The Role of Kif4A in Doxorubicin-Induced Apoptosis in Breast Cancer Cells

This study was to investigate the mechanism and role of Kif4A in doxorubicin-induced apoptosis in breast cancer. Using two human breast cancer cell lines MCF-7 (with wild-type p53) and MDA-MB-231 (with mutant p53), we quantitated the expression levels of kinesin super-family protein 4A (Kif4A) and poly (ADP-ribose) Polymerase-1 (PARP-1) by Western blot after doxorubicin treatment and examined the apoptosis by flow cytometry after treatment with doxorubicin and PARP-1 inhibitor, 3-Aminobenzamide (3-ABA). Our results showed that doxorubicin treatment could induce the apoptosis of MCF-7 and MDA-MB-231 cells, the down-regulation of Kif4A and upregulation of poly(ADP-ribose) (PAR). The activity of PARP-1 or PARP-1 activation was significantly elevated by doxorubicin treatment in dose- and time-dependent manners (P < 0.05), while doxorubicin treatment only slightly elevated the level of cleaved fragments of PARP-1 (P > 0.05). We further demonstrated that overexpression of Kif4A could reduce the level of PAR and significantly increase apoptosis. The effect of doxorubicin on apoptosis was more profound in MCF-7 cells compared with MDA-MB-231 cells (P < 0.05). Taken together, our results suggest that the novel role of Kif4A in doxorubicin-induced apoptosis in breast cancer cells is achieved by inhibiting the activity of PARP-1.     

Effects of triptolide from Tripterygium wilfordii on ERα and p53 expression in two human breast cancer cell lines

The aim of the study was to discover possible differential cytotoxicity of triptolide towards estrogen-sensitive MCF-7 versus estrogen-insensitive MDA-MB-231 human breast cancer cells. Considering that MCF-7 cells express functional Estrogen receptor α (ERα) and wild-type p53, whereas MDA-MB-231 cells which are ERα-negative express mutant p53, the anti-proliferation effect of triptolide on MCF-7 and MDA-MB-231 cells were examined, the apoptotic effect and cell cycle arrest caused by triptolide were investigated, ERα and p53 expression were also observed in this paper. The results showed that the anti-proliferation effects were induced by triptolide in both cell lines. But the value of IC₅₀ in MCF-7 cells for its anti-proliferation effect was about one tenth of that in MDA-MB-231 cells, which indicated that the effect is more potent in MCF-7 cells. Condensed chromatin or fragmented nuclei could be found in MCF-7 cells treated with only 40 nM triptolide but in MDA-MB-231 cells they couldn’t be observed until the concentration reached to 400 nM. Triptolide induced significant S cell cycle arrest along with the presence of sub-G0/G1 peak in MDA-MB-231 cells, whereas there was only slightly S cell cycle arrest on cell cycle distribution in MCF-7 cells. The role of p53 in two breast cancer cells was examined, the results showed that the mutant p53 in MDA-MB-231 cells was suppressed and the wild-type p53 in MCF-7 was increased. Moreover, triptolide could down regulate the expression of ERα in MCF-7 cells. The results showed that triptolide is much more sensitive to ERα-positive MCF-7 cells than to ERα-negative MDA-MB-231 cells, and the sensitivity is significantly associated with the ERα and p53 status.     

Different effects of genistein on molecular markers related to apoptosis in two phenotypically dissimilar breast cancer cell lines

The association between consumption of genistein-containing soybean products and lower risk of breast cancer suggests a cancer chemopreventive role for genistein. Consistent with this suggestion, exposing cultured human breast cancer cells to genistein inhibits cell proliferation, although this is not completely understood. To better understand how genistein works, the ability of genistein to induce apoptosis was compared in phenotypically dissimilar MCF-7 and MDA-MB-231 human breast cancer cells that express the wild-type and mutant p53 gene, respectively. After 6 days of incubation with 50 microM genistein, MCF-7 but not MDA-MB-231 cells, showed morphological signs of apoptosis. Marginal proteolytic cleavage of poly-(ADP-ribose)-polymerase and significant DNA fragmentation were also detected in MCF-7 cells. In elucidating these findings, it was determined that after 2 days of incubation with genistein, MCF-7 but not MDA-MB-231 cells, had significantly higher levels of p53. Accordingly, the expression of certain proteins modulated by p53 was studied next. Levels of p21 increased in both of the genistein-treated cell lines, suggesting that p21 gene expression was activated but in a p53-independent manner, whereas no significant changes in levels of the pro-apoptotic protein, Bax, were found. In MCF-7 cells, levels of the anti-apoptotic protein, Bcl-2, decreased slightly at 18-24 h but then increased considerably after 48 h. Hence, the Bax:Bcl-2 ratio initially increased but later decreased. These data suggest that at the genistein concentration tested, MCF-7 cells in contrast to MDA-MB-231 cells were sensitive to the induction of apoptosis by genistein, but Bax and Bcl-2 did not play clear roles.     

Critical role of p53 upregulated modulator of apoptosis in benzyl isothiocyanate-induced apoptotic cell death

Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, decreases viability of cancer cells by causing apoptosis but the mechanism of cell death is not fully understood. The present study was undertaken to determine the role of Bcl-2 family proteins in BITC-induced apoptosis using MDA-MB-231 (breast), MCF-7 (breast), and HCT-116 (colon) human cancer cells. The B-cell lymphoma 2 interacting mediator of cell death (Bim) protein was dispensable for proapoptotic response to BITC in MCF-7 and MDA-MB-231 cells as judged by RNA interference studies. Instead, the BITC-treated MCF-7 and MDA-MB-231 cells exhibited upregulation of p53 upregulated modulator of apoptosis (PUMA) protein. The BITC-mediated induction of PUMA was relatively more pronounced in MCF-7 cells due to the presence of wild-type p53 compared with MDA-MB-231 with mutant p53. The BITC-induced apoptosis was partially but significantly attenuated by RNA interference of PUMA in MCF-7 cells. The PUMA knockout variant of HCT-116 cells exhibited significant resistance towards BITC-induced apoptosis compared with wild-type HCT-116 cells. Attenuation of BITC-induced apoptosis in PUMA knockout HCT-116 cells was accompanied by enhanced G2/M phase cell cycle arrest due to induction of p21 and down regulation of cyclin-dependent kinase 1 protein. The BITC treatment caused a decrease in protein levels of Bcl-xL (MCF-7 and MDA-MB-231 cells) and Bcl-2 (MCF-7 cells). Ectopic expression of Bcl-xL in MCF-7 and MDA-MB-231 cells and that of Bcl-2 in MCF-7 cells conferred protection against proapoptotic response to BITC. Interestingly, the BITC-treated MDA-MB-231 cells exhibited induction of Bcl-2 protein expression, and RNA interference of Bcl-2 in this cell line resulted in augmentation of BITC-induced apoptosis. The BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with the induction of PUMA protein in the tumor. In conclusion, the results of the present study indicate that Bim-independent apoptosis by BITC in cancer cells is mediated by PUMA.     

Novel quinuclidinone derivatives induced apoptosis in human breast cancer via targeting p53

Small molecules that can target human cancers have been highly sought to increase the anticancer efficacy, the present work describes the design and synthesis of novel series of five quinuclidinone derivatives (2a-2e). Their anticancer activities were investigated against breast cancer cells MCF-7, MDA-MB-231 breast cancer cells harboring mutant p53 and normal breast counterpart MCF-12a. Derivative 2e reduced proliferation of MCF-7 and MCF-12a while it has no effect on MDA-MB-231. Derivative 2e induced apoptosis in MCF-7 cells which is further confirmed by TUNEL assay and it reduced the percentage of cell in G2/M phase as confirmed by increased expression of cyclin B and reduced expression of cyclin D1. Derivative 2e reduced expression levels of Mdm2, Akt and ERK1/2 by and increased expression level of p53. Moreover, the apoptosis induction by 2e was also inhibited by PFT-α as evidenced by non-significant induction of apoptosis after treatment of MCF-7 cells with both derivative 2e and PFT-α. In addition, docking study reveals that derivative 2e has a binding pattern close to the pattern observed in the structure of the lead fragment 5,6-dimethoxy-2-methylbenzothiazole bound to T-p53C-Y220C. The above findings demonstrate that derivative 2e induces apoptosis in MCF-7 cells via targeting p53 which merits further development.     

p53-Independent Down-Regulation of Mdm2 in Human Cancer Cells Treated with Adriamycin

Mdm2 is a nuclear phosphoprotein which functions as a negative feedback regulator of the p53 tumor suppressor gene. In this study, we investigated the alteration of Mdm2 and p53 in three human cancer cell lines containing either a wild-type or mutant p53 gene after treatment with Adriamycin (doxorubicin, ADR), a DNA damaging agent. We found that human breast cancer MCF-7 cells containing wild-type p53 were much more susceptible to ADR compared to human breast cancer MDA-MB-231 and human prostate cancer Du-145 cells which contain mutant p53. ADR resulted in a significant dose-dependent accumulation of p53 protein in MCF-7 cells, whereas little or no influence was observed on p53 protein of the two mutant p53 cell lines. However, a significant down-regulation of Mdm2 at protein and mRNA levels was observed in these three cell lines following ADR treatment. Moreover, the decrease of Mdm2 was in both a dose- and time-dependent manner. It is interestingly noted that 5 μM is a critical dose for significant down-regulation of the Mdm2 protein. Selected proteasome inhibitors did not rescue the ADR-caused decline in the expression of Mdm2 protein. Therefore, our present results reveal that ADR can induce a down-regulation of Mdm2 via a p53-independent pathway in human cancer cells and the ubiquitin-proteasome degradation mechanism may not be involved in the decreased expression of Mdm2 protein.     

Molecular Modification of Metadherin/MTDH Impacts the Sensitivity of Breast Cancer to Doxorubicin

BackgroundBreast cancer is a leading cause of death in women and with an increasing worldwide incidence. Doxorubicin, as a first-line anthracycline-based drug is conventional used on breast cancer clinical chemotherapy. However, the drug resistances limited the curative effect of the doxorubicin therapy in breast cancer patients, but the molecular mechanism determinants of breast cancer resistance to doxorubicin chemotherapy are not fully understood. In order to explore the association between metadherin (MTDH) and doxorubicin sensitivity, the differential expressions of MTDH in breast cancer cell lines and the sensitivity to doxorubicin of breast cancer cell lines were investigated.MethodsThe mRNA and protein expression of MTDH were determined by real-time PCR and Western blot in breast cancer cells such as MDA-MB-231, MCF-7, MDA-MB-435S, MCF-7/ADR cells. Once MTDH gene was knocked down by siRNA in MCF-7/ADR cells and overexpressed by MTDH plasmid transfection in MDA-MB-231 cells, the cell growth and therapeutic sensitivity of doxorubicin were evaluated using MTT and the Cell cycle assay and apoptosis rate was determined by flow cytometry.ResultsMCF-7/ADR cells revealed highly expressed MTDH and MDA-MB-231 cells had the lowest expression of MTDH. After MTDH gene was knocked down, the cell proliferation was inhibited, and the inhibitory rate of cell growth and apoptosis rate were enhanced, and the cell cycle arrest during the G0/G1 phase in the presence of doxorubicin treatment. On the other hand, the opposite results were observed in MDA-MB-231 cells with overexpressed MTDH gene.ConclusionMTDH gene plays a promoting role in the proliferation of breast cancer cells and its high expression may be associated with doxorubicin sensitivity of breast cancer.     

Antineoplastic Effects of α-Santalol on Estrogen Receptor-Positive and Estrogen Receptor-Negative Breast Cancer Cells through Cell Cycle Arrest at G2/M Phase and Induction of Apoptosis

Anticancer efficacy and the mechanism of action of α-santalol, a terpenoid isolated from sandalwood oil, were investigated in human breast cancer cells by using p53 wild-type MCF-7 cells as a model for estrogen receptor(ER)-positive and p53 mutated MDA-MB-231 cells as a model for ER-negative breast cancer. α-Santalol inhibited cell viability and proliferation in a concentration and time-dependent manner in both cells regardless of their ER and/or p53 status. However, α-santalol produced relatively less toxic effect on normal breast epithelial cell line, MCF-10A. It induced G2/M cell cycle arrest and apoptosis in both MCF-7 and MDA-MB-231 cells. Cell cycle arrest induced by α-santalol was associated with changes in the protein levels of BRCA1, Chk1, G2/M regulatory cyclins, Cyclin dependent kinases (CDKs), Cell division cycle 25B (Cdc25B), Cdc25C and Ser-216 phosphorylation of Cdc25C. An up-regulated expression of CDK inhibitor p21 along with suppressed expression of mutated p53 was observed in MDA-MB-231 cells treated with α-santalol. On the contrary, α-santalol did not increase the expression of wild-type p53 and p21 in MCF-7 cells. In addition, α-santalol induced extrinsic and intrinsic pathways of apoptosis in both cells with activation of caspase-8 and caspase-9. It led to the activation of the executioner caspase-6 and caspase-7 in α-santalol-treated MCF-7 cells and caspase-3 and caspase-6 in MDA-MB-231 cells along with strong cleavage of poly(ADP-ribose) polymerase (PARP) in both cells. Taken together, this study for the first time identified strong anti-neoplastic effects of α-santalol against both ER-positive and ER-negative breast cancer cells.     

Ras puts the brake on doxorubicin-mediated cell death in p53-expressing cells

Doxorubicin is one of the most effective molecules used in the treatment of various tumors. Contradictory reports often open windows to understand the role of p53 tumor suppressor in doxorubicin-mediated cell death. In this report, we provide evidences that doxorubicin induced more cell death in p53-negative tumor cells. Several cells, having p53 basal expression, showed increase in p53 DNA binding upon doxorubicin treatment. Doxorubicin induced cell death in p53-positive cells through expression of p53-dependent genes and activation of caspases and caspase-mediated cleavage of cellular proteins. Surprisingly, in p53-negative cells, doxorubicin-mediated cell death was more aggressive (faster and intense). Doxorubicin increased the amount of Fas ligand (FasL) by enhancing activator protein (AP) 1 DNA binding in both p53-positive and p53-negative cells, but the basal expression of Fas was higher in p53-negative cells. Anti-FasL antibody considerably protected doxorubicin-mediated cell death in both types of cells. Activation of caspases was faster in p53-negative cells upon doxorubicin treatment. In contrast, the basal expression of Ras oncoprotein was higher in p53-positive cells, which might increase the basal expression of Fas in these cells. Overexpression of Ras decreased the amount of Fas in p53-negative cells, thereby decreasing doxorubicin-mediated aggressive cell death. Overall, this study will help to understand the much studied chemotherapeutic drug, doxorubicin-mediated cell signaling cascade, that leads to cell death in p53-positive and -negative cells. High basal expression of Fas might be an important determinant in doxorubicin-mediated cell death in p53-negative cells.     

Involvement of p53 in gemcitabine mediated cytotoxicity and radiosensitivity in breast cancer cell lines

Gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdCyd) is one of the anti-metabolites drugs that target DNA replication. We evaluated dFdCyd cytotoxicity and its radiosensitizing ability in human breast cancer cell lines, MCF-7 (wild-type p53) and MDA-MB-231 (mutant-type p53) along with normal mammary epithelial cell line (MCF-12) for comparison. Radiosensitivity and cytotoxicity were measured by the clonogenic survival assays. DNA DSBs was studied by Pulse Field Gel Electrophoresis (PFGE) and cell cycle distribution was analyzed by flow cytometry. MDA-MB-231 cells were the most sensitive to the cytotoxicity of dFdCyd (IC(50) 5 nM) then MCF-7 (IC(50) 10nM), whereas MCF-12 cells were the most resistant to the cytotoxicity of dFdCyd (IC(50) 70 nM). MCF-12 and MCF-7 cell lines did not show any radiosensitization to dFdCyd, whereas the MDA-MB-231 cells showed significantly increased radioresistant to dFdCyd at equimolar concentration (p=0.002) and at IC(50) concentration (p<0.001). The DNA double strand breaks (DSBs) repair showed that dFdCyd neither increases DNA DSBs nor decreases the rate of their repair in MCF-12 and MCF-7 cell lines, while the same treatment in MDA-MB-231 cell line led to decrease the rate of DSBs or increase the rate of DNA repair (p=0.034). Therefore, dFdCyd is a cytotoxic agent, especially in the cancer cells irrespective of having wild-type or mutated p53 protein, but it is not effective as radiosensitizer in the cell lines used in this study. dFdCyd combined with radiation reduces the efficacy of chemo-radiotherapy in p53 mutated cells. Therefore, p53-mutated cancer could be a counter-indication for radiation-gemcitabine combined treatment.     

Lactoferrin inhibits G1 cyclin-dependent kinases during growth arrest of human breast carcinoma cells.

Lactoferrin inhibits cell proliferation and suppresses tumor growth in vivo. However, the molecular mechanisms underlying these effects remain unknown. In this in vitro study, we demonstrate that treatment of breast carcinoma cells MDA-MB-231 with human lactoferrin induces growth arrest at the G1 to S transition of the cell cycle. This G1 arrest is associated with a dramatic decrease in the protein levels of Cdk2 and cyclin E correlated with an inhibition of the Cdk2 kinase activity. Cdk4 activity is also significantly decreased in the treated cells and is accompanied by an increased expression of the Cdk inhibitor p21(CIP1). Furthermore, we show that lactoferrin maintains the cell cycle progression regulator retinoblastoma protein pRb in a hypophosphorylated form. Additional experiments with synchronized cells by serum depletion confirm the anti-proliferative activity of human lactoferrin. These effects of lactoferrin occur through a p53-independent mechanism both in MDA-MB-231 cells and other epithelial cell lines such as HBL-100, MCF-7, and HT-29. These findings demonstrate that lactoferrin induces growth arrest by modulating the expression and the activity of key G1 regulatory proteins.     

Differential regulation of signal transduction pathways in wild type and mutated p53 breast cancer epithelial cells by copper and zinc

Previous studies have suggested that cells may differ in their response to metal stress. This study was undertaken to investigate the role of PI3K/Akt signaling pathway in metal resistance in human breast cancer epithelial cells with different p53 and estrogen receptor status. Exposure to copper and zinc increased Akt phosphorylation with its nuclear localization only in MDA-MB-231 cells with no estrogen receptor and mutated p53. Cyclin D1 expression and cell-cycle progression followed the metal-induced Akt phosphorylation. Treatment with LY294002 abrogated these effects, suggesting the essential role of PI3-kinase. In contrast, in MCF-7 cells with wild type p53 and estrogen receptor, there was no change in Akt activation, while suppression of p53 activity by pifithrin-alpha increased phosphorylation of Akt after the treatment with copper. In MCF-7 cells, the metal treatment increased the phosphorylation of p53 at serine 15, up-regulated p21 expression, and resulted in cell-cycle arrest in G1 phase with apoptosis. These results demonstrate that copper-induced apoptosis in MCF-7 cells is p53 dependent, whereas the metal resistance in MDA-MB-231 cells may be due to activation of Akt in the absence of a functional p53.     

Breast cancer cell behaviors on staged tumorigenesis-mimicking matrices derived from tumor cells at various malignant stages

Extracellular matrix (ECM) has been focused to understand tumor progression in addition to the genetic mutation of cancer cells. Here, we prepared “staged tumorigenesis-mimicking matrices” which mimic in vivo ECM in tumor tissue at each malignant stage to understand the roles of ECM in tumor progression. Breast tumor cells, MDA-MB-231 (invasive), MCF-7 (non-invasive), and MCF-10A (benign) cells, were cultured to form their own ECM beneath the cells and formed ECM was prepared as staged tumorigenesis-mimicking matrices by decellularization treatment. Cells showed weak attachment on the matrices derived from MDA-MB-231 cancer cells. The proliferations of MDA-MB-231 and MCF-7 was promoted on the matrices derived from MDA-MB-231 cancer cells whereas MCF-10A cell proliferation was not promoted. MCF-10A cell proliferation was promoted on the matrices derived from MCF-10A cells. Chemoresistance of MDA-MB-231 cells against 5-fluorouracil increased on only matrices derived from MDA-MB-231 cells. Our results showed that the cells showed different behaviors on staged tumorigenesis-mimicking matrices according to the malignancy of cell sources for ECM preparation. Therefore, staged tumorigenesis-mimicking matrices might be a useful in vitro ECM models to investigate the roles of ECM in tumor progression.     

MicroRNA-3646 Contributes to Docetaxel Resistance in Human Breast Cancer Cells by GSK-3β/β-Catenin Signaling Pathway

Acquisition of resistance to docetaxel (Doc) is one of the most important problems in treatment of breast cancer patients, but the underlying mechanisms are still not fully understood. In present study, Doc-resistant MDA-MB-231 and MCF-7 breast cancer cell lines (MDA-MB-231/Doc and MCF-7/Doc) were successfully established in vitro by gradually increasing Doc concentration on the basis of parental MDA-MB-231 and MCF-7 cell lines (MDA-MB-231/S and MCF-7/S). The potential miRNAs relevant to the Doc resistance were screened by miRNA microarray. We selected 5 upregulated miRNAs (has-miR-3646, has-miR-3658, has-miR-4438, has-miR-1246, and has-miR-574-3p) from the results of microarray for RT-qPCR validation. The results showed that expression level of miR-3646 in MDA-MB-231/Doc cells was significantly higher than in MDA-MB-231/S cells. Compared to MCF-7/S cells, miR-3646 expression was up-regulated in MCF-7/Doc cells. Further studies revealed that transfection of miR-3646 mimics into MDA-MB-231/S or MCF-7/S cells remarkably increased their drug resistance, in contrast, transfection of miR-3646 inhibitors into MDA-MB-231/Doc or MCF-7/Doc cells resulted in significant reduction of the drug resistance. By the pathway enrichment analyses for miR-3646, we found that GSK-3β/β-catenin signaling pathway was a significant pathway, in which GSK-3β was an essential member. RT-qPCR and Western blot results demonstrated that miR-3646 could regulate GSK-3β mRNA and protein expressions. Furthermore, a marked increase of both nuclear and cytoplasmic β-catenin expressions (with phosphorylated-β-catenin decrease) was observed in MDA-MB-231/Doc cells compared with MDA-MB-231/S cells, and their expression were positively related to miR-3646 and negatively correlated with GSK-3β. Taken together, our results suggest that miR-3646-mediated Doc resistance of breast cancer cells maybe, at least in part, through suppressing expression of GSK-3β and resultantly activating GSK-3β/β-catenin signaling pathway.     

NHE1 mediates MDA-MB-231 cells invasion through the regulation of MT1-MMP

Na⁺/H⁺ exchanger 1 (NHE1), an important regulator of intracellular pH (pH_i) and extracellular pH (pH_e), has been shown to play a key role in breast cancer metastasis. However, the exact mechanism by which NHE1 mediates breast cancer metastasis is not yet well known. We showed here that inhibition of NHE1 activity, with specific inhibitor Cariporide, could suppress MDA-MB-231 cells invasion as well as the activity and expression of MT1-MMP. Overexpression of MT1-MMP resulted in a distinguished increase in MDA-MB-231 cells invasiveness, but treatment with Cariporide reversed the MT1-MMP-mediated enhanced invasiveness. To explore the role of MAPK signaling pathways in NHE1-mediated breast cancer metastasis, we compared the difference of constitutively phosphorylated ERK1/2, p38 MAPK and JNK in non-invasive MCF-7 cells and invasive MDA-MB-231cells. Interestingly, we found that the phosphorylation levels of ERK1/2 and p38 MAPK in MDA-MB-231 cells were higher than in MCF-7 cells, but both MCF-7 cells and MDA-MB-231 cells expressed similar constitutively phosphorylated JNK. Treating MDA-MB-231 cells with Cariporide led to decreased phosphorylation level of both p38 MAPK and ERK1/2 in a time-dependent manner, but JNK activity was not influenced. Supplementation with MAPK inhibitor (MEK inhibitor PD98059, p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125) or Cariporide all exhibited significant depression of MDA-MB-231 cells invasion and MT1-MMP expression. Furthermore, we co-treated MDA-MB-231 cells with MAPK inhibitor and Cariporide. The result showed that Cariporide synergistically suppressed invasion and MT1-MMP expression with MEK inhibitor and p38 MAPK inhibitor, but not be synergistic with the JNK inhibitor. These findings suggest that NHE1 mediates MDA-MB-231 cells invasion partly through regulating MT1-MMP in ERK1/2 and p38 MAPK signaling pathways dependent manner.     

Context dependent reversion of tumor phenotype by connexin-43 expression in MDA-MB231 cells and MCF-7 cells: Role of β-catenin/connexin43 association

Connexins (Cx), gap junction (GJ) proteins, are regarded as tumor suppressors, and Cx43 expression is often down regulated in breast tumors. We assessed the effect of Cx43 over-expression in 2D and 3D cultures of two breast adenocarcinoma cell lines: MCF-7 and MDA-MB-231. While Cx43 over-expression decreased proliferation of 2D and 3D cultures of MCF-7 by 56% and 80% respectively, MDA-MB-231 growth was not altered in 2D cultures, but exhibited 35% reduction in 3D cultures. C-terminus truncated Cx43 did not alter proliferation. Untransfected MCF-7 cells formed spherical aggregates in 3D cultures, and MDA-MB-231 cells formed stellar aggregates. However, MCF-7 cells over-expressing Cx43 formed smaller sized clusters and Cx43 expressing MDA-MB-231 cells lost their stellar morphology. Extravasation ability of both MCF-7 and MDA-MB-231 cells was reduced by 60% and 30% respectively. On the other hand, silencing Cx43 in MCF10A cells, nonneoplastic human mammary cell line, increased proliferation in both 2D and 3D cultures, and disrupted acinar morphology. Although Cx43 over-expression did not affect total levels of β-catenin, α-catenin and ZO-2, it decreased nuclear levels of β-catenin in 2D and 3D cultures of MCF-7 cells, and in 3D cultures of MDA-MB-231 cells. Cx43 associated at the membrane with α-catenin, β-catenin and ZO-2 in 2D and 3D cultures of MCF-7 cells, and only in 3D conditions in MDA-MB-231 cells. This study suggests that Cx43 exerts tumor suppressive effects in a context-dependent manner where GJ assembly with α-catenin, β-catenin and ZO-2 may be implicated in reducing growth rate, invasiveness, and, malignant phenotype of 2D and 3D cultures of MCF-7 cells, and 3D cultures of MDA-MB-231 cells, by sequestering β-catenin away from nucleus.