1 expression in both HN30 and HN31 HNSCC lines \u003csup\u003e(\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e)\u003c/sup\u003e and in mouse colon carcinoma cell line \u003csup\u003e(\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e)\u003c/sup\u003e. Celecoxib suppressed growth and promoted cell-cycle arrest at the G0/G1 phase in nasopharyngeal cell lines \u003csup\u003e(\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e)\u003c/sup\u003e, BC cell lines \u003csup\u003e(\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e)\u003c/sup\u003e, murine mammary tumor cell lines \u003csup\u003e(\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e)\u003c/sup\u003e, human pancreatic cancer cell lines \u003csup\u003e(\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e)\u003c/sup\u003e, and human ovarian cancer cell lines \u003csup\u003e(\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e)\u003c/sup\u003e. Celecoxib inhibited cell cycle progression via the G1-S transition in SKOV-3 cells \u003cem\u003ein-vivo\u003c/em\u003e \u003csup\u003e(\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e)\u003c/sup\u003e. Celecoxib inhibited proliferation via the PGE2 pathway in BC, HCC \u003csup\u003e(\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e)\u003c/sup\u003e, and human cholangiocarcinoma cell lines \u003csup\u003e(\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e)\u003c/sup\u003e. 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 \u003csup\u003e(\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur 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 \u003csup\u003e(\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e)\u003c/sup\u003e. It was found that the blockade of caspase activation is enough to suppress apoptosis \u003csup\u003e(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)\u003c/sup\u003e due to decreased AKT phosphorylation and increased Bax expression \u003csup\u003e(\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e)\u003c/sup\u003e. It was reported that Celecoxib prevented colon tumorigenesis by promoting apoptosis via both COX-dependent and COX-independent mechanisms \u003csup\u003e(\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e)\u003c/sup\u003e. It induced apoptosis in cervical cancer cells via Fas-ligand independent FADD activation in a cell type-specific manner \u003csup\u003e(\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e)\u003c/sup\u003e and via an apoptosome-dependent pathway, independent of death receptor pathways in lymphoma \u003csup\u003e(\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e)\u003c/sup\u003e. 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 \u003csup\u003e(\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e)\u003c/sup\u003e. Celecoxib also induces a p53-independent apoptotic response which may be highly relevant in treating human neoplasms \u003csup\u003e(\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e)\u003c/sup\u003e and promoted apoptosis in lung cancer cells which seems to be dose-dependent \u003csup\u003e(\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur results were in line with previous studies that reported that VEGF is reduced by Celecoxib \u003csup\u003e(\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e)\u003c/sup\u003e. It was reported that \u003cem\u003eCOX-2\u003c/em\u003e 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 \u003csup\u003e(\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e)\u003c/sup\u003e. Inhibition of COX-2 activity by Celecoxib reduces all these effects and leads to inhibition of angiogenesis and reduction of tumor growth \u003csup\u003e(\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e)\u003c/sup\u003e. 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 \u003csup\u003e(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)\u003c/sup\u003e. The involvement of COX-2 inhibition in the antiangiogenic effect of Celecoxib was seen in a rat cornea model \u003csup\u003e(\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e)\u003c/sup\u003e. 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 \u003csup\u003e(\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e)\u003c/sup\u003e. COX-2 independent mechanisms contributing to the antiangiogenic effects of Celecoxib was also described in rat hepatoma cells \u003csup\u003e(\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e)\u003c/sup\u003e, human umbilical vein endothelial cells \u003csup\u003e(\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e)\u003c/sup\u003e, ovarian SKOV-3 carcinoma xenografts \u003csup\u003e(\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e)\u003c/sup\u003e, human colon carcinoma cells in nude mice \u003csup\u003e(\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e)\u003c/sup\u003e, and human BC cells \u003csup\u003e(\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTaken 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.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;We declare that no part of the work referred to in here has been published before.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u003csup\u003e\u0026rsquo;\u003c/sup\u003es Contributions:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;N.M,\u0026nbsp;M.M, M.W.H contributed to the research idea.\u0026nbsp;N.M,\u0026nbsp;M.M, M.W.H conceived and designed the experiments.\u0026nbsp;N.M,\u0026nbsp;M.M, M.W.H conducted the experiments.\u0026nbsp;N.M,\u0026nbsp;M.M, M.W.H,\u0026nbsp;M.H\u0026nbsp;contributed to data analysis and presentation.\u0026nbsp;N.M wrote the manuscript. N.M,\u0026nbsp;M.M, M.W.H,\u0026nbsp;M.H revised the manuscript and \u003cstrong\u003edeclared that the data were generated in-ho\u003c/strong\u003eu\u003cstrong\u003ese and that no paper\u0026nbsp;\u003c/strong\u003em\u003cstrong\u003eill was\u0026nbsp;\u003c/strong\u003eu\u003cstrong\u003esed.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that this research was conducted in the absence of any commercial or financial funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll supplementary data are available upon request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Ferlay J, Soerjomataram I et al (2018) Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. 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Proceedings of the National Academy of Sciences of the United States of America 101(2):591\u0026ndash;596\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Triple-negative breast cancer, Src signaling cascade, COX-2/PGE2 pathway, PI3K/AKT/ mTOR pathway, Dasatinib, Celecoxib","lastPublishedDoi":"10.21203/rs.3.rs-904306/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-904306/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDespite the tremendous efforts to implement new paradigms for breast cancer, the disease still remains a major challenge worldwide. Genetic deregulation is evident in all breast cancer subtypes and comprises a multitude of mutated genes and deregulated signaling cascades. In this sense, co-targeting Src and COX-2 signaling cascades have attracted fervent interest. This work explored the probable anti-carcinogenic effects of Dasatinib as a Src inhibitor, Celecoxib as a selective COX-2 inhibitor, and their combination in MDA-MB-231 triple-negative breast cancer cell line. Drug growth inhibition 50 (GI50) was determined using the MTT assay and the obtained results were analyzed using CompuSyn 3.0.1 software. MDA-MB-231 cells were divided into four treatment groups including a positive control, Dasatinib-treated, Celecoxib-treated, and combination-treated groups. Standard sandwich ELISA was used for the determination of the protein levels of c-Src, Bcl-2, p-AKT, FAK, PGE2, VEGF, and cyclin D1. Active caspase-3 was determined colorimetrically and the expression of \u003cem\u003eCOX-2\u003c/em\u003e and \u003cem\u003ec-Src\u003c/em\u003e genes was quantitatively determined via quantitative real-time polymerase chain reaction. The GI50 for Dasatinib was 0.05699 \u0026micro;M while that for Celecoxib was 69.0976 \u0026micro;M. Dasatinib up-regulated c-Src gene while Celecoxib and Dasatinib/Celecoxib combination down-regulated such expression level. COX-2 gene was down-regulated by Celecoxib while it was up-regulated by both Dasatinib and Dasatinib/Celecoxib combination. On one hand, Dasatinib, Celecoxib, and their combination significantly reduced the protein levels of c-Src, Bcl-2, p-AKT, FAK, PGE2, VEGF, and cyclin D1. On the other hand, they elevated active caspase-3. To sum up, Dasatinib/Celecoxib combination increased the capability for apoptosis and suppressed proliferation, angiogenesis, migration, and invasion suggesting a strong cross-talk between Src signaling cascade and COX-2/PGE2 via the intermediate PI3K/AKT/mTOR pathway. Further in-vitro and in-vivo studies are warranted to verify the present findings.\u003c/p\u003e","manuscriptTitle":"Dasatinib/Celecoxib combination: A new hope in triple negative breast cancer treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-11-12 15:47:09","doi":"10.21203/rs.3.rs-904306/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2021-12-31T07:13:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-11-22T23:42:38+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-11-10T11:00:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-11-10T00:47:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Naunyn-Schmiedeberg's Archives of Pharmacology","date":"2021-09-13T08:37:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"be298268-97dd-4eef-a35c-c569aa1cc501","owner":[],"postedDate":"November 12th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":8466100,"name":"Cancer Biology"},{"id":8466101,"name":"Oncology"}],"tags":[],"updatedAt":"2022-03-28T20:59:38+00:00","versionOfRecord":[],"versionCreatedAt":"2021-11-12 15:47:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-904306","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-904306","identity":"rs-904306","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}