Human Osteosarcoma U2OS Cells Research Articles

Aurora-A inactivation causes mitotic spindle pole fragmentation by unbalancing microtubule-generated forces

BackgroundAurora-A is an oncogenic kinase playing well-documented roles in mitotic spindle organisation. We previously found that Aurora-A inactivation yields the formation of spindles with fragmented poles that can drive chromosome mis-segregation. Here we have addressed the mechanism through which Aurora-A activity regulates the structure and cohesion of spindle poles.ResultsWe inactivated Aurora-A in human U2OS osteosarcoma cells either by RNA-interference-mediated silencing or treating cultures with the specific inhibitor MLN8237. We show that mitotic spindle pole fragmentation induced by Aurora-A inactivation is associated with microtubule hyperstabilisation. Silencing of the microtubule-stabilising factor ch-TOG prevents spindle pole fragmentation caused by inactivation of Aurora-A alone and concomitantly reduces the hyperstabilisation of microtubules. Furthermore, decreasing pole-directed spindle forces by inhibition of the Eg5 kinesin, or by destabilisation of microtubule-kinetochore attachments, also prevents pole fragmentation in Aurora-A-inactivated mitoses.ConclusionsOur findings indicate that microtubule-generated forces are imbalanced in Aurora-A-defective cells and exert abnormal pressure at the level of spindle poles, ultimately causing their fragmentation. This study therefore highlights a novel role of the Aurora-A kinase in regulating the balance between microtubule forces during bipolar spindle assembly.

Exportin 4 interacts with Sox9 through the HMG Box and inhibits the DNA binding of Sox9.

Sox9 is a transcription factor that is required for tissue development in mammals. In general, such transcription factors require co-regulators for precise temporal and spatial control of the activation and inactivation of the numerous genes necessary for precise development during embryogenesis. Here we identify a new Sox9 co-regulator: Using affinity chromatography with immobilized Sox9 protein, we identified exportin 4 (Exp4) as an interacting protein of Sox9 in human cultured cells. Interaction between endogenous Exp4 and Sox9 proteins was confirmed in the human osteosarcoma U2OS cells by immunoprecipitation experiments using anti-Sox9 antibody. siRNA depletion of Exp4 enhanced transcription of Sox9 target genes in U2OS cells, but did not affect nuclear localization of Sox9. These results suggest that Exp4 regulates Sox9 activity in the nucleus. Furthermore we found that the HMG box of Sox9 was responsible for binding to Exp4, and the HMG box was required for suppression of Sox9-mediated transcription. This contrasts with the known Sox9 co-regulators which bind to its transcriptional activation domain. Chromatin immunoprecipitation analyses revealed that Exp4 prevents Sox9 binding to the enhancers of its target genes. These results demonstrate that Exp4 acts as a Sox9 co-regulator that directly regulates binding of Sox9 to its target genes.

Sodium Selenite-Induced Apoptosis Mediated by ROS Attack in Human Osteosarcoma U2OS Cells

Sodium selenite (Na(2)SeO(3), SSE) is an inorganic Se compound that is widely used in cancer chemoprevention studies. SSE has been shown to have anti-proliferative effects on several types of human cancer cells, but its effect on osteosarcoma cells has thus far not been reported. In this study, the cytotoxic effect of SSE on osteosarcoma cells U2OS was investigated in vitro and found to be higher than on comparable non-cancer cell lines 293 and L6. Treatment with SSE decreased cell growth in a dose- and time-dependent manner and altered cellular morphology. SSE also inhibited cell viability by inducing apoptosis, as evidenced by the formation of apoptotic bodies, generation of reactive oxygen species (ROS), and accumulation of cells during the advanced phase of apoptosis. SSE-induced apoptosis correlated with the activation of CASP 3, downregulation of BCL-2, and upregulation of P53 and PTEN in U2OS cells. These results indicated that SSE induces apoptosis in U2OS cells mainly through an ROS-mediated caspase pathway. This is the first report to show a possible mechanism of the anti-proliferative effect of SSE for the prevention of osteosarcoma in cell culture models.

P53 siRNA inhibits apoptosis of U2OS cells treated with azurin

The bacterial redox protein azurin selectively induces apoptosis in human osteosarcoma U2OS cells. We constructed a p53 siRNA to test the role that p53 plays in the apoptosis-inducing role of azurin in U2OS cells. Cells treated with p53 siRNA and azurin showed more viable cells, a lower apoptosis rate, lower caspase-3 activity, and up-regulation of bcl-2, downregulation of bax compared to cells treated with negative siRNA and azurin. Cells treated with negative siRNA and azurin yielded positive TUNEL dying, whereas cells treated with p53 siRNA and azurin yielded few positive cells. These results suggested that p53 siRNA was capable of inhibi-ting the apoptosis induced by azurin 2 days after treatment with p53 siRNA and azurin. Azurin may induce apoptosis through combination with p53. The decrease in p53 protein levels did not inhibit cell apoptosis rates, but rather increased these rates in U2OS osteosarcoma cells 3 days after treatment with p53 siRNA. Since U2OS cells are p53 wild-type cancer cells, p53 potentially acts as an oncogene in U2OS osteosarcoma cells. Treatment with azurin may transform the function of p53 from inhibiting to inducing apoptosis.

Optical measurement of cycle-dependent cell growth

Determining the growth patterns of single cells offers answers to some of the most elusive questions in contemporary cell biology: how cell growth is regulated and how cell size distributions are maintained. For example, a linear growth in time implies that there is no regulation required to maintain homeostasis; an exponential pattern indicates the opposite. Recently, there has been great effort to measure single cells using microelectromechanical systems technology, and several important questions have been explored. However, a unified, easy-to-use methodology to measure the growth rate of individual adherent cells of various sizes has been lacking. Here we demonstrate that a newly developed optical interferometric technique, known as spatial light interference microscopy, can measure the cell dry mass of many individual adherent cells in various conditions, over spatial scales from micrometers to millimeters, temporal scales ranging from seconds to days, and cell types ranging from bacteria to mammalian cells. We found evidence of exponential growth in Escherichia coli, which agrees very well with other recent reports. Perhaps most importantly, combining spatial light interference microscopy with fluorescence imaging provides a unique method for studying cell cycle-dependent growth. Thus, by using a fluorescent reporter for the S phase, we measured single cell growth over each phase of the cell cycle in human osteosarcoma U2OS cells and found that the G2 phase exhibits the highest growth rate, which is mass-dependent and can be approximated by an exponential.

Induction of G2/M arrest and apoptosis by sulforaphane in human osteosarcoma U2-OS cells

Sulforaphane is one of the most abundant isothiocyanates found in certain cruciferous vegetables, particularly broccoli. To date, sulforaphane has gained attention as a chemopreventive compound. The mechanism responsible for the anticancer effects of sulforaphane in osteosarcoma, however, is not clear. In this study, we demonstrate an anti-proliferative mechanism of sulforaphane in human osteosarcoma cells. The treatment of cells with sulforaphane resulted in a concentration- and time‑dependent inhibition of growth and G2/M phase arrest of the cell cycle. This effect was associated with a decrease in protein expression of cyclin A and B1 and their activating partners, cyclin-dependent kinases (CDKs) 1 and 2, with concomitant up-regulation of p21, a CDK inhibitor. Sulforaphane treatment also resulted in apoptosis as evidenced by an increase in annexin V+/propidium iodide- (V+/PI-) cells, the cleavage of 116-kDa poly (ADP-ribose) polymerase (PARP) and ICAD and oligonucleosomal DNA fragmentation. Taken together, these findings indicate that the molecular mechanisms underlying sulforaphane-mediated growth inhibition in U2-OS cells may be the modulation of the cell cycle machinery and the induction of apoptosis.

Caffeine lengthens circadian rhythms in mice

Although caffeine alters sleep in many animals, whether or not it affects mammalian circadian clocks remains unknown. Here, we found that incubating cultured mammalian cell lines, human osteosarcoma U2OS cells and mouse fibroblast NIH3T3 cells, with caffeine lengthened the period of circadian rhythms. Adding caffeine to ex vivo cultures also lengthened the circadian period in mouse liver explants from Per2::Luciferase reporter gene knockin mice, and caused a phase delay in brain slices containing the suprachiasmatic nucleus (SCN), where the central circadian clock in mammals is located. Furthermore, chronic caffeine consumption ad libitum for a week delayed the phase of the mouse liver clock in vivo under 12 h light–dark conditions and lengthened the period of circadian locomotor rhythms in mice under constant darkness. Our results showed that caffeine alters circadian clocks in mammalian cells in vitro and in the mouse ex vivo and in vivo.

HMLH1Gene is not Methylated in Osteosarcoma

Background: Epigenetic silencing of the hMLH1 gene by promoter methylation plays a role in initiation and progression of cancers by leading to increased genomic instability. There is no report on methylation status of the hMLH1 gene in osteosarcoma. Methods: In this study, we aimed to determine the methylation status of the hMLH1 gene in human osteosarcoma cells. The methylation status of the promoter region of the hMLH1 gene was determined by bi-sulfite sequencing (BSS) in U2OS human osteosarcoma and normal human diploid fibroblast cells (NHDFc). Methylated human gDNA by Sss I methylase enzyme was also used as a positive control. Results: Methylation was not detected in the promoter of the hMLH1 gene—neither in U2OS nor in NHDF cells. Conclusions: Our data showed the hMLH1 gene is not methylated in osteosarcoma, which may imply the genomic instability caused by hMLH1 inactivation does not have a role in osteosarcomas, unlike most other tumors.

Discovery of Novel Small Molecule Activators of β-Catenin Signaling

Wnt/β-catenin signaling plays a major role in embryonic development and adult stem cell maintenance. Reduced activation of the Wnt/β-catenin pathway underlies neurodegenerative disorders and aberrations in bone formation. Screening of a small molecule compound library with a β-galactosidase fragment complementation assay measuring β-catenin nuclear entry revealed bona fide activators of β-catenin signaling. The compounds stabilized cytoplasmic β-catenin and activated β–catenin-dependent reporter gene activity. Although the mechanism through which the compounds activate β-catenin signaling has yet to be determined, several key regulators of Wnt/β-catenin signaling, including glycogen synthase kinase 3 and Frizzled receptors, were excluded as the molecular target. The compounds displayed remarkable selectivity, as they only induced β-catenin signaling in a human osteosarcoma U2OS cell line and not in a variety of other cell lines examined. Our data indicate that differences in cellular Wnt/β-catenin signaling machinery can be exploited to identify cell type-specific activators of Wnt/β-catenin signaling.

USP4 inhibits p53 through deubiquitinating and stabilizing ARF-BP1

Tumour suppressor p53 levels in the cell are tightly regulated by controlled degradation through ubiquitin ligases including Mdm2, COP1, Pirh2, and ARF-BP1. The ubiquitination process is reversible via deubiquitinating enzymes, such as ubiquitin-specific peptidases (USPs). In this study, we identified ubiquitin-specific peptidase 4 (USP4) as an important regulator of p53. USP4 interacts directly with and deubiquitinates ARF-BP1, leading to the stabilization of ARF-BP1 and subsequent reduction of p53 levels. Usp4 knockout mice are viable and developmentally normal, but showed enhanced apoptosis in thymus and spleen in response to ionizing radiation. Compared with wild-type mouse embryonic fibroblasts (MEFs), Usp4-/- MEFs exhibited retarded growth, premature cellular senescence, resistance to oncogenic transformation, and hyperactive DNA damage checkpoints, consistent with upregulated levels and activity of p53 in the absence of USP4. Finally, we showed that USP4 is overexpressed in several types of human cancer, suggesting that USP4 is a potential oncogene.

Abstract 2424: Interferon consensus sequence binding protein promotes epithelial to mesenchymal transition and cell motility in human osteosarcoma cells

Abstract Interferon consensus sequence binding protein (ICSBP) is a regulatory factor induced by interferon gamma (IFN-γ) and a member of interferon regulatory family (IRF). ICSBP is mainly expressed in hematopoietic cell and regulates immune response and differentiation but its role in non-hematopoietic cells is largely unknown. Here we investigated a role of ICSBP in human osteosarcoma U2OS cell line. Basal level of ICSBP expression was low but was induced by interferon-γ in U2OS cells. In addition, induced ICSBP promoted U2OS cell proliferation. To further elucidate ICSBP functions in osteosarcoma cells, we generated the U2OS stable cell line that overexpresses ICSBP. ICSBP overexpressing cells grew faster compared with mock cells and showed morphological changes. ICSBP upregulated TGF-βRI expression resulting in activation of TGF-β signaling pathways. In addition, ICSBP expression altered cell morphology and increased cell adhesion and motility. Furthermore ICSBP increased vimentin expression but decreased ZO-1 and E-cadherin expression, which is EMT related phenomena. These motility and EMT related phenomena were inhibited by TGF-βRI inhibitor. Taken together, these findings indicate that ICSBP promotes EMT and cell motility via TGF-β pathways in U2OS cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2424. doi:10.1158/1538-7445.AM2011-2424

Non-B DNA-forming Sequences and WRN Deficiency Independently Increase the Frequency of Base Substitution in Human Cells

Although alternative DNA secondary structures (non-B DNA) can induce genomic rearrangements, their associated mutational spectra remain largely unknown. The helicase activity of WRN, which is absent in the human progeroid Werner syndrome, is thought to counteract this genomic instability. We determined non-B DNA-induced mutation frequencies and spectra in human U2OS osteosarcoma cells and assessed the role of WRN in isogenic knockdown (WRN-KD) cells using a supF gene mutation reporter system flanked by triplex- or Z-DNA-forming sequences. Although both non-B DNA and WRN-KD served to increase the mutation frequency, the increase afforded by WRN-KD was independent of DNA structure despite the fact that purified WRN helicase was found to resolve these structures in vitro. In U2OS cells, ∼70% of mutations comprised single-base substitutions, mostly at G·C base-pairs, with the remaining ∼30% being microdeletions. The number of mutations at G·C base-pairs in the context of NGNN/NNCN sequences correlated well with predicted free energies of base stacking and ionization potentials, suggesting a possible origin via oxidation reactions involving electron loss and subsequent electron transfer (hole migration) between neighboring bases. A set of ∼40,000 somatic mutations at G·C base pairs identified in a lung cancer genome exhibited similar correlations, implying that hole migration may also be involved. We conclude that alternative DNA conformations, WRN deficiency and lung tumorigenesis may all serve to increase the mutation rate by promoting, through diverse pathways, oxidation reactions that perturb the electron orbitals of neighboring bases. It follows that such "hole migration" is likely to play a much more widespread role in mutagenesis than previously anticipated.

Human cytomegalovirus immediate early gene expression in the osteosarcoma line U2OS is repressed by the cell protein ATRX

The control of human cytomegalovirus (HCMV) immediate early (IE) gene expression in infected human fibroblasts was compared with that in the U2OS human osteosarcoma cells. Viral IE expression was stimulated by the virion protein pp71 and repressed by the cell protein hDaxx in fibroblasts, as expected from published data. Neither of these events occurred in infected U2OS cells, suggesting that this cell line lacks one or more factors that repress HCMV IE expression. The chromatin remodeling factor ATRX is absent from U2OS cells, therefore the effect of introducing this protein by electroporation of plasmid DNA was investigated. Provision of ATRX inhibited HCMV IE expression, and the presence of the HCMV-specified virion phosphoprotein pp71 overcame the repression. The experiments demonstrate that ATRX can act as a cellular intrinsic antiviral defense in U2OS cells by blocking gene expression from incoming HCMV genomes. In contrast, ATRX did not affect the replication of herpes simplex virus type 1, showing that there are differences in the way U2OS cells respond to the presence of the herpesviral genomes.

Novel molecular mechanisms by inorganic phosphate in osteosarcoma U2OS cells

Osteosarcoma is the most common malignant primary bone tumor in children and adolescents and is characterized by a high metastatic potential. Its clinical outcome remains discouraging despite aggressive treatments. Thus, novel therapeutic approaches are needed. Recent results indicate that inorganic phosphate (Pi) is capable of affecting specific signal transduction pathways and of acting as an active regulator of cell behaviour. Previously, we found that Pi inhibits proliferation of human osteosarcoma U2OS cells via an adenylate cyclase/cAMP mediated mechanism. Here, we report that upon Pi treatment, U2OS cells become extremely hard to dislodge with trypsin. The lack of sensitivity to the trypsin action was paralleled by relevant changes in integrin subunits expression and accompanied by an increase of cell adhesion in cell-matrix adhesion assays. Interestingly, exposure of U2OS cells to Pi results also in a strong activation and protein level up-regulation of Rap1 small GTPase and in an early increase followed by a sustained inhibition of Erk1/2 phosphorylation. Importantly, the Pi-induced increase of cell adhesion was enforced by a cAMP analogue which specifically activated Epac/Rap1 and insensitive to PKA and MEK1/2 inhibitors. Our results enforce the evidences of inorganic phosphate as a signalling molecule, identify beta3 integrin, Rap1, ERK1/2 as proteins whose expression and function are relevantly affected by Pi in osteosarcoma U2OS cells The clinical significance and potential therapeutic applications by our data will be discussed.

Dihydroptychantol A, a macrocyclic bisbibenzyl derivative, induces autophagy and following apoptosis associated with p53 pathway in human osteosarcoma U2OS cells

Dihydroptychantol A (DHA), a novel macrocyclic bisbibenzyl compound extracted from liverwort Asterella angusta, has antifungal and multi-drug resistance reversal properties. Here, the chemically synthesized DHA was employed to test its anti-cancer activities in human osteosarcoma U2OS cells. Our results demonstrated that DHA induced autophagy followed by apoptotic cell death accompanied with G 2/M-phase cell cycle arrest in U2OS cells. DHA-induced autophagy was morphologically characterized by the formation of double membrane-bound autophagic vacuoles recognizable at the ultrastructural level. DHA also increased the levels of LC3-II, a marker of autophagy. Surprisingly, DHA-mediated apoptotic cell death was potentiated by the autophagy inhibitor 3-methyladenine, suggesting that autophagy may play a protective role that impedes the eventual cell death. Furthermore, p53 was shown to be involved in DHA-meditated autophagy and apoptosis. In this connection, DHA increased nuclear expression of p53, induced p53 phosphorylation, and upregulated p53 target gene p21 Waf1/Cip1. In contrast, cytoplasmic p53 was reduced by DHA, which contributed to the stimulation of autophagy. In relation to the cell cycle, DHA decreased the expression of cyclin B 1, a cyclin required for progression through the G 2/M phase. Taken together, DHA induces G 2/M-phase cell cycle arrest and apoptosis in U2OS cells. DHA-induced apoptosis was preceded by the induction of protective autophagy. DHA-mediated autophagy and apoptosis are associated with the cytoplasmic and nuclear functions of p53.

Solamargine induces apoptosis associated with p53 transcription-dependent and transcription-independent pathways in human osteosarcoma U2OS cells

Aims Solamargine, a steroidal glycoalkaloid isolated from S. incanum, has been shown to induce apoptosis in several cancer cell lines. In this study, the involvement of p53 in the pro-apoptotic action of solamargine was investigated in human osteosarcoma U2OS cells. Main methods The cytotoxicity of solamargine was evaluated by MTT assay. Solamargine-induced apoptosis was evidenced by chromatin condensation, formation of apoptotic bodies and exposure of phosphatidylserine on the extracellular surface as revealed by DAPI nuclear staining, transmission electron microscopy and flow cytometry, respectively. mRNA expressions of p53 and Bax were investigated using real time-PCR. Western blot was used to examine the changes in the expression levels of p53, Bax, Bcl-2, caspase-3, caspase-9 and cytochrome c. Subcellular localization of p53 was verified by immunofluorescence staining and cell fractionation. Key findings Solamargine substantially reduced cell viability and induced apoptosis in osteosarcoma U2OS cells. In this connection, solamargine increased the mRNA and protein expressions of p53 and Bax (a pro-apoptotic protein downstream to p53). The expression of Bcl-2 (an anti-apoptotic protein) was also reduced. Furthermore, solamargine induced mitochondrial translocation of p53, loss of mitochondrial membrane potential, cytochrome c release and activation of caspase-9 and -3. p53-specific transcriptional inhibitor pifithrin-α or mitochondrial translocation inhibitor pifithrin-μ partially reversed solamargine-induced apoptosis. Significance Solamargine activates the mitochondria-mediated apoptotic pathway in U2OS cells via both p53 transcription-dependent and -independent mechanisms. This compound may merit further investigation as a potential therapeutic agent for the treatment of cancer.

Bufalin induces apoptosis in osteosarcoma U-2OS and U-2OS methotrexate 300-resistant cell lines in vitro

To study the growth inhibition and apoptosis induction effects of bufalin on human osteosarcoma cell lines in vitro. U-2OS and U-2OS/methotrexate (MTX) 300-resistant cell lines were treated with bufalin. Cell viability was assessed by MTT assay. Cell-cycle status, apoptosis-inducing effects, and the expression of apoptosis-related proteins were evaluated by flow cytometry, fluorescent staining, DNA fragmentation assay, and Western blotting. Bufalin inhibited cell growth in both U-2OS and U-2OS/MTX300 cells. The IC(50) values of bufalin for U-2OS and U-2OS/MTX300 cells were (8.49 ± 2.1) ng/ml and (10.19 ± 1.7) ng/ml, respectively. The induction of G(2)/M cell-cycle arrest was also seen in the bufalin-treated cells. The bufalin-induced apoptosis was confirmed by increased expression of tumor suppressor protein p53, bax and decreased expression of bcl-2. Bufalin inhibits the growth of and induces apoptosis in both MTX-sensitive and MTX-resistant human osteosarcoma U-2OS cell lines. The apoptosis-inducing effect of bufalin is not influenced by the presence of high levels of DHFR.

Estrogenic properties of prenylated isoflavones in U2OS human osteosarcoma cells: structure-activity relationships

In a continuation study aiming to discover novel phytoestrogens, we recently reported the isolation and characterization of novel and known isoflavonoids from Erythrina poeppigiana (Walp.) O.F. Cook. (Fabaceae), among which eight appear to be structurally related to genistein with isoprenyl and/or 7-methoxy substitution. In addition, significant binding affinities for the two isotypes of the estrogen receptor ERα and ERβ were demonstrated [1]. These isoflavones derivatives have been investigated for their estrogenic properties in receptor subtype specific reporter gene assay, with a particular focus on their estrogen receptor alpha and beta (ERα and ERβ) selectivity, and their structure-activity relationships using a bone-derived human osteosarcoma cell line (U2OS cells) stably expressing ERα or transiently expressing ERβ. According to our results, an isoprenyl substitution at position 3? together with a 7-methoxy substitution on the genistein skeleton is associated with a statiscally significant activation of the ERα- and ERβ-dependent reporter gene expression in U2OS cells starting from 0.1 nM; while for genistein itself a statistically significant activation was observable at 1 nM. On the other hand, a double prenylation at position 8 and 3? is associated with an almost complete loss of function on the reporter gene activation in U2OS-ERα, but in ERβ expressing system, the effectiveness remains on a statistically significant level, demonstrating an „exclusive ERβ-selectivity“ in U2OS human osteosarcoma cells.

Estrogenic properties of naturally occurring prenylated isoflavones in U2OS human osteosarcoma cells: Structure–activity relationships

Eight isoflavones derivatives, with isoprenyl and/or 7-methoxy substitution, isolated from Erythrina poeppigiana (Fabaceae) have been investigated for their estrogenic properties in receptor subtype-specific reporter gene assays. First we focused on their estrogen receptor alpha and beta (ERα and ERβ) selectivity, second we addressed structure–activity relationships, using bone-derived human osteosarcoma cell line (U2OS cells) stably expressing ERα or transiently expressing ERβ. Our results show that a substitution at position 3′ together with a 7-methoxy substitution on the genistein skeleton is associated with a statistically significant activation of the ERα- and ERβ-dependent reporter gene expression in U2OS cells starting from 0.1nM. Particularly, the 7-methoxy-3′-isoprenyl (1) and the 7-methoxy-3′-(3-methyl-2-hydroxybuten-3-yl) (3) derivatives of genistein induces an ERα- and ERβ-coupled luciferase activity at a concentration ten times lower than that of genistein, for which a statistically significant effect was observable at 1nM. On the other hand, isoprenyl substitution at position 6 of the A ring, compound 5, seems to have very little impact on the genistein ability to induce ER-coupled luciferase activity in U2OS cells, while a double prenylation at positions 8 and 3′, compound 7, is associated with an almost complete loss of function on the reporter gene activation in U2OS-ERα, but in ERβ expressing system the effectiveness remains on a statistically significant level, demonstrating an “exclusive ERβ-selectivity” in U2OS human osteosarcoma cells, and therefore 7 can be considered as an isotype-selective ER ligand. Finally all the tested isoflavones derivatives appear to exhibit a slightly pronounced ERβ preference, depending upon the position and the nature of the substituent moiety on the isoflavone skeleton. The estrogen-like effect of these prenylated isoflavone derivatives could be inhibited by the pure ER antagonist ICI 182 780, indicating that these effects were primarily mediated through ERs.

Nutlin‐3, a MDM2 antagonist, induces Erk1/2 activation in U2OS cells

Nutlin‐3 is a small molecular antagonist of MDM2 which is a critical negative regulator of p53. Activation of p53 by nutlin‐3 has been reported to trigger cell cycle arrest and apoptosis in cancer cells. Here, we present that nutlin‐3 activates anti‐apoptotic pathway as well as apoptosis in human osteosarcoma U2OS cells.Nutlin‐3 inhibited growth of U2OS cells, which was associated with apoptosis. Concomitantly, Erk1/2 was phosphorylated by nutlin‐3 treatment. Pretreatment of MEK inhibitors such as U0126 and PD98059 enhanced cell death by nutlin‐3, suggesting that activated Erk1/2 protects U2OS cells from apoptosis. In addition, nutlin‐3 induced transcription of EGFR ligands including HB‐EGF and Erk1/2 phosphorylation by nutlin‐3 was prevented by cycloheximide, implying that Erk1/2 activation might be resulted from autostimulation of EGFR by upregulated EGFR ligands.Taken together, p53 activation by nutlin‐3 may induce both apoptotic and survival pathways consisted of Erk1/2. Therefore, combined treatment of nutlin‐3 and Erk1/2 inhibitors may enhance therapeutic efficacy of p53 activation to induce cancer cell apoptosis. This research was supported by MRC from cell death disease research center funded by KOSEF.