Mitotane, also known as o,p'‑DDD or (RS)‑1‑chl-oro‑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.

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Abstract. Degeneration of the intervertebral disc (IVD) is the main cause of age-related damage of spinal tissues. Using multipotent mesenchymal stromal cells (MSCs) regenerative medicine intends to restore the IVD components of annulus fibrosus (AF) and nucleus pulposus (NP). In the present study NP cells (NPCs) and MSCs obtained from adolescent patients suffering from scoliosis were used. IVDs and vertebrae were obtained during surgery and subsequently processed in order to establish cultures of NPCs and MSCs. The two cell types were co-cultured in 1-µm pore size insert system (indirect co-culture) or on one surface (direct co-culture). Prior to co-culture in these systems one of the cell types was stained by lipophilic fluorescent dye DiD (red). The results demonstrated that regardless of the cell type, the flow of DiD from stained to non-stained cells was more efficient in the direct co-culture in comparison with the insert system. Moreover, in the direct system the DiD flow was more efficient from MSCs towards NPCs compared with that in the opposite direction. These data indicated that the membrane interchange between the two cell types was asymmetric. To discriminate the subpopulation of cells that underwent membrane interchange, cells were double stained with DiD and DiO (green). In the first part of the experiment NPCs were stained by DiO and MSCs by DiD. In the second, NPCs were stained by DiD and MSCs by DiO. The cells were co-cultured in the direct system for 8 days and subsequently analyzed by flow cytometry and confocal microscopy.This analysis revealed that >50% of cells were stained by the DiO and DiD dyes. NPCs and MSCs formed structures similar to tunnelling nanotubes (TnT). In conclusion, the formation of TnT-like structures is able to promote, phenotypic changes during the direct co-culture of NPCs with MSCs.

Intervertebral disc (IVD) consists of surrounding tissue annulus fibrosus and central nucleus pulposus, which are partially degenerative in scoliotic IVDs. Successful regeneration of scoliotic alterations requires cognition of critical paracrine mediators of cell-to-cell contact in the IVD. In this work, we hypothesized that transforming growth factor β (TGF-β) is involved in the intercellular communication of nucleus pulposus cells (NPCs) and mesenchymal stem cells (MSCs). We observed that in cultured NPCs TGF-β1 stimulated COL1A1 expression, encoding collagen I, and in MSCs stimulated COL1A1 and SOX9 expressions. We subsequently co-cultured NPCs and MSCs together using direct and indirect transwell systems. The expression of miR-140 and miR-145 were decreased in co-cultured NPCs. We observed that direct co-culture system stronger than the indirect system decreased expression of three miRNA. The expression of COL1A1, ACAN, encoding aggrecan, and SOX9 genes was increased in MSCs co-cultured with NPCs. Co-cultures were incubated with two inhibitors of TGF-β type I receptor: SB-431542 and SB-525334. In co-cultured NPCs, SB-431542 and SB-525334 annulated downregulation of miR-140 and miR-145. In MSCs these inhibitors diminished stimulation of COL1A1, ACAN, and SOX9. We concluded that stimulation of COL1A1, ACAN, and SOX9 in co-cultured MSCs and regulation of miR-140 and miR-145 in NPCs were TGF-β-dependent and TGF-β is involved in the communication of NPCs and MSCs in co-culture. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.