Mitotane, also known as o,p'‑DDD or (RS)‑1‑chl­oro‑2‑[2,2‑dichloro‑1‑(4‑chlorophenyl)‑ethyl]‑benzene, is an adrenal cortex-specific cytotoxic drug used in the therapy of adrenocortical carcinoma (ACC). The drug also inhibits steroidogenesis, however, the mechanisms of its anticancer and antisteroidogenic effects remain unknown. At present, data on the impact of mitotane on cell viability and the regulation of genes encoding proteins associated with steroids synthesis in the adrenal cortex, including cortisol and dehydroepiandrosterone sulfate (DHEAS), are limited and contradictory. In the present study, the effect of 24‑h mitotane treatment on viability of the ACC cell line, NCI‑H295R, was analyzed, identifying a decrease in cell viability and an increase in caspase‑3 and ‑7 activities. Mitotane treatment also led to decreased cortisol and DHEAS concentration in the culture media. Concomitantly, mitotane resulted in decreased mRNA levels of two cytochromes P450 (CYP11A1 and CYP17A1), mRNAs encoding proteins involved in the synthesis of cortisol and DHEAS. Mitotane did not affect mRNA levels of cyclin dependent kinase inhibitor 1A (encoding p21) and MYC (encoding cMyc). cMyc and p21 are key transcription factors associated with cell cycle regulation. However, mitotane inhibited expression of transforming growth factor β1 gene, encoding a potent inhibitor of cell proliferation and steroidogenesis. PRKAR1A, a protein kinase A regulatory subunit, is involved in the activation of steroidogenesis. PRKAR1A mRNA levels were reduced following 24‑h treatment with mitotane. Results indicate that mitotane markedly inhibited expression of genes involved in steroidogenesis, secretion of cortisol and DHEAS. Reduced expression of TGFB1 cannot account fully for the effect of mitotane on CYP11A1 and CYP17A1. We hypothesized that reduced viability of NCI‑H295R cells in the presence of mitotane may be a result of apoptosis triggered by increased caspase‑3 and ‑7 activities. Since p21 and cMyc mRNA levels were stable in the presence of mitotane, the mechanism by which caspase‑3 and ‑7 are induced remains unknown.

Expression of MIR125A is diminished in breast tumors, however the reason for the hsa‑mir‑125a decrease in the cancer is not known. HER2 is encoded by ERBB2, a target for hsa‑miR‑125a which interacts with the 3'UTR of ERBB2 mRNA. The present study reveals that a polymorphism (rs12976445) within the pri‑miR‑125a sequence correlates with the amount of mature hsa‑miR‑125a in breast tumor samples. miRNA, RNA and DNA were extracted from breast cancer samples obtained from 26 patients. Following immunohistological evaluation of the samples, the ERBB2, PGR and ESR1 mRNA profiles were also analyzed using real‑time PCR. Genomic DNA was sequenced using MIR125A flanking primers. PCR products were analyzed using a BaeGI restriction enzyme specific to the rs12976445 variant. The rs12976445 variant (C/T and C/C) correlated with a lower level of hsa‑miR‑125a in comparison with the T/T variant. The expression of HER2 mRNA was increased in tumors with the rs12976445 variant (C/T and C/C) compared with T/T. We conclude that rs12976445 may be a potential prognostic marker of HER2 expression in breast cancer. Its predictive value on the efficacy of trastuzumab treatment in patients with HER2‑positive breast cancer warrants further study.

DiO and DiD are lipophilic cell labelling dyes used in the staining of cells in vivo and in vitro. The aim of the present study was to quantify the asymmetrical distribution of dyes in co‑cultured cells and to measure the intercellular transfer of DiO and DiD. DiO and DiD were applied separately to stain two identical populations of SW‑1353 human chondrosarcoma cells that were subsequently co‑cultured (homotypic co‑culture). The intercellular migration of dyes in the co‑cultured cells was measured by flow cytometry and recorded under a fluorescent microscope. DiD and DiO caused no effect on the proliferation of cells, the degradation rate of the two dyes was comparable and crossover effects between dyes were negligible. The results of the present study suggested that asymmetrical intercellular migration of DiD and DiO was responsible for the asymmetrical distribution of these dyes in co‑cultured cells. To take advantage of the lipophilic dyes migration in the double-stained co-cultured cells we suggest to apply mixed-dyes controls prior to the flow cytometric analysis. These controls are performed by staining cells with a 1:1 mix of the two dyes and would enable the estimation of the intensity of intercellular contact in co‑culture systems. A 1:1 premix of DiO and DiD was applied to estimate cellular effect on intercellular exchange of lipid dyes in co‑cultures incubated with cycloheximide and cytochalasin B. The cellular effect contributed 6‑7% of intercellular migration of the lipophilic dyes, DiO and DiD. The majority of the observed intercellular transfer of these dyes was due to non‑cellular, passive transfer.

Many cell types secrete spherical membrane bodies classified as extracellular vesicles (EVs). EVs participate in intercellular communication and are present in body fluids, including blood, lymph, and cerebrospinal fluid. The time of EVs survival in the body varies depending on the body's localisation. Once the EVs reach cells, they trigger a cellular response. Three main modes of direct interaction of EVs with a target cell were described: receptor-ligand interaction mode, a direct fusion of EVs with the cellular membrane and EVs internalisation. Studies focused on the medical application of EVs. Medical application of EVs may require modification of their surface and interior. EVs surface was modified by affecting the parental cells or by the direct amendment of isolated EVs. The interior modification involved introducing materials into the cells or direct administrating isolated EVs. EVs carry proteins, lipids, fragments of DNA, mRNA, microRNA (miRNA) and long non-coding RNA. Because of EVs availability in liquid biopsy, they are potential diagnostic markers. Modified EVs could enhance the treatment of diseases such as colorectal cancer, Parkinson's disease, leukaemia or liver fibrosis. EVs have specific tissue tropisms, which makes them convenient organ-directed carriers of nucleic acids, drugs and vaccines. In conclusion, recently published works have shown that EVs could become biomarkers and modern vehicles of advanced drug forms.

SPX (spexin) and its receptors GalR2 and GalR3 (galanin receptor subtype 2 and galanin receptor subtype 3) play an important role in the regulation of lipid and carbohydrate metabolism in human and animal fat tissue. However, little is still known about the role of this peptide in the metabolism of muscle. The aim of this study was to determine the impact of SPX on the metabolism, proliferation and differentiation of the skeletal muscle cell line C2C12. Moreover, we determined the effect of exercise on the SPX transduction pathway in mice skeletal muscle. We found that increased SPX, acting via GalR2 and GalR3 receptors, and ERK1/2 phosphorylation stimulated the proliferation of C2C12 cells (p < 0.01). We also noted that SPX stimulated the differentiation of C2C12 by increasing mRNA and protein levels of differentiation markers Myh, myogenin and MyoD (p < 0.01). SPX consequently promoted myoblast fusion into the myotubule (p < 0.01). Moreover, we found that, in the first stage (after 2 days) of myocyte differentiation, GalR2 and GalR3 were involved, whereas in the last stage (day six), the effect of SPX was mediated by the GalR3 isoform. We also noted that exercise stimulated SPX and GalR2 expression in mice skeletal muscle as well as an increase in SPX concentration in blood serum. These new insights may contribute to a better understanding of the role of SPX in the metabolism of skeletal muscle.