Human Epithelial Cell Line MCF-10A Research Articles

MiR-381 inhibited breast cancer cells proliferation, epithelial-to-mesenchymal transition and metastasis by targeting CXCR4

BackgroundMicroRNAs act as posttranscriptional regulators of gene expression in many biological processes, which played a vital role in regulation cancer cells epithelial-to-mesenchymal transition and metastasis. The deregulation of miR-381 has been identified in breast cancer. However, the role and mechanism of miR-381 in breast cancer have not been completely unexplored. MethodsTotal RNA was extracted from the tissues of 27 patients with breast cancer and two breast cancer cell lines, respectively. The expression levels of miR-381 were examined by quantitative real-time PCR. The stable overexpress or silence miR-381 expression cells lines and control cells line were constructed by lentivirus infection. Subsequently, cell proliferation, cell migration, invasion assay and western blot assay were performed to detect the biological functions of miR-381 in vitro. Moreover, a luciferase reporter assay was conducted to confirm target associations. ResultsIn this study, we validated the lower expression of miR-381 in breast cancer tissues than their adjacent non-neoplastic tissues in 27 breast cancer patients. The result also showed that miR-381 was lowly expressed in breast cancer cell lines MCF-7 and MDA-MB-231 than human epithelial cell line MCF-10A. The miR-381 expression was significantly up-regulated under exogenous miRNA-381 treatment in MCF-7 and MDA-MB-231 cells analyzed by quantitative real-time PCR. The results also indicated that an inverse correlation existed between miR-381 expression level and breast cancer cell proliferation, epithelial-to-mesenchymal transition and metastasis. Furthermore, miR-381 was predicted as a regulatory miRNA of CXCR4 in breast cancer, and the data analysis revealed that there was a negatively relationship between miR-381 and CXCR4 expression in breast cancer tissues from the patients. miR-381 played an important role in breast cancer cells proliferation, epithelial-to-mesenchymal transition and metastasis by targeting CXCR4. ConclusionsThis present study revealed that miR-381 might be considered as a novel therapeutic target for breast cancer treatment.

Fractal analysis of cell boundary ultrastructure imaged by atomic force microscopy

Fractal analysis has been widely used to determine the complexity of irregular shapes of various natural objects including biological systems. Here we report an investigation into the nature of the cell boundary nanostructure (less than 200 nm in thickness) in an aqueous environment using atomic force microscopy. The box-counting method revealed the fractal nature of detailed contours of cells cultured on a solid substratum. The fractal dimensions (Df) of individual cells of the human epithelial cell line MCF10A converged on a narrow range (Df = 1.352 ± 0.044) despite their morphological diversity. Treatment with cytochalasin D, an inhibitor of actin polymerization, was shown to decrease both the ruggedness and the Df of cell boundaries. These findings suggest that the Df value is a useful parameter for describing the complexity of cellular ultrastructure dependent on the actin cytoskeleton.

Label-free in vivo molecular imaging of underglycosylated mucin-1 expression in tumour cells.

Alterations in mucin expression and glycosylation are associated with cancer development. Underglycosylated mucin-1 (uMUC1) is overexpressed in most malignant adenocarcinomas of epithelial origin (for example, colon, breast and ovarian cancer). Its counterpart MUC1 is a large polymer rich in glycans containing multiple exchangeable OH protons, which is readily detectable by chemical exchange saturation transfer (CEST) MRI. We show here that deglycosylation of MUC1 results in >75% reduction in CEST signal. Three uMUC1+ human malignant cancer cell lines overexpressing uMUC1 (BT20, HT29 and LS174T) show a significantly lower CEST signal compared with the benign human epithelial cell line MCF10A and the uMUC1− tumour cell line U87. Furthermore, we demonstrate that in vivo CEST MRI is able to make a distinction between LS174T and U87 tumour cells implanted in the mouse brain. These results suggest that the mucCEST MRI signal can be used as a label-free surrogate marker to non-invasively assess mucin glycosylation and tumour malignancy.

The chemokine CXCL9 exacerbates chemotherapy-induced acute intestinal damage through inhibition of mucosal restitution.

Acute intestinal damage induced by chemotherapeutic agent is often a dose-limiting factor in clinical cancer therapy. The aim of this study was to investigate the effect of chemokine CXCL9 on the intestinal damage after chemotherapy and explore the therapeutic potential of anti-CXCL9 agents. In vitro cell proliferation assay was performed with a non-tumorigenic human epithelial cell line MCF10A. Multiple pathway analysis was carried out to explore the pathway that mediated the effect of CXCL9, and the corresponding downstream effector was identified with enzyme-linked immunosorbent assays. Chemotherapy-induced mouse model of intestinal mucositis was prepared by a single injection of the chemotherapeutic agent 5-fluorouracil (5-FU). In vivo expression of cxcl9 and its receptor cxcr3 in intestinal mucosa after chemotherapy was determined by quantitative real-time PCR. Therapeutic treatment with anti-CXCL9 antibodies was investigated to confirm the hypothesis that CXCL9 can contribute to the intestinal epithelium damage induced by chemotherapy. CXCL9 inhibited the proliferation of MCF10A cells by activating phosphorylation of p70 ribosomal S6 kinase (p70S6K), which further promotes the secretion of transforming growth factor beta (TGF-β) as the downstream effector. A blockade of phospho-p70S6K with inhibitor abolished the effect of CXCL9 on MCF10A cells and reduced the secretion of TGF-β. The expression levels of cxcl9 and cxcr3 were significantly up-regulated in intestinal mucosa after 5-FU injection. Neutralizing elevated CXCL9 with anti-CXCR9 antibodies successfully enhanced reconstitution of intestinal mucosa and improved the survival rate of mice that received high-dose chemotherapy. CXCL9 inhibits the proliferation of epithelial cells via phosphorylation of p70S6K, resulting in the excretion of TGF-β as downstream mediator. CXCL9/CXCR3 interaction can exacerbate chemotherapeutic agent-induced intestinal damage, and anti-CXCL9 agents are potential novel therapeutic candidates for promoting mucosal restitution.

Sulfotransferase 1A1 (SULT1A1) gene expression is regulated by members of the NFI transcription factors in human breast cancer cells

BackgroundSulfotransferase 1A1 (SULT1A1) gene expression is tissue specific, with little to no expression in normal breast epithelia. Expression in breast tumors has been documented, but the transcriptional regulation of SULT1A1 in human breast tissue is poorly understood. We identified Nuclear Factor I (NFI) as a transcription factor family involved in the regulation of SULT1A1 expression.MethodsTranscription Factor Activation Profiling Plate Array assay was used to identify the possible transcription factors that regulate the gene expression of SULT1A1in normal breast MCF-10A cells and breast cancer ZR-75-1 cells. Expression levels of NFI-C and SULT1A1 were determined by real-time RT-PCR using total RNA isolated from 84 human liver samples. Expression levels of SULT1A1, NFI-A, NFI-B, NFI-C, and NFI-X were also determined in different human breast cancer cell lines (MCF-7, T-47D, ZR-75-1, and MDA-MB-231), in the transformed human epithelial cell line MCF-10A, and in ZR-75-1 cells that were transfected with siRNAs directed against NFI-A, NFI-B, NFI-C, or NFI-X for 48 h. The copy numbers of SULT1A1 in cell lines ZR-75-1, MCF-7, T-47D, MDA-MB-231, and MCF-10A were determined using a pre-designed Custom Plus TaqMan® Copy Number kit from Life Technologies.ResultsIn normal human liver samples, SULT1A1 mRNA level was positively associated with NFI-C. In different human breast cancer and normal epithelial cell lines, SULT1A1 expression was positively correlated with NFI-B and NFI-C. SULT1A1 expression was decreased 41% and 61% in ZR-75-1 cells treated with siRNAs against NFI-A and NFI-C respectively. SULT1A1 gene expression was higher in cells containing more than one SULT1A1 copy numbers.ConclusionsOur data suggests that SULT1A1 expression is regulated by NFI, as well as SULT1A1 copy number variation in human breast cancer cell lines. These data provide a mechanistic basis for the differential expression of SULT1A1 in different tissues and different physiological states of disease.

Abstract B23: Sulfotransferase isoform 1A1 (SULT1A1) gene expression is regulated by transcription factor NF1 in human breast cancer cell lines.

Abstract Sulfotransferase isoform 1A1 (SULT1A1), a cytosolic phase II detoxification enzyme, plays a central role in drug metabolism, and breast cancer treatment and prevention drugs such as tamoxifen (TAM) and raloxifene (RAL) are among its substrates. Thus, regulation of this gene in target tissues could influence the efficacy of therapy. SULT1A1 activity varies several-fold in human populations, and genetic variants within the gene only account for a small proportion of the observed variability. Expression also varies with tissue type, with SULT1A1 being more highly expressed in breast tumors than in normal breast tissue. We have identified human nuclear factor 1(NF1) as a potential transcription factor (TF) regulating SULT1A1 gene expression in the ZR-75 breast cancer cell line. The NF1 gene family consists of NF1A, NF1B, NF1C, and NF1X. In order to determine the potential transcriptional regulation of SULT1A1 by NF1 genes in breast cancer in vitro, we measured and compared the mRNA levels of SULT1A1, NF1A, NF1B, NF1C, and NF1X in different human breast cancer cell lines (MCF7, T47D, ZR-75, and MDA-231) and the transformed human epithelial cell line MCF10A. SULT1A1 gene expression in each cell line was highly positively correlated with the gene expression of NF1B (p<0.01), NF1C (p<0.01), and NF1X (p<0.05). To determine if NF1s had a direct effect on SULT1A1 gene expression, ZR-75 cells, which have highest SULT1A1 gene expression among the cell lines tested, were transfected with siRNAs directed against NF1A, NF1B, NF1C, or NF1X for 48 hr. Real-time RT-PCR showed that mRNA levels of NF1A, NF1B, NF1C, and NF1X were decreased 70% ± 0.02, 87% ± 0.08, 81% ± 0.08, and 86% ± 0.12, respectively after transfection. SULT1A1 gene expression decreased 41% ± 0.05 (p=0.05) in siNF1A transfected cells and 64% ± 0.08 (p=0.05) in siNF1C transfected cells. ZR-75 cells were also co-transfected with siNF1A, siNF1B, and siNF1C for 48 hr. SULT1A1 mRNA level was decreased approximately 40%, indicating that there was no additive transcription regulatory effect on SULT1A1 gene expression among those three NF1s. Interestingly, SULT1A1 gene expression was decreased more than four-fold in siNF1B transfected MCF7 cells. In summary, our data demonstrates that SULT1A1 activity is partially regulated at the transcription level by transcription factors NF1A, NF1B, and NF1C. We are currently examining the expression levels of these genes in both normal and breast tumor tissue to identify a mechanistic explanation for the differential expression of SULT1A1 between normal breast tissue and breast tumors. This knowledge will lead to better predictors of therapeutic efficacy for chemopreventive agents that are substrates of SULT1A1. Citation Format: Aiwei Yao-Borengassar, Lora Rogers, Vinay Raj, Susan Kadlubar. Sulfotransferase isoform 1A1 (SULT1A1) gene expression is regulated by transcription factor NF1 in human breast cancer cell lines. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B23.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Downregulation of aryl hydrocarbon receptor function and cytochrome P450 1A1 induction by expression of Ha-ras oncogenes.

The immortalized human epithelial cell line MCF10A has the phenotypic characteristics of normal breast cells. Exposure of MCF10A cultures to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) stimulated the transcriptional activation of cytochrome P450 1A1 (CYP1A1), and CYP1B1, and NAD(P)H:quinone oxidoreductase. Northern blot hybridization and nuclear run-on assays demonstrated that transcriptional activation of these genes was suppressed in stably transfected cultures expressing an Ha-ras oncogene (the MCF10A-NeoT line). Similar suppression did not occur in stably transfected lines carrying the expression vector or a normal c-Ha-ras protooncogene. Western blot analyses and immunofluorescence microscopy demonstrated that the lack of inducibility in MDF10A-NeoT cells reflected neither reductions in aryl hydrocarbon receptor (AHR) and aryl hydrocarbon nuclear translocator protein nor prevention of TCDD-induced AHR translocation to the nucleus. Suppression did correlate with reductions in DNA-AHR complex formation, as analyzed by gel retardation assays of soluble cell extracts treated in vitro with TCDD. The induction of Cyp1a-1 by TCDD was also analyzed in transgenic mice that expressed a v-Ha-ras oncogene exclusively in their keratinocytes. Relative to littermates lacking the transgene, the induction of Cyp1a-1 by TCDD was partially suppressed (about 50%) in the epidermises of v-Ha-ras-positive transgenic mice. However, normal levels of Cyp1a-1 induction occurred in the livers of the same mice. induction of Cyp1a-1 by TCDD was also suppressed (more than 98%) in chemically induced skin papillomas having Ha-ras mutations, relative to uninvolved surrounding skin. These studies suggest that the p21-ras protein controls signal transduction pathways capable of modulating AHR function.