Inactivation of GPR30 reduces growth of triple-negative breast cancer cells: possible application in targeted therapy

Triple-negative breast cancers lack estrogen receptor α (ERα), progesterone receptor, and do not overexpress human epidermal growth factor receptor 2 (Her-2). They are neither susceptible to endocrine therapy nor to a therapy using the anti-Her-2 antibody, trastuzumab. Therefore, an efficient targeted therapy is warranted. Triple-negative breast tumors frequently express membrane bound estrogen receptor G-protein coupled receptor (GPR30). As proof of principle, we analyzed the consequences of a knock-down of GPR30 expression on the growth regulation of triple-negative breast cancer cell lines. Cells of triple-negative breast cancer cell lines were transfected with siRNA against GPR30 or control siRNA, and cell growth was stimulated either with 10(−9) M 17β-estradiol or 10(−6) M 4-hydroxytamoxifen. Cell proliferation was measured using Alamar blue staining. Activation of c-Src and epidermal growth factor (EGF)-receptor was assessed using western blot. Expression of c-fos was quantified by reverse transcription polymerase chain reaction. Seven days after transfection with siRNA, GPR30 mRNA in triple-negative breast cancer cell lines MDA-MB-435 and HCC1806 was reduced by 74 and 90%, respectively. 10(−8) M 17β-estradiol enhanced proliferation of MDA-MB-435 to 129.6 ± 5.4% of control (p < 0.05) and HCC1806 to 156.9 ± 15.4% of control (p < 0.05), respectively. 10(−6) M 4-hydroxytamoxifen increased cell number of MDA-MB-435 to 121.0 ± 6.9% of control (p < 0.05) and HCC1806 to 124.5 ± 12.1% of control (n.s.), respectively. This increased proliferation by the two estrogenic compounds was completely prevented by knock-down of GPR30 expression in both cell lines. In control cells, activity of Src kinase was increased 3-fold by estradiol and 3.8-fold using 4-hydroxytamoxifen. Transactivation of the EGF-receptor was similarly increased in both cell lines by 17β-estradiol and 4-hydroxytamoxifen. Both compounds increased c-fos expression 1.5- and 3.1-fold, respectively. Knock-down of GPR30 expression completely abolished activation of all these signaling pathways responsible for enhanced proliferation. A pharmacological inhibition of GPR30 by specific small molecular inhibitors might prove to be an appropriate targeted therapy of triple-negative breast cancer in the future

Organoprotective Effects of Spironolactone on Top of Ramipril Therapy in a Mouse Model for Alport Syndrome

Angiotensin-converting enzyme inhibitors (ACEi) delay progression of the inherited renal disease Alport syndrome. However, the effect of ACEis weakens gradually due to an “aldosterone escape”. Here, we investigate if an aldosterone antagonist can counteract loss of ACEi-efficacy. COL4A3-/- mice were treated with ramipril (ACEi), starting at 4.5 weeks of age, and spironolactone was added at 7 weeks of age. Lifespan until renal failure, as well as kidney function parameters, were investigated. Dual therapy decreased proteinuria levels compared to ACEi monotherapy. Matrix accumulation, as well as tubulointerstitial and glomerular scar-tissue formation, were significantly reduced compared to untreated mice and ACEi-monotherapy at 75 and 100 days. Lifespan in dual treated mice was extended compared to untreated mice. However, lifespan was not superior to ACEi monotherapy–despite improved urea-nitrogen levels in the dual therapy group. In conclusion, adding the aldosterone-antagonist spironolactone to ACEi therapy further improved kidney function and reduced proteinuria and fibrosis. However, survival was not improved further, possibly due to premature death from side effects of dual therapy such as hyperkalemia. Thus, dual therapy could offer an effective therapy option for Alport syndrome patients with progressive proteinuria. However, the risks of adverse events require close monitoring

Induction of tamoxifen resistance in breast cancer cells by ELF electromagnetic fi elds

Abstract The incidence of breast cancer in western societies has been rising ever since the Second World War. Besides the exposure to a multitude of new chemical compounds, electromagnetic field exposure has been linked to breast cancer through a radiation-mediated antimelatonin pathway. We investigated, whether low-frequency electromagnetic field exposure interferes with the anti-estrogenic activity of tamoxifen. Two different clones of the breast cancer cell line MCF-7 were exposed to highly homogeneous 50 Hz electromagnetic fields and IC 50 values were calculated from dose-response curves of tamoxifen at various field intensities. An intensity-dependent shift of tamoxifen dose-response curves to higher concentrations with a maximal response at 1.2 lT was observed. Hypothetically, electromagnetic field exposure could contribute to tamoxifen resistance observed in breast cancer after long-term treatment. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Breast cancer; Estrogen receptor; Electromagnetic fields; Tamoxifen resistance; Dose-response The effect of extremely low-frequency electromagnetic field (ELF/EMF) exposure on human health has been widely debated. A number of epidemiological studies have pointed to a slight increase in malignant diseases in populations exposed to electromagnetic fields through the vicinity of power lines. A significant positive association was observed between childhood leukemia and exposure of children to magnetic fields during the night [1]. In two studies, premenopausal women exposed to environmental fields stronger than 0.2 lT had an increased risk of breast cancer (BC) These epidemiological observations prompted the examination of the impact of electromagnetic fields on breast cancer incidence in an animal model. Sprague-Dawley rats suffer of a high rate of mammary tumors if treated with the chemical carcinogen 7,12-dimethylbenz[a]-anthracene (DMBA). Exposure of these rats to a 100 lT electromagnetic field for 27 weeks increased the number of tumor bearing rats to 65% compared to 50% in sham exposed rat

Collagen receptors integrin alpha2beta1 and discoidin domain receptor 1 regulate maturation of the glomerular basement membrane and loss of integrin alpha2betal delays kidney fibrosis in COL4A3 knockout mice

Maturation of the glomerular basement membrane (GBM) is essential for maintaining the integrity of the renal filtration barrier. Impaired maturation causes proteinuria and renal fibrosis in the type IV collagen disease Alport syndrome. This study evaluates the role of collagen receptors in maturation of the GBM, matrix accumulation and renal fibrosis by using mice deficient for discoidin domain receptor 1 (DDR1), integrin subunit alpha 2 (ITGA2), and type IV collagen alpha 3 (COL4A3). Loss of both collagen receptors DDR1 and integrin alpha 2 beta 1 delays maturation of the GBM: due to a porous GBM filtration barrier high molecular weight proteinuria that more than doubles between day 60 and day 100. Thereafter, maturation of the GBM causes proteinuria to drop down to one tenth until day 200. Proteinuria and the porous GBM cause accumulation of glomerular and tubulointerstitial matrix, which both decrease significantly after GBM-maturation until day 250. In parallel, in a disease with impaired GBM-maturation such as Alport syndrome, loss of integrin alpha 2 beta 1 positively delays renal fibrosis: COL4A3(-/-) ITGA2(-/-) double knockouts exhibited reduced proteinuria and urea nitrogen compared to COL4A3(-/-)/ITGA2(-/-) and COL4A3(-/-)/ITGA2(+/+) mice. The double knockouts lived 20% longer and showed less glomerular and tubulointerstitial extracellular matrix deposition than the COL4A3(-/-) Alport mice with normal integrin alpha 2 beta 1 expression. Electron microscopy illustrated improvements in the glomerular basement membrane structure. MMP2, MMP9, MMP12 and TIMP1 were expressed at significantly higher levels (compared to wild-type mice) in COL4A3(-/-)/ITGA2(+/+) Alport mice, but not in COL4A3(+/+)/ITGA2(-/-) mice. In conclusion, the collagen receptors DDR1 and integrin alpha 2 beta 1 contribute to regulate GBM-maturation and to control matrix accumulation. As demonstrated in the type IV collagen disease Alport syndrome, glomerular cell-matrix interactions via collagen receptors play an important role in the progression of renal fibrosis. (C) 2014 Elsevier B.V. All rights reserved