The inhibitory effect of a novel phosphodiesterase 4 inhibitor ZL-n-91 on proliferation of osteosarcoma U20S cells / 中国药理学通报

Aim To explore the anti-cancer effects of ZL-n-91, a novel and highly selective phosphodiesterase 4 inhibitor, on the osteosarcoma U2OS cells.Methods CCK-8 assay was used to detect the inhibitory effect of ZL-n-91 with different concentrations(0, 20, 40, 80, 160, 240, 320, 400, 480 μmol·L-1)and different intervention time(0, 24, 48, 72, 96 h)on the proliferation of U2OS cells.Tablet clone forming experiment was used to detect the effect of ZL-n-91 on the clonality of U2OS cells.Flow cytometry was used to detect the cell apoptosis and cell cycle distribution.Western blot was employed to detect the expression of Bcl-2, CDK2, CDK4, CyclinD1, CyclinE1 protein.Results The inhibitory rate of ZL-n-91 on U2OS cells was concentration- and time-dependent(P<0.05), and its half inhibition rate IC50 was 174.1 μmol·L-1.ZL-n-91 significantly inhibited the clonality of U2OS cells(P<0.01).ZL-n-91 significantly induced cell apoptosis, and caused cell cycle arrest at G0/G1 phase in U2OS cells(P<0.01).The results of Western blot showed that ZL-n-91 significantly down-regulated the expression of Bcl-2, CDK2, CDK4, CyclinD1, CyclinE1 proteins in U2OS cells(P<0.05).Conclusions The novel selective phosphodiesterase 4 inhibitor, ZL-n-91, can significantly inhibit the proliferation of osteosarcoma U2OS cells with induction of cell cycle arrest and cell apoptosis, and may become a potential anti-cancer agent.

Role of FOXO3a in hypoxia-induced cisplatin resistance in osteosarcoma U-2OS cells / 中国肿瘤生物治疗杂志

@# Objective:To investigate the role of FOXO3a in hypoxia-induced cisplatin (DDP) resistance in osteosarcoma cells. Methods: The FOXO3a expression was detected by RT-PCR and Western blotting in osteosarcoma U-2OS cell line under normoxia and hypoxia conditions. The effects of HIF-1α-siRNAand FOXO3a-siRNAon the expressions of HIF-1αand FOXO3a were detected by Western blotting. CCK-8 and Annexin V/PI assays were used to detect the function of FOXO3a in hypoxia-induced DDP resistance of U20S cells. Results: Hypoxia could significantly increase the mRNAand protein levels of FOXO3a in U-2OS cells (All P<0.05). The expression of FOXO3a was regulated by HIF-1α; compared with control group and HIF-1α-NC group, the FOX03a protein expression was significantly down-regulated in HIF-1α-siRNA group [(0.38±0.03) vs (0.89±0.08)， (0.91±0.07), all P<0.01]. Under hypoxia condition, FOXO3a-siRNA could decrease the tolerance of U-2OS cells to DDP [(38.50±2.83)% vs (61.75±5.73)%, P<0.01], and increase DDP-induced apoptosis of U-2OS cells [(73.41±6.13)% vs (32.38±2.23)%, (55.89±4.46)%,All P<0.05]. Conclusions: Hypoxia significantly enhanced DDP-resistance of U-2OS cells by increasing FOXO3a expression in a HIF-1-dependent manner.

The effect of isosteviol on growth of human osteosarcoma cell lines U-2OS / 中国癌症杂志

Background and purpose:Osteosarcoma, a highly malignant bone tumor, develops rapidly. The current medicines for osteosarcoma present some limitations with serious side effects of long-term use. Isosteviol has the structure of tetracyclic diterpene which is the starting material of many anti-cancer drugs. However, its anti-tumor activity has been rarely reported. This study investigated the effect of isosteviol on proliferation of human osteosarcoma cell line U-2OS.Methods:The effect of isosteviol on U-2OS cell proliferation was assayed by MTT method. Cellular morphologic changes were observed under an inverted phase contrast microscope. The cell condition was observed with Hoechst 33342 and PI staining. Generation of reactive oxygen species and cell membrane potential were detected as well. The cell cycles were analyzed with lfow cytometry. The expressions of apoptosis-related proteins Bcl-2 and Bax were measured by Western blot assay.Results:The result indicates that isosteviol suppressed the growth of U-2OS cells in time- and concentration-dependent manner. Isosteviol could cause S phase cell cycle arrest at 24 h and apoptosis at 48 h. With the increased drug concentration, reactive oxygen species increased significantly, and the membrane potential gradually reduced. In addition, isosteviol treatment enhanced the expression of Bax but reduced that of Bcl-2.Conclusion:The inhibition of isosteviol on cell growth of U-2OS cells was possibly caused by promoting apoptosis through regulating the apoptosis-related protein expressions, such as the enhancement of Bax and reduction of Bcl-2 expression.

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.

Establishment of an orthotopic transplantation model of osteosarcoma labeled with three reporter genes / 军事医学

Objective To establish an orthotopic osteosarcoma nude mice model that co-expresses green fluorescent protein( GFP) , red fluorescent protein ( RFP) and luciferase for the purpose of monitoring the growth of osteosarcoma and screening drug candidates against osteosarcoma .Methods Human osteosarcoma cells of U 2-OS were infected with lentivirus carrying reporter gene .The reporter gene expression was verified by fluorescent microscopy and bioluminescence imaging.The cells were transplanted into tibia of the nude mice and monitored by bioluminescence imaging .Results The reporter gene was stably expressed in U 2-OS cells.The growth and metastsis of osteosarcoma could be detected in nude mice.Conclusion The established orthotopic osteosarcoma nude mice model is an ideal model for investigating the mechanism of growth and metastasis of osteosarcoma and for screening drug candidates against osteosarcoma .

Expression of Gab2 in human osteosarcoma U2-OS cells / 中国病理生理杂志

AIM:To investigate the expression of Grb2-associated binding protein 2 (Gab2) in human osteo-sarcoma cells and its relationship with the invasion and metastases of human osteosarcoma cells.METHODS: The tech-nique of small RNA interference was used to transfect human osteosarcoma U2-OS cell lines.Western blotting and RT-PCR were used to detect the protein and mRNA expression of Gab2 in transfected U2-OS cells.After transfection, through chem-otaxis and invasion assays in vitro, the cell migration and invasion abilities were detected.RESULTS:After transfection, the expression of Gab2 at mRNA and protein levels in Gab2 siRNA transfected cells ( SiGab2/U2-OS) was lower than that in scrambled siRNA transfected cells ( Scr/U2-OS ) and U2-OS cells.After stimulation with epidermal growth factor ( EGF) at concentration of 10 μg/L, the migration SiGab2/U2-OS cells was significantly less than Scr/U2-OS cells and U2-OS cells ( P<0.01 ) .The number of invasion cells of SiGab2/U2-OS group was significantly lower than the other 2 control groups ( P<0.01) .CONCLUSION:Inhibition of Gab2 expression obviously attenuates the migration and invasion abilities of human osteosarcoma U2-OS cell line.

MK-2206, an inhibitor of Akt, induced cell apoptosis and autophagy in U2 OS cells / 中国病理生理杂志

AIM:To observe the effect of MK-2206, an inhibitor of Akt, on the cell apoptosis and autophagy of U2OS cells.METHODS:The cell viability was detected by MTT assay .The cell apoptosis was analyzed by TdT-media-ted dUTP nick end labeling assay .The expression of LC3-II was examined by Western blotting .RESULTS:MK-2206 in-hibited the cell viability in a dose-dependent manner .MK-2206 induced the cell apoptosis via activation of caspase-3, caspase-9 and PARP.MK-2206 treatment substantially induced the U 2OS cell autophagy by increasing in the levels of LC 3-II.Blockage of autophagy using chloroquine magnified MK-2206-induced cell death in U2OS cells.CONCLUSION:The Akt inhibitor MK-2206 induces cell apoptosis and autophagy .Blocking autophagy magnifies MK-2206-induced the inhibi-tion of the viability in U2OS cells.

Effects of MiR-194 on the metastasis of human osteosarcoma cell line U2-OS by recombinant lentivirus vector / 中国肿瘤临床

Objective:This study aimed to construct a lentiviral expression vector for microRNA-194 and investigate its effect on the metastasis of human osteosarcoma cell line U2-OS. Methods:Pri-and mature miR-194 amplified by PCR were inserted into the plenty-GFP vector and identified by restriction endonuclease digestion and nucleotide sequencing. The osteosarcoma cell line U2-OS was transfected with the lentivirus. Then, the stable transfected cells were used in Transwell and wound healing assay. Results:Restric-tion analysis and sequencing showed that the recombinant lentiviral expression vector was constructed correctly. The titers of obtained overexpression and suppression expression recombinant lentivirus were 1.5*108 and 4*108 TU/ml. Cell metastasis ability was signifi-cantly different in different experimental groups (P<0.01). Conclusion:The lentiviral expression vector for microRNA-194 was suc-cessfully constructed. MicroRNA-194 could influence the metastasis of the osteosarcoma cell line U2-OS;thus, it could be further ex-plored as a potential target in osteosarcoma therapy.

The Inhibitory Effect of Down-Regulating Phosphorylated HER2 on Proliferation and Metastasis in U2-OS Osteosarcoma Cells / 天津医药

Objective To investigate the effects of down-regulating phosphorylated human epidermal growth factor receptor 2 (HER2) on the proliferation and metastasis in human osteosarcoma cells (U2-OS) in vitro. Methods Various concentrations of HER2 phosphorylation inhibitor lapatinib ditosylate (5, 10, 20, 30 and 40 μmol/L) were adopted to deal with U2-OS. MTT assay was performed to evaluate the cell proliferation during various times (24, 48 and 72 h), and the IC50 value in 24 h was calculated. The value of 10μmol/L (IC50=22.15μmol/L) was chosen to deal with U2-OS cells. The expres-sion level of phosphorylated HER2 (p-HER2) was measured by Western blot assay. The cell migration and invasion abilities were detected by Wound healing and Transwell invasion assays. Results The cell proliferation of U2-OS was significantly inhibited by HER2 phosphorylation inhibitor lapatinib ditosylate in a concentration- and time-dependent manner. During 24 hours, the p-HER2 level was significantly lower in lapatinib ditosylate group than that of negative control group (0.093± 0.033 vs 0.306±0.033), the cell migration rate was significantly lower in lapatinib ditosylate group than that of negative con-trol group (32.70%±3.00%and 94.52%±4.76%), and the trans-membrane cells were significantly lower than those of nega-tive control group (37/HP±5/HP and 85/HP±10/HP), respectively. Conclusion The down-regulating p-HER2 in U2-OS could efficiently inhibit the cell proliferation, migration and invasion in vitro. HER2 has the potential to become a molecular target for anti-osteosarcoma metastasis.

Triptolide induces apoptosis through extrinsic and intrinsic pathways in human osteosarcoma U2OS cells.

Triptolide, a diterpene derived from Tripterygium wilfordii Hook f., a Chinese medicinal herb, has been reported to inhibit cell proliferation and induce apoptosis in various human cancer cells, but its anticancer effects on human osteosarcoma cells have not yet been elucidated. In this study, we investigated whether triptolide induces apoptosis in human osteosarcoma cells and the underlying molecular mechanisms. We firstly demonstrated that triptolide inhibited cell growth and induced apoptosis in U2OS cells. Western blot analysis showed that the levels of procaspase-8, -9, Bcl-2, Bid and mitochondrial cytochrome c were downregulated in triptolide-treated U2OS cells, whereas the levels of Fas, FasL, Bax, cytosolic cytochrome c, cleaved caspase-3 and cleaved PARP were upregulated. These results suggest that triptolide induces apoptosis in U2OS cells by activating both death receptor and mitochondrial apoptotic pathways.

Effect of Adrenomedullin on Proliferation and Mitogen-Activated Protein Kinase Activity in Osteosarcoma Cell Lines (MG63, U2OS) / 대한정형외과학회잡지

PURPOSE: The aims investigated how how human osteosarcoma cell proliferation and the MAP kinases cascade are regulated, in the MG63 and U2OS human osteosarcoma cell lines after stimulating them with adrenomedullin (AM) with particular focus on extracellular signal-regulated kinase 1/2 (ERK 1 and 2) activation. MATERIALS AND METHODS: A cell proliferation assay was used to examined the effects of AM on the osteosarcoma cell lines (MG63 and U2OS). The effects of AM on ERK1/2 were examed by Western blot analysis. The roles of ERK 1/2 in the AM-induced proliferative signaling pathways in the two cell types were examed using PD98059, a selective inhibitor of the mitogen activated protein-ERK kinase (MEK) pathway. RESULTS: The addition of AM to the medium containing the osteosarcoma MG63 and U2OS cells induced proliferation in a dose-dependent manner. AM strongly activated ERK 1/2 mediated cell proliferation signaling, which was prevented using PD98059. CONCLUSION: These results suggest that AM plays an important role in the proliferation of human osteosarcoma MG63 and U2OS cells, and ERK kinase pathway plays a signal transduction role in AM treated human osteosarcoma MG63 and U2OS cell lines.

In Vitro Inhibitory Effect of Wogonin on Human Osteosarcoma / 中国药房

OBJECTIVE:To study the inhibitory effect of wogonin on human osteosarcoma. METHODS:MTT chromatometry was employed to determine the the influence of wogonin to the activity of U2OS cell line and its time-dependent effect and dose-dependent effect against osteosarcoma cell line detected. The apoptosis of wogonin-treated U2OS cells was determined by flow cytometry together with morphological observation,meanwhile the cell cycle of U2OS cell line was analyzed. RESULTS:The growth of U2OS cells was inhibited and the apoptosis of which was promoted by wogonin in a time-dependent and dose-dependent manner. The U2OS cells were arrested at G0/G1 phase with conspicuous apoptotic peak,nuclear fragmentation and chromosome agglomeration. The IC50 against U2OS cells stood at (37.18?1.57) ?mol?mL-1. CONCLUSION:Wogonin can inhibit cell growth and promote apoptosis of U2OS cells.

Sec13 induces genomic instability in U2OS cells

Sec13p has been known as an endoplasmic reticulum-Golgi transport protein. Recently, it has also been shown to be required for the formation of septation in the fission yeast Schizosaccharomyces pombe. In the present study, we focused on the role of a human homolog of Saccharomyces cerevisiae SEC13, Sec13 protein during mitosis in U2OS cells. We found that the expression of Sec13 was constant throughout the cell cycle, and localized to the kinetochores at metaphase during mitosis. By using green fluorescent protein technology, we observed that Sec13 is required for evasion of mitotic arrest in response to spindle damage, leading to G1-like phase and apoptotic cell death. In addition, cells expressing exogenous Sec13 showed giant nuclei compared to endogenous ones in the absence of nocodazole. These results demonstrate that Sec13 is involved in the regulation of the metaphase/anaphase transition and may be functionally associated with mitotic machinery to maintain genomic stability during mitosis.

Effect of ginsenosides from Panax ginseng on proliferation of human osteosarcoma cell U_2OS / 中草药

Object To find out which of the 27 ginsenosides isolated from Panax ginseng C A Mey that may inhibit the proliferation of human osteosarcoma cell line U 2OS Methods Effects of each individual ginsenoside on the proliferation of U 2OS cell were studied by determining the viability of cancer cells during culture with or without the presence of the test compound DNA assay was determined by flow cytometry Results Ginsonosides Ro, Rh 1, Rh 2, F 1 and L 8 at concentrations of 5 ?mol/L could obviously suppress the proliferation of U 2OS cells while ginsenosides Rg 1, F 3, Rf, PPT and PT significantly inhibited the cancer cells Flow cytometry revealed that ginsenosides Ro, Rg 1, Rf, F 1, Rh 2 ,PPT and PT induced cell cycle arrest at G 0/G 1 phase with obvious decrease of cell count at S and G 2+M phase Moreover, ginsenosides Rf 1, Rg 1, F 1 and PPT induced significantly high rates of cell death as compared with the control Conclusion These data suggested that ginsenosides inhibited U 2OS proliferation via cell cycle arrest or induction of cell death