Direct effects of vitamin D3 analogues on G-protein mediated signalling systems in rat osteosarcoma cells and rat pituitary adenoma cells

In normal rats treated with 1,25(OH)₂D₃ or 24,25(OH)₂D₃, serum Ca²⁺, ALP, PRL and GH are significantly altered. In order to study the primary effect of vitamin D₃ analogues on target organ function, rat UMR 106 osteosarcoma and GH₃ pituitary adenoma cells in monolayer culture were exposed accordingly.Surprisingly, prolonged exposure of these cell lines to physiological levels of either 1,25(OH)₂D₃ or 24,25(OH)₂D₃ did not significantly affect the secretory parameters (ALP, PRL or GH) tested. However, 1,25(OH)₂D₃ exposure significantly reduced PTH- and Gpp(NH)p-elicited AC as well as Gpp(NH)p-stimulated PLC activities in the UMR 106 cells. These changes were accompanied by an increase and decrease in the membrane contents of the G-protein subunits G₃₆ and G_q/11, respectively. In contrast, 24,25(OH)₂D₃ remained without significant biological effect on these signalling systems despite concomitantly augmented levels of G₃₆. TRH- and Gpp(NH)p-elicited PLC activities in the GH₃ cells were significantly reduced by 1,25(OH)₂D₃ with a concurrent reduction in cellular amounts of G_q/11, however, 24,25(OH)₂D₃ did not significantly alter any signalling systems nor G-proteins analyzed.It is concluded that the osteoblastic and pituitary cell secretion of ALP, PRL and GH remain unaffected by the presence of 1,25(OH)₂D₃ and 24,25(OH)₂D₃, despite distinct alterations in components of G-protein mediated signalling pathways. Hence, other factors like ambient Ca²⁺ may be responsible for the perturbed secretory patterns of ALP and PRL seen in vitamin D₃ treated rats.Abbreviations AC adenylate cyclase - ALP alkaline phosphatase - BGP osteocalcin - BSA bovine serum albumin - DA dopamine - DAG diacylglycerol - GH growth hormone - GHRH growth hormone releasing hormone - Gpp(NH)p guanosine 5-[-imido]triphosphate - G-protein guanine nucleotide-binding regulatory protein - G_s etc. G_s protein -subunit - IP₃ inositol 1,4,5 trisphosphate - OAF osteoclast activating factor - PGE₂ prostaglandin E₂ - PKA & PKC protein kinase A & C - PLC phospholipase C - PRL prolactin - PTH parathyroid hormone - SRIF somatostatin - TRH thyrotropin releasing hormone - VIP vasoactive intestinal peptide - 25(OH)D₃ 25 hydroxy vitamin D₃ - 1,25(OH)₂D₃ 1·25 dihydroxy vitamin D₃ - 24,25(OH)₂D₃ 24,25 dihydroxy vitamin D₃     

Multidrug-resistance (MDR) phenotype of human osteosarcoma cells evaluated by quantitative morphological and electron microscopy analyses

Summary— Multidrug-resistant (MDR) variants of a human osteosarcoma cell line (U-2 OS) have been recently obtained by continuous exposure to doxorubicin (DX). The growth and phenotypic characteristics of these cell lines have been demonstrated to be related to the level of expression of P-glycoprotein. In this work, the morphological changes associated with MDR have been evaluated by quantitative image analysis and transmission electron microscopy. Resistant cells present morphological changes with respect to sensitive cells at both cytoplasmic and nuclear level. Some of these changes appear to be related to the degree of resistance but not to the direct presence of DX, since deprived cells maintain some modified characters, while others are partly lost. These findings suggest that DX exposure affects cell metabolism causing progressive changes of the cell morphotype.     

1α,25-dihydroxyvitamin D3 rapidly alters phospholipid metabolism in the nuclea envelope of osteoblasts

1α,25-Dihydroxyvitamin D₃ (1α, 25-(OH)₂D₃) has been shown to increase cytosolic calcium and inositol trophosphate levels in rat osteosarcoma cells (ROS 17/2.8) and to increase nuclear calcium in these cells. To determine the mechanism(s) of 1α, (OH)₂D₃-induced changes in the calcium, the effect of the hormone on phospholipid metabolism in isolated osteoblast nuclei wa assessed. 1α,25 (OH)₂D₃, 20 nM, increased inositol triphosphate levels in the nuclei after 5 min of treatment. The biologically inactive epimer, 1β,25-(OH)₂D₃, had no significant effect on inositol triphosphate levels. ATP, 1 mM, also increased inositol triphosphate levels in the isolated nuclei after 5 min. 1α,25-(OH)₂D₃, 20 nM, increased calcium in the isolated nuclei in the presence but not in the absence of extranuclear calcium with 5 min. Nuclear calcium was also increased within 5 min by ATP, 1 mM, and inositol triphosphate, 1 mM. The effects of ATP on nuclear calcium was not additive with 1α, 25-(OH)₂D₃, suggesting that these two agents increase nuclear calcium in these osteoblast-like cells by similar mechanisms. In summary, 1α,25-(OH)₂D₃ amd ATP rapidly increase inositol triphosphate levels in isolated from ROS 17/2.8 cells. The hormone, the nucleotide, and the inositol phospholipid nuclear calcium. Thus, the 1α,25-(OH)₂D₃ and ATP effects of nuclear calcium may be mediated by changes in phospholipid metabolism in the nuclei of these osteoblastlike cells. © Wiley-Liss, Inc.     

Extracellular nanovesicles‐transmitted circular RNA has_circ_0000190 suppresses osteosarcoma progression

Under the microenvironment, tumour progression is substantially affected by cell‐cell communication. In spite of the mediating effect of extracellular nanovesicles (EVs) on cell‐cell communication by packaging into circRNAs, the effect of EVs circRNA hsa_circ_0000190 (circ‐0000190) in osteosarcoma is still not clear. Circ‐0000190 expressions in tissues and EVs from plasma were compared between osteosarcoma patients and controls. Thereafter, receiver operating characteristic (ROC) curve was drawn and area under the curve was calculated to examine whether the diagnostic results were accurate, and the effect of EVs circ‐0000190 was dug out via the determination of cell phenotypes and animal assays. Results showed circ‐0000190 exhibited an obvious reduction in EVs and tissues of osteosarcoma patients (P < .05). It was also discovered that EVs encapsulated the majority of circ‐0000190, and EVs‐encapsulated circ‐0000190 could be applied to make a distinction between osteosarcoma patients and controls. Besides, EVs circ‐0000190 in osteosarcoma cells transported from normal cells weakened the capacities of osteosarcoma cells to migrate, proliferate and invade, so as to block their biological malignant behaviours (P < .05). In addition, under the action of EVs circ‐0000190, tumour growth was impeded and the expression of TET1 was inhibited via the competitive binding to miR‐767‐5p. In all, EVs circ‐0000190 has a good prospect as it can be regarded as a new biomarker for detecting osteosarcoma. EVs circ‐0000190 transported from normal cells to osteosarcoma cells impeded the in vitro and in vivo development of osteosarcoma, implying that EVs circ‐0000190 exerts an effect on communication between normal cells and osteosarcoma cells in the carcinogenesis process of osteosarcoma.     

HDAC6 inhibitor WT161 performs anti-tumor effect on osteosarcoma and synergistically interacts with 5-FU

Background: WT161, as a selective HDAC6 inhibitor, has been shown to play anti-tumor effects on several kinds of cancers. The aim of the present study is to explore the roles of WT161 in osteosarcoma and its underlying mechanisms.Methods: The anti-proliferative effect of WT161 on osteosarcoma cells was examined using MTT assay and colony formation assay. Cell apoptosis was analyzed using flow cytometer. The synergistic effect was evaluated by isobologram analysis using CompuSyn software. The osteosarcoma xenograft models were established to evaluate the anti-proliferative effect of WT161 in vivo.Results: WT161 suppressed the cell growth and induced apoptosis of osteosarcoma cells in a dose- and time-dependent manner. Mechanistically, we found that WT161 treatment obviously increased the protein level of PTEN and decreased the phosphorylation level of protein kinase-B (AKT). More importantly, WT161 showed synergistic inhibition with 5-FU on osteosarcoma cells in vitro and in vivo.Conclusions: These results indicate that WT161 inhibits the growth of osteosarcoma through PTEN and has a synergistic efficiency with 5-FU.     

Bone morphogenetic protein 9 overexpression reduces osteosarcoma cell migration and invasion

Transforming growth factor-β (TGF-β) is known to promote tumor migration and invasion. Bone morphogenetic proteins (BMPs) are members of the TGF-β family expressed in a variety of human carcinoma cell lines. The role of bone morphogenetic protein 9 (BMP9), the most powerful osteogenic factor, in osteosarcoma (OS) progression has not been fully clarified. The expression of BMP9 and its receptors in OS cell lines was analyzed by RT-PCR. We found that BMP9 and its receptors were expressed in OS cell lines. We further investigated the influence of BMP9 on the biological behaviors of OS cells. BMP9 overexpression in the OS cell lines 143B and MG63 inhibited in vitro cell migration and invasion. We further investigated the expression of a panel of cancer-related genes and found that BMP9 overexpression increased the phosphorylation of Smad1/5/8 proteins, increased the expression of ID1, and reduced the expression and activity of matrix metalloproteinase 9 (MMP9) in OS cells. BMP9 silencing induced the opposite effects. We also found that BMP9 may not affect the chemokine (C-X-C motif) ligand 12 (CXCL12)/C-X-C chemokine receptor type 4 (CXCR4) axis to regulate the invasiveness and metastatic capacity of OS cells. Interestingly, CXCR4 was expressed in both 143B and MG63 cells, while CXCL12 was only detected in MG63 cells. Taken together, we hypothesize that BMP9 inhibits the migration and invasiveness of OS cells through a Smad-dependent pathway by downregulating the expression and activity of MMP9.