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Purpose: To determine the effect of Lonicerae japonicae Caulis extract (LJCE) on the proliferation, apoptosis, and cycle distribution of human osteosarcoma U2OS cells. Methods: Human osteosarcoma U2OS cells were treated with LJCE at doses of 10, 20, 30 and 40 mg/mL, with untreated U2OS cells as the control group. Cell proliferation was measured using MTT method, while flow cytometric analysis was used for determination of apoptosis and cell cycle distribution. Results: As LJCE concentration increased, the viability of U2OS cells gradually decreased, while the apoptosis rate gradually increased. The LJCE treatment had no marked effect on G1 phase cells (p > 0.05). However, the population of G2 cells in each group increased with increasing LJCE concentration, while the S phase cells in each group decreased with increasing concentration. The cells were significantly blocked in the G2 phase (p < 0.05). Conclusion: Lonicerae japonicae Caulis extract inhibits the proliferation of U2OS cells and promotes apoptosis of U2OS cells. Therefore, LJCE possesses potentials as a novel therapeutic agent for osteosarcoma. [ABSTRACT FROM AUTHOR]

Aim: This study evaluates the cytotoxic effect of Potentilla fulgens root extract on MCF7, U2OS, and RPE-1 cell lines and investigates its impact on cell migration. Materials and Methods: The effect of Potentilla fulgens root extract on the cell viability of MCF-7, U2OS, and RPE-1 cells was analyzed using an MTT assay. The effect of the compounds on cell migration was evaluated at 24 and 48 hours using the wound healing assay. A wound-healing assay was used to measure the metastatic ability of cells in vitro. Results: MTT assay showed that PRE had a cytotoxic effect on all three cell lines, depending on dose and time, and this effect was statistically significant. The wound healing assay results showed that PRE slowed the migration of live MCF-7 and U2OS cancer cells, and these effects increased over time. Conclusion: As a result, this study indicates that PRE may be very useful in treating human osteosarcoma and breast cancer. [ABSTRACT FROM AUTHOR]

The search for alternative methods in cancer treatments has been going on for many years. In the current study conducted for this purpose, selenium nanoparticles (ML-SeNPs) were produced from the aqueous leaf extract of Mentha longifolia L. easily and inexpensively, without harming the environment. The anticancer potential of ML-SeNPs on glioblastoma cell (U373), osteosarcoma cell (U2OS), and healthy retinal pigment epithelial cell (RPE-1) lines was determined by MTT (3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium bromid) test. For the test, ML-SeNPs were applied at 100, 300, and 600 µg/mL levels and interaction was provided for 24 and 48 hours. The survival rates (%) in RPE-1, U373, and U2OS cell lines in the 24-hour application were 107.49-98.89, 97.66-86.82, and 87.81-83.37, respectively. The viability rates (%) of the cells in 48 hours of application were 72.27-87.39, 68.17-73.48, and 81.00-84.67, respectively. In general, it was discovered that the cytotoxic effect of ML-SeNPs on RPE-1, U373, and U2OS cell lines was greater at low doses and increased over time. In-vivo studies that support the antiproliferative action of ML-SeNPs may boost the prospect of using them as therapeutic agents in potential cancer treatment procedures in the following years. [ABSTRACT FROM AUTHOR]

Alginate–gelatin hydrogels are extensively used in bioengineering. However, despite different formulations being used to grow different cell types in vitro, their pH and its effect, together with the crosslinking ions of these formulations, are still infrequently assessed. In this work, we study how these elements can affect hydrogel stability and printability and influence cell viability and metabolism on the resulting 3D prints. Our results show that both the buffer pH and crosslinking ion (Ca2+ or Ba2+) influence the swelling and degradation rates of prints. Moreover, buffer pH influenced the printability of hydrogel in the air but did not when printed directly in a fluid-phase CaCl2 or BaCl2 crosslinking bath. In addition, both U2OS and NIH/3T3 cells showed greater cell metabolic activity on one-layer prints crosslinked with Ca2+. In addition, Ba2+ increased the cell death of NIH/3T3 cells while having no effect on U2OS cell viability. The pH of the buffer also had an important impact on the cell behavior. U2OS cells showed a 2.25-fold cell metabolism increase on one-layer prints prepared at pH 8.0 in comparison to those prepared at pH 5.5, whereas NIH/3T3 cells showed greater metabolism on one-layer prints with pH 7.0. Finally, we observed a difference in the cell arrangement of U2OS cells growing on prints prepared from hydrogels with an acidic buffer in comparison to cells growing on those prepared using a neutral or basic buffer. These results show that both pH and the crosslinking ion influence hydrogel strength and cell behavior. [ABSTRACT FROM AUTHOR]

Abstract Background Proteolysis of the histone H3 N-terminal tail (H3NT) is an evolutionarily conserved epigenomic feature of nearly all eukaryotes, generating a cleaved H3 product that is retained in ~ 5–10% of the genome. Although H3NT proteolysis within chromatin was first reported over 60 years ago, the genomic sites targeted for H3NT proteolysis and the impact of this histone modification on chromatin structure and function remain largely unknown. The goal of this study was to identify the specific regions targeted for H3NT proteolysis and investigate the consequence of H3NT “clipping” on local histone post-translational modification (PTM) dynamics. Results Leveraging recent findings that matrix metalloproteinase 2 (MMP-2) functions as the principal nuclear H3NT protease in the human U2OS osteosarcoma cell line, a ChIP-Seq approach was used to map MMP-2 localization genome wide. The results indicate that MMP-2 is selectively targeted to the transcription start sites (TSSs) of protein coding genes, primarily at the + 1 nucleosome. MMP-2 localization was exclusive to highly expressed genes, further supporting a functional role for H3NT proteolysis in transcriptional regulation. MMP-2 dependent H3NT proteolysis at the TSSs of these genes resulted in a > twofold reduction of activation-associated histone H3 PTMs, including H3K4me3, H3K9ac and H3K18ac. One of genes requiring MMP-2 mediated H3NT proteolysis for proficient expression was the lysosomal cathepsin B protease (CTSB), which we discovered functions as a secondary nuclear H3NT protease in U2OS cells. Conclusions This study revealed that the MMP-2 H3NT protease is selectively targeted to the TSSs of active protein coding genes in U2OS cells. The resulting H3NT proteolysis directly alters local histone H3 PTM patterns at TSSs, which likely functions to regulate transcription. MMP-2 mediated H3NT proteolysis directly activates CTSB, a secondary H3NT protease that generates additional cleaved H3 products within chromatin.

Background: Senescent cells are characterized by an arrest in proliferation. In addition to replicative senescence resulting from telomere exhaustion, sub-lethal genotoxic stress resulting from DNA damage, oncogene activation or mitochondrial dysfunction also elicits a senescence phenotype. Methods: Senescence was induced in an osteocarcinoma cancer cell line in response to sub-lethal doses of a genotoxic chemotherapeutic agent, followed by quantitative SWATH proteomics and RNA-seq analyses. Results: We present here an integrative multi-omic analysis of proteomic and RNA-seq from proliferating and senescent osteosarcoma cells. Senescence is a controlled program affecting a wide variety of biological processes with some core hallmarks of senescence as well as cell type specific changes. Conclusions: This study presents an integrated analysis and makes available both RNA-seq and proteomic data from proliferating and senescent cells in appropriate FAIR data repositories to aid reuse by the community.

Telomeres play an essential role in protecting the ends of linear chromosomes and maintaining the integrity of the human genome. One of the key hallmarks of cancers is their replicative immortality. As many as 85–90% of cancers activate the expression of telomerase (TEL+) as the telomere maintenance mechanism (TMM), and 10–15% of cancers utilize the homology-dependent repair (HDR)-based Alternative Lengthening of Telomere (ALT+) pathway. Here, we performed statistical analysis of our previously reported telomere profiling results from Single Molecule Telomere Assay via Optical Mapping (SMTA-OM), which is capable of quantifying individual telomeres from single molecules across all chromosomes. By comparing the telomeric features from SMTA-OM in TEL+ and ALT+ cancer cells, we demonstrated that ALT+ cancer cells display certain unique telomeric profiles, including increased fusions/internal telomere-like sequence (ITS+), fusions/internal telomere-like sequence loss (ITS−), telomere-free ends (TFE), super-long telomeres, and telomere length heterogeneity, compared to TEL+ cancer cells. Therefore, we propose that ALT+ cancer cells can be differentiated from TEL+ cancer cells using the SMTA-OM readouts as biomarkers. In addition, we observed variations in SMTA-OM readouts between different ALT+ cell lines that may potentially be used as biomarkers for discerning subtypes of ALT+ cancer and monitoring the response to cancer therapy. [ABSTRACT FROM AUTHOR]

Background: Proteolysis of the histone H3 N-terminal tail (H3NT) is an evolutionarily conserved epigenomic feature of nearly all eukaryotes, generating a cleaved H3 product that is retained in ~ 5–10% of the genome. Although H3NT proteolysis within chromatin was first reported over 60 years ago, the genomic sites targeted for H3NT proteolysis and the impact of this histone modification on chromatin structure and function remain largely unknown. The goal of this study was to identify the specific regions targeted for H3NT proteolysis and investigate the consequence of H3NT "clipping" on local histone post-translational modification (PTM) dynamics. Results: Leveraging recent findings that matrix metalloproteinase 2 (MMP-2) functions as the principal nuclear H3NT protease in the human U2OS osteosarcoma cell line, a ChIP-Seq approach was used to map MMP-2 localization genome wide. The results indicate that MMP-2 is selectively targeted to the transcription start sites (TSSs) of protein coding genes, primarily at the + 1 nucleosome. MMP-2 localization was exclusive to highly expressed genes, further supporting a functional role for H3NT proteolysis in transcriptional regulation. MMP-2 dependent H3NT proteolysis at the TSSs of these genes resulted in a > twofold reduction of activation-associated histone H3 PTMs, including H3K4me3, H3K9ac and H3K18ac. One of genes requiring MMP-2 mediated H3NT proteolysis for proficient expression was the lysosomal cathepsin B protease (CTSB), which we discovered functions as a secondary nuclear H3NT protease in U2OS cells. Conclusions: This study revealed that the MMP-2 H3NT protease is selectively targeted to the TSSs of active protein coding genes in U2OS cells. The resulting H3NT proteolysis directly alters local histone H3 PTM patterns at TSSs, which likely functions to regulate transcription. MMP-2 mediated H3NT proteolysis directly activates CTSB, a secondary H3NT protease that generates additional cleaved H3 products within chromatin. [ABSTRACT FROM AUTHOR]

The purpose of the research is to evaluate the anti-cancer outcomes of aqueous black rice (BR) extract on human cancer cell lines, HeLa, T47D, and U2OS. To begin the investigation, the total phenol and flavonoid content of the BR extract was determined. Additionally, the antioxidant activity of the extract was determined using the ferric reducing antioxidant power (FRAP), total antioxidant capacity (TAC), and 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) assays. The cell viability, apoptosis and cell cycle on several human cancer cells were determined after BR extract administration in several doses (BR1; 100mg/ml, BR2; 200mg/ml, BR3; 300mg/ml, BR4; 400mg/ml, and BR5, 500mg/ml) using flow cytometry analysis. According to the analysis, the extract had total phenol and flavonoid content are 66.42 mg/g and 11.12 mg/g, respectively. Based on the assay of DPPH (53.19 μg/ml), FRAP (49.86 mg/g), and TAC (96.70 mg/g), BR extract showed strong antioxidant activity. This study also revealed that BR extract significantly reduced human cancer cell lines viability and selectively(shown to have no effect on PBMC cells) (p<0.05)especially BR5 group (500 g/mL). The administration of BR extract was most responsive to induce apoptosis in T47D cells, up to 93.64% in BR5 close to cisplatin-induced apoptosis effect, compared to HeLa and U2OS cells. The study also found BR extract induced the cell (HeLa, T47D, and U2OS) to arrest or in the phase G0/G1. Hence, BR extract was a food-based cancer treatment with a promising effect to inhibit cancer cell growth and progression. [ABSTRACT FROM AUTHOR]

Salvia triloba (S. triloba) is an herb inherently linked to traditional medicine systems in the Eastern Mediterranean region. There is minimal experimental evidence however, regarding the anticancer effects of S. triloba in both osteosarcoma and ovarian cancer. In this study, we investigated the effects of crude (macerated) S. triloba ethanol and acetone leaf extracts on viability, migratory ability, and the expression of genes regulating these activities in U2OS and SKOV3 cells using MTT assay, scratch-wound healing/trans-well migration assay, and RT-qPCR respectively. MTT assay results indicated that the acetone extract significantly reduced both U2OS and SKOV3 cell viability with half-maximal inhibitory concentrations (IC50) of 54.51 ± 1.10 µg/mL and 75.96 ± 1.0237 µg/mL respectively; these concentrations further displayed negligible hemolytic activity. The combination of acetone extract (19 µg/mL) and paclitaxel (0.787 µg/mL) displayed synergy and reduced SKOV3 cell viability by over 90%. Additionally, the trans-well migration assay illustrated that the acetone extract (IC50) inhibited both U2OS and SKOV3 cell migration by more than 50%. Moreover, S. triloba acetone extract significantly downregulated the steady-state mRNA expression of key genes involved in driving select cancer hallmarks. Four fractions were generated from the acetone extract by thin layer chromatography (TLC), and the obtained retention factors (Rf) (ranging from 0.2 to 0.8) suggested a mixture of high and moderately polar compounds whose bioactivities require further investigation. In addition, FTIR measurements of the extract revealed peaks corresponding to OH, aliphatic CH, and ester groups suggesting the presence of phenolic compounds, terpenes, and polysaccharides. Altogether, these results suggest that S. triloba possesses potential therapeutic compounds that inhibit cell proliferation and migration, and modulate several genes involved in osteosarcoma and ovarian carcinoma progression. [ABSTRACT FROM AUTHOR]

Alginate–gelatin hydrogels are extensively used in bioengineering. However, despite different formulations being used to grow different cell types in vitro, their pH and its effect, together with the crosslinking ions of these formulations, are still infrequently assessed. In this work, we study how these elements can affect hydrogel stability and printability and influence cell viability and metabolism on the resulting 3D prints. Our results show that both the buffer pH and crosslinking ion (Ca2+ or Ba2+) influence the swelling and degradation rates of prints. Moreover, buffer pH influenced the printability of hydrogel in the air but did not when printed directly in a fluid-phase CaCl2 or BaCl2 crosslinking bath. In addition, both U2OS and NIH/3T3 cells showed greater cell metabolic activity on one-layer prints crosslinked with Ca2+. In addition, Ba2+ increased the cell death of NIH/3T3 cells while having no effect on U2OS cell viability. The pH of the buffer also had an important impact on the cell behavior. U2OS cells showed a 2.25-fold cell metabolism increase on one-layer prints prepared at pH 8.0 in comparison to those prepared at pH 5.5, whereas NIH/3T3 cells showed greater metabolism on one-layer prints with pH 7.0. Finally, we observed a difference in the cell arrangement of U2OS cells growing on prints prepared from hydrogels with an acidic buffer in comparison to cells growing on those prepared using a neutral or basic buffer. These results show that both pH and the crosslinking ion influence hydrogel strength and cell behavior.

Background: Colchicine is a traditional medication that is currently approved to treat gout and familial Mediterranean fever (FMF). However, colchicine has a wide range of anti-inflammatory activities, and several studies have indicated that it may be useful in a variety of other conditions, such as rheumatic disease, cardiac disease, and cancer. Osteosarcoma, the most common type of bone sarcoma, is derived from primitive bone-forming mesenchymal cells. In this study, we investigated whether colchicine could be used to treat osteosarcoma through the regulation of cell cycle signaling. Methods: Two human osteosarcoma cell lines, U2OS and Saos-2, were used. A clonogenic assay was used to determine the antiproliferative effects of colchicine on osteosarcoma cells. Reactive oxygen species (ROS) production and apoptosis were measured by flow cytometry. Migration and invasion assays were performed to investigate the inhibitory effects of colchicine. The signaling pathways related to colchicine treatment were verified by GO biological process (GOBP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Results: Colchicine was selected as the lead compound based on the results of initial screening and cell viability assays conducted in Saos-2 and U2Os cells. Colchicine reduced the viability of Saos-2 and U2OS cells in a concentration-dependent manner. It also significantly inhibited colonyforming ability and induced ROS production and apoptosis. It also inhibited the migration and invasion of both Saos-2 and U2OS cells. GOBP and KEGG enrichment analyses indicated the involvement of microtubule-based processes and cancer-related pathways. Conclusions: These findings suggest that colchicine has therapeutic potential in osteosarcoma. [ABSTRACT FROM AUTHOR]

Objective To explore whether anti-tumor drug FTY720 participates in regulation of p53-dependent gene transcription and to evaluate the potential roles of FTY720-p53 axis in the modulation of cellular biological processes. Methods CRISPR-Cas9 technique was used to generate a p53-knockout (p53-KO) cell strain with human osteosarcoma cell line U2OS that expressed the wildtype p53. Control cells or p53-KO cells were treated with or without FTY720 and the transcriptional was then assessed by RNA-sequencing (RNA-seq) analysis. The differentially expressed genes as well as their potential functions were annotated through bioinformatic analysis. Results The U2OS p53-KO cell strain was successfully established. 220 p53-dependent downstream genes were identified in response to FTY720 treatment. These genes were functionally enriched in the regulation of multiple biological processes including negative regulation of platelet activation, cellular response to chemokine, positive regulation of Rho protein signal transduction, multicellular organismal homeostasis, mucin type O-glycan biosynthesis, ECM-receptor interaction, viral protein interaction with cytokine and cytokine receptor. Conclusions p53 activation is involved in FTY720-mediated tumor suppressive functions.

Background: Although osteosarcoma (OS) is the most common malignant bone tumor, the biological mechanism underlying its incidence and improvement remains unclear. This study investigated early diagnosis and treatment objectives using bioinformatics strategies and performed experimental verification. Methods and results: The top 10 OS hub genes—CCNA2, CCNB1, AURKA, TRIP13, RFC4, DLGAP5, NDC80, CDC20, CDK1, and KIF20A—were screened using bioinformatics methods. TRIP13 was chosen for validation after reviewing literature. TRIP13 was shown to be substantially expressed in OS tissues and cells, according to Western blotting (WB) and quantitative real-time polymerase chain reaction data. Subsequently, TRIP13 knockdown enhanced apoptosis and decreased proliferation, migration, and invasion in U2OS cells, as validated by the cell counting kit-8 test, Hoechst 33,258 staining, wound healing assay, and WB. In addition, the levels of p-PI3K/PI3K and p-AKT/AKT in U2OS cells markedly decreased after TRIP13 knockdown. Culturing U2OS cells, in which TRIP13 expression was downregulated, in a medium supplemented with a PI3K/AKT inhibitor further reduced their proliferation, migration, and invasion and increased their apoptosis. Conclusions: TRIP13 knockdown reduced U2OS cell proliferation, migration, and invasion via a possible mechanism involving the PI3K/AKT signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract Background Telomeric DNA is typically comprised of G-rich tandem repeat motifs and maintained by telomerase (Greider CW, Blackburn EH; Cell 51:887–898; 1987). In eukaryotes lacking telomerase, a variety of DNA repair and DNA recombination based pathways for telomere maintenance have evolved in organisms normally dependent upon telomerase for telomere elongation (Webb CJ, Wu Y, Zakian VA; Cold Spring Harb Perspect Biol 5:a012666; 2013); collectively called Alternative Lengthening of Telomeres (ALT) pathways. By measuring (TTAGGG) n tract lengths from the same large DNA molecules that were optically mapped, we simultaneously analyzed telomere length dynamics and subtelomere-linked structural changes at a large number of specific subtelomeric loci in the ALT-positive cell lines U2OS, SK-MEL-2 and Saos-2. Results Our results revealed loci-specific ALT telomere features. For example, while each subtelomere included examples of single molecules with terminal (TTAGGG) n tracts as well as examples of recombinant telomeric single molecules, the ratio of these molecules was subtelomere-specific, ranging from 33:1 (19p) to 1:25 (19q) in U2OS. The Saos-2 cell line shows a similar percentage of recombinant telomeres. The frequency of recombinant subtelomeres of SK-MEL-2 (11%) is about half that of U2OS and Saos-2 (24 and 19% respectively). Terminal (TTAGGG) n tract lengths and heterogeneity levels, the frequencies of telomere signal-free ends, and the frequency and size of retained internal telomere-like sequences (ITSs) at recombinant telomere fusion junctions all varied according to the specific subtelomere involved in a particular cell line. Very large linear extrachromosomal telomere repeat (ECTR) DNA molecules were found in all three cell lines; these are in principle capable of templating synthesis of new long telomere tracts via break-induced repair (BIR) long-tract DNA synthesis mechanisms and contributing to the very long telomere tract length and heterogeneity characteristic of ALT cells. Many of longest telomere tracts (both end-telomeres and linear ECTRs) displayed punctate CRISPR/Cas9-dependent (TTAGGG) n labeling patterns indicative of interspersion of stretches of non-canonical telomere repeats. Conclusion Identifying individual subtelomeres and characterizing linked telomere (TTAGGG) n tract lengths and structural changes using our new single-molecule methodologies reveals the structural consequences of telomere damage, repair and recombination mechanisms in human ALT cells in unprecedented molecular detail and significant differences in different ALT-positive cell lines.

Salvia triloba (S. triloba) is an herb inherently linked to traditional medicine systems in the Eastern Mediterranean region. There is minimal experimental evidence however, regarding the anticancer effects of S. triloba in both osteosarcoma and ovarian cancer. In this study, we investigated the effects of crude (macerated) S. triloba ethanol and acetone leaf extracts on viability, migratory ability, and the expression of genes regulating these activities in U2OS and SKOV3 cells using MTT assay, scratch-wound healing/trans-well migration assay, and RT-qPCR respectively. MTT assay results indicated that the acetone extract significantly reduced both U2OS and SKOV3 cell viability with half-maximal inhibitory concentrations (IC50) of 54.51 ± 1.10 µg/mL and 75.96 ± 1.0237 µg/mL respectively; these concentrations further displayed negligible hemolytic activity. The combination of acetone extract (19 µg/mL) and paclitaxel (0.787 µg/mL) displayed synergy and reduced SKOV3 cell viability by over 90%. Additionally, the trans-well migration assay illustrated that the acetone extract (IC50) inhibited both U2OS and SKOV3 cell migration by more than 50%. Moreover, S. triloba acetone extract significantly downregulated the steady-state mRNA expression of key genes involved in driving select cancer hallmarks. Four fractions were generated from the acetone extract by thin layer chromatography (TLC), and the obtained retention factors (Rf) (ranging from 0.2 to 0.8) suggested a mixture of high and moderately polar compounds whose bioactivities require further investigation. In addition, FTIR measurements of the extract revealed peaks corresponding to OH, aliphatic CH, and ester groups suggesting the presence of phenolic compounds, terpenes, and polysaccharides. Altogether, these results suggest that S. triloba possesses potential therapeutic compounds that inhibit cell proliferation and migration, and modulate several genes involved in osteosarcoma and ovarian carcinoma progression.

Background/Aim. Targeting cell proliferation may be an important therapeutic purpose in tumor growth, tissue remodeling or degenerative diseases. Insulin-like growth factor IGF-1 is known as a proliferative agent, but its effect in different cellular models may be contradictory due to crosstalk of multiple cellular regulatory pathways [1, 2], and need to be elucidated. The aim was to characterize the proliferative effects of IGF-1 in cells with high and low proliferative activity. Methods. In cultivated tumor U2OS cells or RPE cells with low proliferative activity, doseand time-dependent effects of recombinant IGF-1 were studied. Proliferative effects were assayed using S-phase analysis with EdU uptake by fluorescence-activated cell sorting (FACS), DNA fiber analysis, and evaluation of ATR-CHK1 kinase signaling activity by immunoblotting. Results. The assay of EdU incorporation by FACS showed an increase in proliferative response after 24 h incubation with IGF-1 in attached (P < 0.0001), but not suspended (P = 0.8607) RPE cells. In contrast, IGF-1 did not increase the proliferation (P = 0.3713) in the attached U2OS cells, and suppressed in suspended cells (P = 0.0064). DNA fiber analysis demonstrated decreasing replication fork velocity in response to 24h IGF-1 treatment in both attached RPE or suspended U2OS cells compared to control (P < 0.0001). Evaluation of the cell cycle kinases activation demonstrated that U2OS cells had a higher expression of pChk-1 than RPE cells, and IGF-1 caused an increase the indicators in both cell lines. The proliferative response to IGF-1 inversely depended on cell concentration in the culture. An increase of IGF-1 dose/time resulted mainly in suppression of proliferative response, more pronounced in U2OS cells, which is consistent with previous data obtained on mesenchymal stem cells with high proliferative capacity [2]. In RPE, IGF-1 may shift cell population to have more proliferative capacity [3] that can be important for the treatment of retinopathies, especially related to aging. Conclusions. IGF-1 causes different proliferative response in the cells with high and low proliferative capacity with nonlinear dependence on time and dose. The tumor U2OS cell proliferation was most suppressed in suspended cells especially with the IGF-1 overdose. In RPE, IGF-1 may be used in low concentrations to increase proliferative capacities and prevent cellular senescence. [ABSTRACT FROM AUTHOR]

Introduction: Osteosarcoma poses a serious therapeutic challenge, mainly due to heterogeneous nature of the cells, which is aggressive and progressive. Doxorubicin has shown efficacy as a therapeutic agent in osteosarcoma, but there has been no significant improvement in the prognosis of osteosarcoma in four decades. Several drugs have been studied for their anti-cancer effects. Salinomycin is believed to have anti-cancer effects against a variety of malignancies. Methods: In this study, the expression of Bax and Bcl2 was examined using the RT-PCR method on the osteosarcoma cell line culture U2OS, which had previously been carried out for cytotoxic tests using the MTT assay method to determine the concentration dose of salinomycin and doxorubicin. Bax and Bcl2 gene expression were calculated using the Livak method. The statistical significance level for analyses carried out as part of this study was p<0.05. Result: This study reported that Bax expression at a salinomycin concentration of 0.005 µM was 1.85- fold higher compared to control cells, while at the same concentration, Bcl2 expression was found to be 0.61-fold lower when compared to control cells. Conclusion: This underscores the potential of salinomycin as a standalone anti-apoptotic pathway modulator in the management of osteosarcoma. Consequently, this investigation not only contributes to our comprehension of these therapeutic agents but also situates their collective impact on apoptosis regulators, providing valuable insights for the refinement of osteosarcoma treatment paradigms. [ABSTRACT FROM AUTHOR]

Three-dimensional (3D) (bio)printing technology has boosted the advancement of the biomedical field. However, tissue engineering is an evolving field and (bio)printing biomimetic constructions for tissue formation is still a challenge. As a new methodology to facilitate the construction of more complex structures, we suggest the use of the fluid-phase 3D printing to pattern the scaffold's properties. The methodology consists of an exchangeable fluid-phase printing medium in which the constructions are fabricated and patterned during the printing process. Using the fluid-phase methodology, the biological and mechanical properties can be tailored promoting cell behaviour guidance and compartmentalization. In this study, we first assessed different formulations of alginate/gelatin to create a stable substrate capable to promote massive cell colonization in vitro over time. Overall, formulations with lower gelatin content and 2-(N-morpholino)ethanesulfonic acid (MES) buffer as a solvent showed better stability under cell culture conditions and enhanced U2OS cell growth. Next, the fluid-phase showed better printing fidelity and resolution in comparison to air printing as it diminished the collapsing and the spread of the hydrogel strand. In sequence, the fluid-phase methodology was used to create functionalized alginate-gelatin-arginylglycylaspartic acid peptide (RGD) hydrogels via carbodiimides chemistry. The alginate-gelatin-RGD hydrogels showed an increase of 2.97-fold in cell growth and more spread substrate colonization in comparison to alginate-gelatin hydrogel. Moreover, the fluid-phase methodology was used to add RGD molecules to pre-determined parts of the alginate-gelatin substrate during the printing process promoting U2OS cell compartmentalization. In addition, different substrate stiffnesses were also created via fluid-phase by crosslinking the hydrogel with different concentrations of CaCl 2 during the printing process. As a result, the U2OS cells were also compartmentalized on the stiffer parts of the printings. Finally, our results showed that by combining stiffer hydrogel with RGD increasing concentrations we can create a synergetic effect and boost cell metabolism by up to 3.17-fold. This work presents an idea of a new printing process for tailoring multiple parameters in hydrogel substrates by using fluid-phase to generate more faithful replication of the in vivo environment., (Creative Commons Attribution license.)

Cell cycle is a cellular process that is subject to stringent control. In contrast to the wealth of knowledge of proteins controlling the cell cycle, very little is known about the molecular role of lncRNAs (long noncoding RNAs) in cell-cycle progression. By performing genome-wide transcriptome analyses in cell-cycle-synchronized cells, we observed cell-cycle phase-specific induction of >2000 lncRNAs. Further, we demonstrate that an S-phase-upregulated lncRNA, SUNO1, facilitates cell-cycle progression by promoting YAP1-mediated gene expression. SUNO1 facilitates the cell-cycle-specific transcription of WTIP, a positive regulator of YAP1, by promoting the co-activator, DDX5-mediated stabilization of RNA polymerase II on chromatin. Finally, elevated SUNO1 levels are associated with poor cancer prognosis and tumorigenicity, implying its pro-survival role. Thus, we demonstrate the role of a S-phase up-regulated lncRNA in cell-cycle progression via modulating the expression of genes controlling cell proliferation.

Background: Telomeric DNA is typically comprised of G-rich tandem repeat motifs and maintained by telomerase (Greider CW, Blackburn EH; Cell 51:887–898; 1987). In eukaryotes lacking telomerase, a variety of DNA repair and DNA recombination based pathways for telomere maintenance have evolved in organisms normally dependent upon telomerase for telomere elongation (Webb CJ, Wu Y, Zakian VA; Cold Spring Harb Perspect Biol 5:a012666; 2013); collectively called Alternative Lengthening of Telomeres (ALT) pathways. By measuring (TTAGGG) n tract lengths from the same large DNA molecules that were optically mapped, we simultaneously analyzed telomere length dynamics and subtelomere-linked structural changes at a large number of specific subtelomeric loci in the ALT-positive cell lines U2OS, SK-MEL-2 and Saos-2. Results: Our results revealed loci-specific ALT telomere features. For example, while each subtelomere included examples of single molecules with terminal (TTAGGG) n tracts as well as examples of recombinant telomeric single molecules, the ratio of these molecules was subtelomere-specific, ranging from 33:1 (19p) to 1:25 (19q) in U2OS. The Saos-2 cell line shows a similar percentage of recombinant telomeres. The frequency of recombinant subtelomeres of SK-MEL-2 (11%) is about half that of U2OS and Saos-2 (24 and 19% respectively). Terminal (TTAGGG) n tract lengths and heterogeneity levels, the frequencies of telomere signal-free ends, and the frequency and size of retained internal telomere-like sequences (ITSs) at recombinant telomere fusion junctions all varied according to the specific subtelomere involved in a particular cell line. Very large linear extrachromosomal telomere repeat (ECTR) DNA molecules were found in all three cell lines; these are in principle capable of templating synthesis of new long telomere tracts via break-induced repair (BIR) long-tract DNA synthesis mechanisms and contributing to the very long telomere tract length and heterogeneity characteristic of ALT cells. Many of longest telomere tracts (both end-telomeres and linear ECTRs) displayed punctate CRISPR/Cas9-dependent (TTAGGG) n labeling patterns indicative of interspersion of stretches of non-canonical telomere repeats. Conclusion: Identifying individual subtelomeres and characterizing linked telomere (TTAGGG) n tract lengths and structural changes using our new single-molecule methodologies reveals the structural consequences of telomere damage, repair and recombination mechanisms in human ALT cells in unprecedented molecular detail and significant differences in different ALT-positive cell lines. [ABSTRACT FROM AUTHOR]

The miR-320a regulates a number of genes involved in various physiological processes. In particular, it has been reported as a tumor suppressor in several types of human cancers and involved in osteoporotic fracture and osteoblast function. Hence, the role of miR-320a has been evaluated in tumor cells and in primary cells in a separated context, but its effect has never been explored in a comparative manner. The present study aims to evaluate the cellular effects of miR-320a on human osteosarcoma cell lines (MG-63 and U2OS) compared to that on primary human osteoblasts (hOBs). miR-320a was either overexpressed or inhibited in all cell lines, and cell proliferation and viability were analyzed. Additionally, the effects of miR-320a on matrix mineralization, alkaline phosphatase activity, and oxidative stress were also evaluated in order to assess osteoblast functionality. In osteosarcoma cells, miR-320a overexpression reduced cell viability and proliferation, while in hOB cell viability was not affected and proliferation even was increased. The overexpression of miR-320a in both osteosarcoma cells and hOBs reduced the mineralization capacity. Finally, an increased oxidative stress was detected in all cells after miR-320a overexpression mainly in osteosarcoma. In conclusion, the overexpression of miR-320a increased stress oxidation levels, which could be involved in the reduced osteoblast performance, even though the cell viability was only affected in osteosarcoma cells. [ABSTRACT FROM AUTHOR]

INF2 is a formin protein that accelerates actin polymerization. A common mechanism for formin regulation is autoinhibition, through interaction between the N-terminal diaphanous inhibitory domain (DID) and C-terminal diaphanous autoregulatory domain (DAD). We recently showed that INF2 uses a variant of this mechanism that we term "facilitated autoinhibition," whereby a complex consisting of cyclase-associated protein (CAP) bound to lysine-acetylated actin (KAc-actin) is required for INF2 inhibition, in a manner requiring INF2-DID. Deacetylation of actin in the CAP/KAc-actin complex activates INF2. Here we use lysine-to-glutamine mutations as acetylmimetics to map the relevant lysines on actin for INF2 regulation, focusing on K50, K61, and K328. Biochemically, K50Q- and K61Q-actin, when bound to CAP2, inhibit full-length INF2 but not INF2 lacking DID. When not bound to CAP, these mutant actins polymerize similarly to WT-actin in the presence or absence of INF2, suggesting that the effect of the mutation is directly on INF2 regulation. In U2OS cells, K50Q- and K61Q-actin inhibit INF2- mediated actin polymerization when expressed at low levels. Directbinding studies show that the CAPWH2 domain binds INF2-DIDwith submicromolar affinity but has weak affinity for actin monomers, while INF2-DAD binds CAP/K50Q-actin 5-fold better than CAP/WTactin. Actin in complex with full-length CAP2 is predominately ATPbound. These interactions suggest an inhibition model whereby CAP/KAc-actin serves as a bridge between INF2 DID and DAD. In U2OS cells, INF2 is 90-fold and 5-fold less abundant than CAP1 and CAP2, respectively, suggesting that there is sufficient CAP for full INF2 inhibition. [ABSTRACT FROM AUTHOR]

During the S-phase, the DNA replication process is finely orchestrated and regulated by two programs: the spatial program that determines where replication will start in the genome (Cadoret et al. (2008 Oct 14), Cayrou et al. (2011 Sep), Picard et al. (2014 May 1) [1–3]), and the temporal program that determines when during the S phase different parts of the genome are replicated and when origins are activated. The temporal program is so well conserved for each cell type from independent individuals [4] that it is possible to identify a cell type from an unknown sample just by determining its replication timing program. Moreover, replicative domains are strongly correlated with the partition of the genome into topological domains (determined by the Hi-C method, Lieberman-Aiden et al. (2009 Oct 9), Pope et al. (2014 Nov 20) [5,6]). On the one hand, replicative areas are well defined and participate in shaping the spatial organization of the genome for a given cell type. On the other hand, studies on the timing program during cell differentiation showed a certain plasticity of this program according to the stage of cell differentiation Hiratani et al. (2008 Oct 7, 2010 Feb) [7,8]. Domains where a replication timing change was observed went through a nuclear re-localization. Thus the temporal program of replication can be considered as an epigenetic mark Hiratani and Gilbert (2009 Feb 16) [9]. We present the genomic data of replication timing in 6 human model cell lines: U2OS (GSM2111308), RKO (GSM2111309), HEK 293T (GSM2111310), HeLa (GSM2111311), MRC5-SV (GSM2111312) and K562 (GSM2111313). A short comparative analysis was performed that allowed us to define regions common to the 6 cell lines. These replication timing data can be taken into account when performing studies that use these model cell lines.

The miR-320a regulates a number of genes involved in various physiological processes. In particular, it has been reported as a tumor suppressor in several types of human cancers and involved in osteoporotic fracture and osteoblast function. Hence, the role of miR-320a has been evaluated in tumor cells and in primary cells in a separated context, but its effect has never been explored in a comparative manner. The present study aims to evaluate the cellular effects of miR-320a on human osteosarcoma cell lines (MG-63 and U2OS) compared to that on primary human osteoblasts (hOBs). miR-320a was either overexpressed or inhibited in all cell lines, and cell proliferation and viability were analyzed. Additionally, the effects of miR-320a on matrix mineralization, alkaline phosphatase activity, and oxidative stress were also evaluated in order to assess osteoblast functionality. In osteosarcoma cells, miR-320a overexpression reduced cell viability and proliferation, while in hOB cell viability was not affected and proliferation even was increased. The overexpression of miR-320a in both osteosarcoma cells and hOBs reduced the mineralization capacity. Finally, an increased oxidative stress was detected in all cells after miR-320a overexpression mainly in osteosarcoma. In conclusion, the overexpression of miR-320a increased stress oxidation levels, which could be involved in the reduced osteoblast performance, even though the cell viability was only affected in osteosarcoma cells.

Background/Aims: Phytoestrogens are known to prevent tumor progression by inhibiting proliferation and inducing apoptosis in cancer cells. Formononetin is one of the main components of red clover plants, and is considered as a typical phytoestrogen. This study investigates formononetin induction of apoptosis of human osteosarcoma cell line U2OS by regulating Bcl-2 and Bax expression in vitro and in vivo. Methods: U2OS cells were treated with different concentrations of formononetin and the proliferation of the cells was measured using an MTT assay. Cell apoptosis was examined by flow cytometry. The levels of miR-375, Bax and Bcl-2 protein expression in treated cells were determined by Western blot and RT-PCR. The antitumor activity of formononetin was also evaluated in vivo in nude mice bearing orthotopic tumor implants. Results: High concentrations of formononetin significantly suppress the proliferation of U2OS cells and induce cell apoptosis. Moreover, compared to control group the expression of Bcl-2 and miR-375 decreases with formononetin in the U2OS cells, while Bax increases. Conclusion: Formononetin has inhibitory effects on the proliferation of U2SO cells, both in vitro and in vivo. This antitumor effect is directly correlated with formononetin concentration.

Steviol is the colonic metabolite of the natural sweetener steviol glycosides. It does not diffuse to the blood and the half maximal inhibitory concentration of steviol is longer compared with that of current chemotherapy agents, including 5-fluorouracil and doxorubicin. The present study demonstrated that steviol inhibits the proliferation of the human osteosarcoma U2OS cell line in a dose- and time-dependent manner, and that the inhibition rate is comparative with that of doxorubicin and 5-fluorouracil. The mechanism of this anticancer activity is also investigated. The results indicated that steviol inhibits U2OS cells through inducing G1 phase cell cycle arrest, downregulating the ability of colony formation via a mitochondrial apoptotic pathway, which was indicated by an increase of the Bax/Bcl-2 ratio and activation of cyclin-dependent kinase inhibitor 1, tumor protein 53 and cyclin-dependent kinase; whereas a Survivin and Caspase 3-independent mechanism was involved. Considering that steviol appears minimally in the plasma during metabolism, and possesses a median lethal dose of 100-fold greater compared with that of 5-fluorouracil, it may become a potential chemotherapy agent. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the anti-cancer effects of the natural plant flavonoid, taxifolin, on human osteosarcoma cancer cells. Taxifolin was demonstrated to exhibit anti-cancer effects on U2OS and Saos-2 osteosarcoma cell lines. Treatment of cells with taxifolin inhibited proliferation and diminished colony formation in soft agar in a dose-dependent manner. In vivo, intraperitoneal administration of taxifolin in nude mice bearing U2OS xenograft tumors, significantly inhibited tumor growth. In addition, taxifolin treatment was demonstrated to promote G1 cell cycle arrest and cell apoptosis in U2OS and Saos-2 cell lines, as demonstrated by flow cytometry analysis. Western blot analysis demonstrated that taxifolin treatment was associated with a reduction in the expression levels of AKT serine/threonine kinase 1 (AKT), phosphorylated (p-Ser473) AKT, v-myc avian myelocytomatosis viral oncogene homolog (c-myc) and S-phase kinase associated protein 2 (SKP-2) in U2OS and Saos-2 cell lines. Overexpression of AKT considerably reversed the taxifolin-induced decrease in AKT, c-myc and SKP-2 protein expression and the decrease in AKT phosphorylation, suggesting that inactivation of AKT was a mediator of taxifolin-induced inhibition of c-myc and SKP-2. Furthermore, overexpression of SKP-2 in U2OS cells partially reversed the growth inhibition mediated by taxifolin. Finally, taxifolin treatment repressed cell migration and invasion in U2OS cells and this effect was markedly reversed by SKP-2 overexpression. The results of the present study indicate that taxifolin may present a potential novel therapeutic agent for osteosarcoma treatment. [ABSTRACT FROM AUTHOR]

Genotoxic stress causes proliferating cells to activate the DNA damage checkpoint, to assist DNA damage recovery by slowing cell cycle progression. Thus, to drive proliferation, cells must tolerate DNA damage and suppress the checkpoint response. However, the mechanism underlying this negative regulation of checkpoint activation is still elusive. We show that human Cyclin-Dependent-Kinases (CDKs) target the RAD9 subunit of the 9-1-1 checkpoint clamp on Thr292, to modulate DNA damage checkpoint activation. Thr292 phosphorylation on RAD9 creates a binding site for Polo-Like-Kinase1 (PLK1), which phosphorylates RAD9 on Thr313. These CDK-PLK1-dependent phosphorylations of RAD9 suppress checkpoint activation, therefore maintaining high DNA synthesis rates during DNA replication stress. Our results suggest that CDK locally initiates a PLK1-dependent signaling response that antagonizes the ability of the DNA damage checkpoint to detect DNA damage. These findings provide a mechanism for the suppression of DNA damage checkpoint signaling, to promote cell proliferation under genotoxic stress conditions.

Aloperine (ALO) is a novel type of alkaloid drug that is extracted from S. alopecuroide , and exert an anti-inflammatory, anti-allergenic, antitumor and antiviral effects. In our study, we evaluated the effects and underlying mechanisms of ALO on MG-63 and U2OS osteosarcoma (OS) cells. ALO suppressed the proliferation and clonogenecity of both cell lines in a dose- and time-dependent manner as observed by CCK-8 and clonogenic survival assays. Data of morphologic changes, DAPI assays and flow cytometry showed that ALO induced apoptosis of OS cells, and the results of western blotting and qRT-PCR indicated that ALO upregulated protein and mRNA of Bax and cleaved caspase-3, while downregulated Bcl-2. Besides, ALO inhibited the invasion of MG-63 and U2OS cells as shown by transwell invasion assay. The protein and mRNA of MMP-2 and MMP-9 were decreased with ALO treatment. ALO also downregulated the protein and mRNA expression of PI3K and p-AKT1. In conclusion, ALO induced apoptosis and inhibited invasion in MG-63 and U2OS cells, which maybe through suppression of PI3K/AKT signaling pathway. [ABSTRACT FROM AUTHOR]

Background: Large-scale image sets acquired by automated microscopy of perturbed samples enable a detailed comparison of cell states induced by each perturbation, such as a small molecule from a diverse library. Highly multiplexed measurements of cellular morphology can be extracted from each image and subsequently mined for a number of applications. Findings: This microscopy dataset includes 919 265 five-channel fields of view, representing 30 616 tested compounds, available at "The Cell Image Library" (CIL) repository. It also includes data files containing morphological features derived from each cell in each image, both at the single-cell level and population-averaged (i.e., per-well) level; the image analysis workflows that generated the morphological features are also provided. Quality-control metrics are provided as metadata, indicating fields of view that are out-of-focus or containing highly fluorescent material or debris. Lastly, chemical annotations are supplied for the compound treatments applied. Conclusions: Because computational algorithms and methods for handling single-cell morphological measurements are not yet routine, the dataset serves as a useful resource for the wider scientific community applying morphological (image-based) profiling. The dataset can be mined for many purposes, including small-molecule library enrichment and chemical mechanism-of-action studies, such as target identification. Integration with genetically perturbed datasets could enable identification of small-molecule mimetics of particular disease- or gene-related phenotypes that could be useful as probes or potential starting points for development of future therapeutics. [ABSTRACT FROM AUTHOR]

Objective: To explore the effect of Cinobufacini plus Cisplatin (CDDP) on the proliferation and apoptosis of U2OS cell line, and explore the possible molecular mechanism of combination therapy. Methods: U2OS cells were treated with different concentrations of Cinobufacini and CDDP or in combination for one to three days. The proliferation of U2OS was tested by CCK-8 assay. The clonality of U2OS was assessed by clone formation assay. The cell apoptosis was detected by flow cytometer. The expression of apoptosis related genes and proteins was detected by RT-PCR and Western blot. Results: Cinobufacini or CDDP could inhibit the proliferation and induce the apoptosis of U2OS cells in a dose- and time-dependent manner. However, combination of two drugs could inhibit the cell clone formation and markedly promote the cell apoptosis. Combination of two drugs had synergistic effect on the cell proliferation and apoptosis. The pro-apoptosis gene Bax was up-regulated and suppression apoptosis Bcl-2 was significantly down regulated in the combination group. The expression of apoptosis associated proteins caspase-3 and caspase-9 in the combined administration group were obviously increased. Conclusion: Cinobufacini and cisplatin could significantly inhibit the proliferation and induce apoptosis of osteosarcoma cell, combination these two drugs had synergistic effect, its mechanism might be related to the activation of apoptosis pathway. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Wnt/β-catenin signal pathway plays a role in regulating cell proliferation and apoptosis and is correlated with tumor onset, progression and drug resistance. B-cell lymphoma 2 (Bcl-2) is an anti-apoptotic factor inducing tumor cell drug resistance. Wnt/β-catenin signal pathway can modulate Bcl- 2 expression. This study established a cell model of drug resistance using adriamycin (ADM) treatment. Wnt/β-catenin signal pathway was intervened to discuss its role in drug resistance of osteosarcoma cells. MATERIALS AND METHODS: Expression of β-catenin and Bcl-2 was compared between U2OS and hFOB1.19 cells. ADM resistant cell line U2OS/ADM was established for comparing β-catenin and Bcl-2 expression. Cell counting kit-8 (CCK-8) assay was used to test cell proliferation, followed by flow cytometry for apoptotic rate under ADM concentrations. U2OS/ADM cells were further treated with si-β-catenin and/or β-catenin inhibitor XAV939. β-catenin and Bcl-2 expression were measured, followed by CCK-8 and flow cytometry. RESULTS: Comparing to hFOB1.19 cells, U2OS cells had significantly elevated β-catenin and Bcl-2 expression. U2OS/ADM cells had higher β-catenin and Bcl-2 expression than U2OS, plus lower ADM sensitivity and suppressed apoptotic rate. Transfection of si-β-catenin and XAV939 suppressed β-catenin and Bcl-2 expression, and significantly enhanced ADM sensitivity and ADM-induced apoptosis. CONCLUSIONS: Up-regulation of β-catenin plays a role in potentiating expression and downstream anti-apoptotic factor Bcl-2 and in enhancing ADM resistance of osteosarcoma U2OS cells. [ABSTRACT FROM AUTHOR]

Background: Withaferin-A (WF-A) is a well-known dietary compound isolated from Withania sominifera. It has tremendous pharmacological potential and has been shown to exhibit antiproliferative activity against several types of cancerous cells. Currently, the main focus of anti-cancer therapeutic development is to identify apoptosis inducing drug-like molecules. Osteosarcoma is a rare type of osteocancer, affecting human. The present study therefore focused on the evaluation of antitumor potential of WF-A against several osteosarcoma cell lines. Materials and Methods: MTT assay was used to evaluate WF-A against osteosarcoma cell lines and to calculate the IC50. DAPI staining was used to confirm the apoptosis inducing potential of WF-A. Mitochondrial membrane potential, reactive oxygen species (ROS) assay, and Western blotting were used to confirm the basis of apoptosis. Results: The results revealed that that WF-A exhibited strong antiproliferative activity against all the cells lines, with IC50 ranging from 0.32 to 7.6 μM. The lowest IC50 (0.32 μM) was observed against U2OS cell line and therefore it was selected for further analysis. DAPI staining indicated that WF-A exhibited antiproliferative activity via induction of apoptosis. Moreover, WF-A induced ROS-mediated reduction in mitochondrial membrane potential ΔΨm) in a dose-dependent manner and activation of caspase-3 in osteosarcoma cells. Conclusion: We propose that WF-A may prove a potent therapeutic agent for inducing apoptosis in osteosarcoma cell lines via generation of ROS and disruption of mitochondrial membrane potential. [ABSTRACT FROM AUTHOR]

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This study explored whether valproic acid (VPA, a histone deacetylase inhibitor) could radiosensitize osteosarcoma and primary-culture tumor cells, and determined the mechanism of VPA-induced radiosensitization. The working system included osteosarcoma cells (U2OS) and primary-culture cells from chemical carcinogen (DMBA)-induced breast cancer in rats; and clonogenic survival, immunofluorescence, fluorescent in situ hybridization (FISH) for chromosome aberrations, and comet assays were used in this study. It was found that VPA at the safe or critical safe concentration of 0.5 or 1.0 mM VPA could result in the accumulation of more ionizing radiation (IR)-induced DNA double strand breaks, and increase the cell radiosensitivity. VPA-induced radiosensitivity was associated with the inhibition of DNA repair activity in the working systems. In addition, the chromosome aberrations including chromosome breaks, chromatid breaks, and radial structures significantly increased after the combination treatment of VPA and IR. Importantly, the results obtained by primary-culture cells from the tissue of chemical carcinogen-induced breast cancer in rats further confirmed our findings. The data in this study demonstrated that VPA at a safe dose was a radiosensitizer for osteosarcoma and primary-culture tumor cells through suppressing DNA-double strand breaks repair function. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Withaferin-A (WF-A) is a well-known dietary compound isolated from Withania somnifera. It has marked pharmacological potential and has been shown to exhibit antiproliferative activity against several types of cancerous cells. Currently, the main focus of anti- cancer therapeutic development is to identify apoptosis-inducing drug-like molecules. Osteosarcoma is a rare type of bone cancer affecting humans. The objective of the present study was therefore to evaluate the antitumor potential of WF-A against several osteosarcoma cell lines. MATERIALS AND METHODS: MTT assay was used to evaluate WF-A against osteosarcoma cell lines and to calculate the IC50. DAPI staining was used to confirm the apoptosis-inducing potential of WF-A. Mitochondrial membrane potential, reactive oxygen species (ROS) assay, and Western blotting were used to confirm the basis of apoptosis. RESULTS: The results of the present study revealed that WF-A exhibited strong antiproliferative activity against all the cells lines, with IC50 ranging from 0.32 to 7.6 μM. The lowest IC50 (0.32 μM) was observed against U2OS cell line and, therefore, it was selected for further analysis. DAPI staining indicated that WF-A exhibited antiproliferative activity via induction of apoptosis. Moreover, WF-A induced a ROS-mediated reduction in mitochondrial membrane potential in a dose-dependent manner and activation of caspase-3 in osteosarcoma cells. CONCLUSIONS: We suggest that WF-A may prove a potent therapeutic agent for inducing apoptosis in osteosarcoma cell lines via generation of ROS and disruption of mitochondrial membrane potential. [ABSTRACT FROM AUTHOR]

Osteosarcoma chemotherapy has improved survival rates, however, chemoresistance and drug toxicity still limit therapy. Drug combinations may overcome these limitations by allowing fewer chemoresistant cells to survive. The aim of this study was to evaluate the cytotoxic potential of hesperetin to osteosarcoma and to analyze the cell cycle effects of combinations of hesperetin with chemotherapeutic agents. For this, the U2OS human osteosarcoma cell line was exposed to hesperetin or hesperetin combined with etoposide or doxorubicin in defined proportions. Hesperetin was less cytotoxic compared to chemotherapeutic agents, as shown by cell growth, viability and clonogenic assays. Notwithstanding, hesperetin combined with etoposide showed additive effects on the inhibition of cell growth. Furthermore, hesperetin induced G2-phase arrest, associated with decreased gene expression of cyclins B1 and E1 and cyclin-dependent kinases 1 and 2. The combination with higher additive effect resulted in higher percentage of cells in G2-phase, showing that G2-phase arrest is associated with cytotoxicity. Moreover, hesperetin induced cytostatic effects. In conclusion, our results suggest that G2-phase arrest is an important step for hesperetin-induced cytotoxicity in U2OS cells. Hesperetin shows potential cytotoxicity when combined with etoposide, which may have implications on therapeutic developments for osteosarcoma. [ABSTRACT FROM AUTHOR]

Costunolide, an active sesquiterpene lactone, is derived from many herbal medicines and it exhibits a broad spectrum of bioactivities such as anti-inflammatory, potential anti-tumor activity. Herein we assessed the anti-cancer effects of costunolide on U2OS cells and explored the underlying molecular mechanisms. The experiment data show that Costunolide exhibited significant anti-tumor activity by apoptosis related assays including Annexin V-FITC/PI flow cytometric analysis and 4,6-diamino-2-phenyl indole (DAPI) staining morphological analysis. Furthermore, we found Costunolide induced the loss of mitochondrial transmembrane potential, down-regulated Bcl-2/Bax ratio, encouraged Cyt-c release and caspase activation. All those effects are contributed by reactive oxygen species (ROS) generation and ER stress-induced mitochondrial dysfunction which are also responsible for c-Jun N-terminal kinase (JNK) activation. After the treatment of JNK inhibitor SP600125, it obviously reversed costunolide-induced apoptosis. Given N -acetyl- l -cysteine (NAC) effectively blocked the activation of JNK. Taken together, our results demonstrate that costunolide induces apoptosis in human U2OS cells through ROS generation and p38 MAPK/JNK activation. [ABSTRACT FROM AUTHOR]

Osteosarcoma (OS) is the most common primary malignant tumor of bone, occurring most frequently in children and adolescents. The mechanism of formation and development of OS have been studied for a long time. Tumor suppressor pathway governed by p53 gene are known to be involved in the pathogenesis of osteosarcoma. Moreover, loss of wild-type p53 activity is thought to be a major predictor of failure to respond to chemotherapy in various human cancers. In previous studies, we described the activity of a new indole derivative, NSC743420, belonging to the tubulin inhibitors family, capable to induce apoptosis and arrest of the cell cycle in the G2/M phase of various cancer cell lines. However, this molecule has never been tested on OS cell line. Here we address the activity of NSC743420 by examine whether differences in the p53 status could influence its effects on cell proliferation and death of OS cells. In particular, we compared the effect of the tested molecule on p53-wild type and p53-silenced U2OS cells, and on SaOS2 cell line, which is null for p53. Our results demonstrated that NSC743420 reduces OS cell proliferation by p53-dependent and p53-independent mechanisms. In particular, the molecule induces proliferative arrest that culminate to apoptosis in SaOS2 p53-null cells, while it brings a cytostatic and differentiating effect in U2OS cells, characterized by the cell cycle arrest in G0/G1 phase and increased alkaline phosphatase activity. [ABSTRACT FROM AUTHOR]

Background/Aims: Phytoestrogens are known to prevent tumor progression by inhibiting proliferation and inducing apoptosis in cancer cells. Formononetin is one of the main components of red clover plants, and is considered as a typical phytoestrogen. This study investigates formononetin induction of apoptosis of human osteosarcoma cell line U2OS by regulating Bcl-2 and Bax expression in vitro and in vivo. Methods: U2OS cells were treated with different concentrations of formononetin and the proliferation of the cells was measured using an MTT assay. Cell apoptosis was examined by flow cytometry. The levels of miR-375, Bax and Bcl-2 protein expression in treated cells were determined by Western blot and RT-PCR. The antitumor activity of formononetin was also evaluated in vivo in nude mice bearing orthotopic tumor implants. Results: High concentrations of formononetin significantly suppress the proliferation of U2OS cells and induce cell apoptosis. Moreover, compared to control group the expression of Bcl-2 and miR-375 decreases with formononetin in the U2OS cells, while Bax increases. Conclusion: Formononetin has inhibitory effects on the proliferation of U2SO cells, both in vitro and in vivo. This antitumor effect is directly correlated with formononetin concentration. [ABSTRACT FROM AUTHOR]

Background Polyploidy 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. Results By 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 nearhexaploid 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. Conclusions Our 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. [ABSTRACT FROM AUTHOR]

Magnesium-based implants exhibit various advantages such as biodegradability and potential for enhanced in vivo bone formation. However, the cellular mechanisms behind this possible osteoconductivity remain unclear. To determine whether high local magnesium concentrations can be osteoconductive and exclude other environmental factors that occur during the degradation of magnesium implants, magnesium salt (MgCl2) was used as a model system. Because cell lines are preferred targets in studies of non-degradable implant materials, we performed a comparative study of 3 osteosarcoma-derived cell lines (MG63, SaoS2 and U2OS) with primary human osteoblasts. The correlation among cell count, viability, cell size and several MgCl2 concentrations was used to examine the influence of magnesium on proliferation in vitro. Moreover, bone metabolism alterations during proliferation were investigated by analyzing the expression of genes involved in osteogenesis. It was observed that for all cell types, the cell count decreases at concentrations above 10 mM MgCl2. However, detailed analysis showed that MgCl2 has a relevant but very diverse influence on proliferation and bone metabolism, depending on the cell type. Only for primary cells was a clear stimulating effect observed. Therefore, reliable results demonstrating the osteoconductivity of magnesium implants can only be achieved with primary osteoblasts. [ABSTRACT FROM AUTHOR]

The present study investigated the role of extracellular signal-regulated kinase (ERK) activation in the migratory phenotype of human U2OS osteosarcoma (OS) cells in a collagen matrix. The activation of ERK was inhibited by PD98059, a specific inhibitor of ERK kinase. Additionally, no significant differences were observed in the adhesion and proliferation of the cells with or without PD98059 treatment in collagen-coated dishes. The migratory capacity of the U2OS cells was then examined in non-coated and collagen-coated dishes, and the results depicted that collagen I enhanced the migration of the U2OS cells, the effect of which was significantly blocked by the treatment of the cells with PD98059. Furthermore, enhanced gene and protein expression of matrix metalloproteinase 9 (MMP9), but not MMP2, was observed to be involved in the enhanced migratory phenotype of the U20S cells in the collagen-coated plates. This effect was partially abolished by the treatment of the cells in the collagen-coated dishes with ERK inhibitor. Collectively, the data demonstrate that ERK signaling is important for the migration of U2OS cells through the extracellular matrix (ECM), which is comprised mostly of collagen, by enhancing MMP9 production. These results may contribute to the regulation of MMP9 production in metastatic OS. [ABSTRACT FROM AUTHOR]

It has been shown that over-expression of Special AT-rich binding protein 1 (SATB1) in breast cancer predicts a poor prognosis. This study was aimed at investigating the effects of silencing SATB1 on mesenchymal derived human osteosarcoma U2OS cells and the underlying mechanisms. The expressions of SATB1 and the related genes in the cells were detected by qRT-PCR and/or Western Blotting. SATB1 silencing was achieved by stable transfection with the vectors expressing small hairpin RNA versus SATB1. Cell proliferation was detected in a microplate reader with Cell Counting Kit-8 and the cell cycle was analyzed by flow cytometry using a cell cycle detection kit. The study found that SATB1 was particularly over-expressed in human osteosarcoma U2OS. Silencing SATB1 inhibited the proliferation of U2OS. It was found that inhibition of cell proliferation resulted from cell cycle arrest due to down-regulated expression of CFGF and JunB. The over-expression of SATB1 is responsible for abnormal proliferation of mesenchymal derived human Osteosatcoma U2OS cells, indicating that silencing SATB1 expression in the cells might be developed as an efficient osteosarcoma therapy. CTGF and JunB were involved in SATB1-mediated proliferation of U2OS cells. [ABSTRACT FROM AUTHOR]