Marta Tkacz

5‑Azacytidine inhibits human rhabdomyosarcoma cell growth by downregulating insulin‑like growth factor 2 expression and reactivating the H19 gene product miR‑675, which negatively affects insulin‑like growth factors and insulin signaling

Insulin-like growth factor 2 (IGF2) and 1 (IGF1) and insulin (INS) promote proliferation of rhabdomyosarcoma (RMS) cells by interacting with the insulin-like growth factor 1 receptor (IGF1R) and the insulin receptor (INSR). Loss of imprinting (LOI) by DNA hypermethylation at the differentially methylated region (DMR) for the IGF2‑H19 locus is commonly observed in RMS cells and results in an increase in the expression of proliferation-promoting IGF2 and downregulation of proliferation-inhibiting non-coding H19 miRNAs. One of these miRNAs, miR‑675, has been reported in murine cells to be a negative regulator of IGF1R expression. To better address the role of IGF2 and 1, as well as INS signaling in the pathogenesis of RMS and the involvement of LOI at the IGF2‑H19 locus, we employed the DNA demethylating agent 5‑azacytidine (AzaC). We observed that AzaC‑mediated demethylation of the DMR at the IGF2‑H19 locus resulted in downregulation of IGF2 and an increase in the expression of H19. This epigenetic change resulted in a decrease in RMS proliferation due to downregulation of IGF2 and, IGF1R expression in an miR‑675‑dependent manner. Interestingly, we observed that miR‑675 not only inhibited the expression of IGF1R in a similar manner in human and murine cells, but we also observed its negative effect on the expression of the INSR. These results confirm the crucial role of LOI at the IGF2‑H19 DMR in the pathogenesis of RMS and are relevant to the development of new treatment strategies.

A lack of positive effect of enhanced vegetative nervous system tonus on mobilization of hematopoietic stem and progenitor cells in patients suffering from acute psychotic syndromes.

A lack of positive effect of enhanced vegetative nervous system tonus on mobilization of hematopoietic stem and progenitor cells in patients suffering from acute psychotic syndromes

Study on size effect of the silica nanospheres with solid core and mesoporous shell on cellular uptake.

The properties of mesoporous silica nanoparticles including large surface area, large pore volume, easy surface functionalization and control of structure and pore size has made them promising drug carriers. In this study, the effect of different diameters (50 nm, 70 nm, 90 nm, 110 nm and 140 nm) of silica nanospheres with a solid core and mesoporous shell (mSiO2/SiO2) on cellular internalization in mouse fibroblast cells (L929) was evaluated. The physical properties of the nanostructures were characterized with various methods, such as transmission electron microscopy with x-ray dispersion spectroscopy, thermogravimetric analysis, Fourier transform infrared spectroscopy and zeta potential. In order to define the cellular uptake, the nanostructures were labelled with fluorescent dye Alexa647, and imaging and quantitative methods were applied: laser scanning confocal microscopy, flow cytometry and thermogravimetry. Our results indicate that cellular uptake of the studied nanospheres is size-dependent, and nanospheres of 90 nm in diameter showed the most efficient cell internalization. Thus, particle size is an important parameter that determines cellular uptake of nanoparticles and should be considered in designing drug delivery carriers.

Molecular mechanisms regulating metastasis of cancer cells with special emphasis on rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a malignant tumor of soft tissue derived from embryonic mesenchymal and/or neuroectodermal tissues. It is most often associated with other genetic syndromes such as Li-Fraumeni or Bechwith-Wiedeman. RMS cells show morphological similarities to striated muscle and the presence of specific markers of muscle tissue. At the histological level, it is divided into two subtypes (alveolar RMS - ARMS and embryonal RMS - ERMS), which differ in their genetic background, and prognosis. In recent years there has been significant progress in understanding the mechanisms that regulate RMS cell growth and metastasis. Recently, a number of several chemokines, cytokines or growth factors and their receptors were identified involved in RMS pathogenesis as well as animal models of this tumor have been developed. This knowledge is of great importance in the development of potential therapeutic strategies not only in RMS, but also other types of cancer. This paper will discuss the theories of the origin of this rare tumor and the molecular mechanisms involved in its growth and metastasis. The processes and mechanisms described herein, such as chemotaxis, adhesion, proliferation, intracellular signal transduction, seem to universal for number of cancer types.

Plasma concentration of urokinase plasminogen activator receptor is a marker of kidney allograft function

BackgroundUrokinase-type plasminogen activator receptor (uPAR) is found in a variety of cell types including monocytes, lymphocytes, macrophages, and endothelial cells and plays an important role in fibrinolysis and in the activation and chemotaxis of neutrophils and lymphocytes. In this study, we examined the correlation between uPAR plasma concentration and kidney allograft function.AimsThis study enrolled 78 Caucasian deceased-donor renal transplant recipients.MethodsPlasma concentrations of uPAR were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits.ResultsWe observed elevated with borderline significance (p = 0.095) uPAR plasma concentrations in patients with tubular atrophy. Plasma concentrations of uPAR showed strong statistically significant positive correlations with serum creatinine or urea and strong negative correlation with estimated glomerular filtration rate (eGFR). There was also a borderline positive correlation between uPAR plasma concentration and protein concentration in urine as well as the duration of hemodialysis.ConclusionsThe results of our study indicate that uPAR plasma concentrations in kidney allograft recipients are significantly negatively correlated with graft function and may be elevated in patients with tubular atrophy.

Differential effect of adenosine on rhabdomyosarcoma migration and proliferation

Introduction Adenosine and its receptors are involved deeply in the regulation of tumour biology. Purine nucleotides are released from stressed cells in states of hypoxia or radiochemotherapy-induced cell damage. Adenosine exerts its effect through the P1 family of selective receptors. The purpose of the study was to evaluate the exact role of extracellular role on biology of Rhabdomyosarcoma (RMS) cells. Material and methods Series of in vitro studies accompanied by immunohistochemical, RQ-PCR and shRNA methods have characterised adenosine receptor expression on Rhabdomyosarcoma cell lines, normal skeletal muscle and effect of adenosine on Rhabdomyosarcoma growth and migration. Results Extracellular adenosine (highest at 50 μM, p < 0.05) and AMP (highest at 300 μM, p < 0.05) markedly enhanced chemotaxis in the Boyden chamber assay The reaction is mostly governed by the A1 receptor, which is greatly overexpressed in Rhabdomyosarcoma as compared with normal skeletal muscle. Cell migration induced by adenosine and AMP is blocked by pertussis toxin, phospholipase C and MAP kinase inhibitor, which demonstrates the importance of these signalling pathways. High doses of adenosine have a detrimental effect on cellular proliferation, in a receptor-independent manner (≥ 500 μM; p < 0.05). The blockage of adenosine transporter by dipyridamole abolishes this effect, indicating involvement of an intrinsic pathway. Further increase of adenosine concentration, induced by deaminase inhibitors, augment the effect. Conclusions Our results suggest that adenosine and AMP trigger cell migration by binding to P1 receptors and directing cancer cells to the sites of hypoxia or cellular damage. Specifically by A1 receptor which is overexpressed in RMS.

COX2 and NOS3 gene polymorphisms in women with gestational diabetes

Gestational diabetes (GDM) is carbohydrate intolerance occurring in pregnancy. Low‐grade inflammation plays an important role in the pathogenesis of this disorder. The present study aimed to examine the association between COX2 (rs6681231) and NOS3 (rs1799983 and rs2070744) gene polymorphisms and GDM.

Picropodophyllin (PPP) is a potent rhabdomyosarcoma growth inhibitor both in vitro and in vivo.

BackgroundInsulin-like growth factors and insulin are important factors promoting cancer growth and metastasis. The molecules act through IGF1 (IGF1R) and insulin (InsR) receptors. Rhambodmyosarcomas (RMS) overproduce IGF2 – a potent ligand for IGF1R and, at the same time, highly express IGF1 receptor. The purpose of the study was to evaluate possible application of picropodophyllin (PPP) – a potent IGF1R inhibitor.MethodsIn our study we used a number of in vitro assays showing influence of IGF1R blockage on RMS cell lines (both ARMS and ERMS) proliferation, migration, adhesion, cell cycling and signal transduction pathways. Additionally, we tested possible concomitant application of PPP with commonly used chemotherapeutics (vincristine, actinomycin-D and cisplatin). Moreover, we performed an in vivo study where PPP was injected intraperitoneally into RMS tumor bearing SCID mice.ResultsWe observed that PPP strongly inhibits RMS proliferation, chemotaxis and adhesion. What is more, application of the IGF1R inhibitor attenuates MAPK phosphorylation and cause cell cycle arrest in G2/M phase. PPP increases sensitivity of RMS cell lines to chemotherapy, specifically to vincristine and cisplatin. In our in vivo studies we noted that mice treated with PPP grew smaller tumors and displayed significantly decreased seeding into bone marrow.ConclusionsThe cyclolignan PPP effectively inhibits RMS tumor proliferation and metastasis in vitro and in an animal model.

Connexin 43 expression in the testes during postnatal development of finasteride-treated male rat offspring

Introduction Hormone-dependent events that occur throughout the first wave of spermatogenesis, such as cellular communication within seminiferous epithelium during early postnatal testis maturation, are important for adult male fertility. Any changes in the T/DHT ratio in male progeny born from females fertilized by finasteride-treated male rats can result in impairment of testicular physiology. The aim of the study was to verify whether finasteride has a transgenerational effect on the expression of connexin 43 (Cx43), a gap junction protein in testes of the F1 generation. Material and methods The subjects of the study were 7, 14, 21/22, 28, and 90-day-old Wistar male rats born by females fertilized by finasteride-treated rats (F1:Fin). The offspring born by untreated rats were used as controls (F1:Control). Connexin 43 was evaluated in the seminiferous epithelium by immunohistochemistry, and in the testis homogenates by Western blot and qRT-PCR. The Cx43 mRNA and protein expression was correlated with intratesticular levels of T and DHT by Spearman’s rank correlation coefficient. Results We observed a difference in the Cx43 expression in the testis of male rats born by female rats fertilized by finasteride-treated male rats, as compared to the control on following PND (7, 22 and 28 PND, p < 0.001; 14 PND, p < 0.01); and a strong, positive correlation between Cx43 with DHT was only in the F1:Fin group (mRNA: rs = +0.51, p = 0.004; protein: rs = +0.54, p = 0.002). Conclusions Finasteride treatment of male adult rats may cause changes in the communication between the testicular cells of their offspring, leading to a defective course of spermatogenesis.