Expression and localization of serotonin receptors in human breast cancer.

The aim of this study was to examine the expression of serotonin receptors in patients with breast cancer and to explore their utility as diagnostic and prognostic markers. Immunohistochemical analysis was performed to examine the expression of serotonin (5-HT) receptor subtypes 1A, 1B, 2B and 4 in a tissue microarray containing tumor specimens from 102 patients. Statistical analysis was performed to correlate the expression of these proteins with regard to clinical parameters. We found that all four serotonin receptors (5-HTRs) exhibited different expression patterns in breast cancer specimens. In general strong staining for 5-HTR1A was observed on the membrane of cancer cells but it was detected only in the cytoplasm of non-malignant cells. 5-HTR1B and 5-HTR2B were predominantly expressed in the cytoplasm of breast cancer cells, while 5-HTR4 was exclusively found in the nucleus of malignant and non-malignant cells. Correlation analysis revealed a significant correlation of 5-HTR2B with estrogen receptor-α (ER-α) and 5-HTR4 with ER-α and progesterone (PR). In conclusion, the different expression patterns and subcellular localization of 5-HTRs in breast cancer may reflect their role in breast cancer progression

Lipoproteins Are Responsible for the Pro-Inflammatory Property of Staphylococcus aureus Extracellular Vesicles

Staphylococcal aureus (S. aureus), a Gram-positive bacteria, is known to cause various infections. Extracellular vesicles (EVs) are a heterogeneous array of membranous structures secreted by cells from all three domains of life, i.e., eukaryotes, bacteria, and archaea. Bacterial EVs are implied to be involved in both bacteria–bacteria and bacteria–host interactions during infections. It is still unclear how S. aureus EVs interact with host cells and induce inflammatory responses. In this study, EVs were isolated from S. aureus and mutant strains deficient in either prelipoprotein lipidation (Δlgt) or major surface proteins (ΔsrtAB). Their immunostimulatory capacities were assessed both in vitro and in vivo. We found that S. aureus EVs induced pro-inflammatory responses both in vitro and in vivo. However, this activity was dependent on lipidated lipoproteins (Lpp), since EVs isolated from the Δlgt showed no stimulation. On the other hand, EVs isolated from the ΔsrtAB mutant showed full immune stimulation, indicating the cell wall anchoring of surface proteins did not play a role in immune stimulation. The immune stimulation of S. aureus EVs was mediated mainly by monocytes/macrophages and was TLR2 dependent. In this study, we demonstrated that not only free Lpp but also EV-imbedded Lpp had high pro-inflammatory activity

Expression of VEGF and Its receptors VEGFR1/VEGFR2 Is Associated with Invasiveness of Bladder Cancer.

Aim: Vascular endothelial growth factor (VEGF) signaling is frequently altered in invasive tumor cells and is associated with patient outcome. In the present study, we examined VEGF, VEGFR1, and VEGFR2 expression in non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC), and evaluated the association between VEGF and its receptors with disease characteristics and bladder cancer recurrence

Cyclin A1 Modulates the Expression of Vascular Endothelial Growth Factor and Promotes Hormone-Dependent Growth and Angiogenesis of Breast Cancer

<div><p>Alterations in cellular pathways related to both endocrine and vascular endothelial growth factors (VEGF) may contribute to breast cancer progression. Inhibition of the elevated levels of these pathways is associated with clinical benefits. However, molecular mechanisms by which endocrine-related pathways and VEGF signalling cooperatively promote breast cancer progression remain poorly understood. In the present study, we show that the A-type cyclin, cyclin A1, known for its important role in the initiation of leukemia and prostate cancer metastasis, is highly expressed in primary breast cancer specimens and metastatic lesions, in contrasting to its barely detectable expression in normal human breast tissues. There is a statistically significant correlation between cyclin A1 and VEGF expression in breast cancer specimens from two patient cohorts (<i>p</i><0.01). Induction of cyclin A1 overexpression in breast cancer cell line MCF-7 results in an enhanced invasiveness and a concomitant increase in VEGF expression. In addition, there is a formation of protein–protein complexes between cyclin A1 and estrogen receptor ER-α cyclin A1 overexpression increases ER-α expression in MCF-7 and T47D cells. In mouse tumor xenograft models in which mice were implanted with MCF-7 cells that overexpressed cyclin A1 or control vector, cyclin A1 overexpression results in an increase in tumor growth and angiogenesis, which is coincident with an enhanced expression of VEGF, VEGFR1 and ER-α Our findings unravel a novel role for cyclin A1 in growth and progression of breast cancer, and suggest that multiple cellular pathways, including cell cycle regulators, angiogenesis and estrogen receptor signalling, may cooperatively contribute to breast cancer progression.</p> </div

Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia

Abstract Background Chronic lymphocytic leukemia (CLL) has been a good model system to understand the functional role of 5-methylcytosine (5-mC) in cancer progression. More recently, an oxidized form of 5-mC, 5-hydroxymethylcytosine (5-hmC) has gained lot of attention as a regulatory epigenetic modification with prognostic and diagnostic implications for several cancers. However, there is no global study exploring the role of 5-hydroxymethylcytosine (5-hmC) levels in CLL. Herein, using mass spectrometry and hMeDIP-sequencing, we analysed the dynamics of 5-hmC during B cell maturation and CLL pathogenesis. Results We show that naïve B-cells had higher levels of 5-hmC and 5-mC compared to non-class switched and class-switched memory B-cells. We found a significant decrease in global 5-mC levels in CLL patients (n = 15) compared to naïve and memory B cells, with no changes detected between the CLL prognostic groups. On the other hand, global 5-hmC levels of CLL patients were similar to memory B cells and reduced compared to naïve B cells. Interestingly, 5-hmC levels were increased at regulatory regions such as gene-body, CpG island shores and shelves and 5-hmC distribution over the gene-body positively correlated with degree of transcriptional activity. Importantly, CLL samples showed aberrant 5-hmC and 5-mC pattern over gene-body compared to well-defined patterns in normal B-cells. Integrated analysis of 5-hmC and RNA-sequencing from CLL datasets identified three novel oncogenic drivers that could have potential roles in CLL development and progression. Conclusions Thus, our study suggests that the global loss of 5-hmC, accompanied by its significant increase at the gene regulatory regions, constitute a novel hallmark of CLL pathogenesis. Our combined analysis of 5-mC and 5-hmC sequencing provided insights into the potential role of 5-hmC in modulating gene expression changes during CLL pathogenesis

Evaluation of endogenous cyclin A1 and VEGF expression in breast cancer cell lines.

<p>(A) Cyclin A1 mRNA levels are assessed in T47D, BT549, MCF-7, MDA-MB-468, MDA-MB231 and Cama-1 cell lines by semiquantitative RT-PCR and a representative picture is shown (upper panel). Quantification of cyclin A1 mRNA level is shown and mean ± SD represents three independent experiments (lower panel). (B) Western blot analysis of levels of cyclin A1 protein in the cell lines as indicated. A representative picture is shown (upper panel). Quantification of cyclin A1 protein level is shown and mean ± SD represents three independent experiments (lower panel). (C) VEGF mRNA levels are assessed in the indicated cell lines by semiquantitative RT-PCR and a representative picture is shown (upper panel). Quantification of cyclin A1 protein level is shown and mean ± SD represents three independent experiments (lower panel). (D) ELISA assay of VEGF secretion in breast cancer cell lines as indicated. Mean ± SD represents three independent experiments.</p

Effect of cyclin A1 expression on tumor invasion is associated with its effect on VEGF expression in MCF-7 cells.

<p>(A) Evaluation of cyclin A1 and VEGF expression in metastatic lesions from lymph nodes from patients with breast cancer metastasis using immunohistochemical analysis. Representative pictures show the cancer cells are strongly positive to cyclin A1 and VEGF expression. Upper panels represent cores at 20x magnificantion and lower panels represent the higher magnification (40x) of the selected areas. (B) MCF-7 cells that were transfected with cyclin A1pCMS-EGFP or pCMS-EGFP vectors were applied on the Matrigel-coated invasion chamber and were assessed after 48 or 72 hours. Data in graphs are the mean ± SD represents two independent experiments, each performed in duplicates. P value is indicated. (C) MDA-MB-231 cells transfected with cyclin A1pCMS-EGFP or pCMS-EGFP were applied on the Matrigel-coated invasion chamber and were analyzed after 48 or 72 hours. Data in graphs are the mean of two independent experiments, each performed in duplicate, p=0.002 for 48 h and p=0.02 for 72 h. (D) Cell cycle distribution of the cells that were transfected with cyclin A1pCMS-EGFP or pCMS-EGFP. Data in graphs are the mean ± SD represents three independent experiments from flow cytometry analysis. The percentage of cells at onset of each cell cycle phase is indicated. (E) Western blot analysis shows the levels of cyclin D1 and CDK1 protein expression in the cells that were transfected with cyclin A1pCMS-EGFP or pCMS-EGFP. (F) Representative picture shows the VEGF mRNA expression in the cells transfected with cyclin A1pCMS-EGFP or pCMS-EGFP (upper panel). Quantification of VEGF mRNA level in the samples is indicated. P value is shown (lower panel). (G) ELISA assay of VEGF secretion in the cells transfected with cyclin A1pCMS-EGFP or pCMS-EGFP. Mean ± SD represents three independent experiments (lower panel). Breast cancer cell lines used for these studies are T47D, MCF-7 and MDA-MB231 as indicated.</p

The effect of cyclin A1 on growth and vascularization of tumor xenografts <i>in mice.</i>

<p>MCF-7 cells transfected with cyclin A1pCMS–EGFP or pCMS-EGFP vectors were subcutaneous implanted into female nude mice with E2 supplementation. (A, B) Representative microphotographs of xenograft tumor sections stained with Haematoxylin and Eosin are shown. The control tumor “pCMS-control” and cyclin A1 expressing tumor “pCMS-cyclin A1” are indicated. (C, D) Representative pictures show the xenograft tumors stained with antibody against human CD31, the CD31 positive vessels are indicated. (E) Growth curves of the two groups of xenograft tumors. The control tumor “pCMS-control” and cyclin A1 expressing tumor “pCMS-cyclin A1” are indicated. The time is indicated in x-axis and tumor volume in mm³ is indicated in y-axis. (F) Quantification of the tumor vascularizations in cyclin A1 expressing xenograft tumors “pCMS-cyclin A1” and in control xenograft tumors “pCMS-control”. The numbers of CD31-positive blood vessels in the central vs. edge regions of the tumor areas are shown. P values are indicated. Mean ± SD represents three independent experiments.</p

Evaluation of the association between cyclin A1 and ER-α estrogen signaling and the regulation of VEGF expression.

<p>(A) -Evaluation of the effect of estrogen on mRNA expression of cyclin A1, VEGF and ER-α, in T47D, MCF-7 and MDA-MB231 cells using semi-quantitative RT-PCR analysis. The three cell lines mentioned above were cultured in charcoal stripped medium (CSS) or in CSS medium containing E2 at 5 µM for additional 48 hours as indicated. (B) The effect of E2 treatment or cyclin A1 overexpression alone or in combination on VEGF mRNA level was determined in MCF-7 cells. Cells that were transfected with cyclin A1pCMS-EGFP or pCMS-EGFP vectors in the absence or presence of E2 are indicated. (C) Evaluation of the effects of Tamoxifen (Tam), E2, and E2 in combination with Tamoxifen (E2+Tam) on VEGF protein expression in MCF-7 cells. MCF-7 cells were transfected with cyclin A1 vector or control vector as indicated. Data in graphs below are the mean ± SD represents two independent experiments. (D) Immunoblot analysis data obtained in MDA-MB-231 which were treated using the same conditions as mentioned in (C). (E) Immunoprecipitation analysis (IP) shows physical interaction between cyclin A1 and ER-αin MCF-7 cells that were transfected with cyclin A1pCMS-EGFP or pCMS-EGFP vectors. ER-α antibody was used in IP to pull down the immunocomplexes and subsequent Westernblot was performed using cyclin A1 or ER-α antibodies to detect the immunocomplexes as indicated. The input was used as controls as indicated. (F) Evaluation of ER-α mRNA expression in the cells that were transfected with A1pCMS-EGFP or pCMS-EGFP vectors. The representative picture is shown in the upper panel. Quantification of ER-α mRNA level is shown in the lower panel and mean ± SD represents three independent experiments. (G) Western blot analysis shows the expression level of ER-α protein in the cells that were transfected with A1pCMS-EGFP or pCMS-EGFP vectors. Breast cancer cell lines used for these studies are T47D, MCF-7 and MDA-MB231 as indicated.</p

Long-term effect of elevated level of cyclin A1 on growth and angiogenesis phenotype of xenograft tumors <i>in mice.</i>

<p>MCF-7 cells stable expressing pcDNA–cyclin A1 or pcDNA vectors were subcutaneous implanted into female nude mice with E2 supplementation. (A, B) Representative microphotographs of xenograft tumor sections stained with Haematoxylin and Eosin are shown. (C, D) Representative pictures show the xenograft tumors stained with antibody against human CD31, the CD31 positive vessels are indicated. The control tumor “control-pcDNA” and cyclin A1 expressing tumors “cyclin A1-pcDNA” are indicated. (E) Growth curves of the two groups of xenograft tumors are indicated. The time is indicated in x-axis and tumor volume in mm³ is indicated in y-axis. (F) Quantification of the tumor vascularizations in cyclin A1 expressing xenograft tumors “cyclin A1-pcDNA” and in control xenograft tumors “control-pcDNA”. The numbers of CD31-positive blood vessels in the central vs. edge regions of the tumor areas are shown. P values are indicated. Mean ± SD represents three independent experiments. (G–N) Xenograft tumors from “cyclin A1-pcDNA” and “control-pcDNA” groups were immunostained with antibodies against VEGF, VEGFR1, ER-α and Ki67. The representative microphotographs are shown.</p