Endothelial colony forming cells from human umbilical cord blood improved severe erectile dysfunction in obese type II diabetic rats

Aim:To investigate the effect of intracavernous injection of human umbilical cord blood derived endothelial colony forming cells (HUCB ECFCs) on erectile dysfunction (ED) in Zucker Diabetic Fatty (ZDF) rat model. Methods:Erectile function was assessed by cavernous nerve electrostimulation in ZDF rats aged 20–28 weeks. Following confirmation of severe ED at the age of 28 weeks, 21 ZDF rats were randomly assigned to three experimental groups: 1 million ECFCs, 2 million ECFCs, and phosphate buffered saline (PBS). Four weeks after intracavernous injection, the efficacy of ECFCs was quantified by intracavernous pressure (ICP) measurement, Masson's trichrome staining, immunohistologic and immunoblot analyses and TUNEL assay. Key findings:Intracavernous ECFC administration improved ICP in a dose-dependent manner in comparison to the age-matched PBS group. Functional improvement in ICP was accompanied by a significant restoration of the cavernosal endothelial and smooth muscle cell content and cavernosal nerve function. The percentage eNOS and nNOS positive cavernosal cells, and their respective protein expression levels and nNOS positive cells in the dorsal penile nerve in 2 million ECFCs treated groups were significantly higher than the PBS group. TUNEL stain quantification showed a significant decrease in cavernosal apoptosis following ECFC treatment. Significance:The results are expected to provide a scientific basis to further study the clinical application of HUCB ECFCs in ameliorating ED in human. Conclusions:HUCB ECFCs significantly improved severe ED in ZDF rats through improvement of the nerve and endothelium function and restoration of smooth muscle in the cavernosum by overcoming the cavernosal apoptosis.MOE (Min. of Education, S’pore

Short-chain fatty acid receptors inhibit invasive phenotypes in breast cancer cells

This article evaluates the effects of enforced overexpression of two known G protein-coupled receptors in two phenotypically distinct breast cancer cell lines

Reduced expression of FFAR2 and FFAR3 in invasive and triple negative breast cancer tissues.

<p>The Curtis breast dataset available sourced from Oncomine.org was analyzed for the expression of (A) <i>FFAR2</i> and (B) <i>FFAR3</i> mRNA in 1992 breast carcinoma and 144 normal breast samples. Whiskers represent the minimum to maximum values. One-way ANNOVA was performed to calculate significance. <i>FFAR2</i> expression is reduced in some classes, while <i>FFAR3</i> was reduced in all invasive tumor types. The dataset was analyzed for (C) <i>FFAR2</i> and (D) <i>FFAR3</i> expression level in 144 normal breast tissue samples and in 250 triple negative breast tumors. Expression of both <i>FFAR2</i> and <i>FFAR3</i> were significantly reduced in triple negative breast tumors. (E) Expression data obtained from the Cancer Cell Line Encyclopedia show that among 57 breast cancer cell lines, there is a significant positive correlation between <i>FFAR2</i> and <i>CDH1</i> mRNA levels (Pearson r = 0.516, P <0.001). (F) Expression data obtained from the Human Protein Atlas show that among 1075 primary breast tumor samples, there is a significant positive correlation between <i>FFAR2</i> and <i>CDH1</i> mRNA levels (Pearson r = 0.138, P <0.001).</p

Anti-invasive phenotype in FFAR2- and FFAR3-overexpressing MDA-MD-231 cells.

<p>(A) MDA-MB-231 cells were seeded in to the upper chambers of Biocoat Matrigel transwells, then treated with vehicle control or with 1 mM propionate for 24 hours. Representative images show cells that have invaded through the matrix. (B) Quantitation of the invasion assay in (A) shows a significant ligand-mediated reduction in the invasiveness in cells overexpressing FFAR2 and FFAR3, but not vector control cells (N = 3). (C) MDA-MB-231 cells were labeled with rhodamine-phalloidin in order to visualize actin filaments. Representative images are shown. (D) Quantification of cells from (C) that demonstrate the presence of stress fibers (N = 3). (*p< 0.05, **p< 0.01).</p

FFAR2 regulates E-cadherin.

<p>(A) qRT-PCR analysis of EMT-related genes was performed on MDA-MB-231 cells overexpressing FFAR2 and FFAR3. Expression of <i>CDH1</i> was selectively increased in FFAR2-overexpressing cells relative to vector controls, while no changes in EMT-related genes were observed in FFAR3-overexpressing cells (N = 3). (B) Cells were treated with vehicle control or with 100 μM propionate for 24 hours, then evaluated for <i>CDH1</i> expression by qRT-PCR. FFAR2-overexpressing cells, but not in vector control cells or in FFAR3-overexpressing cells, demonstrated a ligand-dependent increase in <i>CDH1</i> mRNA (N = 3). (C) Cells were treated with vehicle control or with 100 μM propionate for 24 hours, then evaluated for E-cadherin levels by western blot analysis. Results are representative of three independent experiments. (D) Quantitation of the relative density of the data from (C) demonstrates that E-cadherin is increased in FFAR2-overexpressing cells, and is further increased by propionate treatment (N = 3). (*p< 0.05, **p< 0.01, ***p< 0.001.).</p

Schematic summarizing the effect of short chain fatty acid receptor overexpression in breast cancer cells.

<p>We provide novel data that demonstrate that activation of FFAR2 can activate LATS1 (repressor of YAP1), which results in an increase in E-cadherin levels. In contrast, FFAR3 activity inhibits the MAP/ERK pathway. These processes are likely to underlie the repressive effect of short chain fatty acid receptors on breast cancer cell invasion.</p

FFAR3 regulates MAP/ERK signaling.

<p>(A) MDA-MB-231 cells were treated with vehicle control or with 1 mM propionate for 24 hours, then evaluated for ERK1/2, pERK1/2, and β-actin levels by western blot analysis. Results are representative of three independent experiments. (B) Quantitation of the relative density of the data from (A) demonstrates that pERK1/2:ERK1/2 ratio is significantly reduced in FFAR3-overexpressing cells, but not in FFAR2-overexpressing cells (N = 3).</p

Short-chain fatty acid receptors inhibit invasive phenotypes in breast cancer cells

Short chain fatty acids (2 to 6 carbons in length) are ubiquitous lipids that are present in human plasma at micromolar concentrations. In addition to serving as metabolic precursors for lipid and carbohydrate synthesis, they also act as cognate ligands for two known G protein-coupled receptors (GPCRs), FFAR2 and FFAR3. While there is evidence that these receptors may inhibit the progression of colorectal cancer, their roles in breast cancer cells are largely unknown. We evaluated the effects of enforced overexpression of these receptors in two phenotypically distinct breast cancer cell lines: MCF7 and MDA-MD-231. Our results demonstrate that both receptors inhibit cell invasiveness, but through different signaling processes. In invasive, mesenchymal-like MDA-MB-231 cells, FFAR2 inhibits the Hippo-Yap pathway and increases expression of adhesion protein E-cadherin, while FFAR3 inhibits MAPK signaling. Both receptors have the net effect of reducing actin polymerization and invasion of cells through a Matrigel matrix. These effects were absent in the less invasive, epithelial-like MCF7 cells. Correspondingly, there is reduced expression of both receptors in invasive breast carcinoma and in aggressive triple-negative breast tumors, relative to normal breast tissue. Cumulatively, our data suggest that the activation of cognate receptors by short chain fatty acids drives breast cancer cells toward a non-invasive phenotype and therefore may inhibit metastasis. © 2017 Thirunavukkarasan et al