Insulin regulates GLUT1-mediated glucose transport in MG-63 human osteosarcoma cells

Osteosarcoma is the most common type of malignant bone cancer, accounting for 35% of primary bone malignancies. Because cancer cells utilize glucose as their primary energy substrate, the expression and regulation of glucose transporters (GLUT) may be important in tumor development and progression. GLUT expression has not been studied previously in human osteosarcoma cell lines. Furthermore, although insulin and insulin-like growth factor (IGF-I) play an important role in cell proliferation and tumor progression, the role of these hormones on GLUT expression and glucose uptake, and their possible relation to osteosarcoma, have also not been studied. We determined the effect of insulin and IGF-I on GLUT expression and glucose transport in three well-characterized human osteosarcoma cell lines (MG-63, SaOs-2, and U2-Os) using immunocytochemical, RT-PCR and functional kinetic analyses. Furthermore we also studied GLUT isoform expression in osteosarcoma primary tumors and metastases by in situ hybridization and immunohistochemical analyses. RT-PCR and immunostaining show that GLUT1 is the main isoform expressed in the cell lines and tissues studied, respectively. Immunocytochemical analysis shows that although insulin does not affect levels of GLUT1 expression it does induce a translocation of the transporter to the plasma membrane. This translocation is associated with increased transport of glucose into the cell. GLUT1 is the main glucose transporter expressed in osteosarcoma, furthermore, this transporter is regulated by insulin in human MG-63 cells. One possible mechanism through which insulin is involved in cancer progression is by increasing the amount of glucose available to the cancer cell.     

Thrombospondin is synthesized and secreted by human osteoblasts and osteosarcoma cells. A model to study the different effects of thrombospondin in cell adhesion

In this study we have shown by both immunofluorescence and immunoprecipitation techniques that human osteoblasts and osteosarcoma cells synthesize and secrete thrombospondin, a 450-kDa glycoprotein initially found in platelets. Immunofluorescence with a mouse monoclonal antibody to human platelet thrombospondin yielded specific granular staining within the cytoplasm of human osteoblasts. SDS/polyacrylamide gel electrophoresis analysis of immunoprecipitates obtained with polyclonal and monoclonal anti-thrombospondin antibodies allows the identification of thrombospondin in the cellular lysates and the culture media of biosynthetically labelled osteoblasts and osteosarcoma cells. Kinetic and dose/response studies of osteoblasts and of two osteosarcoma cell lines (MG-63, SaOs-2) were performed to assess the ability of these cells to adhere to thrombospondin and type-I collagen. Thrombospondin promoted the attachment of human osteoblasts whereas it inhibited the adhesion of MG-63 and SaOs-2 cells, both when it was directly adsorbed to plastic and when it was bound to type-I collagen. Therefore osteoblasts and osteosarcoma cells may be valuable tools to study the role of thrombospondin in cell adhesion.     

Inhibition of proliferation and induction of differentiation of osteoblastic cells by a novel 1,25-dihydroxyvitamin D3 analog with an extensively modified side chain (CB1093)

1,25-Dihydroxyvitamin D₃ (1,25D) is involved in the regulation of proliferation and differentiation of a variety of cell types including cancer cells. In recent years, numerous new vitamin D₃ analogs have been developed in order to obtain favorable therapeutic properties. The effects of a new 20-epi analog, CB1093 (20-epi-22-ethoxy-23-yne-24a,26a,27a-trihomo-1α,25(OH)₂D₃), on the proliferation and differentiation of human MG-63 osteosarcoma cell line were compared here with those of the parent compound 1,25D. Proliferation of the MG-63 cells was inhibited similarly by 22%, 50% and 59% after treatment with 0.1 μM 1,25D or CB1093 for 48 h, 96 h, and 144 h, respectively. In transfection experiments, the compounds were equipotent in stimulating reporter gene activity under the control of human osteocalcin gene promoter. In cell culture experiments, however, CB1093 was more potent than 1,25D at low concentrations and more effective for a longer period of time in activating the osteocalcin gene expression at mRNA and protein levels. Also, a 6-h pretreatment and subsequent culture for up to 120 h without 1,25D or CB1093 yielded higher osteocalcin mRNA and protein levels with analog-treated cells than with 1,25D-treated cells. The electrophoretic mobility shift assay (EMSA) revealed stronger VDR-VDRE binding with analog-treated MG-63 cells than with 1,25D-treated cells. The differences in the DNA binding of 1,25D-bound vs. analog-bound VDR, however, largely disappeared when the binding reactions were performed with recombinant hVDR and hRXRβ proteins. These results demonstrate that the new analog CB1093 was equally or even more effective than 1,25D in regulating all human osteosarcoma cell functions ranging from growth inhibition to marker gene expression and that the differences in effectivity most probably resulted from interactions of the hVDR:hRXRβ-complex with additional nuclear proteins. J. Cell. Biochem. 70:414–424, 1998. © 1998 Wiley-Liss, Inc.     

Hierarchical Clustering of Breast Cancer Methylomes Revealed Differentially Methylated and Expressed Breast Cancer Genes

Oncogenic transformation of normal cells often involves epigenetic alterations, including histone modification and DNA methylation. We conducted whole-genome bisulfite sequencing to determine the DNA methylomes of normal breast, fibroadenoma, invasive ductal carcinomas and MCF7. The emergence, disappearance, expansion and contraction of kilobase-sized hypomethylated regions (HMRs) and the hypomethylation of the megabase-sized partially methylated domains (PMDs) are the major forms of methylation changes observed in breast tumor samples. Hierarchical clustering of HMR revealed tumor-specific hypermethylated clusters and differential methylated enhancers specific to normal or breast cancer cell lines. Joint analysis of gene expression and DNA methylation data of normal breast and breast cancer cells identified differentially methylated and expressed genes associated with breast and/or ovarian cancers in cancer-specific HMR clusters. Furthermore, aberrant patterns of X-chromosome inactivation (XCI) was found in breast cancer cell lines as well as breast tumor samples in the TCGA BRCA (breast invasive carcinoma) dataset. They were characterized with differentially hypermethylated XIST promoter, reduced expression of XIST, and over-expression of hypomethylated X-linked genes. High expressions of these genes were significantly associated with lower survival rates in breast cancer patients. Comprehensive analysis of the normal and breast tumor methylomes suggests selective targeting of DNA methylation changes during breast cancer progression. The weak causal relationship between DNA methylation and gene expression observed in this study is evident of more complex role of DNA methylation in the regulation of gene expression in human epigenetics that deserves further investigation.     

DNA methylation patterns associated with asparagine synthetase expression in asparagine-overproducing and -auxotrophic cells.

In Chinese hamster ovary cells, the gene for asparagine synthetase, which spans 20 kilobase pairs, was found to contain a cluster of potential sites for CpG methylation in a 1-kilobase-pair region surrounding the first exon. Fourteen of the sites that could be assayed for methylation by MspI-HpaII digestions were found in this region, with an additional nine MspI sites spread throughout the remainder of the gene. The methylation status of the gene was analyzed in a series of cell lines that differed in the amount of asparagine synthetase activity. The level of expression showed a direct correlation with the extent of methylation of a subset of the MspI sites found in the 5' region of the gene. The rest of the gene was completely methylated in most cell lines. Wild-type cells, which expressed a basal level of asparagine synthetase activity, were partially demethylated in the 5' region. In contrast, asparagine-requiring N3 cells, which lacked detectable mRNA for asparagine synthetase, were methylated throughout the entire gene. Spontaneous revertants of strain N3, selected for growth in asparagine-free medium, exhibited extensive hypomethylation of the asparagine synthetase gene. The methylation pattern of the gene in cell lines that overproduced the enzyme was also examined. Albizziin-resistant cell lines, which had amplified copies of the gene, were extensively demethylated in the 5' region. Overexpression of asparagine synthetase in beta-aspartyl hydroxamate-resistant lines without amplified copies of the gene was also correlated with DNA hypomethylation.     

Stimulation of prostaglandin E2 (PGE2) production by arachidonic acid, oestrogen and parathyroid hormone in MG-63 and MC3T3-E1 osteoblast-like cells

Polyunsaturated fatty acids (PUFAs) as well as oestrogen (E2) and parathyroid hormone (PTH) affect bone cells. The aim of the study was to determine whether arachidonic acid (AA), E2, and PTH increase prostaglandin E₂ (PGE₂) synthesis in MG-63 and MC3T3-E1 osteoblastic cells and the level of mediation by COX-1 and COX-2. PGE₂ levels were determined in the conditioned culture media of MG-63 and MC3T3-E1 osteoblasts after exposure to AA, PTH and E2. Cells were pre-incubated in some experiments with the unselective COX inhibitor indomethacin or the COX-2 specific blocker NS-398. Indirect immunofluorescence was performed on MG-63 cells to detect the presence and location of the two enzymes involved. AA increased PGE₂ secretion in both cell lines; production by MC3T3-E1 cells, however, was significantly higher than that of MG-63 cells. This could be due to autoamplification via the EP₁ subtype of PGE receptors in mouse MC3T3-E1 osteoblasts. Both COX-1 and COX-2 affected the regulation of PGE₂ synthesis in MG-63 cells. E2 had no effect on PGE₂ secretion in both cell lines, while PTH caused a slight increase in PGE₂ synthesis in the MG-63 cell line.     

The effect of titanium with heparin/BMP-2 complex for improving osteoblast activity

The objective of this study was to investigate the enhanced osteoblast activity of MG-63 cells cultured on titanium (Ti) with a heparin/BMP-2 (Hep/BMP-2) complex. The Ti substrates were initially modified by chemical grafting poly-l-lysine (PLL) using condensing agent, followed by immobilizing the heparin/BMP-2 complex to the PLL-grafted Ti substrate via electrostatic interactions. The surface modification of Ti substrates with PLL and/or Hep/BMP-2 complex were confirmed with scanning electron microscopy, contact angle measurements, and X-ray photoelectron spectroscopy. Immobilized BMP-2 was released from the Hep/BMP-2/Ti substrate in a sustained manner. In vitro studies revealed that osteoblasts grown on Hep/BMP-2/Ti substrate increased ALP activity, calcium deposition, ALP and osteocalcin levels as compared to those grown on pristine Ti or PLL-Ti. These results indicated that heparin/BMP-2 complex immobilized Ti substrate can be useful to effectively improve osteoblast activity.     

Rapid and specific hypomethylation of enhancers in endothelial cells during adaptation to cell culturing

Epigenetics, including DNA methylation, is one way for a cell to respond to the surrounding environment. Traditionally, DNA methylation has been perceived as a quite stable modification; however, lately, there have been reports of a more dynamic CpG methylation that can be affected by, for example, long-term culturing. We recently reported that methylation in the enhancer of the gene encoding the key fibrinolytic enzyme tissue-type plasminogen activator (t-PA) was rapidly erased during cell culturing. In the present study we used sub-culturing of human umbilical vein endothelial cells (HUVECs) as a model of environmental challenge to examine how fast genome-wide methylation changes can arise. To assess genome-wide DNA methylation, the Infinium HumanMethylation450 BeadChip was used on primary, passage 0, and passage 4 HUVECs. Almost 2% of the analyzed sites changed methylation status to passage 4, predominantly displaying hypomethylation. Sites annotated as enhancers were overrepresented among the differentially methylated sites (DMSs). We further showed that half of the corresponding genes concomitantly altered their expression, most of them increasing in expression. Interestingly, the stroke-related gene HDAC9 increased its expression several hundredfold. This study reveals a rapid hypomethylation of CpG sites in enhancer elements during the early stages of cell culturing. As many methods for methylation analysis are biased toward CpG rich promoter regions, we suggest that such methods may not always be appropriate for the study of methylation dynamics. In addition, we found that significant changes in expression arose in genes with enhancer DMSs. HDAC9 displayed the most prominent increase in expression, indicating, for the first time, that dynamic enhancer methylation may be central in regulating this important stroke-associated gene.     

Effect of 17β-estradiol on the human osteosarcoma cell line MG-63

We present evidence that 17β-estradiol (17β-E₂) regulates 1,25(OH)₂D₃-induced alkaline phosphatase synthesis and osteocalcin secretion by the human osteosarcoma cell line MG-63. When cells were pre-treated with 17β-E₂ for 48 h prior to treatment with 1,25(OH)₂D₃ (50 nM) for another 48 h, alkaline phosphatase activity increased by 40% (P < 0.025) with 2 nM 17β-E₂ and plateaued at levels of 20 and 200 nM 17β-E₂. Under the same experimental conditions, osteocalcin secretion was enhanced by 37% (P < 0.005) with 2 nM E₂. However, 17β-E₂ had no effect on basal alkaline phosphatase or on osteocalcin secretion. Moreover, simultaneous addition of 17β-E₂ and 1,25(OH)₂D₃ to cells did not result in any additional effect over l,25(OH)₂D₃ treatment alone. Tamoxifen (10 nM) inhibited 17β-E₂-induced activities in l,25(OH)₂D₃-treated cells while not affecting control cells. Dexamethasone pretreat-ment (100 nM, 48 h) also stimulated alkaline phosphatase activity in MG-63 cells. Moreover, dexamethasone pretreatment followed by treatment with 17β-E₂ and l,25(OH)₂D₃ gave an additive effect for alkaline phosphatase activity. 17α-Estradiol (17α-E₂), a less active form of estrogen, failed to modify, at low concentrations, control or l,25(OH)₂D₃-induced alkaline phosphatase synthesis and osteocalcin secretion. In fact, a 100–1000-fold higher concentration of 17α-E₂ was necessary to reproduce the effects of 17β-E₂ on osteocalcin secretion. The addition of insulin-like growth factor I (IGF-I) for 24 h (1–50 ng/ml) to MG-63 cells did not modify 1,25(OH)₂D₃-induced osteocalcin release from these cells. However, longer incubations with 50 ng/ml IGF-I did reproduce some of the effects observed with 17β-E₂. Thus, the effects of 17β-E₂ are probably not related to IGF-I production in MG-63 cells since under these conditions the addition of IGF-I alone should have produced a response at shorter incubation times and in the presence of lower concentrations of IGF-I. Since 17β-E₂ pretreatment was necessary to observe any effects on l,25(OH)₂D₃-induced activities, we hypothesized that 17β-E₂ regulated 1,25(OH)₂D₃ receptors in MG-63 cells. When cells were treated with 100 nM 17β-E₂ for 48 h, the binding affinity was unchanged: 37.3 ± 1.9 versus 35.1 ± 0.4 pM for cells whether treated or not with l7β-E₂, respectively. In contrast, a significant increase in binding capacity (B_max) was noted (15 ± 3.5%; P < 0.025). These results suggest that the estrogen analogue 17β-E₂ induces the differentiation of MG-63 cells into a more osteoblastic-like phenotype while 17α-E₂ is without physiological effect. They also suggest that estrogens may regulate bone remodeling by modulating hormonal-induction of proteins involved in bone mineralization. This effect is indirect since it does not modify basal activities, but involves a regulation of 1,25(OH)₂D₃ receptor levels in these MG-63 cells.     

Effects of lysophosphatidic acid (LPA) signaling via LPA receptors on cellular functions associated with ATP reduction in osteosarcoma cells treated with ethidium bromide

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA₁ to LPA₆) exhibits a variety of malignant properties in cancer cells. Intracellular ATP depletion leads to the development of necrosis and apoptosis. The present study aimed to evaluate the effects of LPA receptor-mediated signaling on the regulation of cancer cell functions associated with ATP reduction. Long-term ethidium bromide (EtBr) treated (MG63-EtBr) cells were established from osteosarcoma MG-63 cells. The intracellular ATP levels of MG63-EtBr cells were significantly lower than that of MG-63 cells. LPAR2, LPAR3, LPAR4 and LPAR6 gene expressions were elevated in MG63-EtBr cells. The cell motile and invasive activities of MG63-EtBr cells were markedly higher than those of MG-63 cells. The cell motile activity of MG-63 cells was increased by LPA₄ and LPA₆ knockdowns. In cell survival assay, cells were treated with cisplatin (CDDP) every 24 h for 3 days. The cell survival to CDDP of MG63-EtBr cells was lower than that of MG-63 cells. LPA₂ knockdown decreased the cell survival to CDDP of MG-63 cells. The cell survival to CDDP of MG-63 cells was inhibited by (2 S)-OMPT (LPA₃ agonist). Moreover, the cell survival to CDDP of MG-63 cells was enhanced by LPA₄ and LPA₆ knockdowns. These results indicate that LPA signaling via LPA receptors is involved in the regulation of cellular functions associated with ATP reduction in MG-63 cells treated with EtBr.

     

基于JAK2/STAT3信号通路探讨白藜芦醇对骨肉瘤MG-63细胞凋亡及迁移的影响

摘要 目的：研究白藜芦醇(RES)通过蛋白酪氨酸激酶2/信号转导子与激活子3（JAK2/STAT3）信号通路对人骨肉瘤体外细胞株MG-63细胞凋亡、侵袭和迁移的影响。方法：体外培养MG-63细胞,以不同浓度的RES作用于MG-63细胞。Annexin V-FITC/PI双染流式细胞术检测不同时间和不同浓度的RES对MG-63细胞凋亡的影响。划痕实验和Transwell实验检测不同时间和不同浓度的RES对MG-63细胞侵袭和迁移能力的影响。免疫印迹实验检测不同时间和不同浓度的RES对MG-63细胞磷酸化蛋白酪氨酸激酶2（p-JAK2）、磷酸化信号转导子与激活子3（p-STAT3）、凋亡相关蛋白B淋巴细胞瘤-2（Bcl-2）、Bcl-2家族促凋亡蛋白（Bax）及基质金属蛋白酶（MMP）-2、MMP-9表达的影响。结果：RES浓度越高,时间越久,MG-63细胞凋亡率越高（P<0.05）。RES浓度越高,MG-63细胞迁移和侵袭能力越弱(P<0.05)。RES处理MG-63细胞后其p-JAK2、p-STAT3、Bcl-2以及MMP-2、MMP-9的表达明显降低,而Bax蛋白表达明显升高,且p-JAK2、p-STAT3、Bax、Bcl-2以及MMP-2、MMP-9的表达水平变化具有RES浓度依赖性（P<0.05）。结论：RES可能通过调控JAK2/STAT3信号通路促使人骨肉瘤MG-63细胞凋亡,并抑制MG-63细胞侵袭和迁移。     

Impact of DNA demethylation of the G0S2 gene on the transcription of G0S2 in squamous lung cancer cell lines with or without nuclear receptor agonists

We recently identified that DNA methylation of the G0S2 gene was significantly more frequent in squamous lung cancer than in non-squamous lung cancer. However, the significance of G0S2 methylation levels on cancer cells is not yet known. We investigated the effect of G0S2 methylation levels on cell growth, mRNA expression, and chromatin structure using squamous lung cancer cell lines and normal human bronchial epithelial cells. DNA methylation and mRNA expression of G0S2 were inversely correlated, and in one of the squamous lung cancer cell lines, LC-1 sq, G0S2 was completely methylated and suppressed. Overexpression of G0S2 in LC-1 sq did not show growth arrest or apoptosis. The G0S2 gene has been reported to be a target gene of all-trans retinoic acid and peroxisome proliferator-activated receptor agonists. We treated LC-1 sq with 5-Aza-2′-deoxycytidine, Trichostatin A, all-trans retinoic acid, Wy 14643, or Pioglitazone either alone or in combination. Only 5-Aza-2′-deoxycytidine restored mRNA expression of G0S2. Chromatin immunoprecipitation revealed that histone H3 lysine 9 was methylated regardless of DNA methylation or mRNA expression. In summary, mRNA expression of G0S2 was regulated mainly by DNA methylation in squamous lung cancer cell lines. When the G0S2 gene was methylated, nuclear receptor agonists could not restore mRNA expression of G0S2 and did not show any additive effect on mRNA expression of G0S2 even after the treatment with 5-Aza-2′-deoxycytidine.     

Regional methylation of the 5' end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells.

In mammalians, demethylation of specific promoter regions often correlates with gene activation; inversely, dense methylation of CpG islands leads to gene silencing, probably mediated by methyl-CpG binding proteins. In cell lines and cancers, inhibition of tissue-specific genes and tumor suppressor genes expression seems to be related to such hypermethylation. The 5' end of the breast cancer predisposition gene BRCA1 is embedded in a large CpG island of approximately 2.7 kb in length. In human sporadic breast cancers, the down-regulation of BRCA1 does not seem to be related to BRCA1 gene alterations. Southern blot analysis and the bisulfite sequencing method indicate that the BRCA1 CpG island is regionally methylated in all human tissues analyzed and unmethylated in the gametes, suggesting a role for DNA methylation in the control of gene expression. We have therefore investigated the potential role of methyl-CpG binding proteins in the regulation of BRCA1 gene expression. In vitro, partial methylation of constructs containing this region strongly inhibits gene expression in the presence of MeCP2 protein. Moreover, in the five human cell lines analyzed, chemically induced hypomethylation is associated with BRCA1 gene activation. These data suggest that methyl-CpG binding proteins might be associated with the control of BRCA1 gene expression and that methyl-DNA binding proteins may participate in the regulation of gene expression in mammalian cells.     

The importance of hormone receptor analysis in osteosarcoma cells growth submitted to treatment with estrogen in association with thyroid hormone

Bone tumor incidence in women peaks at age 50-60, coinciding with the menopause. That estrogen (E2) and triiodothyronine (T3) interact in bone metabolism has been well established. However, few data on the action of these hormones are available. Our purpose was to determine the role of E2 and T3 in the expression of bone activity markers, namely alkaline phosphatase (AP) and receptor activator of nuclear factor kappaB ligand (RANKL). Two osteosarcoma cell lines: MG-63 (which has both estrogen (ER) and thyroid hormone (TR) receptors) and SaOs-29 (ER receptors only) were treated with infraphysiological E2 associated with T3 at infraphysiological, physiological, and supraphysiological concentrations. Real-time RT-PCR was used for expression analysis. Our results show that, in MG-63 cells, infraphysiological E2 associated with supraphysiological T3 increases AP expression and decreases RANKL expression, while infraphysiological E2 associated with either physiological or supraphysiological T3 decreases both AP and RANKL expression. On the other hand, in SaOs-2 cells, the same hormone combinations had no significant effect on the markers' expression. Thus, the analysis of hormone receptors was shown to be crucial for the assessment of tumor potential growth in the face of hormonal changes. Special care should be provided to patients with T3 and E2 hormone receptors that may increase tumor growth.