Celecoxib Inhibits Triple-negative Breast Cancer Cell Line MDA-MB-231 By Downregulation NF-κB Pathway

Objective: Breast cancer is a heterogeneous disease that encompasses several distinct entities with remarkably different biological characteristics and clinical behavior, and threatened to female public health. The incidence of breast cancer is increasing, and current therapy is unable to achieve clinical responses in patients with highly invasive metastatic disease. It is estimated that, in the past decade, Chinese urban cancer registries have documented increased incidence rates of between 20% and 30% for breast cancer. Breast cancer has acclaimed to be the first leading cause of death among 30 to 54 year old women. A new subtype of particular interest is triple-negative breast cancer, namely TNBC, which is characterized by estrogen receptor negative (ER), progesterone receptor negative (PR), and human epidermal growth factor receptor-2 (HER-2) negative using histochemical staining. Although TNBC accounts for 11%-20% of breast cancer cases, it is responsible for a disproportionate number of breast cancer deaths, especially in younger female before menopause. However, there is still absence of suitable treatment for this tumor.Currently, studies with nonsteroidal anti-inflammatory drugs (NSAIDs) have shown significant effects in reducing the incidence and progression of tumors in both animal models and in treatment of cancer patients. It was therefore of extreme interest when recent epidemiological studies suggested the presence of an inverse association between regular intake of NSAIDs and the relative risk of breast cancer. Celecoxib is a new NSAID that specifically inhibits cyclooxygenase-2 (COX-2). It has significant anti-inflammatory and analgesic properties but lesser side effects than other NSAIDs such as aspirin and ibuprofen, which inhibit both COX-1 and COX-2. Because of emerging evidence suggesting that NSAIDs inhibition of COX reduces the risk of breast cancer, Harris et al conducted a great deal of studies to evaluate the antitumor effects of a specific COX-2 blockade by this compound. Results showed that celecoxib produced striking reductions in the incidence of mammary cancer, tumor burden and tumor volume. Debates have raged continuously for a long period over whether the antitumor effects of celecoxib occur in COX-2-dependent or COX-2-independent manner. However, this mechanism has yet to be clearly defined.Nuclear factor kappaB (NF-κB) is a pleiotropic transcription factor which was initially found to bind enhancer of immunoglobinκchain extracted from mature B lymphocyte. Regulation of NF-κB activity plays a critical role in tumor development. Inactivation of NF-κB may lead to significant reduction in cancer cell proliferation, tumor angiogenesis, inflammation and enhancement of sensitivity to apoptosis and chemotherapeutics. Preclinical and laboratory data showed that constitutively activated NF-κB was preferentially involved in human breast cancer cell lines, mammary breast cancer tissue sample and carcinogen-induced animal models. Furthermore, highly constitutive NF-κB activation was categorized in the ER negative breast cancer cells compared with those ER positive counterparts, suggesting that NF-κB might become a useful therapeutic target for this subtype of cancer. The family of NF-κB comprises of five members, while p65 subunit contains an extremely active C-terminal transcriptional activation domain (TAD), required for its cellular function. Substantial evidence verified that blocking p65-mediated NF-κB activation could inhibit tumorigenic cancer cell line proliferation and promote apoptosis. Thus, these results showed that incapacitating NF-κB mediated by silencing p65 prevents xenograft tumor growth of cells that otherwise readily form tumors in recipient animals, suggesting that silencing p65 may have promising perspective in tumor clinical therapy.Methods: in the present studies, triple-negative and highly invasive breast cancer cell line MDA-MB-231 was selected as study object. Multiple experimental methods, such as flow cytometry (FCM), western blot, electrophoretic mobility shift assay (EMSA), were employed to investigate the possible antitumor mechanisms of celecoxib, and screen its potential signaling transduction pathway. To evaluate the underlying roles of constitutively activated NF-κB play in proliferation, apoptosis and invasion of MDA-MB-231 cells. Finally, our aim is to verify whether celecoxib could become an assistant candidate in synergy with chemotherapy to cure TNBC, and to inactivate NF-κB by interfering p65 could provide an additional target therapy to complement antineoplastic dosages of traditional medicine.Results:1 The effect of celecoxib on MDA-MB-231 cell biological behaviourIn the initial part, we investigated the influential effect of celecoxib on proliferation, apoptosis and invasiveness behavior of MDA-MB-231 cells. The results were as follows:1.1 Celecoxib inhibits MDA-MB-231 cell proliferationCelecoxib at concentrations of 40, 80 and 120μM was used to treat MDA-MB-231 cell line for 24h, 48h and 72h,respectively. The rate of proliferation in response to celecoxib treatment was analyzed by measuring MTT assay. Significant inhibition of proliferation was observed in MDA-MB-231 cells in a dose- and time-dependent manner (P<0.05). The IC50 of celecoxib at 24h, 48h and 72h was 139.76,68.39,48.27μM.Prostaglandin E2 (PGE2) is the major prostaglandin that COX-2 derived arachidonic acid into, which can be used to determine the COX-2 activity in quantity. To determine whether celecoxib-induced growth inhibition could be reversed by exogenous PGE2, we added PGE2 to the culture of MDA-MB-231 cells treated with constant dose (80μM) of celecoxib for 48h. In MDA-MB-231 cells, growth inhibition induced by 80μM celecoxib could not be restored by addition of exogenous PGE2, thereby suggesting that celecoxib suppressed MDA-MB-231 cell growth was independent of PGE2 levels and COX-2 pathway (P>0.05).1.2 Celecoxib induces MDA-MB-231 cell apoptosisTo investigate whether the observed growth inhibition mediated by celecoxib was associated with induction of programmed cell death. We used Hoechst 33258 staining, FCM analysis and DNA fragmentation assay to observe nuclear fragmentation, and monitor the changes of cells after celecoxib treatment. Under the phase contrast microscope, cells exhibited a dramatic morphologic change after 48h of drug treatment. Celecoxib at concentrations of 80μM and 120μM caused significant increase in the percentage apoptotic cells (21.08±4.18%, 43.51±6.06%, P<0.05, respectively). It is showed that celecoxib treatment results in the formation of DNA fragments in MDA-MB-231 cells, as determined by agarose gel electrophoresis at 72h. As the intrinsic mitochondrial apoptotic pathway is relatively more important than the death receptor pathway for the induction of apoptosis by chemotherapeutic drugs, we examined the executioners, caspase-3 and poly-ADP-ribose polymerase (PARP) by immunoblotting. Results showed that in celecoxib-treated cells, they were both cleaved into their specific active forms. The effector caspases-3 proteolytically cleaves and activates several other caspases as well as several other apoptotic proteins including PARP, which is rapidly activated during the cellular response to DNA damage, and is a part of safeguard mechanisms protecting cells from genotoxic damage. These results suggested that celecoxib-induced apoptosis in MDA-MB-231 cells is due to activation of caspases-3 and its subsequent cleaved apoptotic protein PARP.1.3 Celecoxib blocks MDA-MB-231 cell migration and invasionCancer cell migration and invasion play very important roles in cancer metastasis. Consequently, we further studied the effects of celecoxib on migration and invasion as well as the related angiogenic factors, such as vascular endothelial growth factor (VEGF), matrix metalloproteinases-2 (MMP-2) and interleukin-8 (IL-8) mRNA expression of MDA-MB-231 cells.In vitro, wound healing assay results showed that the migration of MDA-MB-231 mock cells and celecoxib-treated cells differ at 24h after wounding with mock cells migrating almost twice as slowly as 80μM celecoxib-treated cells (P<0.05). These data suggested that celecoxib could affect MDA-MB-231 cells on horizontal migratory activity induced by wound, which was consistent with the prognostic value of celecoxib in tumoural disease and metastatic potential.Next, we examined the motility of different concentration celecoxib-treated cells with transwell cell invasion assay. Results revealed that with the presence of 120μM celecoxib significantly reduced the invasion of MDA-MB-231 cells (P<0.05). The level of celecoxib-induced cell invasion was significantly reduced by 67% of control level (P<0.05). These experiments indicated that treatment of celecoxib in MDA-MB-231 cells inhibits cell motility and invasion activity in vitro.The expression of VEGF, MMP-2 and IL-8 mRNA in MDA-MB-231 cells was examined by RT-PCR analysis. As shown, all cells expressed the same level ofβ-actin without difference (P>0.05), but celecoxib could significantly lower these cytokines mRNA expression (P<0.05).The secretion of IL-8 was further confirmed by enzyme-linked immunosorbent assay (ELISA). The expression level of celecoxib-treated cells was significantly lower than those in control groups (P<0.05). Besides, celecoxib could abrogate the potential of TNF-α-induced IL-8 secretion (P<0.05).2 Study on the underlying mechanism of celecoxib inhibiting MDA-MB-231 cell growth, invasion and promoting apoptosisIn previous study, we knew that celecoxib had strong inhibition on MDA-MB-231 cell growth and invasion, and could induce cell apoptosis. To explore the underlying mechanism of these phenomena, various pathway signaling inhibitors were employed to screen its possible targets.2.1 Various signaling pathway inhibitors to screen the potential target of celecoxibSignaling pathway inhibitors including SP600125 (JNK inhibitor, Jun N-terminal kinase inhibitor), PD98059 (ERK inhibitor, extracellular signal-regulated kinase inhibitor), SB203580 (p38 inhibitor), PDTC (pirrolidine dithiocarbamate, NF-κB inhibitor) and LY294002 (AKT inhibitor, protein kinase B/AKT inhibitor) were employed to treat cells alone or in combination with celecoxib. MTT assay results showed that PDTC and LY294002 could cause inhibition of MDA-MB-231 cell proliferation (P<0.05). Especially, combinations of PDTC and celecoxib at different concentrations showed a strong synergistic effect on cell proliferation. Basu et al reported that the mechanism of celecoxib-induced growth arrest was by induction of apoptosis, associated with reduced activation of AKT. Hence, we want to figure out whether NF-κB aberrant activation played a crucial part in highly invasive breast carcinoma cell line MDA-MB-231, and whether celecoxib exerted its antitumor effect through inactivation of NF-κB.2.2 Celecoxib inhibits NF-κB p65 expression but not p50Unstimulated MDA-MB-231 cells demonstrated somewhat higher levels of constitutive NF-κB p65 and p50 expression. However, treatment with 80μM celecoxib resulted in additional reduction in p65 expression (P<0.05), but no influence on p50 expression (P>0.05).2.3 Celecoxib inhibits NF-κB p65 nuclear translocationCytoplasmic p65 level was slightly decreased after celecoxib stimulation. In contrast, celecoxib significantly suppressed p65 nuclear translocation after 24h treatment (P<0.05). The data clearly demonstrated that celecoxib could influence the process of p65 from cytoplasm to nucleus.2.4 Celecoxib inhibits IκBαphosphorylationPhosphorylation and degradation of IκBαis the initiating event in NF-κB activation and translocation, and upregulation of IκBαexpression constrains p65/p50 to the cytosol and inhibits in?ammatory NF-κB activity. Accordingly, we also tested the hypothesis that celecoxib acted on p65 localization via effects on IκBαprotein expression. Results showed that celecoxib inhibited IκBαphosphorylation, indicating that intact IκBαcould suppress p65 translocation (P<0.05).2.5 Celecoxib causes inactivation of NF-κB DNA Binding activityTo investigate whether celecoxib modulated NF-κB DNA binding activity in MDA-MB-231 breast cancer cells, EMSA was performed. Autoradiography revealed that NF-κB DNA Binding activity was constitutively activated in unstimulated cells. However, NF-κB DNA binding activity was decreased in cells treated with celecoxib for 24h. Besides, in competition assay, mutant oligonucleotide failed to compete with the specific binding, while unlabeled specific oligonucleotide counteracted the effects of the labeled. The above results indicated that the induction of apoptosis by celecoxib may be potentially mediated by the downregulation of NF-κB transcription factor activity which, in turn, may be responsible for both cell growth inhibition and induction of apoptotic processe.2.6 Celecoxib inhibits the expression levels of Bcl-2To explore the possible role of Bcl-2 family members in celecoxib -induced apoptosis, the effects of celecoxib on the expression level of Bcl-2 by western blot analysis was examined. Exposure of cells to 80μM of celecoxib resulted in downregulation of Bcl-2 significantly (P<0.05).3 Study on the effect of overexpression p65 on cell apoptosis and invasion in MDA-MB-231 cells in vitroPrevious studies showed that celecoxib could inhibit p65 expression, translocation and IκBαphosphorylation, therefore inactivation of NF-κB DNA binding activity. We hypothesized that NF-κB p65 signaling in breast cancer cells MDA-MB-231 could be a selective target of celecoxib treatment. In this part, we conducted p65cDNA transfection into MDA-MB-231 cells and observed the effects of endogenous p65 on cell apoptosis, invasion and its potential influence on celecoxib treatment.3.1 NF-κB p65cDNA transfectionThe recombinant plasmid, designated as pcDNA3.1-p65, eukaryotic expression plasmid encoding p65 gene coding region was constructed. The recombinant p65cDNA transfected into breast cancer cells MDA-MB-231 by the vector of lipofectamine 2000. Then total cell RNA, the cytoplasmic proteins and nuclear proteins were extracted. NF-κB p65 mRNA and protein expression were detected by RT-PCR and western blot analyses, respectively. Results showed that MDA-MB-231 cells which have been transfected p65cDNA recombination plasmids could significantly enhance the mRNA and protein level of p65 gene expression (P<0.05).3.2 p65cDNA transfection enhanced NF-κB DNA binding activityImportantly, EMSA results showed that NF-κB p65cDNA transfection enhanced the NF-κB DNA binding activity significantly in vitro (P<0.05).3.3 p65cDNA transfection promotes cell growthWe applied MTT to detect the proliferation capability of MDA-MB-231 cells after transfection. The results suggested the growth and proliferation of MDA-MB-231 cells transfected with the recombinant of p65cDNA were promoted than those not transfected. Moreover, the growth and proliferation of MDA-MB-231 cells transfected with the recombinant of p65cDNA in combination of celecoxib treatment were faster than those without transfection.3.4 p65cDNA transfection promotes S phase progression of MDA-MB-231 cellsFCM was done to further define the mechanism by which p65cDNA transfection promoted the growth of MDA-MB-231 cells. p65cDNA transfection led to an increase in the percentage of cells in S and a corresponding decrease in the percentage of cells in G0/G1 phase (P<0.05). Results showed that overexpression p65 gene might alter the progression to S phase of MDA-MB-231 cells.Cyclin D1 is involved in the early- to mid- G1 phase of the cell cycle in association with its catalytic partner CDK4. Emerging evidence indicated that cyclin D1 was essential for G1 progression in this breast cancer cell line. Our results showed that overexpression p65 gene could increase both cyclinD1 mRNA and protein expression (P<0.05), but little effects on CDK4 (P>0.05).3.5 p65cDNA transfection inhibits cell apoptosisFCM results revealed that the rate of apoptosis decreased in p65cDNA transfected MDA-MB-231 cells compared with control cells after 80μM celecoxib treatment. It indicated that the constructed recombination plasmids can suppress the apoptosis effect of MDA-MB-231 cells induced by celecoxib. Using western blot analysis, we observed that celecoxib downregulated the expression of caspase-3 and PARP in NF-κB p65cDNA-transfected or parental breast cancer cells (P<0.05). However, there was little change found in Bcl-2 expression (P>0.05). These results are consistent with the cell growth inhibition assay, suggesting that greater cell growth inhibition resulting from the celecoxib treatment is partly mediated through the induction of greater apoptosis in breast cancer cells. These results provide mechanistic support in favor of our claim that the apoptosis-inducing effect of celecoxib is partly mediated through the NF-κB pathway.3.6 p65cDNA transfection promotes cell invasionThe result from transwell cell invasion assay indicated that p65cDNA transfection significantly promoted the invasion of MDA-MB-231 cells, as compared with mock-transfected and control vector–transfected cells (P<0.05). Furthermore, the mRNA levels of VEGF,IL-8 and MMP-2 mRNA were showed great enhancement in MDA-MB-231 cells transfected p65cDNA (P<0.05). MDA-MB-231 cells constitutively expressed a high level of IL-8, however, p65cDNA transfection can significantly accelerate the secretion (P<0.05).4 Study on the effect of p65miRNA by targeting p65 gene on cell apoptosis and invasion in MDA-MB-231 cells in vitroIn the third part, overexpression p65 could abrogate the ability of celecoxib-induced cell apoptosis and promote the invasiveness and metastasis of MDA-MB-231 cells. Accordingly, we investigated the down-modulation of p65 expression and function by artificial microRNA (miRNA) by measuring mRNA and protein levels in the breast cancer cell line MDA-MB-231, which has a high level of p65 expression.4.1 NF-κB p65miRNA transfectionPrimarily, the recombinant expressive plasmid p65miRNA in pcDNATM6.2-GW/EmGFP-miR-based miRNA with a pre-microRNA sequence was constructed and transfected to MDA-MB-231 cells. Fluorescene microscope was applied to observe green fluorescence, which suggested cells transfected successfully. RT-PCR and western blot analyses were used to detect the expression of p65 at mRNA and protein level. The results showed that the MDA-MB-231 cells which have been transfected the p65miRNA recombination plasmids could significantly inhibit the mRNA and protein level of p65 expression (P<0.05).4.2 NF-κB p65miRNA transfection reduced NF-κB DNA binding activityEMSA results clearly demonstrated that NF-κB p65miRNA transfection decreased the NF-κB DNA binding activity significantly (P<0.05), suggesting that silencing p65 gene led to inactivation of NF-κB.4.3 Silencing p65 inhibits MDA-MB-231 cell growthMTT results showed that p65miRNA transfection inhibited cell proliferation of MDA-MB-231 cells in vitro as compared with mock-transfected and control vector–transfected cells (P<0.05).4.4 Silencing p65 sensitizes MDA-MB-231 cells apoptosisFCM results showed that rate of apoptosis increased in cells transfected with the recombinant of p65miRNA than those not transfected. To examine whether silencing p65 promoted caspase activation, we used western blot to detect the changes of caspase-3 and PARP. In consistent with previous data, p65miRNA transfection profoundly reduced the level of precatalytic caspase-3 and PARP (P<0.05). To determine the definite mechanisms by which targeting p65 promoted apoptosis, we examined the effect of p65miRNA transfection on IAPs gene expression. Results revealed that downregulation p65 could lead to reduction in survivin and XIAP protein level as well (P<0.05).4.5 Silencing p65 inhibits MDA-MB-231 cells migration and invasionTranswell cell invasion assay was used to determine invasion capability change of breast cancer cells transfected p65miRNA. Our results demonstrated that lowering levels of p65 mRNA and protein inhibited cell invasion considerably (P<0.05). Our previous investigation has demonstrated that p65 overexperssion could lead to upregulate angiogenic cytokines, which were related to tumor metastasis and angiogenesis. Herein, to verify whether VEGF, MMP-2 and IL-8 were blocked due to silence of p65 expression with this artificial miRNA, RT-PCR for detecting of mRNA was performed. Results revealed that VEGF, MMP-2 and IL-8 were downregulated in the breast cancer cell line MDA-MB-231 transfected with p65miRNA after 24h (P<0.05). Control-miRNA-transfected MDA-MB-231 cells still expressed the same levels. Besides, ELISA assay showed that IL-8 secretion was reduced significantly after blocking p65 expression (P<0.05).Conclusions:1. Celecoxib inhibits MDA-MB-231 cell proliferation in a dose- and time-dependent manner, whereas probably independent of COX-2 pathway. Celecoxib induces apoptosis in MDA-MB-231 cells by increasing DNA ladder formation and the activity of caspase-3 and PARP. The mechanism may be involved in inhibition of the expression, nuclear translocation of p65 and IκBαphosphorylation, which lead to inactivation of NF-κB DNA binding activity and reduction its downstream Bcl-2 expression. Furthermore, celecoxib could suppress the migration and invasion of MDA-MB-231 cells, the mechanism of which might be concerned with downregulation of VEGF, MMP-2 and IL-8 mRNA levels, and reduction in IL-8 secretion.2. Overexpression of p65 promotes p65 expression in both mRNA and protein levels, and results in enhancement of NF-κB DNA binding activity. p65cDNA transfection abrogates the rate of apoptosis induced by celecoxib and promotes the invasiveness ability of MDA-MB-231 cells. Overexpression of p65 overrode caspase-3 and PARP activation, whereas no influence on Bcl-2 expression. Besides, VEGF, MMP-2 and IL-8 mRNA levels plus IL-8 secretion were upregulated caused by p65cDNA transfection.3. Reduction of endogenous p65 by miRNA treatment significantly impaired NF-κB activation, induced apoptosis, and reduced invasion of MDA-MB-231 cells. The mechanism may be related to activation of caspase-3 and PARP. p65miRNA transfection significantly reduced the levels of XIAP and survivin compared with control. Furthermore, p65miRNA suppressed invasive ability of MDA-MB-231 cells, in level with VEGF, MMP-2 and IL-8 downregulation, and reduced IL-8 secretion.