Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: Effects of hypoxia on cell markers, morphology, and migration (A) Expression in breast cancer MCF7 and MDA-MB-231 cells of E-cadherin (epithelial marker), fibronectin (epithelial marker), HIF-1α (hypoxia marker), β-catenin, and GLUT1. Cells were cultured at 37°C in 5% CO 2 atmosphere for normoxic treatment, and in 1% O 2 / 5% CO 2 / 94% N 2 atmosphere for hypoxic treatment. Cells were harvested, lysed in T-PER Reagent, and protein content was determined by BCA assay. Western blotting was performed as described in M&M. (B) Morphological changes under normoxic and hypoxic conditions. Cells (2 × 10 5 per well) were grown in 6-well plates for 24 h under the two conditions. Photos were taken by phase-contrast microscopy at 200× magnification. (C) Cell migration assessed by wound assay. Cell monolayers under the two conditions were scratched with pipette tip. Cells were washed with ice-cold 1× PBS and cultured in serum-free medium. Pictures of wounds were taken at 0 and 24 h by phase-contrast microscopy (100× magnification).

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Migration, Expressing, Marker, Cell Culture, BIA-KA, Western Blot, Microscopy, Transferring

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: MALDI-TOF-MS spectra of N-glycans from MCF7 cells MCF7 cells were cultured in 10-cm dishes under normoxic and hypoxic conditions, and N-glycans were separated and desalted as described in M&M. Lyophilized N-glycans were dissolved in MW, and an aliquot of mixture with DHB solution was spotted on MTP AnchorChip sample target and air-dried. MALTI-TOF-MS was performed in positive-ion mode. Experiments were performed in biological triplicate, and representative N-glycan spectra are shown. Peaks (signal-to-noise ratio > 5) were selected for relative proportion analysis. Detailed structures were analyzed using the GlycoWorkbench program. Proposed structures are indicated by m/z value.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Cell Culture, Glycoproteomics

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: Relative proportions of various types of N-glycans in MCF7 and MDA-MB-231 cells under normoxia and hypoxia.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques:

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: Variation of fine glycan structures detected by lectin microarray analysis (A) Variation of levels of glycans from MCF7 (upper) and MDA-MB-231 (lower) cells, detected by 37 lectins, is presented as a heatmap. Lectin microarray analysis was performed as described as M&M. Red: fluorescence signal activation. Green: signal inhibition. Black: missing data. (B) Altered glycan levels evaluated by lectin histochemistry. Four lectins (Con A, MAL-I, LCA, PHA-E) were applied, and lectin histochemistry was performed as described in M&M. Signals are shown from merge images of Cy3-conjugated lectins and DAPI staining of nuclei in MCF7 (left) and MDA-MB-231 (right) under normoxic and hypoxic conditions (60× magnification). (C) Expression in MCF7 and MDA-MB-231 cells of HIF-1α, MGAT3, and tubulin.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Glycoproteomics, Microarray, Fluorescence, Activation Assay, Inhibition, Staining, Expressing

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: Differential glycopatterns in normoxia- vs. hypoxia-treated MCF7 cells revealed by lectin microarray analysis.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Microarray

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: MGAT3 overexpression suppresses hypoxia-induced EMT in MCF7 cells (A) MGAT3 expression in mock- and MGAT3-transfected MCF7 cells. Cells were stably transduced with a GFP-marked lentivirus carrying mock gene or MGAT3 gene, harvested, and lysed in T-PER Reagent. Western blotting was performed as described in M&M using anti-MGAT3 and anti-GFP antibody. (B) Levels of bisecting GlcNAc structures in mock- and MGAT3-transfectants. Whole cell lysates of the two transfectants were subjected to PHA-E lectin blotting as described in M&M. (C) Proliferation of transfectant cells. The two transfectants were cultured for 24, 36, 48, 60, and 72 h, and proliferation was assessed by MTS assay. (E) Colony formation ability. The two transfectants (2500 cells each) were cultured in 6-cm dishes for 1–2 week, fixed, stained with crystal violet solution, and photographed. Acetic acid was added to dissolve crystal violet, and OD 595 was determined (D) . * p < 0.05; *** p < 0.001. (F) Cell migration. Migration assays of the two transfectants under normoxic and hypoxic conditions were performed as described in M&M, and relative migration rate was shown (H) . * p < 0.05. (G) Expression of HIF-1α, MGAT3, AKT, p-AKT, E-cadherin, fibronectin, and tubulin in the two transfectants under normoxic and hypoxic conditions. Cells were cultured as described in Figure , harvested, and lysed in T-PER Reagent. Protein content was determined by BCA assay. Western blotting was performed as described in M&M.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Over Expression, Expressing, Transfection, Stable Transfection, Transduction, Western Blot, Cell Culture, MTS Assay, Staining, Migration, BIA-KA

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: MGAT3 knockdown promotes hypoxia-induced EMT in MCF7 cells (A) MGAT3 expression in mock- and MGAT3-shRNA-transfected MCF7 cells. Cells were stably transduced with lentivirus carrying anti-MGAT3 shRNAs (MCF7/shMGAT3-1/2) or shNC (MCF7/mock), harvested, and lysed in T-PER Reagent. Western blotting was performed as described in M&M. (B) Cell proliferation. The two transfectants were cultured for 24, 36, 48, 60, and 72 h, and proliferation was assessed by MTS assay. (C) Cell migration. Migration assays of the two transfectants under normoxic and hypoxic conditions were performed as described in M&M, and relative migration rate was shown (E) . * p < 0.05; ** p < 0.01. (D) Expression of HIF-1α, MGAT3, AKT, p-AKT, E-cadherin, fibronectin, β-catenin, and tubulin in the two transfectants under normoxic and hypoxic conditions. Cells were cultured as described in Figure , harvested, and lysed in T-PER Reagent. Protein content was determined by BCA assay. Western blotting was performed as described in M&M.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Knockdown, Expressing, shRNA, Transfection, Stable Transfection, Transduction, Western Blot, Cell Culture, MTS Assay, Migration, BIA-KA

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: LRP6 overexpression defines a class of breast cancer subtype and is a target for therapy.

doi: 10.1073/pnas.0911220107

Figure Lengend Snippet: Fig. 1. LRP6 expression is frequently up-regulated in a subset of human breast cancer tissues and cell lines. (A) Breast cancer TissueScan Real-Time qPCR array, containing seven normal/Stage 0 cDNAs and 41 human breast cancer cDNAs, was analyzed for LRP6 expression by real-time PCR. Averages of relative LRP6 expression from three independent plates are plotted with clinical status indicated. LRP6 mRNA levels are markedly up-regulated in a subset of human breast cancer tissues. #Samples with elevated HER2 tran- scripts. (B and C) Breast cancer tissue microarray was used for IHC staining of LRP6. (B) Representatives of LRP6 staining in normal and malignant breast tissue are shown. LRP6 antibody (C-term T1546, Abgent), which specifically recognizes human LRP6, was used for IHC staining. (C) The quantification of LRP6 IHC staining was determined from three independent experiments. Staining intensity was scored as absent (0), weak (1), moderate (2), or strong (3). Four observations were made on each slide by independent inves- tigators, and a mean score was recorded. (D) Expression of LRP6 in human mammary epithelial cell (MCF-10A) and indicated breast cancer cell lines analyzed by Western blot analysis. *P < 0.05; **P < 0.01.

Article Snippet: MDA-MB-231, MDA-MB-157, SKBR3, MCF-7, MDA-MB-435s, MDA-MB-361, HCC1187, HCC1143, HCC1806, HCC38, HCC1937, HCC1395, T-47D, and CAMA1 breast cancer cell lines and MCF-10A nontransformed cells were all from the American Type Culture Collection (ATCC) and grown according to ATCC recommendations.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Microarray, Immunohistochemistry, Staining, Western Blot

Journal: Journal of Molecular Endocrinology

Article Title: HOXC10 is overexpressed in breast cancer and transcriptionally regulated by estrogen via involvement of histone methylases MLL3 and MLL4

doi: 10.1530/jme-11-0078

Figure Lengend Snippet: Figure 1 Expression of HOXC10 in different malignant and nonmalignant human cell lines and human breast cancer tissue. (A) The total RNA was isolated from T47D (breast cancer), MCF7 (breast cancer), HEPG2 (hepatocellular carcinoma), JAR (choriocarcinoma placenta), HeLa (cervical cancer), SW480 (colorectal adenocarcinoma), K562 (chronic myelogenous leukemia), H358 (bronchoalveolar carcinoma), MCF10 (normal breast epithelial cells), HFL1 (normal lung fibroblast HFL1), and Hs798.Pl (normal placenta) cell lines, reverse transcribed to cDNA, and analyzed by real-time PCR using primers specific to HOXC10. GAPDH was used as a loading control. HOXC10 expression relative to GAPDH is plotted. Each experiment was repeated at least thrice (nZ3). Bars indicate S.E.M. (B–D) Immunohistological analysis of HOXC10 expression in breast cancer tissue: human breast cancer tissue microarray (six cases of breast cancer along with their matched adjacent normal breast tissue) was obtained from US Biomax and subjected to immunohistological staining (DAB staining) with HOXC10 antibody. The relative quantification of HOXC10 expression within the tissue section is presented in panel C. A magnified view of tissue histology showing HOXC10 expression in case 5 is shown in panel D.

Article Snippet: Cell culture, estrogen treatment, and antisense experiment Human choriocarcinoma placenta cells (JAR), human adenocarcinoma mammary (MCF7), ER-negative human adenocarcinoma mammary (MDA-MB-231), ductal carcinoma mammary (T47D), hepatocellular carcinoma (HEPG2), cervical cancer (HeLa), colorectal adenocarcinoma (SW480), chronic myelogenous leukemia (K562), bronchoalveolar carcinoma (H358), normal breast epithelial (MCF10), normal lung fibroblast (HFL1), and normal placenta (Hs 798.Pl) cells were obtained from ATCC (Manassas, VA, USA) and grown in Phenol Red-free DMEM-F-12 (or RPMI as Journal of Molecular Endocrinology (2012) 48, 61–75 needed, Sigma), supplemented with 10% charcoalstripped fetal bovine serum, 2 mM L-glutamine, and penicillin/streptomycin (100 units and 0.1 mg/ml).

Techniques: Expressing, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Microarray, Staining

Journal: Molecular Cancer

Article Title: SALL1 functions as a tumor suppressor in breast cancer by regulating cancer cell senescence and metastasis through the NuRD complex

doi: 10.1186/s12943-018-0824-y

Figure Lengend Snippet: SALL1 expression is down-regulated in human breast cancer. a and b Gene expression levels of SALL1 in different cancer cell lines (in a ) and in tumor tissues (in b ) using Real-time PCR analyses. Tumor cell lines include breast cancer (human MDA-MB-231, MCF7, BC80, 31, 30, 29, 16, 12, and 10), melanoma (human Mel1938, Mel1586, Mel1860, Mel1363, Mel1526 and Mel1628), prostate cancer (PC3 and DU145), colon cancer (SW480), and lymphoma (L428 and L504). Normal breast cell lines (BN6, BN16, MCF10A and MCF12A), Fibroblasts (F163, F160, F158 and F112) and 293 T cells were included as controls. mRNA levels in each cell line and tissue were normalized to the relative quantity of GAPDH expression and then adjusted to SALL1 levels in 293 T cells (set as 1). Results shown in the histogram are mean ± SD from three independent experiments. c and d Association analyses of SALL1 expression with specific breast cancer subtypes. The data sets were accessed from the TCGA breast cancer Argilent microarray expression database downloaded from the cBioPortal ( http://www.cbioportal.org /). The box plot indicated the log 2 transformed mRNA median expression level of SALL1 in the tissues. N indicated the number of sample size of each tissue type. Mann-Whitney analysis was used to compare the SALL1 expression across the different breast cancer subtypes and normal tissues, and ** p < 0.01 within the comparison groups. e SALL1 expression in tumor cells in breast cancer tissues was determined using the immunohistochemical staining. f and g SALL1 expression levels in breast cancer tissues with different ER and HER2 status. SALL1 + cell population in ER + patients was significantly higher than that in ER − patients. Furthermore, SALL1 + cell numbers in HER2 + patients were much higher than that in HER2 − patients. Tissue immunohistochemical staining and cell number counting were identical as in ( e ). Significance was determined by unpaired T test

Article Snippet: Breast tumor cell lines (human MDA-MB-231, MCF7, BC80, 31, 30, 29, 16, 12, 10, and murine 4 T1 and E0771), Melanoma cell lines (Mel1938, Mel1586, Mel1860, Mel1363, Mel1526 and Mel1628, and murine B16F0), prostate cell line PC3 and DU145, colon cancer cell line SW480 and lymphoma L428 and L504, as well as normal breast cells and fibroblast cells, were either obtained from the American Tissue Culture Collection (ATCC) or established by our group, and maintained in RPMI 1640 medium containing 10% fetal calf serum (FCS) and penicillin-streptomycin (Invitrogen, Inc. San Diego, CA).

Techniques: Expressing, Gene Expression, Real-time Polymerase Chain Reaction, Microarray, Transformation Assay, MANN-WHITNEY, Comparison, Immunohistochemical staining, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: Tumor-endothelial interaction links the CD44(+)/CD24(-) phenotype with poor prognosis in early-stage breast cancer.

doi: 10.1593/neo.09670

Figure Lengend Snippet: Figure 1. Effect of heterotypic interaction between an endothelial cell and a breast cancer cell line. (A) Biologically independent repli- cates of the monocultured HDMEC, the breast cancer cell line Hs578T, and the mixed coculture of HDMEC and Hs578T were grown for 48 hours at low serum conditions and characterized by DNA microarray hybridization. Hierarchical clustering of a total of 1140 elements that display a greater than three-fold variance in expression in more than two different experimental samples. Data from individual ele- ments or genes are represented as single rows, and different experiments are shown as columns. Red and green denote the expres- sion levels of the samples. The intensity of the color reflects the magnitude of the deviation from baseline. Unsupervised hierarchical clustering of the experiments grouped the biologic replicates together. Gene expression varied considerably between HDMEC and Hs578T cultures as expected for cells of mesenchymal or epithelial origin, respectively. The coculture profile showed mainly inter- mediate expression levels. However, the vertical black bar marks a cluster of genes that were induced in all cocultures when compared with both monocultures, which indicated that they were induced by the heterotypic interaction. Zooming in on the genes that were upregulated in coculture revealed that they were specific for proliferation and mitosis. (B) Correlation of the measured coculture gene expression levels and their estimated expression levels based on the proportional contribution of each cell type as determined by a linear regression fit of the monoculture to the coculture data. (C) Fold change of each gene that was associated with coculturing of HDMEC and Hs578T. Genes of the “proliferation and mitosis” cluster are indicated in red. Dashed lines indicate 95% confidence limits.

Article Snippet: MCF-7, T47D, MDA-MB-231, SKBR-3, Hs578T, and BT549 (ATCC, Atlanta, GA) were propagated in Dulbecco’s modified Eagle medium that was supplemented with 10% FBS (HyClone, Logan, UT), glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco, Grand Island, NY).

Techniques: Microarray, Hybridization, Expressing, Gene Expression

Journal: Neoplasia (New York, N.Y.)

Article Title: Tumor-endothelial interaction links the CD44(+)/CD24(-) phenotype with poor prognosis in early-stage breast cancer.

doi: 10.1593/neo.09670

Figure Lengend Snippet: Figure 3. Proliferation of tumor and endothelial cells is due to recip- rocal stimulation. (A) Proliferation of HUVECs and Hs578T monocul- tures and their 1:1 coculture as determined by measuring the increase in cell number by direct cell counting after 36 hours. (B) Box-and-whisker diagrams of relative cell numbers of MCF-7, Hs578T, and MDA-MB-231 after incubation with conditioned me- dium from HUVECs compared with a normalized negative control of the same cells incubated with autologous medium as measured by the colorimetric cell proliferation assay with the WST-1 com- pound. (C) Proliferation of HUVECs that was induced by conditioned medium from Hs578T and MCF-7 cells as measured by WST-1. Rel- ative absorbance values of colorful formazan, which has been con- verted by HUVECs, correspond to relative cell numbers. A single column represents average absorbance values for a minimum of eight independent replicates. y Axis error bars correspond to SD. The HUVECs that were treated with the Hs578T supernatant grew significantly faster than the same cells that were treated with the MCF-7 supernatant, the fresh vehicle medium, or the autologous (HUVEC-derived) medium.

Article Snippet: MCF-7, T47D, MDA-MB-231, SKBR-3, Hs578T, and BT549 (ATCC, Atlanta, GA) were propagated in Dulbecco’s modified Eagle medium that was supplemented with 10% FBS (HyClone, Logan, UT), glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco, Grand Island, NY).

Techniques: Cell Counting, Whisker Assay, Incubation, Negative Control, Proliferation Assay, Derivative Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Tumor-endothelial interaction links the CD44(+)/CD24(-) phenotype with poor prognosis in early-stage breast cancer.

doi: 10.1593/neo.09670

Figure Lengend Snippet: Figure 5. Stimulatory effect of Hs578T conditioned medium on en- dothelial cells can be partially blocked by bevacizumab. (A) Rela- tive expression of the “tumor-endothelial cell–induced M-phase cell cycle” genes in different monocultures of breast cancer cell lines and endothelial cells and in their respective cocultures. (B) Blocking of the stimulatory effect of Hs578T conditioned medium by bevacizumab. Absolute absorbance values of formazan dye that was converted by HUVECs, which corresponds to cell numbers, are shown in columns, with the y axis bars corresponding to SD. Bevacizumab (100 ng/ml) depleted the stimulatory effect of Hs578T cell culture supernatant in a significant manner, whereas it had no effect on the MCF-7 cell culture supernatant. Recombinant VEGF-A (5 ng/ml) and 5% FBS served as positive and negative controls, respectively. (C) Dose-dependent blocking of HUVEC proliferation by bevacizumab and trastuzumab. Absolute absorbance values of formazan dye that was converted by HUVECs are shown. HUVECs that were treated with the Hs578T cell culture supernatant repre- sent the baseline stimulatory effect. Bevacizumab depleted the stimulatory effect of the Hs578T conditioned medium in a signifi- cant, dose-dependent manner. Trastuzumab, which is a monoclonal antibody against HER2, did not influence HUVEC stimulation by the Hs578T conditioned medium.

Article Snippet: MCF-7, T47D, MDA-MB-231, SKBR-3, Hs578T, and BT549 (ATCC, Atlanta, GA) were propagated in Dulbecco’s modified Eagle medium that was supplemented with 10% FBS (HyClone, Logan, UT), glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco, Grand Island, NY).

Techniques: Expressing, Blocking Assay, Cell Culture, Recombinant