G-292 Cells Research Articles

The miR-34a-5p promotes the multi-chemoresistance of osteosarcoma via repression of the AGTR1 gene

BackgroundChemoresistance hinders the curative cancer chemotherapy. MicroRNAs (miRNAs) are key players in diverse biological processes including the chemoresistance of cancers.MethodsA RNA-seq-based miR-omic analysis of osteosarcoma (OS) cells was performed to detect the levels of miR-34a-5p. Bioinformatics analysis revealed that AGTR1 is one of the target genes of miR-34a-5p. The mRNA and protein levels of AGTR1 were detected in both the miR-34a-5p-mimic transfected G-292 and miR-34a-5p-antagomiR transfected SJSA-1 cells. The involvement of AGTR1 with OS chemoresistance was validated by the experiments with siRNA-mediated repression or overexpression of the AGTR1 gene.ResultsWe showed that miR-34a-5p promotes the multi- chemoresistance of OS. The angiotensin II type 1 receptor (AGTR1) gene, is one of the targets of miR-34a-5p in OS and thus negatively correlates with OS chemoresistance by systematic investigations of a multi-drug sensitive (G-292) and resistant (SJSA-1) OS cell lines. Down-regulation of the AGTR1 expression by siRNA passivates G-292 cells and suppresses cell apoptosis, while over-expression of AGTR1 sensitizes SJSA-1 cells and thus promotes the drug-triggered cell death.ConclusionsThe miR-34a-5p and its target gene AGTR1 are the potential targets for an effective chemotherapy of OS. Our results also provide novel insights into the effective chemotherapy for OS patients.

MiR-20a-5p represses multi-drug resistance in osteosarcoma by targeting the KIF26B gene.

BackgroundChemoresistance hinders curative cancer chemotherapy in osteosarcoma (OS), resulting in only an approximately 20 % survival rate in patients with metastatic disease at diagnosis. Identifying the mechanisms responsible for regulating chemotherapy resistance is crucial for improving OS treatment.MethodsThis study was performed in two human OS cell lines (the multi-chemosensitive OS cell line G-292 and the multi-chemoresistant OS cell line SJSA-1). The levels of miR-20a-5p and KIF26B mRNA expression were determined by quantitative real-time PCR. KIF26B protein levels were determined by western blot analysis. Cell viability was assessed by MTT assay. Apoptosis was evaluated by flow cytometry.ResultsWe found that miR-20a-5p was more highly expressed in G-292 cells than in SJSA-1 cells. Forced expression of miR-20a-5p counteracted OS cell chemoresistance in both cell culture and tumor xenografts in nude mice. One of miR-20a-5p’s targets, kinesin family member 26B (KIF26B), was found to mediate the miR-20a-5p-induced reduction in OS chemoresistance by modulating the activities of the MAPK/ERK and cAMP/PKA signaling pathways.ConclusionsIn addition to providing mechanistic insights, our study revealed that miR-20a-5p and KIF26B contribute to OS chemoresistance and determined the roles of these genes in this process, which may be critical for characterizing drug responsiveness and overcoming chemoresistance in OS patients.

Abstract 4433: Genome-wide CpG island methylation profiling analysis identifies an epigenetically dysregulated necroptosis mediator in osteosarcoma

Abstract BACKGROUND Aberrant CpG island (CGI) methylation with dysregulation of gene expression is a hallmark of cancer. To identify biologically-relevant targets of aberrant epigenetic silencing in osteosarcomas, we analyzed CGI genes exhibiting disproportionate high-frequency gain of methylation. METHODS Infinium HumanMethylation 450 Beadchip profiling quantitated methylation at 485,578 CpG sites in bisulfite-modified DNA of 16 primary human osteosarcomas (OS) samples. Diff scores (D), reflecting difference in prevalence of site-specific methylation for OS samples compared to pooled non-neoplastic controls, were analyzed for gene-associated CGI sites. Genes exhibiting disproportionate high frequency of gain of methylation (+D), were identified utilizing a program to rank order gene CGI loci along an observed/expected (Obs/Exp) hit distribution where “hits” were defined as DAverage > DThreshold. RIPK3 CGI hypermethylation was quantitated by Combined Bisulfite Restriction Analysis (COBRA) and by bisulfite sequencing in 5 OS cell lines (HOS, MNNGHOS, MG-63, G-292, U2OS) and in human mesenchymal cells (hMSC). RIPK3 expression was analyzed by q-RT PCR. To evaluate the effect of RIPK3 expression on cisplatin-associated cytotoxicity, G-292 cells were stably transfected with RIPK3 or the corresponding empty vector (EV). Cisplatin cytotoxicity was assayed in RIPK3 transfectants versus vector-only controls. RESULTS At DTh = 50, >2500/15,000 CGI genes in the OS samples exhibited gain of CGI methylation in excess of what would be expected based on a uniform distribution of methylation (i.e., Obs/Exp > 1). 169 genes exhibited hypermethylation “hits” at all CGI sites assayed (Obs/Exp = 11.95) including the receptor interacting serine-threonine kinase 3 gene (RIPK3). RIPK3 mediates necroptotic cell death and has been associated with cisplatin sensitivity. COBRA analysis of RIPK3 CGI showed extensive methylation in MNNGHOS, MG63 and G292 cells and partial methylation in HOS and U2OS. hMSCs were minimally methylated. COBRA data were congruent with the prevalence of methylation at 18 CpG sites within the CGI determined by bisulfite sequencing. Quantitation of RIPK3 mRNA expression by q-RT PCR confirmed transcriptional silencing in MNNGHOS, MG63, U2OS and G292 cells. G292 stable transfectants expressing RIPK3 were more sensitive (lower LD50) when cultured in cisplatin compared to vector-only controls. CONCLUSIONS Analysis of genome-wide methylation profiling data to identify CGI genes exhibiting a disproportionate gain of methylation in OS identified RIPK3 hypermethylation. Extensive RIPK3 CGI hypermethylation is associated with transcriptional silencing in OS cells. We further show that expression of RIPK3 is associated with enhanced cisplatin cytotoxicity in G-292 cells. These results suggest that methylation of RIPK3 may contribute to cisplatin resistance in OS. Citation Format: Aditya Sharma, Elaine Notis, Christine Mella, Steven J. Kuerbitz. Genome-wide CpG island methylation profiling analysis identifies an epigenetically dysregulated necroptosis mediator in osteosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4433.

Evaluation of biphasic calcium phosphate/nanosized 3YSZ composites as toughened materials for bone substitution

In this research, biphasic calcium phosphate (BCP), comprising 70wt% of beta tricalcium phosphate and 30wt% of hydroxyapatite, was mixed with different amounts of 3mol% yttria-stabilized zirconia (3YSZ) and sintered at 1200°C to produce toughened bone substitutes. The fracture toughness (KIc) of the obtained bodies was determined using the indentation-strength fracture method. Scanning electron microscopy and energy dispersive X-ray spectroscopy analysis were utilized to study the microstructure of the samples. The phase composition of the samples was also determined using X-ray diffraction technique. In order to investigate the cell supporting ability of the samples, G-292 cells were cultured on them and cell morphology was evaluated after 48h. Based on the results, the maximum fracture toughness and compressive strength values (i.e., 2.11MPam0.5 and 150MPa, respectively) were obtained for the sample containing 3vol% of 3YSZ. The obtained fracture toughness value was approximately two times higher than that of the original BCP (1.07MPam0.5) and also was comparable with that of the cortical human bone. The following mechanisms for the improved KIc of the β-tricalcium phosphate were determined: Grain bridging of 3YSZ particles during crack growth resistance, formation of microcracks on the tip of the larger cracks, absorbing crack extension energy due to the volume expansion during 3YSZ tetragonal-monoclinic transformation and crack deflection by the presence of 3YSZ particles. Also, 3YSZ additive encourages transformation of HA phase into β-TCP during sintering BCP. Finally, based on the cell studies, the samples exhibited an adequate cell attachment and a good cell spreading condition.

A novel synthetic derivative of the natural product berbamine inhibits cell viability and induces apoptosis of human osteosarcoma cells, associated with activation of JNK/AP-1 signaling

Osteosarcoma is the most common primary bone tumor in children and adolescents. There is a critical need to find more potent drugs for patients with metastatic or recurrent disease. Berbamine (BBM) is a natural compound derived from the Berberis amurensis plants. BBM and its derivatives have been shown to have antitumor effects in several cancers. Here, we report that a novel synthetic berbamine derivative, BBMD3, inhibits cell viability and induces apoptosis of G292, KHOS, and MG-63 human osteosarcoma cells. Induction of apoptosis in these tumor cells depends on activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP). Since pan-caspase inhibitor (Z-VAD-FMK) and caspase-9 inhibitor (Z-LEHD-FMK) could block the cleavage of PARP, the apoptosis induced by BBMD3 is through intrinsic signaling pathway. BBMD3 increased phosphorylation of c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), resulting in increase of phosphorylated c-Jun and total c-Fos, the major components of transcriptional factor AP-1. JNK inhibitor could partially suppress antitumor effect of BBMD3 on osteosarcoma cells. BBMD3 increased the production of reactive oxygen species (ROS) and ROS scavenger, N-acetylcysteine (NAC), could block the phosphorylation of JNK and c-Jun induced by BBMD3. BBMD3 increased the expression of the pro-apototic gene Bad, associated with apoptosis induction. Finally, BBMD3 also decreased the expression of cyclin D1 and D2, the positive cell cycle regulators, which is correlated with growth inhibition in osteosarcoma cells. Collectively, these findings indicate that BBMD3 is a potentially promising drug for the treatment of human osteosarcoma.

Dual effect of capsaicin on cell death in human osteosarcoma G292 cells

Thirty percent of osteosarcoma patients die within 5 years. New agents that induce apoptosis of osteosarcoma cells might be therapeutically useful. Here, we characterized the apoptotic mechanism induced by capsaicin in G292 osteosarcoma cells. Our results show that capsaicin induces an increase in the cytosolic Ca2+ concentration which is independent of the extracellular Ca2+ concentration and depletes intracellular Ca2+ stores, suggesting the presence of endoplasmic reticulum transient receptor potential vanilloid receptor type 1. Capsaicin also activates the mitochondrial caspase 3-dependent death cascade. Rapamycin, an inhibitor of mammalian target of rapamycin, evokes autophagy, as do capsaicin or thapsigargin, a sarco(endo)plasmic reticulum Ca2+ ATPase inhibitor that causes Ca2+ store depletion. Capsaicin-induced cell death is completely inhibited by co-treatment with the pan-caspase inhibitor Z-VAD-fmk and increased by the autophagy inhibitor 3-methyladenine, suggesting the existence of an autophagy-dependent anti-apoptotic mechanism. Capsaicin also induces ERK phosphorylation, which acts as a downstream effector of autophagy. 3-Methyladenine or PD98059, an ERK kinase inhibitor, restores capsaicin-induced cell death in the presence of Z-VAD-fmk, suggesting that inhibition of autophagy activates a second cell death pathway that is caspase-independent. Taken together, our data show that capsaicin causes Ca2+ depletion of intracellular Ca2+ stores and simultaneously activates the mitochondrial caspase-dependent death cascade and autophagy-dependent ERK activation and that the latter counteracts a second death signaling pathway that is caspase-independent.

The cancer‐related transcription factor Runx2 modulates cell proliferation in human osteosarcoma cell lines

Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre-osteoblast proliferation by affecting cell cycle progression in the G(1) phase. The growth suppressive potential of Runx2 is normally inactivated in part by protein destabilization, which permits cell cycle progression beyond the G(1)/S phase transition, and Runx2 is again up-regulated after mitosis. Runx2 expression also correlates with metastasis and poor chemotherapy response in osteosarcoma. Here we show that six human osteosarcoma cell lines (SaOS, MG63, U2OS, HOS, G292, and 143B) have different growth rates, which is consistent with differences in the lengths of the cell cycle. Runx2 protein levels are cell cycle-regulated with respect to the G(1)/S phase transition in U2OS, HOS, G292, and 143B cells. In contrast, Runx2 protein levels are constitutively expressed during the cell cycle in SaOS and MG63 cells. Forced expression of Runx2 suppresses growth in all cell lines indicating that accumulation of Runx2 in excess of its pre-established levels in a given cell type triggers one or more anti-proliferative pathways in osteosarcoma cells. Thus, regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumor growth.

Fabrication of biocompatible titanium scaffolds using space holder technique

Open-pore titanium scaffolds were fabricated by sintering of compressed mixtures of TiH(1.924) and urea. Spherical and irregular shaped space holders were used to investigate the effect of pore shape on cellular behavior. After removal of the space holder, the shape of the spacers was replicated to the pores. Average diameter of the pores was in the range of 300-600 μm. SEM images showed that titanium hydride resulted in higher surface roughness and larger micro porosities than pure titanium. In vitro evaluations were carried out by using MTT assay, measuring alkaline phosphatase activity and alizarin red staining in flow perfusion bioreactor for cell culture. Observations revealed excellent attachment and proliferation of G-292 cells to the highly porous scaffolds fabricated with titanium hydride and urea of this research.

Abstract 2069: A novel berbamine derivative induces apoptosis of chemotherapy-resistant human osteosarcoma cells through activation of ROS/JNK signaling

Abstract Osteosarcoma is the most common primary bone tumor in children and adolescence. There is a critical need to find more potent drugs for patients with metastatic or recurrent disease. Berbamine (BBM) is a natural compound derived from the Berberis amurensis plants. BBM and its derivatives have been shown to have anti-tumor effects in several cancers. Here, we report that a new berbamine derivative, BBMD3 (5 µM), induces apoptosis of human osteosarcoma cells, which are resistant or insensitive to conventional chemotherapy including cisplatin, doxorubicin and methotrexate. BBMD3 inhibited cell viability and induced apoptosis of G292, KHOS and MG-63 human osteosarcoma cells in a time- and dose-dependent manner. BBMD3 activated caspase-3, and cleaved poly(ADP-ribose) polymerase (PARP). Caspase inhibitor (Z-VAD-FMK) could rescue tumor cells from the cytotoxic effects of BBMD3 on cell survival. Importantly, BBMD3 increased phosphorylation of c-Jun N-terminal kinase (JNK) in these tumor cells, resulting in activation of its substrates, c-Jun and c-Fos. Moreover, reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC), blocked the phosphorylation of JNK and c-Jun induced by BBMD3. However, Z-VAD-FMK treatment did not inhibit the induction of phosphorylated JNK and c-Jun by BBMD3, suggesting that phosphorylation of JNK and c-Jun occurred before activation of caspases. BBMD3 increased the expression of the pro-apototic gene Bad, associated with apoptosis induction. Finally, BBMD3 also decreased the expression of cyclin D1, a positive cell cycle regulator, which is correlated with growth suppression in osteosarcoma cells. Collectively, these findings indicate that BBMD3 is a potentially promising drug for the treatment of chemotherapy-resistant osteosarcomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2069. doi:1538-7445.AM2012-2069

High-Frequency Canonical Wnt Activation in Multiple Sarcoma Subtypes Drives Proliferation through a TCF/β-Catenin Target Gene, CDC25A

Wnt canonical signaling is critical for normal development as well as homeostasis of several epithelial tissues, and constitutive activation of this pathway is commonly observed in carcinomas. We show here that 50% of human sarcomas (n = 45) and 65% of sarcoma cell lines (n = 23) of diverse histological subtypes exhibit upregulated autocrine canonical Wnt signaling. Furthermore, in Wnt autocrine cell lines, we identify alterations including overexpression or gene amplification of Wnt ligands and/or LRP5/6 coreceptors and epigenetic silencing of different cell surface Wnt antagonists. Mutations in adenomatous polyposis coli (APC) gene were observed in two nonautocrine Wnt-positive sarcoma cell lines. Finally, downregulation of the activated Wnt pathway inhibited sarcoma cell proliferation both in vitro and in vivo by a mechanism involving the downregulation of CDC25A.

Methylation of histone H3 lysine 27 associated with apoptosis in osteosarcoma cells induced by staurosporine.

The relationship between histone methylation and apoptosis, programmed cell death, is beginning to be explored. The objective of this study was to investigate the effects of staurosporine, a PKC inhibitor on the methylation of histone H3 in osteosarcoma cells. Following stimulation by staurosporine in vitro of G292 cells, a human osteosarcoma cell line with fibroblast-like phenotype, methylation of histone H3 was evaluated by western blotting and immunocytochemistry. G292 cells revealed the expression of cleaved PARP after incubation with staurosporine for 3 hours. Monomethyl lysine (K) 27 was induced by staurosporine at a concentration of 1, but no monomethyl K4 or K9 in histone H3 was seen. Dimethyl and trimethyl histone H3 K27 were also identified. There was no expression of dimethyl or trimethyl histone H3 K4 and K9. Expression of monomethyl histone H3 K27 was dose-dependent. The morphologic changes of apoptosis induced by staurosporine were observed under microscopy. Immunocytochemistry of monomethyl histone H3 K27 showed a weak signal in controls, a strong signal in staurosporine-treated tumor cells and a denser signal in the apoptotic cells. Our studies demonstrated that monomethyl histone H3 lysine 27 is expressed in staurosporine-induced apoptotic osteosarcoma cells. The findings may provide novel bridge information between the epigenetic episodes and apoptotic process.

Pharmacological properties of ATP-sensitive purinergic receptors expressed in human G292 osteoblastic cells

We characterized the pharmacological properties of P2 receptors expressed in G292 osteoblastic cells by studying the responses or changes in intracellular Ca 2+ level to P2 receptor agonists, antagonists and modulators. ATP induced robust responses in a concentration-dependent manner with EC 50 of 0.5 ± 0.07 μM. While α,β-methylene-ATP (αβmeATP) and 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) were ineffective, ADP mimicked the action of ATP with EC 50 of 0.7 ± 0.2 μM. UTP and UDP also evoked responses with EC 50 of 2.0 ± 0.4 μM and 0.5 ± 0.1 μM respectively, but their responses were much smaller, resulting in an order of the response magnitude: ATP ~ ADP >> UTP ~ UDP. The responses evoked by ATP and ADP were blocked by pyridoxal-5'-phosphate-6-azophenyl-2,4,-disulfonate (PPADS) with IC 50 of 3.0 ± 0.05 μM and 5.0 ± 0.4 μM respectively, but not by suramin up to 30 μM. ATP-evoked responses were insensitive to inhibition by trinitrophenyl-ATP (TNP-ATP) and brilliant blue G. ADP-evoked responses were significantly inhibited by 2'-deoxy- N 6-methyladenosine-3',5'-biphosphate (MRS2179) and 2-chloro- N 6-methyl-( N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate (MRS2279) with IC 50 of 48 ± 1.9 μM and 7.7 ± 0.9 μM respectively. Taken together, these results provide strong evidence for functional expression of ATP-sensitive P2Y receptors and particularly P2Y 1-like receptor in G292 cells.

Estrogen regulation of apoptosis in osteoblasts

Dysregulated apoptosis is a critical failure associated with prominent degenerative diseases including osteoporosis. In bone, estrogen deficiency has been associated with accelerated osteoblast apoptosis and susceptibility to osteoporotic fractures. Hormone therapy continues to be an effective option for preventing osteoporosis and bone fractures. Induction of apoptosis in G-292 human osteoblastic cells by exposure to etoposide or the inflammatory cytokine TNF-α promoted acute caspase-3/7 activity and this increased activity was inhibited by pretreatment with estradiol. Etoposide also increased the expression of a battery of apoptosis-promoting genes and this expression was also inhibited by estradiol. Among the apoptotic genes whose expression was inhibited by estradiol was ITPR1, which encodes the type 1 InsP3R. InsP3Rs are intracellular calcium channels and key proapoptotic mediators. Estradiol via estrogen receptor β1 suppresses ITPR1 gene transcription in G-292 cells. These analyses suggest that an underlying basis of the beneficial activity of estrogens in combating osteoporosis may involve the prevention of apoptosis in osteoblasts and that a key event in this process is the repression of apoptotic gene expression and inhibition of caspase-3/7.

Biological evaluation of partially stabilized zirconia added HA/HDPE composites with osteoblast and fibroblast cell lines

In the present study, the biocompatibility of partially stabilized zirconia (PSZ) added hydroxyapatite (HA)--high density polyethylene (HDPE) composites was evaluated by proliferation and cell attachment assays on two osteoblast cell lines (G-292, Saos-2) and a type of fibroblast cell isolated from bone tissue namely HBF in different time intervals. Cell-material interactions on the surface of the composites were observed by scanning electron microscopy (SEM). The effect of composites on the behavior of osteoblast and fibroblast cells was compared with those of HDPE and Tissue Culture Poly Styrene (TPS) (as negative control) samples. Results showed that the composite samples supported a higher proliferation rate of osteoblast cells in the presence of composite samples as compared to the HDPE and TPS samples after 3, 7 and 14 days of incubation period. It was showed that an equal or in some cases an even higher proliferation rate of G-292 and Saos-2 osteoblast cells on composite samples in compare to negative controls in culture period (P < 0.05). The number of adhered cells on the composite samples was equal and in some cases higher than the number adhered on the HDPE and TPS samples after the above mentioned incubation periods (P < 0.05). Adhered cells presented a normal morphology by SEM and many of the cells were seen to be undergoing cell division.

Phytoestrogens activate estrogen receptor β1 and estrogenic responses in human breast and bone cancer cell lines

Plant-derived phytoestrogens and estrogens in hormone replacement therapies have overlapping yet sometimes divergent effects on the incidence of breast cancer and osteoporosis. Using human MCF-7 breast carcinoma and G-292 osteosarcoma cell lines, it was investigated whether the phytoestrogens genistein and daidzein affect reporter gene transcription via the estrogen receptors (ERs) ERalpha and ERbeta1 as well as whether they affect the expression of estrogen-responsive genes in MCF-7 cells and the secretion of the cytokine IL-6 from G-292 cells. The results showed that genistein and daidzein potently trigger transactivation with ERbeta1 from estrogen response element-reporter genes (EC50s of 1.7-16 nM) although they were 400- to 600-fold less potent than 17beta-estradiol (E2) (EC50 of 0.02-0.04 nM). E2 was the only potent activator of ERalpha (EC50 of 0.1-0.4 nM). The rank order potency (E2 > genistein > daidzein) is maintained in MCF-7 cells as well as G-292 cells with both receptor subtypes, with a strong receptor selectivity of the phytoestrogens for ERbeta1 over ERalpha. Genistein and daidzein increased the expression of estrogen-responsive genes in MCF-7 cells. Daidzein, like E2, inhibited IL-1beta- and hormone-mediated IL-6 secretion from G-292 cells. The results provide a basis for understanding how dietary phytoestrogens protect bone without increasing the risks for breast cancer.

The effect of partially stabilized zirconia on the biological properties of HA/HDPE composites in vitro

The effect of partially stabilized zirconia (PSZ) on the biological properties of the hyroxyapatite - high density polyethylene (HA/HDPE) composites was studied by investigating the simultaneous effect of hydroxyapatite and PSZ volume fractions on the in vitro response of human osteoblast cells. The biocompatibility of composite samples with different volume fraction of HA and PSZ powders was assessed by proliferation, alkaline phosphatase (ALP) and cell attachment assays on the osteoblast cell line (G-292) in different time periods. The effect of composites on the behavior of G-292 cells was compared with those of HDPE and TPS (Tissue Culture Poly Styrene as negative control) samples. Results showed a higher proliferation rate of G-292 cells in the presence of composite samples as compared to the HDPE sample after 7 and 14 days of incubation period. ALP production rate in all composite samples was higher than HDPE and TPS samples. The number of adhered cells on the composite samples was higher than the number adhered on the HDPE and TPS samples after the above mentioned incubation periods. These findings indicates that the addition of PSZ does not have any adverse affect on the biocompatibility of HA/HDPE composites. In fact in some experiments PSZ added HA/HDPE composites performed better in proliferation, differentiation and attachment of osteoblastic cells.

In vitro evaluation of biocompatibility of beta‐tricalcium phosphate‐reinforced high‐density polyethylene; an orthopedic composite

Beta-tricalcium phosphate-reinforced high-density polyethylene (beta-TCP/HDPE) is a new biomaterial which was made as a copy of bone composition with the aim of replacement of bony tissues. The composite samples were prepared using medical grade TCP powder and granular polyethylene. The raw materials were first compounded and the resulting composite preforms were compression molded into desired shape. The biocompatibility of composite samples with different volume fractions of TCP (20, 30, and 40 vol %) was assessed by proliferation, alkaline phosphatase (ALP), and cell adhesion assays using G-292 osteoblast cells. Cell-material interaction on the surface of the composites was observed by scanning electron microscopy (SEM). The effect of beta-TCP/HDPE on the behavior of G-292 cells was compared with those of a composite and a negative control samples. Results showed the composite samples had a higher proliferation rate of G-292 cells in the presence of composite samples as compared to the composite control sample after 3, 7, and 14 days of incubation period. ALP production after incubation in the presence of composite samples was seen to peak on the day 7. The number of adhered cells on the composite samples was higher than the numbers adhered on composite and negative control samples after the above incubation periods. Morphology investigation of adhered cells by SEM indicated a normal morphology and also many of the cells were in the process of cell division. The above results indicate that beta-TCP/HDPE samples are biocompatible, nontoxic, and in some cases show an increase in the proliferation rate of the cells, ALP production, and cell adhesion as compared to the control counterparts.

Effects of sphingosine-1-phosphate and lysophosphatidic acid on human osteoblastic cells

The effects of the lysophospholipids, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) were studied in human primary osteoblastic cells and the human osteosarcomal cell lines, G292 and MG-63. The studies focused on the role of the Gi protein in the regulation of S1P and LPA-induced proliferation, the effects of the phospholipids on alkaline phosphatase, an early marker of osteoblastic cell proliferation, and the presence of edg receptors. Proliferation was assessed by 3H-thymidine incorporation. Short-term incubation with S1P or LPA induced increases in proliferation that were attenuated in the presence of the Gi inhibitor, pertussis toxin. Alkaline phosphatase activity was measured with a spectrophotometric assay. Biphasic effects of S1P and LPA were observed with the nature of the response dependent upon the cell type, concentration of test agent and the time period of incubation. RTPCR studies revealed that edg-1,2,4,5 receptors are present in the primary normal osteoblastic cells, the MG63 and G292 cells. Only the G292 cells expressed the edg-3 receptor to any significant extent.

IL-1β Increases Type 1 Inositol Trisphosphate Receptor Expression and IL-6 Secretory Capacity in Osteoblastic Cell Cultures

Acute IL-6 secretion from osteosarcoma cells induced by the PI-linked hormones PTH(1–34) and endothelin-1 is potentiated by IL-1β. The present findings indicate that this potentiation is accompanied by increased signal transduction capacity. Specifically, IL-1β (30 pM) increased the Bmax of InsP3 receptor binding (2.7-fold) and immunoblot showed a 2.4-fold increase specifically in the type 1 InsP3 receptor protein. Northern analyses of IL-1β-treated G-292 cells showed an 1.8-fold increase in type 1 InsP3 receptor mRNA and, in IL-1β-treated murine MC3T3-E1 osteoblastic cells, an 8.4-fold enhancement of the type 1 InsP3 receptor gene transcription. Promoter reporter assays confirmed the mRNA measurements and showed the effect of IL-1β to be mediated by the major transcriptional regulatory region of the type 1 InsP3 receptor promoter. The findings support the hypothesis that chronic regulators of osteoblast function, such as IL-1β, affect the capacity of cellular signal transduction through changes in InsP3 receptor levels.

The effect of bone morphogenetic protein-7 on the expression of type I inositol 1,4,5-trisphosphate receptor in G-292 osteosarcoma cells and primary osteoblast cultures

Bone morphogenetic protein-7 (BMP-7) affects differentiation of preosteoblasts enabling the resultant cells to respond optimally to acutely acting regulators. As the phosphoinositide cascade and, particularly, the calcium-mobilizing inositol 1,4,5-trisphosphate (InsP 3) receptor are integral to stimulus–secretion coupling in osteoblasts, the hypothesis that BMP-7 affects InsP 3 receptor expression was examined in the G-292 human osteosarcoma cell line and in primary cultures of human osteoblasts. G-292 osteosarcoma cells were found to be a valid experimental model for primary human osteoblasts, expressing osteoblastic mRNAs encoding osteocalcin, bone sialoprotein, alkaline phosphatase, α1-collagen, epidermal growth-factor receptor, and BMP type II receptor. When cultured long term in the presence of ascorbic acid and β-glycerophosphate, G-292 cells underwent further osteoblastic differentiation, forming nodules and exhibiting restricted mineralization. G-292 cells responded to BMP-7 with an increase in InsP 3 receptor density. Ligand-binding studies established that BMP-7 (50 ng/ml) treatment of G-292 cells increased InsP 3 receptor density 2.4-fold with no apparent change in affinity. Immunoblot analysis with antibodies specific for type I, type II, and type III InsP 3 receptors revealed that BMP-7 (50 ng/ml) treatment resulted in a specific increase (206±8%) in the type I receptor. Reverse transcription-polymerase chain reaction and Northern blot analyses of G-292 and primary human osteoblasts confirmed an increase in type I InsP 3 receptor mRNA upon BMP-7 treatment. These results demonstrate that G-292 cells respond to BMP-7 with an increase InsP 3 receptor density, consistent with the enhanced capacity of these cells to respond to Ca 2+-mobilizing secretory hormones during osteoblast differentiation.