Genistein Induces Antiproliferative Activity and Apoptosis in Human Osteosarcoma Saos-2 Cells.

BACKGROUND/AIM: Genistein (4', 5, 7-trihydroxyisoflavone) and daidzein (4', 7-dihydroxyisoflavone) are isoflavones derived from soybean and have anti-cancer effects in various cells. However, the effects of genistein and daidzein on the human osteosarcoma cell line Saos-2 has not been investigated before. MATERIALS AND METHODS: Human osteosarcoma Saos-2 cells were treated with genistein for 24 and 48 hours. Cytotoxicity and apoptosis were measured. RESULTS: Genistein significantly inhibited proliferation of Saos-2 cells stronger than daidzein in a dose-dependent manner (0 to 80 µM). Genistein also significantly suppressed Saos-2 cell viability in a dose-dependent manner (0 to 100 µM). In contrast, daidzein did not affect Saos-2 cell viability. Real-time PCR revealed that genistein caused G1-arrest by increasing the expression of p21 and p27 mRNAs in Saos-2 cells. In addition, genistein induced apoptosis through the up-regulation of effector caspase-3/7 activity in Saos-2 cells. Genistein also enhanced initiator caspase-9 and TNF-α mRNA expression in cells. CONCLUSION: Genistein may inhibit proliferation through the up-regulation of p21 and p27 and viability by inducing apoptosis in Saos-2 cells.

Comparative Study of Ozonated Glycerol and Macrogol Ointment on Bone Matrix Production by Human Osteosarcoma Cell Line Saos-2.

Ozonated glycerol is glycerol containing ozone, has no unpleasant odor, and has a long half-life. To apply ozonated glycerol for clinical use, ozonated macrogol ointment has been developed by adding macrogol ointment to ozonated glycerol to increase the retention in the affected area. However, the effects of ozone on this macrogol ointment were unclear. The viscosity of the ozonated macrogol ointment was approximately two times higher than that of ozonated glycerol. The effect of the ozonated macrogol ointment on the human osteosarcoma cell line Saos-2 (Saos-2 cells) proliferation, type 1 collagen production, and alkaline phosphatase (ALP) activity were studied. The proliferation of Saos-2 cells was assessed using MTT and DNA synthesis assays. Type 1 collagen production and ALP activity were studied using ELISA and ALP assays. Cells were treated for 24 h with or without 0.05, 0.5, or 5 ppm ozonated macrogol ointment. The 0.5 ppm ozonated macrogol ointment significantly elevated Saos-2 cell proliferation, type 1 collagen production, and ALP activity. These results also showed almost the same trend as for ozonated glycerol.

Relative Biological Effectiveness Values of Spot-scanning Proton Beam Therapy at Shonan Kamakura General Hospital.

BACKGROUND/AIM: This study aimed to confirm the relative biological effectiveness (RBE) values of the proton beam therapy (PBT) system installed in Shonan Kamakura General Hospital. MATERIALS AND METHODS: Clonogenic cell-survival assays were performed with a human salivary gland (HSG) cell line, a human tongue squamous-cell carcinoma cell line (SAS), and a human osteosarcoma cell line (MG-63). Cells were irradiated with proton beams and X-rays with different doses (1.8, 3.6, 5.5, and 7.3 Gy for proton beams, and 2, 4, 6, and 8 Gy for X-rays). Proton beam irradiation used spot-scanning methods and three different depths (at the proximal, center, and distal sides of the spread-out Bragg peak). RBE values were obtained from a comparison of the dose that resulted in a surviving fraction of 10% (D10). RESULTS: D10 of proton beams at the proximal, center, and distal sides and X-rays in HSG were 4.71, 4.71, 4.51, and 5.25 Gy, respectively; those in SAS were 5.08, 5.04, 5.01, and 5.59 Gy, respectively; and those in MG-63 were 5.36, 5.42, 5.12, and 6.06 Gy, respectively. The RBE10 values at the proximal, center, and distal sides in HSG were 1.11, 1.11, and 1.16 respectively; those in SAS were 1.10, 1.11, and 1.12, respectively; and those in MG-63 were 1.13, 1.12, and 1.18, respectively. CONCLUSION: RBE10 values of 1.10-1.18 were confirmed by in vitro experiments using the PBT system. These results are considered acceptable for clinical use in terms of therapeutic efficacy and safety.

Mitophagy Effects of Protodioscin on Human Osteosarcoma Cells by Inhibition of p38MAPK Targeting NIX/LC3 Axis.

Protodioscin (PD) is a steroidal saponin with various pharmacological activities, including neuro-protective, anti-inflammatory, and anti-tumor activities. However, the effect of PD on human osteosarcoma (OS) cells is unclear. In this study, we found that PD significantly inhibits the growth of human HOS and 143B OS cells through the upregulation of apoptotic-related proteins (cleaved caspase-3, cleaved caspase-9, and cleaved PARP) and mitophagy-related proteins (LC3B and NIX), which contribute to the induction of apoptosis, and MMP (mitochondrial membrane potential) dysfunction and mitophagy. The inhibition of LC3 or NIX was shown to decrease apoptosis and mitophagy in PD-treated OS cells. The knockdown of p38MAPK by siRNA decreased mitochondrial dysfunction, autophagy, mitophagy, and the NIX/LC3B expression in the PD-treated OS cells. A binding affinity analysis revealed that the smaller the KD value (-7.6 Kcal/mol and -8.9 Kcal/mol, respectively), the greater the binding affinity in the PD-NIX and PD-LC3 complexes. These findings show the inhibitory effects of PD-induced mitophagy in human OS cells and may represent a novel therapeutic strategy for human OS, by targeting the NIX/LC3 pathways.

Synergistic Antitumor Interaction of Risedronate Sodium and Standard Anticancer Agents in Canine (D-17) and Human Osteosarcoma (U-2 OS) Cell Lines.

The study discusses in vitro cytotoxicity of a combination of cytostatic drugs (doxorubicin, cisplatin, carboplatin, etoposide) and risedronate sodium against canine and human osteosarcoma (D-17 and U-2 OS). Standard protocols were used for the preparation of cell cultures and evaluation of their viability and apoptosis. MTT assay assessed the culture viability and EC50, while the apoptotic effect of the drugs was checked with a TUNEL assay. Doxorubicin alone showed the strongest cytotoxicity against D-17 (0.056 ± 0.019 µg/mL) and U-2 OS (0.051 ± 0.003 µg/mL), while the lowest cytotoxicity was observed for carboplatin (D-17, 6.45 ± 0.2 µg/mL and U2-OS, 27.5 ± 2.3 µg/mL). Risedronate sodium at 100, 10 and 1 µg/mL lowered viability in OS cell lines by 53.38 ± 1.46 and 49.56 ± 0.7%, 97.08 ± 3.32 and 74.92 ± 4.01%, and 102.67 ± 3.56 and 94.56 ± 3.52%, respectively. In all analyzed drug combinations, risedronate sodium significantly (* p < 0.05) increased the cytotoxicity against tested osteosarcoma cell lines. The decrease in cell viability caused by the studied compound combinations was weaker in canine than in human cell cultures. A combination of doxorubicin (all concentrations), cisplatin (1 µg/mL) and etoposide (1 µg/mL) with 100 µg/mL of risedronate sodium significantly improved the cytotoxicity of the drugs against canine and human osteosarcoma. Administration of carboplatin (1 µg/mL) and risedronate sodium (100 µg/mL), compared to carboplatin per se, produced no significant differences in cytotoxicity against the D-17 cell culture but significantly enhanced cytotoxicity in the U-2 OS line. The strongest apoptosis in both lines was detected for 0.01 µg/mL doxorubicin combined with 100 µg/mL risedronate sodium or 1 µg/mL cisplatin and 100 µg/mL risedronate sodium. In all combinations, the tested compounds revealed a synergistic mechanism of action.

Identification of Conserved Pappalysin 1-Derived Circular RNA-Mediated Competing Endogenous RNA in Osteosarcoma.

The etiology of osteosarcoma (OS) is complex and not fully understood till now. This study aimed to identify the miRNAs, circRNAs, and genes (mRNAs) that are differentially expressed in OS cell lines to investigate the mechanism of circRNA-associated competing endogenous RNAs (ceRNAs) in OS. Microarray datasets reporting mRNA (GSE70414), miRNA (GSE70367), and circRNA changes (GSE96964) in human OS cell lines were downloaded, differentially expressed (DE) RNAs were identified, and DEmRNAs were used for the annotation of Gene Ontology (GO) biological processes (BP), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The mechanisms of DEcircRNA-mediated ceRNAs were identified in a step-by-step process. A total of 326 DEmRNAs, 45 DEmiRNAs, and 110 DEcircRNAs were identified from 3 datasets. The DEmRNAs were associated with GO BP terms, including cholesterol biosynthetic process, angiogenesis, extracellular matrix organization and KEGG pathways, including p53 signaling pathway and biosynthesis of antibiotics. The final ceRNA network consisted of 8 DEcircRNAs, including 5 pappalysin (PAPPA) 1-derived DEcircRNAs (hsa_circ_0005456, hsa_circ_0088209, hsa_circ_0002052, hsa_circ_0088214 and has_circ_0008792, all downregulated), 3 DEmiRNAs (hsa-miR-760, hsa-miR-4665-5p and hsa-miR-4539, all upregulated), and downregulated genes (including MMP13 and HMOX1). The ceRNA regulation network of OS was built, which played important roles in the pathogenesis of OS and might be of great importance in therapy.

Potential Antimetastatic Effect of Timosaponin AIII against Human Osteosarcoma Cells through Regulating the Integrin/FAK/Cofilin Axis.

Timosaponin AIII (TSAIII) is a steroidal saponin which demonstrates anti-tumour activities. However, the effect of TSAIII on human osteosarcoma cells remains largely unknown. In this study, we demonstrated that TSAIII exerted a significant inhibitory effect on the distribution of cytoskeletal F-actin and cytoskeletal-related proteins, which contributed to the suppression of cell migration and invasion, without inhibiting cell growth or apoptosis. In the synergistic inhibitory analysis, cotreatment of TSAIII with αVß3 integrin inhibitor [Cyclo(RGDyK)] or focal adhesion kinase (FAK) inhibitor (PF-573228) exerted greater synergistic inhibitory effects on the expression of Intergin αVß3/FAK/cofilin axis, thus inhibiting the migration and invasion capacities of human osteosarcoma cells. TSAIII was demonstrated to significantly inhibit the pulmonary metastasis formation of human osteosarcoma cells in vivo in metastasis animal models. These findings reveal the inhibitory effects of TSAIII on the metastasis progression of human osteosarcoma cells and the regulation of integrin-αVß3-FAK-Src and TESK1/p-cofilin mediated cytoskeletal F-actin pathway. Therefore, TSAIII might represent a novel strategy for the auxiliary treatment of human osteosarcoma cells.

Anti-osteosarcoma activity and underlying molecular mechanism of a novel small molecule spermine oxidase inhibitor SI-4650 / 药学学报

The purpose of this study is to further explore the effects of SI-4650, a newly discovered small molecule inhibitor of spermine oxidase (SMO) in our laboratory, on proliferation and migration of human osteosarcoma 143B cells and its underlying molecular mechanism. Chemiluminescence and high performance liquid chromatograph were used to analyze the effect of SI-4650 on SMO activity in 143B cells. DCFH-DA-staining/FCM was used to analyze the accumulation of cellular reactive oxygen species (ROS), whereas MTT and FCM were used to detect proliferation and cell cycle. Transwell culture and Western blot were used to analyze the expression levels of migration-related proteins. PI/FITC-Annexin V/FCM, fluorescence microscopy and Western blot were used to analyze apoptosis and autophagy. Our results showed that SI-4650 could significantly decrease SMO activity, inhibit cell proliferation or migration, and induce a S-phase cell cycle arrest in 143B human osteosarcoma cells. The mechanism may be related to interfering with polyamine metabolism, activating mitochondrial-mediated apoptosis and causing autophagic death. These results suggest that SI-4650 has the potential for clinical use in treatment of osteosarcoma.

In vitro biocompatibility of orthodontic miniscrews with human gingival fibroblast and SAOS-2 osteoblast cultures.

PURPOSE: Miniscrews are an important choice for orthodontic anchorage. Yet reports on failures do exist, and attempts have been made to elucidate the causes. Clinical outcomes may be compromised not only by the mechanical implications of miniscrew design and the location of anchorage but also by poor biocompatibility. Hence, this study deals with the surface roughness and elemental composition of miniscrews and how these properties may affect the in vitro biocompatibility of four commercially available miniscrews. METHODS: Most of the currently available miniscrews are made of TiAl6V4, an alloy widely considered to be biocompatible. The samples tested in this study included four similarly dimensioned TiAl6V4 products from different manufacturers: tomas® by Dentaurum, OrthoEasy® by Forestadent®, Dual Top™ by Jeil Medical/Promedia, and LOMAS by Mondeal®. The surface properties of these products were characterized by scanning electron microscopy (SEM) and energy-dispersive X­ray spectroscopy (EDX). Cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and agar overlay assays according to ISO 10993-5. RESULTS: The miniscrew products were found to show variations in surface-finish quality pertaining to topography and chemical composition, with the latter departing slightly from the manufacturers' specifications. MTT assays yielded rates of cell culture viability in excess of 90%, and agar overlay assays did not reveal decoloration beyond the specimen outlines in any of the experimental groups tested. CONCLUSIONS: The four miniscrew products exhibited some minor, but statistically significant, differences in microtopography, alloy composition, and biological inertness. Cytotoxicity testing revealed that all four products should be considered non-cytotoxic, thus, ruling out poor biocompatibility as a cause of miniscrew failure.

Green and cost-effective synthesis of carbon dots from date kernel and their application as a novel switchable fluorescence probe for sensitive assay of Zoledronic acid drug in human serum and cellular imaging.

A new label-free, sensitive and selective off and on signaling fluorescence platform for assay of trace levels of Zoledronic acid (ZA) drug in human biological samples based on nitrogen doped carbon dots (N-CDs) - ferric ions (Fe3+) was designed. The fluorescence probe, N-CDs, was synthesized for the first time through a facile, eco-friendly and one-step hydrothermal treatment using date kernel as the precursor without any need to use chemical reagents. These CDs exhibited excellent water solubility, ionic and photo stability in various circumstances and a highly relative quantum yield of 12.5%. In the presence of Fe3+, the fluorescence intensity (FL) for N-CDs was strongly quenched due to the interaction between ferric ions and the functional groups at the N-CDs (switch off). Afterwards, by the addition of ZA, the fluorescence sensor status turned to "ON" (switch on) due to the dominance of ZA in the competition between functional groups on the surface of N-CDs and phosphate groups in ZA in the interaction with Fe3+ results in removing Fe3+ from the surface of N-CDs. Under the optimized conditions, the proposed fluorescence probe (N-CDs-Fe3+) exhibited good sensing performance for ZA assay with a linearity from 0.1 µM to 10.0 µM, a detection limit of 0.04 µM and the precision of 2.70%. The developed N-CDs-Fe3+ sensor was successfully used for the assay of ZA contents with good recoveries and selectivity in human serum samples. Meanwhile, the in vitro cytotoxic activity and cellular uptake of N-CDs were investigated on human osteosarcoma (MG-63) cell line.

Baicalin induces apoptosis in human osteosarcoma cell through ROS-mediated mitochondrial pathway.

Baicalin is extracted from a traditional Chinese herb, Scutellaria baicalensis. In this study, the anticancer activity and underlying mechanisms of baicalin towards human osteosarcoma cell (HOS) were investigated. Baicalin could inhibit HOS cell proliferation in a concentration-dependent manner. Mitochondrial membrane potential decreased obviously after treated with different concentration of baicalin by flow cytometry assay and revealed that baicalin triggered a significant generation of reactive oxygen species (ROS). Western blotting assay further revealed that baicalin-induced cell apoptosis by suppressing Bcl-2 level, then activating caspase-9 and caspase-3. In vivo experiment, baicalin significantly suppressed tumour growth in female BALB/C nude mice bearing HOS tumours. In addition, baicalin did show toxicity to treated animal by comparing the body weight increase and mortality. In general, the present results demonstrated that baicalin-induced apoptosis in human osteosarcoma cell via a ROS-mediated mitochondrial pathway. The paper indicated that baicalin is a promising candidate for the treatment of HOS.

Chimaphilin inhibits human osteosarcoma cell invasion and metastasis through suppressing the TGF-ß1-induced epithelial-to-mesenchymal transition markers via PI-3K/Akt, ERK1/2, and Smad signaling pathways.

Epithelial-to-mesenchymal transition is a cellular process associated with cancer invasion and metastasis. However, the antimetastatic effects of chimaphilin remain elusive. In this study, we attempted to investigate the potential use of chimaphilin as an inhibitor of TGF-ß1-induced epithelial-to-mesenchymal transition in U2OS cells. We found that TGF-ß1 induced epithelial-to-mesenchymal transition to promote U2OS cell invasion and metastasis. Western blotting demonstrated that chimaphilin inhibited U2OS cell invasion and migration, increased the expression of the epithelial phenotype marker E-cadherin, repressed the expression of the mesenchymal phenotype marker vimentin, as well as decreased the level of epithelial-to-mesenchymal-inducing transcription factors Snail1 and Slug during the initiation of TGF-ß1-induced epithelial-to-mesenchymal transition. In this study, we revealed that chimaphilin up-regulated the E-cadherin expression level and inhibited the production of vimentin, Snail1, and Slug in TGF-ß1-induced U2OS cells by blocking PI-3K/Akt and ERK 1/2 signaling pathway. Additionally, the TGF-ß1-mediated phosphorylated levels of Smad2/3 were inhibited by chimaphilin pretreatment. Above all, we conclude that chimaphilin represents an effective inhibitor of the metastatic potential of U2OS cells through suppression of TGF-ß1-induced epithelial-to-mesenchymal transition.

14-3-3ε is a nuclear matrix protein, and its altered expression and localization are associated with curcumin-induced apoptosis of MG-63 cells.

The 14-3-3 protein family may regulates protein interaction, transportation and cellular localization. The regulatory role of 14-3-3ε is influenced by its altered localization. In the present study, human osteosarcoma MG-63 cells were treated with curcumin to induce apoptosis. Subsequently, the altered expression and localization of 14-3-3ε and its co-localization with other apoptosis-associated proteins during apoptosis was investigated. Analysis of nuclear matrix proteins (NMPs), using two-dimensional gel electrophoresis with matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry, revealed that 14-3-3ε existed on the nuclear matrix of MG-63 cells, and its expression was decreased compared with that in control cells following curcumin treatment. In addition, western blot analysis validated that the expression level of 14-3-3ε was downregulated during curcumin-induced apoptosis of MG-63 cells compared with that in control cells. Using immunofluorescence labeling, it was observed that 14-3-3ε was located on the nuclear matrix of MG-63 cells and the distribution of 14-3-3ε on the nuclear matrix was decreased following treatment with curcumin, compared with that in control cells. Double immunofluorescence staining and laser-scanning confocal microscopy demonstrated that 14-3-3ε was co-localized with B-cell lymphoma-2 (Bcl-2), Bcl-2-associated-X protein, p53 and c-FOS transcription factor in MG-63 cells. Furthermore, following treatment with curcumin, these co-localization regions were decreased. The results of the present study revealed that 14-3-3ε is an NMP in MG-63 cells, and its altered expression and co-localization with apoptosis-associated proteins indicated an important function of 14-3-3ε in apoptosis of MG-63 cells. Additional studies are required to investigate the results of the present study.

Evaluation of Apoptosis and Autophagy Inducing Potential of Berberis aristata, Azadirachta indica, and Their Synergistic Combinations in Parental and Resistant Human Osteosarcoma Cells.

Cancer is a multifactorial disease and hence can be effectively overcome by a multi-constituently therapeutic strategy. Medicinal plant extracts represent a perfect example of such stratagem. However, minimal studies have been done till date that portray the effect of extraction techniques on the phyto-constituent profile of plant extracts and its impact on anticancer activity. In the present study, we have evaluated the anticancer potential of methanolic extracts of Berberis aristata root and Azadirachta indica seeds prepared by various extraction techniques in human osteosarcoma (HOS) cells. Soxhlation extract of B. aristata (BAM-SX) and sonication extract of A. indica (AIM-SO) were most effective in inducing apoptosis in parental drug sensitive, as well as resistant cell type developed by repeated drug exposure. Generation of reactive oxygen species and cell cycle arrest preceded caspase-mediated apoptosis in HOS cells. Interestingly, inhibition of autophagy enhanced cell death suggesting the cytoprotective role of autophagy. Combination studies of different methanolic extracts of BAM and AIM were performed, among which, the combination of BAM-SO and AIM-SO (BAAISO) was found to show synergism (IC50 10.27 µg/ml) followed by combination of BAM-MC and AIM-MC (BAAIMC) with respect to other combinations in the ratio of 1:1. BAAISO also showed synergism when it was added to cisplatin-resistant HOS cells (HCR). Chromatographic profiling of BAM-SX and AIM-SO by high performance thin layer chromatography resulted in identification of berberine (Rf 0.55), palmitine (Rf 0.50) in BAM-SX and azadirachtin A (Rf 0.36), azadirachtin B (Rf 0.56), nimbin (Rf 0.80), and nimbolide (Rf 0.43) in AIM-SO. The cytotoxic sensitivity obtained can be attributed to the above compounds. Our results highlight the importance of extraction technique and subsequent mechanism of action of multi-constituential B. aristata and A. indica against both sensitive and drug refractory HOS cells.

Chimaphilin inhibits proliferation and induces apoptosis in multidrug resistant osteosarcoma cell lines through insulin-like growth factor-I receptor (IGF-IR) signaling.

Chimaphilin, an active compound separated from pyrola, possesses the highly efficient antitumor activities. Insulin-like growth factor-I receptor (IGF-IR) plays an important role in tumor cell survival. To look for effective strategies for interrupting IGF-IR signaling pathway, we found that chimaphilin can inhibit the receptor tyrosine kinase activity of IGF-IR. Chimaphilin inhibited the growth of both drug-sensitive and drug-resistant osteosarcoma cell lines in a time and dose-dependent manner; however, it showed relatively little toxicity in normal osteoblast cell lines. Chimaphilin can increase the sensitivity of doxorubicin in doxorubicin-resistant osteosarcoma cell lines. Additionally, small interfering RNA downregulation of IGF-IR expression in drug-resistant cell lines also caused resensitization to doxorubicin. Above all, we conclude that chimaphilin represents a valuable natural source and may potentially be applicable for reversing the drug-resistant phenotype in osteosarcoma therapy.

Experimental research of Hedyotic diffusa injection on the inhibition of the proliferation of human osteosar- coma cell line MG-63 in vitro / 中国基层医药

Objective To investigate the inhibition of Hedyotic diffusa injection on the proliferation of hu-man osteosarcoma cell line MG -63 in vitro.Methods The human osteosarcoma cell line MG -63 was subcultivated aseptically in vitro.Different concentration of Hedyotic diffusa injection (50μL/mL,100μL/mL,200μL/mL,400μL/mL) successively acted on such cell .A total three time points were selected to determine the activity and numbers of cells by MTT assay including 12h,24h and 48h.Results The cellular proliferation inhibition rates of human osteosarcoma cell line MG-63 in drug groups of 50μL/mL concentration holes were (2.87 ±2.22)%,(13.42 ±2.14)% and (30.80 ±3.67)%after 12 h,24 h and 48 h.The rates of 100μL/mL concentration holes were (22.25 ±1.58)%, (43.34 ±2.84)%and (66.46 ±2.64)%,after 12 h,24 h and 48 h.The rates gradually increased and had statis-tical meaning,t12h =12.319,t24h =14.573,t48h =12.319,P<0.05;the cell in drug groups of 200μL/ml and 400μL/ml concentration holes was totally dead , and pathological findings showed that there were circular and floating cells in which nucleoplasmic ratio decreased and small fragments of cells increased .Conclusion Hedyotic diffusa extract has a certain inhibition in vitro on the proliferation of human osteosarcoma cell line MG -63.Moreover,the strength of its inhibition is relevant probably with drug concentration ,which deserves a further research .

Knockdown of Notch-1 augments inhibitory effect of dihydroartemisinin on viability of human osteosarcoma cell line U-2OS / 中国病理生理杂志

AIM:To investigate the effect of Notch-1 knockdown on the growth of dihydroartemisinin-inhibited human osteosarcoma cell line U-2OS.METHODS:U-2OS cells treated with different concentrations of dihydroartemisinin (5, 10, 15 and 20μmol/L) were collected.The expression of Notch-1, MMP-2, MMP-9 and Hes-1 at mRNA and protein levels was measured by real-time PCR and Western blotting, respectively.U-2OS cells were transfected with Notch-1 siRNA for 24 h and incubated with dihydroartemisinin for another 24 h.The cell apoptotic rate , protein expression of MMP-2, MMP-9 and Hes-1, and the migration ability were measured by MTT assay , Western blotting and Transwell experiment , respectively.RESULTS:Dihydroartemisinin (5, 10, 15 and 20 μmol/L) decreased the expression of Notch-1, MMP-2, MMP-9 and Hes-1 at mRNA and protein levels in a dose-dependent manner .Down-regulation of Notch-1 significantly en-hanced the effect of dihydroartemisinin on the cell apoptosis , the protein expression of MMP-2, MMP-9 and Hes-1, and mi-gration ability ( P<0.05 ) .CONCLUSION: Notch-1 pathway is involved in the process of dihydroartemisinin-inhibited U-2OS cell growth.Knockdown of Notch-1 augments the inhibitory effect of dihydroartemisinin on U-2OS cell viability.

miR-30 a suppresses migration, invasion and vitality of human osteosarcoma cell line 143 B / 基础医学与临床

Objective To investigate the effect of miR-30a on human osteosarcoma cell 143B in migration,invasion andcellviability.Methods 143BcellswereinfectedortransfectedwithrecombinantadenovirusmiR-30a(Ad-miR30a) and miR-30a inhibitor respectively .Wound healing assay was performed to detect the cell healing ability ( P<0.05 ) .Cell migration and invasion ability were determined by Transwell assay ( P<0.05 ) .The cell viability was analyzed by MTT assay ( P<0.01 ) .Real-time quantitative PCR was performed to analyze the expression of RUNX2 mRNA level and confirmed the adenovirus miR-30a expressed in 143B cells.The expression of RUNX2 was analyzed by Western blot .miR-30a target to RUNX2 was verified by luciferase reported gene assay .Results The ability of migration and invasion was suppressed in osteosarcoma cell 143B by overexpression miR-30a,and the cell viability also decreased .After the endogenous miR-30 a being inhibited , the cell motility and invasion enhanced and the cell viability was promoted .The RUNX2 protein decreased after overexpression miR-30 a as compared with controlgroup.TheluciferaseactivityofRUNX2decreasedbyaddingmiR-30a.Conclusions 143Bcellmigration, invasion and viability were suppressed by miR-30a,and this process is potentially achieved via suppressing RUNX 2 protein expression .

Effect of Ginsenoside Rh2 on apoptosis of human osteosarcoma cell line MG-63 / 中国病理生理杂志

AIM:To investigate the effect of Ginsenoside Rh2 (Rh2) on the apoptosis of human osteosarcoma cell line MG-63.METHODS:The cell viability was determined by MTT assay .MG-63 cell apoptotic rate was examined by flow cytometry with Annexin V-PI double staining.The expression of Bcl-2, Bax, cytochrome C ( Cyt C) and cleaved caspase-3 were measured by Western blot .RESULTS: Rh2 enhanced the apoptosis of MG-63 cells in a dose-dependent manner.Furthermore, after treatment with Rh2, the release of mitochondrial Cyt C and Bax expression were increased , while Bcl-2 and the ratio of Bcl-2/Bax were decreased as compared with control group ( P<0.05 ) .The protein level of cleaved caspase-3 was also increased (P<0.05).CONCLUSION:Ginsenoside Rh2 accelerates the apoptosis of MG-63 cells through mitochondria-dependent pathway , suggesting that Rh2 is a novel approach for the treatment of osteosarcoma .

DNA demethylation in the PTEN gene promoter induced by 5-azacytidine activates PTEN expression in the MG-63 human osteosarcoma cell line.

This study used the MG-63 osteosarcoma cell line to investigate the demethylation of the phosphate and tension homolog (PTEN) gene promoter and the change in PTEN gene expression levels, which are caused by the methylation inhibitor 5-azacytidine (5-Zac), and the association between the two. Different concentrations of 5-Zac (0, 5 and 10 µmol/l) were added into the MG-63 cell culture medium and the cells were cultured for 72 h. The following techniques were performed on the cells: Western blot analysis to detect the PTEN protein; reverse transcription-polymerase chain reaction (PCR) to detect the mRNA transcription levels of the PTEN gene; flow cytometry to detect the cell apoptotic rate; and sodium bisulfate to deal with the DNA of each group. The genes of the PTEN promoter and the transcription factors specificity protein 1 (Sp1) and Myc were PCR amplified and transformed into Escherichia coli, then a number of clones were selected for sequencing and the methylation status of the amplified PTEN promoter fragment was detected. Following culture of the MG-63 cells with 5-Zac at concentrations of 0, 5 and 10 µmol/l for 72 h, the expression levels of PTEN protein in each group were gradually increased, presenting a concentration-dependent effect: Group 0 µmol/l compared with groups 5 and 10 µmol/l, P<0.05; and group 5 µmol/l compared with group 10 µmol/l, P=0.007. The mRNA expression levels of the PTEN gene significantly increased. The apoptotic rates of groups 0, 5 and 10 µmol/l were 0.69±0.42, 2.50±0.30 and 6.59±0.62%, and significant differences (P<0.01) were observed between every two groups. The bisulfate DNA sequencing results of three groups showed that, following the treatment with 5-Zac, the binding of the CG site to transcription factors was affected by demethylation. The average rate of demethylation indicated a statistical difference among the three groups. In conclusion, the methylation inhibitor 5-Zac leads to a significant increase in the expression levels of the tumor suppressor gene PTEN in the MG-63 osteosarcoma cell line in vitro. The expression levels of mRNA and the cellular apoptotic rate were also increased. The elevated activation and expression levels of the PTEN gene may be associated with the low methylation levels of the CG site that binds to the transcription factors Sp1 and Myc in the PTEN gene promoter, and they promote the combination of the transcription factors and the gene promoter.