Invasion Of U2OS Cells Research Articles

FKBP51 regulates cell motility and invasion via RhoA signaling.

FK506 binding protein 51 (FKBP51), a member of the immunophilin family, is involved in multiple signaling pathways, tumorigenesis, and chemoresistance. FKBP51 expression correlates with metastatic potential in melanoma and prostate cancer. However, the functions of FKBP51, particularly involving the regulation of cell motility and invasion, are not fully understood. We discovered two novel interacting partner proteins of FKBP51, i.e., deleted in liver cancer 1 (DLC1) and deleted in liver cancer 2 (DLC2), using immunoprecipitation and mass spectrometry. DLC1 and DLC2 are Rho GTPase‐activating proteins that are frequently downregulated in various cancers. Next, we demonstrated that overexpression of FKBP51 enhances cell motility and invasion of U2OS cells via upregulation of RhoA activity and enhanced Rho‐ROCK signaling. Moreover, FKBP51‐depleted cells displayed a cortical distribution of actin filaments and decreased cell motility and invasion. Consistent with this phenotype, FKBP51 depletion caused a downregulation of RhoA activity. Considered together, our results demonstrate that FKBP51 positively controls cell motility by promoting RhoA and ROCK activation; thus, we have revealed a novel role for FKBP51 in cytoskeletal rearrangement and cell migration and invasion.

CXCL5 Plays a Promoting Role in Osteosarcoma Cell Migration and Invasion in Autocrine- and Paracrine-Dependent Manners.

CXCL5, a CXC-type chemokine, is an important attractant for granulocytic immune cells by binding to its receptor CXCR2. Recently, CXCL5/CXCR2 has been found to play an oncogenic role in many human cancers. However, the exact role of CXCL5 in osteosarcoma cell migration and invasion has not been revealed. Here we found that the protein expression of CXCL5 was significantly increased in osteosarcoma tissues compared with that in matched adjacent nontumor tissues. Moreover, the expression of CXCL5 was significantly associated with advanced clinical stage and metastasis. Further investigation showed that the CXCL5 expression levels were also significantly increased in osteosarcoma cell lines, including Saos-2, MG63, U2OS, and SW1353, when compared with those in normal osteoblast hFoB1.19 cells. U2OS cells were further transfected with CXCL5-specific siRNA or overexpression plasmid. Knockdown of CXCL5 significantly suppressed U2OS cell migration and invasion. On the contrary, overexpression of CXLC5 remarkably promoted the migration and invasion of U2OS cells. Interestingly, both exogenous CXCL5 treatment and the conditioned medium of CXCL5-overexpressing hFoB1.19 cells could also enhance the migration and invasion of U2OS cells, suggesting that the promoting role of CXCL5 in U2OS cell migration and invasion is also in a paracrine-dependent manner. According to these data, our study demonstrates that CXCL5 is upregulated in osteosarcoma and may play an oncogenic role in osteosarcoma metastasis. Therefore, CXCL5 may become a potential therapeutic target for osteosarcoma treatment.

17β-Estradiol treatment drives Sp1 to upregulate MALAT-1 expression and epigenetically affects physiological processes in U2OS cells

Osteosarcoma is the most common primary bone tumor characterized by high risk of metastasis, thus presents with an overall survival rate of 60%, despite the use of chemotherapy and surgery. Metastasis‑associated lung adenocarcinoma transcript1 (MALAT‑1) has been reported to upregulated and epigenetically regulate the metastasis in osteosarcoma; however, the regulatory mechanisms of MALAT‑1 expression remain unclear. In the current study, significant upregulation of MALAT‑1 was observed subsequent to exposure to low concentrations of 17β‑estradiol (E2) in U2OS cells. Using chromatin immunoprecipitation assays, E2‑activated estrogen receptorα (ERα) was identified to promote the binding of specificity protein1 (Sp1) to the MALAT‑1 promoter. Electrophoretic mobility shift assay and immunoprecipitation results demonstrate that ERα binds indirectly to the MALAT‑1 promoter by binding directly to Sp1 protein. Notably, without E2 stimulation, overexpressed ERα results in no significant promotion of the Sp1/MALAT‑1 promoter, indicating that the translocation of ERα to nuclei stimulated by E2 is necessary. The immunofluorescence assay confirmed that E2 stimulation promotes the translocation of Sp1 to the nuclei in an ERα‑dependent manner. Subsequently, the effects of E2 on osteosarcoma physiological processes were further analyzed. Consistently, E2 treatment was observed to promote proliferation, colony formation, migration and invasion in U2OS cells. Taken together, the results indicate a role for E2 in regulating the physiological processes of osteosarcoma cells by regulating MALAT‑1 expression levels.

AMD3100 combined with triptolide inhibit proliferation, invasion and metastasis and induce apoptosis of human U2OS osteosarcoma cells.

Osteosarcoma (OS) mainly occurs in children and adolescents, and has a high propensity for lung metastasis. Little is known about the role of SDF-1/CXCR4 axis in OS progression. AMD3100 is a specific CXCR4 antagonist. Triptolide can induce apoptosis and proliferation inhibition in various cancer cell lines. This work aimed to investigate the effects of AMD3100 plus triptolide on the proliferation, apoptosis, invasion and metastasis of OS cells. The expression levels of SDF-1 and CXCR4 in five OS cell lines was analyzed by qRT-PCR, western blotting and ELISA assays. The effect of AMD3100 and triptolide on the proliferation, apoptosis and invasion of U2OS cells was evaluated by CCK-8, flow cytometry and transwell assay, respectively. Orthotopic intra-tibial growth and lung metastasis mouse model of OS were employed to evaluate the inhibition effect of AMD3100 and triptolide on primary OS growth and lung metastasis. CXCR4 protein expression was detected in HOS-8603, MG-63, U2OS and 143B but not Saos2 cells, and all these cell lines expressed SDF-1. AMD3100 plus triptolide induced proliferation inhibition and apoptosis of U2OS cells, which was attributed to the downregulation of c-Myc, survivin, cyclin D1 and increased cleaved caspase-3 and PARP. AMD3100 and triptolide also suppressed SDF-1 induced invasion of CXCR4+ U2OS cells, which was validated by decreased expression of MMP-2 and 9, VEGF, m-Calpain and β-catenin. Moreover, the phosphorylation levels of Erk1/2, Akt and STAT3, as well as the nuclear translocation and phosphorylation of NF-κB p65 in U2OS cells were also reduced by AMD3100 and triptolide. In vivo, AMD3100 and triptolide significantly reduced primary tumor growth and lung metastasis of U2OS cells. AMD3100 combined with triptolide can reduce proliferation and metastasis, and induce apoptosis of U2OS cells, which may be related to the Erk1/2, Akt, STAT3 and NF-κB pathways.

Gene silencing of USP1 by lentivirus effectively inhibits proliferation and invasion of human osteosarcoma cells

Osteosarcoma is the most frequent malignant bone tumor, affecting the extremities of adolescents and young adults. Ubiquitin-specific protease 1 (USP1) plays a critical role in many cellular processes including proteasome degradation, chromatin remodeling and cell cycle regulation. In the present study, we discovered that USP1 was overexpressed in 26 out of 30 osteosarcoma tissues compared to cartilage tumor tissues and normal bone tissues. We then constructed a lentiviral vector mediating RNA interference (RNAi) targeting USP1 and demonstrated that it significantly suppressed the mRNA and protein expression of the USP1 gene in U2OS cells. Knockdown of USP1 inhibited the growth and colony-forming, as well as significantly reduced the invasiveness of U2OS cells. Western blot analysis indicated that suppression of USP1 downregulated the expression of many proteins including SIK2, MMP-2, GSK-3β, Bcl-2, Stat3, cyclinE1, Notch1, Wnt-1 and cyclinA1. Most of these proteins are associated with tumor genesis and development. RNAi of SIK2 significantly decreased SIK2 protein expression and inhibited the ability of forming colonies, as well as induced apoptosis and reduced the invasiveness of U2OS cells. Collectively, our results suggest that silencing USP1 inhibits cell proliferation and invasion in U2OS cells. Therefore, USP1 may provide a novel therapeutic target for the treatment of osteosarcoma.

Expression of long non-coding RNA MALAT1 in osteosarcoma and its effect on invasiveness and metastatic potential of osteosarcoma cells

To investigate the significance of long non-coding RNA MALAT1 expression in osteosarcoma, and the potential mechanism by which MALAT1 promotes tumor metastasis. Twenty cases of osteosarcoma in the First Affiliated Hospital of Xinxiang Medical University and Ping Ding Shan First People's Hospital were collected from January 2014 to December 2015. The expression of MALAT1 in osteosarcoma tissue and paired adjacent noncancerous tissue were analyzed by qRT-PCR. Correlation of MALAT1 expression in osteosarcoma with clinical pathologic features was performed by the Mann-Whitney U test. U-2OS cells were transfected with lenti-virus carrying MALAT1-shRNA and nonspecific shRNA (LV-vector). The expression of MALAT1 was detected by qRT-PCR. The cell activity was evaluated by MTT asssy. The impact of MALAT1-shRNA on invasion in U-2OS cells were determined by transwell migration assay. The expression of Wnt/β-catenin signal pathway related proteins were detected by Immunofluorescence stain and Western blot. The expression level of MALAT1 in osteosarcoma tissue was higher than that in paired adjacent noncancerous tissue and correlated significantly with nodal and pulmonary metastasis(P<0.01). MTT assay showed that knockdown of MALAT1 with lenti virus-MALAT1 shRNA inhibited the growth of U-2OS cells, along with marked decrease of invasive ability of U-2OS cells in the transwell migration assay. By immunofluorescence stain and Western blot assay, MALAT1 significantly reduced the expression of β-catenin, MMP7, and c-MYC in U-2OS cells. The expression of MALAT1 is high in osteosarcoma and correlates with tumor metastasis. MALAT1 promotes invasion and metastasis of osteosarcoma cells likely thought the Wnt/β-catenin signal pathway.

MicroRNA-101 has a suppressive role in osteosarcoma cells through the targeting of c-FOS.

MicroRNAs (miRs) have been implicated in the development and progression of osteosarcoma (OS). However, the underlying mechanism of miR-101 in regulating of the proliferation, migration and invasion of OS cells remains to be elucidated. In the present study, reverse transcription-quantitative polymerase chain reaction data revealed that miR-101 was frequently downregulated in the tissue samples of 12 patients with OS compared with their matched adjacent non-tumor tissues. Furthermore, miR-101 was significantly downregulated in three common OS cell lines, Saos-2, MG63 and U2OS, compared with the human osteoblast cell line, hFOB1.19 (P<0.01). A luciferase reporter assay was also performed and identified c-FOS as a novel target of miR-101 in U2OS cells; overexpression of miR-101 significantly suppressed the protein expression levels of c-FOS, while knockdown of miR-101 significantly enhanced the formers' expression levels in U2OS cells (P<0.01). Independent inhibition of c-FOS and overexpression of miR-101 expression levels significantly suppressed U2OS cell proliferation, migration and invasion (P<0.01). However, overexpression of c-FOS reversed the inhibitory effect of miR-101 upregulation on proliferation, migration and invasion of U2OS cells, suggesting that miR-101 acts as a tumor suppressor in OS cells via targeting of c-FOS. Thus, we propose that the miR-101/c-FOS axis may be a potential therapeutic target for OS.

MiR-1 Inhibits Cell Growth, Migration, and Invasion by Targeting VEGFA in Osteosarcoma Cells.

microRNAs (miRNAs) are small noncoding RNAs and have been shown to play a crucial role in the osteosarcoma (OS) tumorigenesis and progression. VEGFA is a key regulator of angiogenesis and plays an important role in regulation of tumor metastasis. The objective of this study was to determine whether VEGFA was involved in miR-1-mediated suppression of proliferation, migration, and invasion of OS cells. The expression levels of miR-1 were significantly lower in OS tumor tissues than those in adjacent normal tissues and in SAOS-2 and U2OS cell lines compared to a normal osteoblast (NHOst) cell line. VEGFA was upregulated in OS tumor tissues and SAOS-2 and U2OS cell lines. The results of CCK-8 assay and transwell assay showed that miR-1 acted as a tumor suppressor by inhibiting cell proliferation, migration, and invasion in U2OS cells. Dual luciferase reporter assay demonstrated that VEGFA was a direct and functional target gene of miR-1. miR-1 directly inhibits the protein expression of VEGFA via its 3′-UTR. Knockdown of VEGFA by siRNA inhibited proliferation, migration, and invasion of U2OS cells. Our study suggested the potential inhibitory function of miR-1 in OS cell proliferation, migration, and invasion via inhibiting VEGFA.

Impact of PTTG1 downregulation on cell proliferation, cell cycle and cell invasion of osteosarcoma and related molecular mechanisms

To downregulate the expression of pituitary tumor transforming gene 1 (PTTG1) in osteosarcoma (OS) cells by siRNA technology and to investigate related biological impact on cell proliferation, cell cycle and cell invasion of OS. Three OS cell lines and osteoblast hFOB1.19 cell line were used in this study. Control siRNA and PTTG1 siRNA were employed to transfect OS U2OS cells, and PTTG1 protein level was detected by Western blot after the transfection. Effects of PTTG1 siRNA on cell proliferation, cell cycle and cell invasion were investigated by CCK-8, flow cytometry and Boyden chamber, respectively. Finally, activity of Akt and its downstream target gene expression were analyzed by Western blot in U2OS cells upon various treatments. Expression of PTTG1 protein in 3 OS cells (MG-63, SaOS-2 and U2OS) was significantly higher than that in osteoblast hFOB1.19, among which U2OS cells displayed the highest level. PTTG1 siRNA markedly downregulated the expression of PTTG1 protein in U2OS cells, leading to obvious inhibition of cell proliferation, altered cell cycle distribution and reduced ability of invasion of U2OS cells. Moreover, downregulation of PTTG1 reduced the expression of p-Akt (S473 and T308), MMP-2 and MMP-9 proteins, along with enhanced expression of p21 and E-cadherin proteins. PTTG1 may be tightly linked to the development of OS and therefore may serve as a novel target for precision therapy of OS.

Abstract 4996: Interferon consensus sequence binding protein (ICSBP) promotes epithelial to mesenchymal transition (EMT)-like phenomena, cell motility, and invasion via TGF-β signaling in U2OS cells

Abstract Interferon consensus sequence binding protein (ICSBP) is a transcription factor induced by interferon gamma (IFN-γ) and a member of the interferon regulatory factor (IRF) family. ICSBP is predominantly expressed in hematopoietic cells and regulates the immune response and cell growth and differentiation. However, little is known about its function in non-hematopoietic cells. Here we show a novel function for ICSBP in epithelial to mesenchymal transition (EMT)-like phenomena (ELP), cell motility, and invasion in human osteosarcoma cell lines, including U2OS cells. IFN-γ treatment induced ICSBP expression and EMT-like morphological change in U2OS cells, which were suppressed by ICSBP knock-down. To further investigate the role of ICSBP in ELP, we established a stable U2OS cell line that overexpresses ICSBP. ICSBP expression caused U2OS cells to have a more elongated shape and an increased vimentin and fibronectin expression. ICSBP expression also promoted adhesiveness, motility, and invasiveness of U2OS cells. ICSBP up-regulated transforming growth factor (TGF)-β receptors and activated TGF-β signaling cascades, which were responsible for ELP as well as increased cell motility and invasion. In addition, ICSBP-induced TGF-β receptor activation resulted in the up-regulation of Snail. Knock-down of Snail attenuated the ICSBP-induced augmentation of cell motility and invasion. Up-regulation of Snail, ELP, and increased invasion by ICSBP expression were also observed in other osteosarcoma cell lines, such as Saos-2 and 143B. Furthermore, ICSBP and TGF- β receptor I were expressed in 45/54 (84%) and 47/54 (87%) of human osteosarcoma tissues, respectively, and showed significant correlation (r= 0.47, p= 0.0007) with respect to their expression levels. Taken altogether, these data demonstrate a novel function for ICSBP in ELP, cell motility, and invasion through the TGF-β and Snail signaling pathways Citation Format: Jee Young Sung, Seog-Yun Park, June Hyuk Kim, Hyun Guy Kang, Jong Hyung Yoon, Yoon Sook Na, Yong-Nyun Kim, Byung-Kiu Park. Interferon consensus sequence binding protein (ICSBP) promotes epithelial to mesenchymal transition (EMT)-like phenomena, cell motility, and invasion via TGF-β signaling in U2OS cells. [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 4996. doi:10.1158/1538-7445.AM2014-4996

Effect of the PTEN gene on adhesion, invasion and metastasis of osteosarcoma cells.

The phosphatase and tensin homolog (PTEN) gene, an important tumor-suppressor gene, has been demonstrated to have the potential for inhibiting proliferation, migration and invasion in various types of cancer cells. The aim of the present study was to investigate the effect of PTEN expression on osteosarcoma (OS) cells. The wild-type PTEN plasmid was transfected into OS U2-OS cells. The effects of PTEN on the adhesion, migration and invasion of U2-OS cells were evaluated by cell adhesion analysis, invitro scratch and Transwell assays, respectively. The levels of MMP-2 and MMP-9, and focal adhesion kinase (FAK) protein regulated by PTEN were detected via western blot analysis. Meanwhile, the level of intracellular FAK phosphorylation was observed. The results from the present study showed that overexpression of PTEN transcription and protein were observed in U2-OS cells following PTEN transfection. Furthermore, the migration, invasion and adhesion capabilities of the cells with PTEN transfection were significantly decreased compared to these capacities in the cells without PTEN. Meanwhile, it was shown that there was downregulation of MMP-9, FAK and p-FAK concomitant with the elevation of the intracellular PTEN level. It is therefore evident that the upregulation of PTEN may attenuate the adhesion, migration and invasion capabilities of OS cells. The mechanisms of the effects of PTEN on OS cells may be correlated with a reduction in the related genes by PTEN regulation.

Interferon consensus sequence-binding protein (ICSBP) promotes epithelial-to-mesenchymal transition (EMT)-like phenomena, cell-motility, and invasion via TGF-β signaling in U2OS cells.

Interferon consensus sequence-binding protein (ICSBP) is a transcription factor induced by interferon gamma (IFN-γ) and a member of the interferon regulatory factor (IRF) family. ICSBP is predominantly expressed in hematopoietic cells and regulates the immune response and cell growth and differentiation. However, little is known about its function in non-hematopoietic cells. Here we show a novel function for ICSBP in epithelial-to-mesenchymal transition (EMT)-like phenomena (ELP), cell motility, and invasion in human osteosarcoma cell lines, including U2OS cells. IFN-γ treatment induced ICSBP expression and EMT-like morphological change in U2OS cells, which were suppressed by ICSBP knockdown. To further investigate the role of ICSBP in ELP, we established a stable U2OS cell line that overexpresses ICSBP. ICSBP expression caused U2OS cells to have a more elongated shape and an increased vimentin and fibronectin expression. ICSBP expression also promoted adhesiveness, motility, and invasiveness of U2OS cells. ICSBP upregulated transforming growth factor (TGF)-β receptors and activated TGF-β signaling cascades, which were responsible for ELP as well as increased cell motility and invasion. In addition, ICSBP-induced TGF-β receptor activation resulted in the upregulation of Snail. Knockdown of Snail attenuated the ICSBP-induced augmentation of cell motility and invasion. Upregulation of Snail, ELP, and increased invasion by ICSBP expression were also observed in other osteosarcoma cell lines, such as Saos-2 and 143B. Furthermore, ICSBP and TGF-β receptor I were expressed in 45/54 (84%) and 47/54 (87%) of human osteosarcoma tissues, respectively, and showed significant correlation (r=0.47, P=0.0007) with respect to their expression levels. Taken altogether, these data demonstrate a novel function for ICSBP in ELP, cell motility, and invasion through the TGF-β and Snail signaling pathways.

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.

Abstract 719: Role of secreted frizzled-related proteins in inhibiting growth and invasion in human osteosarcoma

Abstract Aberrant activation of the canonical Wnt/β-catenin signaling pathway promotes osteosarcoma tumorigenesis and metastasis. Secreted frizzled-related proteins (SFRPs), a family of five glycoproteins (SFRP1-5), antagonize this pathway by competing with Frizzled receptors for Wnt ligands. Down-regulation of SFRPs through epigenetic silencing was commonly found in various malignancies. Specifically, in osteosarcoma, the loss of SFRP-3 expression has previously been observed in osteogenic sarcoma biopsy specimens and several osteosarcoma cells lines. Despite strong evidence of their status as tumor suppressors, the implication of SFRP-1, −2, −4 and −5 in the pathogenesis of osteosarcoma has yet been reported. The objective of this study, therefore, is to understand the role of SFRPs as Wnt antagonists and their functions in affecting key anti-cancer processes such as the suppression of growth and invasion, induction of apoptosis and cell cycle disruption, as well as down-regulation of various Wnt target oncogenes in osteosarcoma. In this study, we first performed western blot analysis and detected abundant levels of β-catenin protein in a panel of metastatic osteosarcoma cell lines, suggesting frequent activation of canonical Wnt/β-catenin signaling in osteosarcoma. Effective inhibitions of the Wnt/β-catenin pathway by restoration of SFRPs in U2OS cells were subsequently shown by marked suppression of both β-catenin/Tcf transcriptional activities and β-catenin protein using luciferase reporter assays and western blot analysis respectively. Immunofluorescence confocal microscopy analysis revealed an abnormal decrease in β-catenin accumulations in the nuclei and a corresponding increase in β-catenin localization to the cell membranes in U2OS cells over-expressing SFRPs. We next performed wound healing and Matrigel invasion assays and observed that ectopic expression of SFRPs significantly decreased osteosarcoma cell migration and invasion. Western blotting showed that the reduced U2OS cell invasion and migration correlated with the protein expression of matrix metalloproteinases-2 and −9. Furthermore, using MTT proliferation and colony formation assays, we found that restoration of SFRPs significant inhibited both anchorage-dependent and -independent growth in U2OS cells. These anti-proliferative effects were attributed to GO/G1 phase arrest in U2OS/SFRP-1, U2OS/SFRP-2 and U2OS/SFRP-5 cells and G2/M phase arrest in U2OS/SFRP-4 cells. At the same time, these anti-proliferative effects correlated with the suppression of major Wnt target oncogenes such as cyclin D1, c-Myc and survivin. Collectively, our findings strongly suggest that SFRPs may be exploited as a therapeutic target in the treatment of osteosarcoma. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 719.

Cyclooxygenase-2 promotes cell proliferation, migration and invasion in U2OS human osteosarcoma cells

Osteosarcoma is the most common primary bone tumor, but the pathogenesis is not well understood. While cyclooxygeanse-2 (COX-2) is known to be closely associated with tumor growth and metastasis in several kinds of human tumors, the function of COX-2 in osteosarcoma is unclear. Therefore, to investigate the function of COX-2 in osteosarcoma, we established stable cell lines overexpressing COX-2 in U2OS human osteosarcoma cells. COX-2 overexpression as well as prostaglandin E2 treatment promoted proliferation of U2OS cells. In addition, COX-2 overexpression enhanced mobility and invasiveness of U2OS cells, which was accompanied by increases of matrix metalloproteinase-2 and -9 (MMP-2 and -9) activities. Selective COX-2 inhibitors, NS-398 and celecoxib, inhibited cell proliferation and abrogated the enhanced mobility, invasiveness and MMP activities induced by COX-2 overexpression. These results suggest that COX-2 is directly associated with cell proliferation, migration and invasion in human osteosarcoma cells, and the therapeutic value of COX-2 inhibitors should be evaluated continuously.

Antitumor effect of nutrient synergy on human osteosarcoma cells U-2OS, MNNG-HOS and Ewing's sarcoma SK-ES.1.

Current treatment of osteosarcoma is associated with poor prognosis, especially due to the increased risk of developing other cancers with chemotherapy. Therefore, new, safe and effective treatment strategies are needed. We investigated the effect of a unique mixture of nutrients containing lysine, proline, arginine, ascorbic acid, and epigallocatechin gallate (EGCG) on human osteosarcoma cell lines U-2OS, MNNG-HOS, and Ewing's sarcoma SK-ES-1 by measuring: cell proliferation, expression of matrix metalloproteinase-2 (MMP-2), MMP-9, and invasive and angiogenesis potential. Cell proliferation was evaluated by MTT assay, matrix metalloproteinases (MMP) expression by gelatinase zymography, VEGF expression by ELISA, and invasion through Matrigel. Cells were also treated with phorbol 12-myristate 13-acetate (PMA) to study enhanced MMP and VEGF expression. The invasion of osteosarcoma U-2OS and MNNG-HOS cells through Matrigel was significantly reduced in a dose-dependent fashion, with 100% inhibition of invasion of U-2OS cells at 100 microg/ml, and MNNG cells at 50 microg/ml concentration of the synergistically acting nutrient mixture. Ewing's sarcoma SK-ES-1 cells were not invasive. Nutrient synergy (NS) exhibited a dose response antiproliferative effect on osteosarcoma U-2OS cells, reaching 67% at 1000 microg/ml of NS; no significant suppression of cell proliferation was seen with MNNG or Ewing's sarcoma cells. Zymography showed dose-dependent inhibition of MMP secretion by all three cell lines in the presence of NS. VEGF secretion by U-2OS cells was completely blocked at 500 microg/ml of NS. Our results suggest NS is an excellent candidate for therapeutic use in the treatment of osteosarcoma, by inhibiting cancer cell invasion, and secretion of MMPs and VEGF, all critical parameters for cancer control and prevention.