Human Osteosarcoma Cell Line U2OS Research Articles

The transcription map of human papillomavirus type 18 during genome replication in U2OS cells.

The human osteosarcoma cell line U2OS is useful for studying genome replication of human papillomavirus (HPVs) subtypes that belong to different phylogenetic genera. In this study, we defined the HPV18 transcription map in U2OS cells during transient replication, stable maintenance and vegetative amplification by identifying viral promoter regions, transcription polyadenylation and splicing sites during HPV18 genome replication. Mapping of the HPV18 transcription start sites in U2OS cells revealed five distinct promoter regions (P102, P520, P811, P1193 and P3000). With the exception of P3000, all of these regions have been previously identified during productive HPV18 infection. Collectively, the data suggest that U2OS cells are suitable for studying the replication and transcription properties of HPVs and to serve as a platform for conducting high-throughput drug screens to identify HPV replication inhibitors. In addition, we have identified mRNA species that are initiated from the promoter region P3000, which can encode two E2C regulator proteins that contain only the C-terminal hinge and DNA-binding and dimerization domains of E2. We show that these proteins regulate the initial amplification of HPV18 by modulating viral transcription. Moreover, we show that one of these proteins can act as a transcriptional activator of promoter P102.

Abstract 762: Prevention and reversion of multidrug resistance (MDR) in cancer by NSC23925

Abstract The major limitation to the success of chemotherapy in cancer is the development of multidrug resistance (MDR). Preventing the emergence of MDR during chemotherapy treatment has been a high priority of clinical and investigational oncology, but remains an elusive goal. NSC23925 has recently been identified as a novel and potent MDR reversal agent. However, whether NSC23925 can prevent the development of MDR in cancer is unknown. Therefore, this study was to evaluate the effects of NSC23925 on prevention and reversion of MDR. Human osteosarcoma cell line U-2OS and Saos were exposed to increasing concentrations of taxol alone or in combination with NSC23925 for 6 months. The results showed that cells selected with increasing concentrations of taxol alone developed MDR with resistance to taxol and other Pgp substrates, while cells cultured with taxol-NSC23925 simultaneously prevented the development of MDR and cells remained sensitive to chemotherapeutic agents. Taxol selected resistant cells showed high expression and activity of drug transporter P-glycoprotein (Pgp), whereas taxol-NSC23925 combination treatment prevented the expression of Pgp. Our findings suggest that NSC23925 may prevent the development of MDR by specifically preventing the overexpression of Pgp. Moreover, NSC23925 can reverse MDR in drug resistant cells by modulating Pgp activity. Given the significant incidence of MDR in cancer and the lack of effective agents for prevention and revision of MDR, NSC23925 and derivatives hold the potential to improve the outcome of cancer patients with poor prognosis due to drug resistance. Citation Format: Xiaoqian Yang, Francis Hornicek, Zhenfeng Duan. Prevention and reversion of multidrug resistance (MDR) in cancer by NSC23925. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 762. doi:10.1158/1538-7445.AM2014-762

Induction of polyploidy by nuclear fusion mechanism upon decreased expression of the nuclear envelope protein LAP2β in the human osteosarcoma cell line U2OS

BackgroundPolyploidy has been recognized for many years as an important hallmark of cancer cells. Polyploid cells can arise through cell fusion, endoreplication and abortive cell cycle. The inner nuclear membrane protein LAP2β plays key roles in nuclear envelope breakdown and reassembly during mitosis, initiation of replication and transcriptional repression. Here we studied the function of LAP2β in the maintenance of cell ploidy state, a role which has not yet been assigned to this protein.ResultsBy knocking down the expression of LAP2β, using both viral and non-viral RNAi approaches in osteosarcoma derived U2OS cells, we detected enlarged nuclear size, nearly doubling of DNA content and chromosomal duplications, as analyzed by fluorescent in situ hybridization and spectral karyotyping methodologies. Spectral karyotyping analyses revealed that near-hexaploid karyotypes of LAP2β knocked down cells consisted of not only seven duplicated chromosomal markers, as could be anticipated by genome duplication mechanism, but also of four single chromosomal markers. Furthermore, spectral karyotyping analysis revealed that both of two near-triploid U2OS sub-clones contained the seven markers that were duplicated in LAP2β knocked down cells, whereas the four single chromosomal markers were detected only in one of them. Gene expression profiling of LAP2β knocked down cells revealed that up to a third of the genes exhibiting significant changes in their expression are involved in cancer progression.ConclusionsOur results suggest that nuclear fusion mechanism underlies the polyploidization induction upon LAP2β reduced expression. Our study implies on a novel role of LAP2β in the maintenance of cell ploidy status. LAP2β depleted U2OS cells can serve as a model to investigate polyploidy and aneuploidy formation by nuclear fusion mechanism and its involvement in cancerogenesis.

ERK5 Silencing Inhibits Invasion of Human Osteosarcoma Cell via Modulating the Slug/MMP-9 Pathway

Background and aim: ERK5 is over expressed in a many of human cancers and this overexpression has been associated with metastasis and invasion. Furthermore, ERK5 siliencing inhibits aggressive phenotypes of cancer cells. However, mechanisms by which ERK5 regulates tumour progression or metastasis have not been elucidated. In this study, using human osteosarcoma cell lines U2OS as a model, we explored the involvement of ERK5 siliencing on invasiveness of U2OS cells. Materials and methods: ERK5 siRNA targeting ERK5 was stably transfected into the human osteosarcoma cell lines U2OS. ERK5 knocked-down U2OS cells was then transfected with slug cDNA or MMP-9 cDNA plasmid to reexpress Slug or MMP-9. Cell proliferation was detected by MTT assay. Cell invasion and metastasis was detected by Matrigel invasion and wound healing assay. An orthotopic nude mouse model of U2OS was applied for in vivo lung metastasis experiments. ERK5, Slug, MMP-9 and E-cadherin were analyzed by real-time PCR, and Western blotting. Results: ERK5 silencing by siRNA in U2OS cells decreased Slug and MMP-9 expression. Compared with the vector-transfected cells, ERK5 knocked-down cells showed reduced migration and invasion in vitro, as well as decreased metastatic potential in experimental metastasis. Re-expression of Slug or MMP-9 in ERK5 knocked-down cells restored the invasive phenotypes. We also discovered that Re-expression of Slug in ERK5 knocked-down cells restored the MMP-9 expression, and re-expression of MMP-9 in ERK5 knocked-down cells did not affect slug and ERK5 expression. CONCLUSIONS: Our data suggest that ERK5 knockdown inhibits aggressive behaviour of human U2OS cells through modulating Slug signalling and MMP-9 expression.

Effects of cyclic AMP response element binding protein–Zhangfei (CREBZF) on the unfolded protein response and cell growth are exerted through the tumor suppressor p53

Zhangfei/CREBZF, a basic region-leucine zipper (bLZip) transcription factor, is a potent suppressor of growth and the unfolded protein response (UPR) in some cancer cell lines, including the canine osteosarcoma cell line, D-17. However, the effects of Zhangfei are not universal, and it has no obvious effects on untransformed cells and some cancer cell lines, suggesting that Zhangfei may act through an intermediary that is either not induced or is defective in cells that it does not affect. Here we identify the tumor suppressor protein p53 as this intermediary. We show the following: in cells ectopically expressing Zhangfei, the protein stabilizes p53 and co-localizes with it in cellular nuclei; the bLZip domain of Zhangfei is required for its profound effects on cell growth and interaction with p53. Suppression of p53 by siRNA at least partially inhibits the effects of Zhangfei on the UPR and cell growth. The effects of Zhangfei on D-17 cells is mirrored by its effects on the p53-expressing human osteosarcoma cell line U2OS, while Zhangfei has no effect on the p53-null osteosarcoma cell line MG63. In U2OS cells, Zhangfei displaces the E3 ubiquitin ligase mouse double minute homolog 2 (Mdm2) from its association with p53, suggesting a mechanism for the effects of Zhangfei on p53.

Study on anti-osteosarcoma activity of ethanol extract of <i>Venenum Bufonis</i> in vitro

Venenum bufonis is the dried white secretion of the auricular and skin glands of Bufo gargarizans Cantor, or Bufo melanostictus Schneider, Bufonidae. It is used in the treatment of deep-rooted carbuncle, boils and swelling; pain in the throat, heart stroke, coma, abdominal pain, vomiting and diarrhea. The objective of this paper is to preliminarily observe the effects of ethanol extract of Venenum bufonis on growth, and proliferation of human osteosarcoma U2OS cell lines, and to provide a theoretical basis for an in-depth study of the clinical application of Venenum bufonis for osteosarcoma inhibition, with its mechanism of action. SRB assay was used to determine the effect of Venenum bufonis ethanol extract on U2OS cell line activity, and to detect its inhibitory dose-effect on osteosarcoma cells. FCM was applied to determine the effect of Venenum bufonis ethanol extract on U2OS cell apoptosis and to perform cell cycle analysis. As results, different Venenum bufonis ethanol extracts showed apparent concentration-effect relationships on U2OS cell lines. FCM analysis showed that it had a U2OS apoptosis promoting effect, which increased with increasing concentration. Cell cycle analysis revealed that the Venenum bufonis ethanol extract mainly arrested U2OS in the G0/G1 phase, preventing the cells from progressing to the S phase. The study concluded that Venenum bufonis ethanol extract has an inhibitory effect on proliferation of osteosarcoma U2OS cells.

Mapping of Betapapillomavirus Human Papillomavirus 5 Transcription and Characterization of Viral-Genome Replication Function

Betapapillomavirus replication and transcription have not been studied in detail because of a lack of suitable cellular systems supporting human papillomavirus (HPV) genome replication. We have recently shown that the human osteosarcoma cell line U2OS provides a useful environment for the genome replication of many different HPVs, including the betapapillomaviruses HPV5 and HPV8. Using mutational analysis and complementation assay, we demonstrated herein that the viral early proteins E1 and E2 are viral transfactors that are necessary and sufficient for HPV5 genome replication. We also identified four HPV5 early promoter regions with transcription start sites (TSSs) at nucleotides (nt) 184/191, 460, 840, and 1254, respectively, and the HPV late promoter with a TSS at nt 7640. In addition, we mapped the HPV5 early polyadenylation cleavage sites via 3' rapid amplification of cDNA ends (3'RACE) to nt 4457 and 4475. In total, 14 different viral mRNA species, originating from the HPV5 genome, were mapped in U2OS cells during transient and stable replication. The main splicing donor and acceptor sites identified herein are consistent with the data previously obtained in HPV5-positive skin lesions. In addition, we identified novel E8 open reading frame (ORF)-containing transcripts (E8^E1C and E8^E2C) expressed from the HPV5 genome. Similar to several other papillomaviruses, the product of the E8^E2C mRNA acts as a repressor of viral genome replication.

Abstract B59: Zhangfei/CREBZF arrests the growth of osteosarcoma cells by displacing Mdm2 and stabilizing p53.

Abstract Osteosarcomas (OS) are the most common malignant bone tumors in humans and dogs. Although there has been dramatic progress in treating OS in both species by surgery and chemotherapy, these therapies often fail, leading to recurrence of the tumor and metastatic spread. Our previous studies showed that Zhangfei/CREBZF, a basic region-leucine zipper (bLZip) transcription factor, is a potent suppressor of growth in some cancer cell lines, including the canine osteosarcoma cell line D-17. However, the mechanisms underlying the suppressive effects of Zhangfei have not been fully elucidated. Furthermore, the effects of Zhangfei are not universal and it has no obvious effects on untransformed cells and some cancer cell lines, suggesting that Zhangfei may act through an intermediary that is either not induced or is defective in cells that it does not affect. The objective of this study was to identify such an intermediary and to determine how Zhangfei exerts its effect. By monitoring the mRNA levels, protein, localization, and transcription activity of tumor suppressor protein p53 and its target genes in D-17 cells infected with adenovirus vectors expressing Zhangfei, we found Zhangfei stabilizes p53 and co-localizes with it in cellular nuclei and that the basic-leucine zipper domain of Zhangfei is required for its profound effects on cell growth and interaction with p53. Suppression of p53 by siRNA at least partially inhibited the effects of Zhangfei on the growth of cells. The effects of Zhangfei on dog osteosarcoma cells (D-17 cells) is mirrored by its effects on the p53 expressing human osteosarcoma cell line U2OS while Zhangfei has no effect on the p53-null osteosarcoma cell line MG63. In U2OS cells Zhangfei displaces the ubiquitin ligase, MDM2, from its association with p53 suggesting a mechanism for the effects of Zhangfei on p53. Overall, we identified that Zhangfei suppressed cell growth in osteosarcoma cells through direct interaction with tumor suppressor protein p53. We demonstrated that Zhangfei stabilized p53 and promoted its nuclear retention by displacing the E3 ubiquitin ligase, Mdm2. Our findings reveal a novel mechanism by which Zhangfei may inhibit tumor growth and metastasis. This may provide a potential application to gene therapy of certain types of osteosarcoma, and perhaps other tumors containing functional p53. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B59. Citation Format: Rui Zhang, Vikram Misra. Zhangfei/CREBZF arrests the growth of osteosarcoma cells by displacing Mdm2 and stabilizing p53. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B59.

Histone deacetylase inhibits the growth and migration of human osteosarcoma cells

To explore the effects of histone deacetylase inhibitors (HDACIs) on human osteosarcoma in vitro and in vivo. HDACI suberoylanilide hydroxamic acid (SAHA) and sodium butyrate (SB) were employed to examine the effects on human osteosarcoma in vitro and in vivo. The in vitro effects of HDACI SAHA and SB were evaluated in SaOS2 and U2OS human osteosarcoma cell lines. Cell growth, cell cycle progression and histone acetylation were examined by methyl tolyl sulfide (MTS), flow cytometry and Western blot respectively.In addition, SAHA or SB was administered for 4 weeks in a murine xenograph model for assessing the in vitro effects. MTS assay revealed that SAHA and SB significantly suppressed the growth of SaOS2 and U2OS cells in a concentration-dependent manner.Western blot analysis indicated that the levels of acetylated H3 increased after HDACI treatment.Flow cytometry showed that SAHA arrested the cell cycle in G1 and G2/M phase, while SB arrested the cell cycle in G2/M phase. The tumor growth of murine xenograph model with SaOS2 was inhibited by SAHA and SB compared with vehicle control. HDACI SAHA and SB significantly inhibit the growth of human osteosarcoma cells and induce cell cycle arrest. Meanwhile, the tumor inhibitory effects were also validated in a murine xenograft model.

Down-regulation of Long Non-coding RNA TUG1 Inhibits Osteosarcoma Cell Proliferation and Promotes Apoptosis

To investigate the expression level of TUG1 and one of its transcript variants (n377360) in osteosarcoma cells and assess the role of TUG1 in proliferation and apoptosis in the U2OS cell line. TUG1 and n377360 expression levels in patients with osteosarcomas and the U2OS human osteosarcoma cell line were evaluated using real-time quantitative PCR. U2OS cells were transected with TUG1 and n377360 siRNA or non-targeting siRNA. MTS was performed to assess the cell proliferation and flow cytometry was applied to analyze apoptosis. We found significantly higher TUG1 and n377360 expression levels in osteosarcoma tissues compared with matched non-tumorous tissues. In line with this, suppression of TUG1 and n377360 expression by siRNA significantly impaired the cell proliferation potential of osteosarcoma cells. Furthermore, inhibition of TUG1 expression significantly promoted osteosarcoma cell apoptosis. The overexpression of TUG1 and n377360 in osteosarcoma specimens and the functional role of TUG1 and n377360 regarding cell proliferation and apoptosis in an osteosarcoma cell line provided evidence that the use of TUG1 or n377360 may be a viable but an as yet unexplored therapeutic strategy in tumors that over express these factors.

DNA-damage response in chromatin of ribosomal genes and the surrounding genome

DNA repair events have functional significance especially for genome stability. Although the DNA damage response within the whole genome has been extensively studied, the region-specific characteristics of nuclear sub-compartments such as the nucleolus or fragile sites have not been fully elucidated. Here, we show that the heterochromatin protein HP1 and PML protein recognize spontaneously occurring 53BP1- or γ-H2AX-positive DNA lesions throughout the genome. Moreover, 53BP1 nuclear bodies, which co-localize with PML bodies, also occur within the nucleoli compartments. Irradiation of the human osteosarcoma cell line U2OS with γ-rays increases the degree of co-localization between 53BP1 and PML bodies throughout the genome; however, the 53BP1 protein is less abundant in chromatin of ribosomal genes and fragile sites (FRA3B and FRA16D) in γ-irradiated cells. Most epigenomic marks on ribosomal genes and fragile sites are relatively stable in both non-irradiated and γ-irradiated cells. However, H3K4me2, H3K9me3, H3K27me3 and H3K79me1 were significantly changed in promoter and coding regions of ribosomal genes after exposure of cells to γ-rays. In fragile sites, γ-irradiation induces a decrease in H3K4me3, changes the levels of HP1β, and modifies the levels of H3K9 acetylation, while the level of H3K9me3 was relatively stable. In these studies, we confirm a specific DNA-damage response that differs between the ribosomal genes and fragile sites, which indicates the region-specificity of DNA repair.

Xiao Jin Wan, a traditional Chinese herbal formula, inhibits proliferation via arresting cell cycle progression at the G2/M phase and promoting apoptosis via activating the mitochondrial-dependent pathway in U-2OS human osteosarcoma cells

Xiao Jin Wan (XJW) is a well-known traditional Chinese folk-medicine, which is commonly used for the treatment of various types of diseases including cancers. However, the mechanism of the anticancer activity of XJW against U-2OS human osteosarcoma cells, have not yet been reported. In the present study, we investigated the cellular effects of the XJW on the U-2OS human osteosarcoma cell line. Our results showed that XJW induced cell morphological changes, reduced cell viability in a dose- and time-dependent manner and arrested in the G2/M phase of the cell cycle suggesting that XJW inhibited the proliferation of U-2OS cells. Hoechst 33258 staining and Annexin V/propidium iodide double staining exhibited the typical nuclear features of apoptosis and increased the proportion of apoptotic Annexin V-positive cells in a dose-dependent manner, respectively. In addition, XJW treatment caused loss of plasma membrane asymmetry, collapse of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and increase of the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Taken together, the results indicate that the U-2OS cell growth inhibitory activity of XJW was due to cell cycle arrested and mitochondrial-mediated apoptosis, which may partly explain the anticancer activity of Xiao Jin Wan.

Effect and Mechanism of Plumbagin in Human Osteosarcoma Cell Line U2OS

Effect and Mechanism of Plumbagin in Human Osteosarcoma Cell Line U2OS

Critical Role of Heat Shock Protein 27 in Bufalin-Induced Apoptosis in Human Osteosarcomas: A Proteomic-Based Research

Bufalin is the primary component of the traditional Chinese herb “Chan Su”. Evidence suggests that this compound possesses potent anti-tumor activities, although the exact molecular mechanism(s) is unknown. Our previous study showed that bufalin inhibited growth of human osteosarcoma cell lines U2OS and U2OS/MTX300 in culture. Therefore, this study aims to further clarify the in vitro and in vivo anti-osteosarcoma effects of bufalin and its molecular mechanism of action. We found bufalin inhibited both methotrexate (MTX) sensitive and resistant human osteosarcoma cell growth and induced G2/M arrest and apoptosis. Using a comparative proteomics approach, 24 differentially expressed proteins following bufalin treatment were identified. In particular, the level of an anti-apoptotic protein, heat shock protein 27 (Hsp27), decreased remarkably. The down-regulation of Hsp27 and alterations of its partner signaling molecules (the decrease in p-Akt, nuclear NF-κB p65, and co-immunoprecipitated cytochrome c/Hsp27) were validated. Hsp27 over-expression protected against bufalin-induced apoptosis, reversed the dephosphorylation of Akt and preserved the level of nuclear NF-κB p65 and co-immunoprecipitated Hsp27/cytochrome c. Moreover, bufalin inhibited MTX-resistant osteosarcoma xenograft growth, and a down-regulation of Hsp27 in vivo was observed. Taken together, bufalin exerted potent anti-osteosarcoma effects in vitro and in vivo, even in MTX resistant osteosarcoma cells. The down-regulation of Hsp27 played a critical role in bufalin-induced apoptosis in osteosarcoma cells. Bufalin may have merit to be a potential chemotherapeutic agent for osteosarcoma, particularly in MTX-resistant groups.

Cell cycle regulation by the PRMT6 arginine methyltransferase through repression of cyclin-dependent kinase inhibitors.

PRMT6 belongs to the family of Protein Arginine Methyltransferase (PRMT) enzymes that catalyze the methylation of guanidino nitrogens of arginine residues. PRMT6 has been shown to modify the tail of histone H3, but the in vivo function of PRMT6 is largely unknown. Here, we show that PRMT6 regulates cell cycle progression. Knockdown of PRMT6 expression in the human osteosarcoma cell line U2OS results in an accumulation of cells at the G2 checkpoint. Loss of PRMT6 coincides with upregulation of p21 and p27, two members of the CIP/KIP family of cyclin-dependent kinase (CDK) inhibitors. Gene expression and promoter analysis show that p21 and p27 are direct targets of PRMT6, which involves methylation of arginine-2 of histone H3. Our findings imply arginine methylation of histones by PRMT6 in cell cycle regulation.

Gallic Acid Induces the Apoptosis of Human Osteosarcoma Cells In Vitro and In Vivo via the Regulation of Mitogen-Activated Protein Kinase Pathways

To examine the antitumor effects of gallic acid (GA) on osteosarcoma, two human osteosarcoma cell lines U-2OS and MNNG/HOS were treated by GA and subjected to cell proliferation and apoptosis assays. In addition, MNNG/HOS xenograft tumors were established in nude BALB/c mice to evaluate the anticancer capacity of GA in vivo. The results showed that GA inhibited the proliferation and induced the apoptosis of osteosarcoma cells, accompanied by the upregulation of p-38 activation and the downregulation of c-Jun N-terminal kinase (JNK) and extracellular signal regulated kinase (ERK1/2) activation. Additionally, p38 MAPK inhibitor abrogated GA-induced growth inhibition of osteosarcoma cells, whereas JNK or ERK1/2 inhibitors sensitized osteosarcoma cells to GA-induced growth inhibition. In vivo studies further showed that GA administration decreased xenograft tumor growth in a dose-dependent manner. Immunohistochemistry analysis demonstrated the downregulation of PCNA and CD31 expression and upregulation of apoptosis in MNNG/HOS tumor tissues following GA treatment. This study demonstrates the antitumor efficacy of GA for osteosarcoma that is mediated by the modulation of cell proliferation, apoptosis, and angiogenesis. Our findings suggest that GA could be a potent agent for osteosarcoma intervention.

Role of reactive oxygen species in the regulation of HIF-1 by prolyl hydroxylase 2 under mild hypoxia

The function and survival of eukaryotic cells depends on a constant and sufficient oxygen supply. Cells recognize and respond to hypoxia by accumulation of the transcription factor hypoxia-inducible factor 1 (HIF-1), composed of an oxygen-sensitive HIF-1α and a constitutive HIF-1β subunit. Besides physiology, HIF-1 induction is involved in major pathological processes such as cardiovascular disease, inflammation and cancer, which are associated with the formation of reactive oxygen species (ROS). ROS have been reported to affect HIF-1 activity but the role for ROS in regulating HIF-1 has not been definitely settled. In order to shed light on the redox-regulation of HIF-1 by ROS, we studied the impact of exogenous ROS treatment (H2O2) on HIF-1α and HIF-1 regulatory protein prolyl hydroxylase 2 (PHD2) in the human osteosarcoma cell line U2OS. At early reaction periods, H2O2 induced HIF-1α but at prolonged observation phases the opposite occurred. Herein, modulation of PHD activity appeared to be the key element, because knockdown and inhibition of the PHD2 prevented reduction of HIF-1α. However, H2O2 treatment constantly suppressed HIF-1 transactivation at all time-points. Our data indicate a dual redox regulation of HIF-1α protein amount with a constant suppression of HIF-1 target gene expression by ROS.

Millimeter wave treatment induces apoptosis via activation of the mitochondrial-dependent pathway in human osteosarcoma cells

Millimeter wave (MW) is an electromagnetic wave with a wavelength between 1 and 10 mm and a frequency of 30-300 GHz that causes multiple biological effects and has been used as a major component in physiotherapies for the clinical treatment of various types of diseases including cancers. However, the precise molecular mechanism of the anticancer activity of millimeter wave remains to be elucidated. In the present study, we investigated the cellular effects of the MW in the U-2OS human osteosarcoma cell line. Our results showed that MW induced cell morphological changes and reduced cell viability in a dose- and time-dependent manner suggesting that MW inhibited the growth of U-2OS cells as demonstrated. Hoechst 33258 staining and Annexin V/propidium iodide double staining exhibited the typical nuclear features of apoptosis and increased the proportion of apoptotic Annexin V-positive cells in a dose-dependent manner, respectively. In addition, MW treatment caused loss of plasma membrane asymmetry, release of cytochrome c, collapse of mitochondrial membrane potential, activation of caspase-9 and -3, and increase of the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Taken together, the results indicate that the U-2OS cell growth inhibitory activity of MW was due to mitochondrial-mediated apoptosis, which may partly explain the anticancer activity of millimeter wave treatment.

Inhibition of fatty acid synthase supresses osteosarcoma cell invasion and migration

Fatty acid synthase (FASN) is overexpressed in a variety of human cancers, and may be involved in cancer metastasis. Hence, the strategies targeted on FASN may have therapeutic potential for treating cancer metastasis. The aim of this study is to investigate the correlation of FASN expression with metastasis in human osteosarcoma. Human osteosarcoma cell lines U2-OS and osteosarcoma biopsy specimens were employed in this study. The expression of FASN protein in osteosarcoma specimens was detected by IHC (immunohistochemistry) and the relationship with metastasis was analyzed. We performed the cerulenin, an inhibitor of FASN, to inhibit FASN expression in U2-OS cells. Western blot and RT-PCR were performed to investigate the expression of FASN in U2-OS cells. Cells mobility was detected by wound healing and Transwell assays. Results showed that the FASN expression level in the cases with pulmonary metastases was significantly higher than in those without metastasis. In vitro, the invasion and migration of U2-OS cells were suppressed by inhibiting FASN. Our findings suggested that FASN may be involved in osteosarcoma metastasis.

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.