Péter Hamar

miR-200-containing extracellular vesicles promote breast cancer cell metastasis.

Metastasis is associated with poor prognosis in breast cancer patients. Not all cancer cells within a tumor are capable of metastasizing. The microRNA-200 (miR-200) family, which regulates the mesenchymal-to-epithelial transition, is enriched in the serum of patients with metastatic cancers. Ectopic expression of miR-200 can confer metastatic ability to poorly metastatic tumor cells in some settings. Here, we investigated whether metastatic capability could be transferred between metastatic and nonmetastatic cancer cells via extracellular vesicles. miR-200 was secreted in extracellular vesicles from metastatic murine and human breast cancer cell lines, and miR-200 levels were increased in sera of mice bearing metastatic tumors. In culture, murine and human metastatic breast cancer cell extracellular vesicles transferred miR-200 microRNAs to nonmetastatic cells, altering gene expression and promoting mesenchymal-to-epithelial transition. In murine cancer and human xenograft models, miR-200-expressing tumors and extracellular vesicles from these tumors promoted metastasis of otherwise weakly metastatic cells either nearby or at distant sites and conferred to these cells the ability to colonize distant tissues in a miR-200-dependent manner. Together, our results demonstrate that metastatic capability can be transferred by the uptake of extracellular vesicles.

Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown.

A central hurdle in developing small interfering RNAs (siRNAs) as therapeutics is the inefficiency of their delivery across the plasma and endosomal membranes to the cytosol, where they interact with the RNA interference machinery. With the aim of improving endosomal release, a poorly understood and inefficient process, we studied the uptake and cytosolic release of siRNAs, formulated in lipoplexes or lipid nanoparticles, by live-cell imaging and correlated it with knockdown of a target GFP reporter. siRNA release occurred invariably from maturing endosomes within ∼5–15 min of endocytosis. Cytosolic galectins immediately recognized the damaged endosome and targeted it for autophagy. However, inhibiting autophagy did not enhance cytosolic siRNA release. Gene knockdown occurred within a few hours of release and required <2,000 copies of cytosolic siRNAs. The ability to detect cytosolic release of siRNAs and understand how it is regulated will facilitate the development of rational strategies for improving the cytosolic delivery of candidate drugs.

Mycophenolate mofetil inhibits lymphocyte binding and the upregulation of adhesion molecules in acute rejection of rat kidney allografts

Mycophenolate mofetil (MMF) interacts with purine metabolism and possibly with the expression of adhesion molecules. In the present study, we analysed the expression of these molecules in transplanted kidney allografts treated with RS LBNF1 kidneys were orthotopically transplanted into Lewis rats and either treated with RS (20 mg/kg/day) or vehicle. Rats were harvested 3, 5 and 7 days following transplantation. For binding studies, fresh-frozen sections of transplanted kidneys were incubated with lymph node lymphocytes (LNL) derived from transplanted rats. Additionally, immunohistology was performed with various monoclonal antibodies. In general, MMF resulted in better preservation of graft structure by 7 days. Cellular infiltration and tubular atrophy were less pronounced. At day 3, macrophages were diminished in MMF-treated animals to a high extent, while the number of T cells was almost identical to that of controls. In addition, the number of cells positive for MHC class II and LFA-1 was reduced in the MMF-treated animals. These findings correlated with the binding results. Three days following engraftment, LNL bound to MMF-treated kidneys to a lesser extent compared to controls. In conclusion, MMF resulted in a markedly reduced leucocytic infiltrate, presumably based on a reduced expression of lymphocytic adhesion molecules and an interaction with macrophages.

Inhibition of matrix metalloproteinases during chronic allograft nephropathy in rats.

Background. Chronic allograft nephropathy (CAN) belongs to the major causes of long-term kidney allograft failure. One of the histologic hallmarks of CAN is interstitial fibrosis, influenced by matrix metalloproteinases (MMPs) that are controlling extracellular matrix (ECM) degradation. Whether MMPs affect the development and progression of CAN is not clear so far. To analyze the role of MMPs in CAN, we investigated the effects of an early and a late application of BAY 12–9566, an inhibitor of MMP-2, -3, and -9 on the development and progression of CAN in a rat kidney-transplantation model. Methods. Fisher kidneys were orthotopically transplanted into Lewis recipients that were treated with BAY 12–9566 (15 mg/kg per day) or vehicle either for the first 10 days after transplantation (early treatment) or from week 12 to week 20 after transplantation (late treatment). Proteinuria was analyzed every 4 weeks up to week 20 after transplantation when kidney grafts were removed for further analysis. Results. Early MMP-inhibition resulted in a significantly reduced 24-hour protein excretion that was paralleled by a lower grade of CAN after 20 weeks. However, late MMP inhibition starting at week 12 after transplantation resulted in significantly higher proteinuria and a higher grade of CAN as compared with controls. Furthermore, transforming growth factor-&bgr; and platelet-derived growth factor-B chain mRNA levels were significantly increased in these animals. Conclusions. Inhibition of MMPs early after transplantation reduced the development and progression of CAN but promoted CAN if initiated at later stages. Thus, MMPs are involved in the development and progression of CAN.

Glutathion s transferase π indicates chemotherapy resistance in breast cancer

BACKGROUND Breast cancer is the most common malignant disease of women. Pathologic response of breast cancer to chemotherapy has a great prognostic importance. Glutathion S Transferases (GSTs) might detoxify chemotherapeutic drugs within the cancer cells, thus contributing to chemotherapy resistance. The pi isoenzyme of GSTs seems to be of great relevance. Thus, we hypothesized that GSTpi expression in cancer biopsy can be a prognostic indicator for resistance to chemotherapy. To test this hypothesis, we evaluated before and after chemotherapy, tumor size, apoptosis of tumor cells with TUNEL assay, and proliferation of tumor cells by determining PCNA expression in biopsy samples, or in the surgically removed tumor tissue of GSTpi (-), and GSTpi (+) cases. MATERIALS AND METHODS GSTpi immunoreactivity was determined in 42 female patients with breast cancer. Patients were divided into two groups according to the expression of GSTpi in the pre-treatment biopsy specimen: (+) (n = 22) and (n = 20) samples were analyzed. Surgery was performed 2 weeks after a single intravenous injection of the chemotherapeutic drugs [5-fluorouracil, adriamycin, mitomycin (FAM protocol)]. RESULTS Pre-chemotherapy values of tumor size, apoptosis, or proliferation did not differ between GSTpi (-) and (+) samples. Chemotherapy significantly inhibited tumor growth, and cell proliferation, and induced apoptosis in GSTpi (-) cases. However, these effects were significantly reduced in GSTpi (+) patients. CONCLUSION These results suggest, that the presence of GSTpi in breast cancer tissue is a bad prognostic indicator, and these tumors are largely resistant to chemotherapy. Thus, GSTpi might be important in inactivating one or more of the chemotherapeutic agents used in this treatment.

Elevated systemic TGF-β impairs aortic vasomotor function through activation of NADPH oxidase-driven superoxide production and leads to hypertension, myocardial remodeling, and increased plaque formation in apoE−/− mice

The role of circulating, systemic TGF-beta levels in endothelial function is not clear. TGF-beta(1) may cause endothelial dysfunction in apolipoprotein E-deficient (apoE(-/-)) mice via stimulation of reactive oxygen species (ROS) production by the NADPH oxidase (NOX) system and aggravate aortic and heart remodeling and hypertension. Thoracic aorta (TA) were isolated from 4-mo-old control (C57Bl/6), apoE(-/-), TGF-beta(1)-overexpressing (TGFbeta(1)), and crossbred apoE(-/-) x TGFbeta(1) mice. Endothelium-dependent relaxation was measured before and after incubation with apocynin (NOX inhibitor) or superoxide dismutase (SOD; ROS scavenger). Superoxide production within the vessel wall was determined by dihydroethidine staining under confocal microscope. In 8-mo-old mice, aortic and myocardial morphometric changes, plaque formation by en face fat staining, and blood pressure were determined. Serum TGF-beta(1) levels (ELISA) were elevated in TGFbeta(1) mice without downregulation of TGF-beta-I receptor (immunohistochemistry). In the aortic wall, superoxide production was enhanced and NO-dependent relaxation diminished in apoE(-/-) x TGFbeta(1) mice but improved significantly after apocynin or SOD. Myocardial capillary density was reduced, fibrocyte density increased, aortic wall was thicker, combined lesion area was greater, and blood pressure was higher in the apoE(-/-) x TGFbeta vs. C57Bl/6 mice. Our results demonstrate that elevated circulating TGF-beta(1) causes endothelial dysfunction through NOX activation-induced oxidative stress, accelerating atherosclerosis and hypertension in apoE(-/-) mice. These findings may provide a mechanism explaining accelerated atherosclerosis in patients with elevated plasma TGFbeta(1).

Male gender predisposes to development of endotoxic shock in the rat.

OBJECTIVE After intravenous (i.v.) injection of lipopolysaccharide (LPS) macrophages release nitric oxide (NO) due to the expression of the inducible NO synthase (iNOS). After LPS NO is abundantly produced also in the cardiovascular system and may contribute to the development of hypotension and shock. Since the immune response, the synthesis of NO and the regulation of blood pressure (BP) differ between males and females, in the present study the effect of LPS on BP, renal function, the plasma and urinary concentration of the metabolites of NO as well as the splenic and aortic expression of the iNOS gene were compared between male and female rats. METHODS BP and renal function were measured in anesthetized rats following the i.v. injection of LPS (E. coli, 4 mg/kg). The NO2- and NO3- (metabolites of NO=NOx) concentration was measured by the Griess reaction. The iNOS gene expression was studied by RT-PCR. RESULTS Four hours after LPS, BP of males (n=9) was reduced by 63+/-12 mmHg versus 10+/-4 in females (n=7, P<0.005). Aminoguanidine, a selective inhibitor of iNOS, prevented the reduction of BP in males. The plasma concentration of NOx (P(NOx)), microM) was lower in hypotensive males (128+/-20) than in normotensive females (235+/-29, P<0.005). Males also exhibited lower urinary NOx excretion (U(NOx)V) after LPS (P<0.001 vs. females). Prior castration of males provided protection against hypotension (fall of BP: -4+/-4 mmHg, n=6, P<0.02 versus males) and resulted in higher P(NOx) as well as U(NOx)V (both P<0.001 versus males and not different from females). Prior ovariectomy (n=5) had no influence on the hemodynamic and NOx response to LPS. Male rats displayed enhanced aortic iNOS/beta-actin ratio relative to females after LPS (n=3 in each group, P<0.05). CONCLUSIONS (1) Male gender may sensitize to LPS-induced shock and (2) sensitivity of males to endotoxin is associated with an attenuated, not exaggerated total rate of NO synthesis.

Can siRNA technology provide the tools for gene therapy of the future

A new era in genetics has started 15 years ago, when co-suppression in petunia has been discovered. Later, co-suppression was identified as RNA interference (RNAi) in many plant and lower eukaryote animals. Although an ancient antiviral host defense mechanism in plants, the physiologic role of RNAi in mammals is still not completely understood. RNAi is directed by short interfering RNAs (siRNAs), one subtype of short double stranded RNAs. In this review we summarize the history and mechanisms of RNAi. We also aim to highlight the correlation between structure and efficacy of siRNAs. Delivery is the most important obstacle for siRNA based gene therapy. Viral and nonviral deliveries are discussed. In vivo delivery is the next obstacle to clinical trials with siRNAs. Although hydrodynamic treatment is effective in animals, it cannot be used in human therapy. One possibility is organ selective catheterization. The known side effects of synthesized siRNAs are also discussed. Although there are many problems to face in this new field of gene therapy, successful in vitro and in vivo experiments raise hope for treating human disease with siRNA.

Proteomic identification of vanin-1 as a marker of kidney damage in a rat model of type 1 diabetic nephropathy

At present, the urinary albumin excretion rate is the best noninvasive predictor for diabetic nephropathy (DN) but major limitations are associated with this marker. Here, we used in vivo perfusion technology to establish disease progression markers in an animal model of DN. Rats were perfused with a reactive ester derivative of biotin at various times after streptozotocin treatment. Following homogenization of kidney tissue and affinity purification of biotinylated proteins, a label-free mass spectrometry-based proteomic analysis of tryptic digests identified and relatively quantified 396 proteins. Of these proteins, 24 and 11 were found to be more than 10-fold up- or downregulated, respectively, compared with the same procedure in vehicle-treated rats. Changes in the expression of selected differentially regulated proteins were validated by immunofluorescence detection in kidney tissue from control and diabetic rats. Immunoblot analysis of pooled human urine found that concentrations of vanin-1, an ectoenzyme pantetheinase, distinguished diabetic patients with macroalbuminuria from those with normal albuminuria. Uromodulin was elevated in the urine pools of the diabetic patients, regardless of the degree of albuminuria, compared with healthy controls. Thus, in vivo biotinylation facilitates the detection of disease-specific changes in the abundance of potential biomarker proteins for disease monitoring and/or pharmacodelivery applications.

Gene Knockdown by EpCAM Aptamer–siRNA Chimeras Suppresses Epithelial Breast Cancers and Their Tumor-Initiating Cells

Effective therapeutic strategies for in vivo siRNA delivery to knockdown genes in cells outside the liver are needed to harness RNA interference for treating cancer. EpCAM is a tumor-associated antigen highly expressed on common epithelial cancers and their tumor-initiating cells (TIC, also known as cancer stem cells). Here, we show that aptamer–siRNA chimeras (AsiC, an EpCAM aptamer linked to an siRNA sense strand and annealed to the siRNA antisense strand) are selectively taken up and knock down gene expression in EpCAM+ cancer cells in vitro and in human cancer biopsy tissues. PLK1 EpCAM-AsiCs inhibit colony and mammosphere formation (in vitro TIC assays) and tumor initiation by EpCAM+ luminal and basal-A triple-negative breast cancer (TNBC) cell lines, but not EpCAM− mesenchymal basal-B TNBCs, in nude mice. Subcutaneously administered EpCAM-AsiCs concentrate in EpCAM+ Her2+ and TNBC tumors and suppress their growth. Thus, EpCAM-AsiCs provide an attractive approach for treating epithelial cancer. Mol Cancer Ther; 14(10); 2279–91. ©2015 AACR.