Stable MCF-7 Cells Research Articles

The Glycosylated N-Terminal Domain of MUC1 Is Involved in Chemoresistance by Modulating Drug Permeation Across the Plasma Membrane.

Mucin 1 (MUC1) is aberrantly expressed in various cancers and implicated in cancer progression and chemoresistance. Although the C-terminal cytoplasmic tail of MUC1 is involved in signal transduction, promoting chemoresistance, the role of the extracellular MUC1 domain [N-terminal glycosylated domain (NG)-MUC1] remains unclear. In this study, we generated stable MCF7 cell lines expressing MUC1 and cytoplasmic tail-deficient MUC1 (MUC1ΔCT) and show that NG-MUC1 is involved in drug resistance by modulating the transmembrane permeation of various compounds without cytoplasmic tail signaling. Heterologous expression of MUC1ΔCT increased cell survival in treating anticancer drugs (such as 5-fluorouracil, cisplatin, doxorubicin, and paclitaxel), in particular by causing an approximately 150-fold increase in the IC50 of paclitaxel, a lipophilic drug, compared with the control [5-fluorouracil (7-fold), cisplatin (3-fold), and doxorubicin (18-fold)]. The uptake studies revealed that accumulations of paclitaxel and Hoechst 33342, a membrane-permeable nuclear staining dye, were reduced to 51% and 45%, respectively, in cells expressing MUC1ΔCT via ABCB1/P-gp-independent mechanisms. Such alterations in chemoresistance and cellular accumulation were not observed in MUC13-expressing cells. Furthermore, we found that MUC1 and MUC1ΔCT increased the cell-adhered water volume by 2.6- and 2.7-fold, respectively, suggesting the presence of a water layer on the cell surface created by NG-MUC1. Taken together, these results suggest that NG-MUC1 acts as a hydrophilic barrier element against anticancer drugs and contributes to chemoresistance by limiting the membrane permeation of lipophilic drugs. Our findings could help better the understanding of the molecular basis of drug resistance in cancer chemotherapy. SIGNIFICANCE STATEMENT: Membrane-bound mucin (MUC1), aberrantly expressed in various cancers, is implicated in cancer progression and chemoresistance. Although the MUC1 cytoplasmic tail is involved in proliferation-promoting signal transduction thereby leading to chemoresistance, the significance of the extracellular domain remains unclear. This study clarifies the role of the glycosylated extracellular domain as a hydrophilic barrier element to limit the cellular uptake of lipophilic anticancer drugs. These findings could help better the understanding of the molecular basis of MUC1 and drug resistance in cancer chemotherapy.

Effect of long noncoding RNA PANDAR on the proliferation and invasion of breast cancer cells

Objective To investigate the effect of long noncoding RNA (lncRNA) PANDAR on the proliferation and invasion of breast cancer cells. Methods We constructed the stable MCF-7 cells with overexpression or knockdown of PANDAR by using lentivirus system and detected the cell proliferation and invasion by cell counting kit-8 (CCK-8) assay and matrigel Transwell assay. Results The relative expression of PANDAR in MCF-7 cells with overexpression of PANDAR was 20.05±1.08, higher than in the control group (P=0.000), and that in MCF-7 cells with knockdown of PANDAR was 0.16±0.02, lower than in the control group (P=0.008). The cell proliferation activity of PANDAR overexpression group (0.489±0.067, 0.945±0.025, 1.236±0.072 ) at 24, 48 and 72 h, were higher than these in control group (0.385±0.024, 0.576±0.059, 0.737±0.041; P=0.045, P=0.024, P=0.012, respectively). The cell proliferation activity of PANDAR knockdown group (0.292±0.055, 0.371±0.041, 0.455±0.068) at 24, 48 and 72 h, were significantly lower than these in control group (P=0.036, P=0.030, P=0.014), respectively. The amount of invasion cells in PANDAR overexpression group were significantly more than control group (48.37±13.08 vs. 25.15±6.75; P=0. 011). While the amount of invasion cells in PANDAR knockdown group were significantly less than control group (23.35±2.96 vs. 50.13±11.87; P=0. 015). Conclusion PANDAR promotes cell proliferation and invasion of breast caner cells. Key words: Long noncoding RNA; Breast cancer

Epidermal Growth Factor Receptor (EGFR)-mediated Positive Feedback of Protein-tyrosine Phosphatase ϵ (PTPϵ) on ERK1/2 and AKT Protein Pathways Is Required for Survival of Human Breast Cancer Cells

Increased tyrosine phosphorylation has been correlated with human cancer, including breast cancer. In general, the activation of tyrosine kinases (TKs) can be antagonized by the action of protein-tyrosine phosphatases (PTPs). However, in some cases PTPs can potentiate the activation of TKs. In this study, we have investigated the functional role of PTPε in human breast cancer cell lines. We found the up-regulation and activation of receptor PTPε (RPTPε) in MCF-7 cells and MDA-MB-231 upon PMA, FGF, and serum stimulation, which depended on EGFR and ERK1/2 activity. Diminishing the expression of PTPε in human breast cancer cells abolished ERK1/2 and AKT activation, and decreased the viability and anchorage-independent growth of the cells. Conversely, stable MCF-7 cell lines expressing inducible high levels of ectopic PTPε displayed higher activation of ERK1/2 and anchorage-independent growth. Our results demonstrate that expression of PTPε is up-regulated and activated in breast cancer cell lines, through EGFR, by sustained activation of the ERK1/2 pathway, generating a positive feedback regulatory loop required for survival of human breast cancer cells.

Mitochondrially targeted ceramides preferentially promote autophagy, retard cell growth, and induce apoptosis

C(6)-pyridinium (D-erythro-2-N-[6'-(1''-pyridinium)-hexanoyl]sphingosine bromide [LCL29]) is a cationic mitochondrion-targeting ceramide analog that promotes mitochondrial permeabilization and cancer cell death. In this study, we compared the biological effects of that compound with those of D-erythro-C(6)-ceramide, its non-mitochondrion-targeting analog. In MCF7 cells it was found that C(6)-pyridinium ceramide preferentially promoted autophagosome formation and retarded cell growth more extensively than its uncharged analog. This preferential inhibition of cell growth was also observed in breast epithelial cells and other breast cancer cells. In addition, this compound could promote Bax translocation to mitochondria. This redistribution of Bax in MCF7 cells could be blocked by the pan-caspase inhibitor zVAD-fmk but via a Bid-independent signaling pathway. Moreover, C(6)-pyridinium ceramide-induced translocation of Bax to mitochondria led to mitochondrial permeabilization and cell death. Overall, we show that mitochondrial targeting of C(6)-pyridinium ceramide significantly enhances cellular response to this compound.

Opposite regulation of estrogen receptor-α and its variant ER-α36 by the Wilms’ tumor suppressor WT1

The genomic and non-genomic signaling pathways are well-known estrogen signaling pathways. The 66-kDa estrogen receptor-α (ER-α66) is a typical ligand-inducible transcription factor that mainly mediates genomic estrogen signaling. Recently, we identified and cloned a 36-kDa variant of ER-α66, known as ER-α36. This variant lacks intrinsic transcription activity and predominantly mediates non-genomic estrogen signaling. Thus, the expression of ER-α66 and ER-α36 should be firmly regulated and carefully correlated to maintain a balance between genomic and non-genomic estrogen signaling. However, the molecular mechanisms underlying this correlation remain poorly understood. The Wilms' tumor suppressor gene, wt1, encodes a zinc-finger protein WT1 that functions as a dual transcription regulator to activate or suppress gene transcription. High levels of WT1 expression are associated with breast cancer malignancy. In the present study, high-passage ER-positive breast cancer MCF7 cells were found to express ER-α66 and WT1 at higher levels and ER-α36 at a very low level. Using the small hairpin RNA method, stable MCF7 cells were established that expressed knocked-down levels of WT1. The cells expressed a reduced level of ER-α66 but an increased level of ER-α36, suggesting that WT1 regulates the expression of ER-α66 and ER-α36 oppositely. Further co-transfection assays showed that all isoforms of WT1 directly activated the promoter activity of the ER-α66 gene while suppressing ER-α36 promoter activity. Our results therefore indicate that WT1 acts as a dual transcription factor that regulates the promoter activity of ER-α66 and ER-α36 oppositely, implicating WT1 as one of the coordinators that orchestrate genomic and non-genomic estrogen signaling.

GCIP Is a Tumor Suppressor in Breast Cancer.

Abstract Background: GCIP protein is a HLH-leucine zipper (HLH-LZ) proteins without basic DNA-binding region, which was identified to be a cyclin D1-binding protein and also was recognized as a human homologue of MAID protein (HHM). Recently, GCIP was found to function as a tumor suppressor in liver and colon tumorigenesis. In this study, we examined in vitro the functional role of GCIP in various breast cancer cell lines.Material and Methods: GCIP-directed siRNA construct was prepared and stable downregulation of GCIP in epithelial-like and nonaggressive/differentiated breast cancer cell line, MCF-7, were selected. In addition, two aggressive/ invasive breast cancer cells (MDA-MB231 and MDA-MB435s) that exhibit low levels of endogenous GCIP expression were transfected with p3x-GCIP-flag, respectively and stable GCIP-re-expressing transfectants were selected. The phalloidin staining cell morphology and functional assays with these stable GCIP downregulating or GCIP re-expressing were performed to characterize the biological effects of GCIP.Results: Stable MCF-7 cells with down-regulated GCIP expression acquired a mesenchymal-like shape and exhibited increased colony formation, migration and invasion capabilities. Furthermore, this was found to be associated with the loss of membrane E-cadherin and increased expression of the mescenchymal marker vimentin. In addition, both the stable MDA-MB231 and MDA-MB435s with GCIP-re-expression acquired an epithelial-like shape and exhibited decreased colony formation, migration and invasion capabilities.Conclusion: our previous study found that decreased expression of GCIP in breast carcinomas compared with normal ductal epithelium (P < 0.001). In this study, we present evidence that GCIP inactivation contributes to the enhanced migratory and invasive capacity of MCF-7 and that GCIP re-expression inhibits a wild range of cellular functions related to the malignant properties of MDA-MB231 and MDA-MB435s, confirming that GCIP is a tumor suppressor in breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3158.

The effects of transfection of CXCL10 gene into breast cancer cell line MCF-7 on expression of tumor related genes

Objective To construct stable transfected MCF-7 cell line by recombinant plasmid CXCL10-pcDNA3.1 (+) and explore the effects of CXCL10 on expression of tumor related genes. Methods CXCL10 gene, cloned from treated PBMCs by IFN-γ, was used to construct recombinant plasmid CXCL10-pcDNA3.1 (+). Recombinant plasmid CXCL10-pcDNA3.1 (+) and control plasmid PcDNA3.1 (+) were separately transfected into MCF-7 cells by lipifection-2000. The stable transfected cell lines were screened by G418. The transcription, expression and secretion of transfected CXCL10 gene was assayed by RT-PCR, Western blot and ELISA in turn. The cell growth activity of stable transfected cell lines was detected by soft agar cloning formation in experimental group [transfected by CXCL10-pcDNA 3.1 (+) ], control group [transfected by pcDNA3. 1 (+)] and blank group (nothing transfected). Furthermore, the reverse transcriptions of three tumor related genes, included MMP9, VEGF and STAT3, were assayed by RT-PCR. Results The stable MCF-7 cell lines transfected by combined plasmid CXCL10-pcDNA3.1 (+) and plasmid pcDNA3.1 (+) were constructed successfully. There was no significant difference in cell growth activity rate between the experimental group and the control group (χ2=3.8, P>0.05, the rates of the 3 groups were 78.2%, 79.9% and 87.8% in turn). There was significant difference in CXCL10 mRNA expression level between the experimental group and the control group (F=50.5, P 0.05, the gray values of the 3 groups were seperately 0.067±0.003, 0.103±0.007 and 0.085±0.026). Comparing with the control group, the experimental group showed significant lower expression level of VEGF (F=2 232, P<0.01, the gray values of the 3 groups were 0.183±0.003, 0.787±0.019 and 0.789±0.011) and STAT3 ( F=115, P<0.01, the gray values of the 3 groups were 0.573±0.016, 0.707±0.008 and 0.711±0.013). Conclusions CXCL10 can express stablely in MCF-7 cell lines, which resulted in down-regulation of expression of VEGF and STAT3 gene. CXCL10 played an important role in anti-tumor effect. Key words: Breast neoplasms; Cell line; tumor; Chemopkine CXCL10; Gene expression

Histone Deacetylase 2 Modulates p53 Transcriptional Activities through Regulation of p53-DNA Binding Activity

Histone deacetylase (HDAC) inhibitors are emerging as promising cancer therapeutics. HDAC inhibitors have been found to induce cellular activities that are strikingly similar to p53-mediated responses to genotoxic stress. For example, HDAC inhibitors induce cell cycle arrest, apoptosis, and cellular senescence. Because at least 11 HDACs are affected by the current HDAC inhibitors, the HDAC critical for tumor cell survival and proliferation remains unknown. Thus, we sought to characterize the distinct roles of HDACs in the p53 pathway. Through the use of stable MCF7 cell lines which inducibly express short hairpin RNA targeting HDAC2, we found that HDAC2 plays important roles in the p53 pathway. Specifically, we found that knockdown of HDAC2 inhibited cellular proliferation in a dose-dependent manner which was also partly p53-dependent. Furthermore, knockdown of HDAC2 induced cellular senescence. Importantly, we found that knockdown of HDAC2 enhanced p53-dependent trans-repression and trans-activation of a subset of target genes. We found that the enhancement was due to increased p53-DNA binding activity but not alterations in p53 stability or posttranslational modification(s). Thus, for the first time, our data suggest that HDAC inhibitors function through the p53 pathway, at least in part, by activating p53-DNA binding activity.

9-cis-Retinoic Acid Up-regulates Expression of Transcriptional Coregulator PELP1, a Novel Coactivator of the Retinoid X Receptor α Pathway

Retinoid X receptor alpha (RXRalpha), functioning as either a homodimer or a heterodimer with peroxisome proliferator receptors, is known to be involved in manifesting antiproliferative effects in cells. Consequently, studies of RXRalpha functions and its coregulators have been in the focus for therapeutic approaches against cancer. Here we have discovered that 9-cis-retinoic acid (9-cis-RA), a RXRalpha-specific ligand, up-regulated the expression of transcriptional coregulatory protein PELP1 (proline-, glutamic acid-, and leucine-rich protein 1). PELP1 functioned as a coactivator of RXRalpha, increasing its transactivation function in response to 9-cis-RA as evident by the retinoid X receptor response element-luciferase assays. PELP1 was found to be a binding partner of RXRalpha, and the binding interactions were confirmed both in vitro and in vivo. An electrophoretic mobility shift assay showed greater formation and stability of RXRalpha homodimers on consensus oligonucleotides in PELP1-overexpressing clones in comparison to the pcDNA clones. The presence of PELP1 in these oligonucleotide-bound RXRalpha homodimers was proved by the supershift of the complex when incubated with PELP1-specific antibody. PELP1-overexpressing stable MCF-7 cells exhibited a significantly higher extent of 9-cis-RA-induced apoptosis than the control pcDNA clones. Silencing of PELP1 expression in parental MCF-7 cells and PELP1-overexpressing clones using PELP1-specific RNA-mediated interference compromised the susceptibility to 9-cis-RA-induced apoptosis. PELP1 could also function as a coactivator of the RXRalpha-peroxisome proliferator-activated receptor (PPARgamma) heterodimer as evident by the peroxisome proliferator-activated receptor response element-luciferase assay in response to both 9-cis-RA and PPARgamma-specific ligands. This was reinforced by the higher propensity of PELP1-overexpressing clones to undergo differentiation in response to PPARgamma-specific ligands. This study has revealed a novel facet of PELP1 functions and identified it to be an important potentiator of the antiproliferative effects of 9-cis-RA and PPARgamma-specific ligands.

Levels of IL-1 beta control stimulatory/inhibitory growth of cancer cells

Different cellular signaling pathways operate in response to varying levels of IL-1 beta leading to genotoxic damage, cell apoptosis or cell growth. At high levels of IL-1 beta, cells receiving genotoxic insults engage apoptotic pathways. The IL-1 beta over expressing stable MCF7 cell secreting high level of IL-1 beta peptides undergo cell apoptosis. Cotreatment with an inhibitor of IL-1 beta and TNF-alpha synthesis prevented stilbene estrogen-induced lesions. In addition to direct effect of 17 beta-estradiol (E2) on mitochondria and redox cycling of catechol estrogens, E2-induced overexpression of IL-1 beta can produce an increase in the level of ROS. Our recent data showed that MCF7 cell growth and cyclin D1 expression are suppressed by antioxidants and mitochondrial blockers. Stably IL-1 beta transfected cells secreting moderate level of IL-1 beta peptides stimulated the clonal expansion of MCF7 cells. These studies support that in addition to ovarian estrogens, mitogenic signals may also come from TNF-alpha and IL-1 beta-generated O2*- and hydrogen peroxide. Further validation of this concept that the concentrations of the peptide interleukin-1 beta within the cells determine its stimulatory or inhibitory signals regulating the growth of estrogen-dependent tumors might result in novel preventive strategies.

Constitutive over expression of serine/threonine protein phosphatase 5 (PP5) augments estrogen-dependent tumor growth in mice

Serine/threonine protein phosphatase 5 (PP5) appears to play an underappreciated role in the regulation of cellular proliferation. In estrogen-responsive cells, PP5 expression is stimulated by 17 β-estradiol, and in a variety of p53 wild-type tumor cells the suppression of PP5 expression with ISIS 15534 inhibits growth. To further explore the relationship between PP5 and the development of human cancer, here we tested the effect of elevated PP5 expression on tumor growth using a mouse xenograph model and a stable MCF-7 cell line in which the expression of wild-type PP5 was placed under the control of tetracycline-off regulated transactivator and operator plasmids. In the xenograph model a modest two fold increase in PP5 protein levels significantly enhanced the growth rate of estrogen-dependent tumors, suggesting PP5 plays a positive role in tumor development.

Identification of an Estrogen-inducible Phosphatase (PP5) That Converts MCF-7 Human Breast Carcinoma Cells into an Estrogen-independent Phenotype when Expressed Constitutively

The proliferation of many estrogen receptor (ER)-positive breast cancer cells depends on estradiol, and tumors arising from these cells are often responsive initially to treatment with selective ER modulators, which produce an antiestrogen effect. However, tumors that are refractory to the antiestrogenic effects of selective ER modulators often reemerge, and the prognosis for these patients is poor because of the lack of additional effective therapy. Accordingly, deciphering the cellular events associated with estrogen-dependent growth and the subsequent outgrowth of tumors with an estrogen-independent phenotype is of considerable interest. Here we show that the expression of PP5, an evolutionarily conserved Ser/Thr phosphatase that functions as an inhibitor of glucocorticoid- and p53-induced signaling cascades leading to growth suppression, is responsive to 17beta-estradiol (E(2)) in ER-positive human breast carcinoma cells (MCF-7). Northern analysis revealed that E(2)-induced PP5 expression is blocked by treatment with tamoxifen, and a consensus ER recognition element was identified in the PP5 promoter. The PP5-ER recognition element associates with human ERs and confers E(2)-induced transcriptional activation to reporter plasmids. The specific inhibition of PP5 expression ablates E(2)-mediated proliferation in MCF-7 cells without having an apparent effect on E(2)-induced expression of c-myc or cyclin D1. Thus, although critical for cell growth, PP5 likely acts either downstream or independently of c-Myc and Cyclin D1. To further characterize the role of PP5 in E(2)-regulated growth control, we constructed stable MCF-7 cell lines in which the expression of PP5 was placed under the control of tetracycline-regulated transactivator and operator plasmids. Studies with these cells revealed that the constitutive overexpression of PP5 affords E(2)-dependent MCF-7 cells with the ability to proliferate in E(2)-depleted media. Together, these studies indicate that E(2)-induced PP5 expression functions to enhance E(2)-initiated signaling cascades leading to cell division and that aberrant PP5 expression may contribute to the development of MCF-7 cells with an estrogen-independent phenotype.