Elucidation of anti-inflammatory effect and osteogenesis of Inchinkoto in human osteosarcoma cell line Saos-2

Human herpes simplex virus 1 (HSV-1) is a widespread pathogen, with 80% of the population being latently infected. To successfully evade the host, the virus has evolved strategies to counteract antiviral responses, including the gene-silencing and innate immunity machineries. The immediately early protein of the virus, infected cell protein 0 (ICP0), plays a central role in these processes. ICP0 blocks innate immunity, and one mechanism is by degrading hostile factors with its intrinsic E3 ligase activity. ICP0 also functions as a promiscuous transactivator, and it blocks repressor complexes to enable viral gene transcription. For these reasons, the growth of a ΔICP0 virus is impaired in most cells, except cells of the human osteosarcoma cell line U2OS, and it is only partially impaired in cells of the human osteosarcoma cell line Saos-2. We found that the two human osteosarcoma cell lines that supported the growth of the ΔICP0 virus failed to activate innate immune responses upon treatment with 2'3'-cyclic GAMP (2'3'-cGAMP), the natural agonist of STING (i.e., stimulator of interferon genes) or after infection with the ΔICP0 mutant virus. Innate immune responses were restored in these cells by transient expression of the STING protein but not after overexpression of interferon-inducible protein 16 (IFI16). Restoration of STING expression resulted in suppression of ΔICP0 virus gene expression and a decrease in viral yields. Overexpression of IFI16 also suppressed ΔICP0 virus gene expression, albeit to a lesser extent than STING. These data suggest that the susceptibility of U2OS and Saos-2 cells to the ΔICP0 HSV-1 is in part due to an impaired STING pathway.IMPORTANCE The DNA sensor STING plays pivotal role in controlling HSV-1 infection both in cell culture and in mice. The HSV-1 genome encodes numerous proteins that are dedicated to combat host antiviral responses. The immediate early protein of the virus ICP0 plays major role in this process as it targets hostile host proteins for degradation with its E3 ligase activity, and it disrupts repressor complexes via protein-protein interaction to enable viral gene transcription. Therefore, the ΔICP0 HSV-1 virus is defective for growth in most cells, except the human osteosarcoma cell lines U2OS and Saos-2. We found that both cell lines that support ΔICP0 virus infection have defects in the STING DNA-sensing pathway, which partially accounts for the rescue of the ΔICP0 virus growth. Restoration of STING expression in these cells rescued innate immunity and suppressed ΔICP0 virus infection. This study underscores the importance of STING in the control of HSV-1.

Objective To investigate the effect of small interference RNA(siRNA) targeting RRM2 on the biological behavior of human osteosarcoma cell line Saos-2 and the molecular mechanisms.Methods RRM2 expression was knocked down in human osteosarcama cell line Saos-2 by RRM2 siRNA.The expression of RRM2 mRNA and protein was determined in human osteosarcoma cell line Saos-2 and human osteoblast-like cell line hFOB1.19 by real time-PCR and Western blot.The cell proliferation was detected by CCK-8.The migration was observed by using transwell system.The apoptotic rate was observed by ELISA.The expression of CyclinD1 and Bcl-2 proteins were detected by Western blot.Results The expression of RRM2 mRNA and protein was higher in Saos-2 than in hFOB1.19.siRRM2 could down-regulate the expression of RRM2 in Saos-2 cells in a time-and concentration-dependent manner.CCK-8 assay showed that si-RRM2 could inhibit the proliferation ability of Saos-2 cells in a time-and concentrationdependent manner,but had no effect on the proliferation of hFOB1.19 cells.Transwell assay indicated that si-RRM2 could inhibit the migration of Saos-2 cells.si-RRM2 combined with adriamycin could increase the apoptosis of Saos-2 cells.Western blot showed that the expression of Cyclin D1 and Bcl-2 were decreased by silencing RRM2.Condusion RRM2 overexpression maybe associate with the osteosarcoma cells proliferation and migration and suppression of its function is a potential therapeutic strategy in osteosarcoma. Key words: RNA interference; Gene, RRM2 ; Osteosarcoma

Garlic is generally used as a therapeutic reagent against various diseases, and numerous studies have indicated that garlic and its derivatives can reduce the risk of various types of human cancer. Diallyl trisulfide (DATS), a major member of garlic derivatives, could inhibit the cell proliferation by triggering either cell cycle arrest or apoptosis in a variety of cancer cell lines as shown in many studies. However, whether DATS has the same effect on human osteosarcoma cells remains unknown. In this study, we have attempted to analyze the effects of DATS on cell proliferation, cell cycle, induction of apoptosis, global protein expression pattern in a human osteosarcoma cell line Saos-2 cells, and the potential molecular mechanisms of the action of DATS. Saos-2 cells, a human osteosarcoma cell line, were treated with or without 25, 50, and 100 micromol/l DATS for various time intervals. The cell proliferation, cell cycle progression, and apoptosis were examined in this study. Then, after treatment with or without 50 micromol/l DATS for 48 h, protein add pattern in Saos-2 cells were systematically studied using two-dimensional electrophoresis and mass spectrometry. DATS could inhibit the proliferation of Saos-2 cells in a dose-dependent and time-dependent manner. Moreover, the percentage of apoptotic cell and cell arrest in G0/G1 phase was also dose-dependent and time-dependent upon DATS treatment. A total of 27 unique proteins in Saos-2 cells, including 18 downregulated proteins and nine upregulated proteins, were detected with significant changes in their expression levels corresponding to DATS administration. Interestingly, almost half of these proteins (13 of 27) are related to either the cell cycle or apoptosis. DATS has the ability to suppress cell proliferation of Saos-2 cells by blocking cell cycle progression and inducing apoptosis in a dose and time-dependent manner. The proteomic results presented, therefore, provide additional support to the hypothesis that DATS is a strong inducer of apoptosis in tumor cells. However, the exact molecular mechanisms, how these proteins significantly changed in the Saos-2 cell line upon DATS treatment, should be further studied.

Introduction Cancer continues to be a significant health issue worldwide, with colorectal cancer (CRC) standing out as one of the most prevalent forms of cancer on a global scale. The lifetime risk of developing CRC is about one in 23 (4.3%) for men and one in 25 (4.0%) for women. Moreover, children and adolescents are frequently reported with osteosarcoma with a low five-year survival rate (69% and 67%, respectively). Aim The aim of the study was to analyze the cytotoxic effects of boldine against human CRC (HCT-116) and osteosarcoma cell lines (Saos-2). Materials and methods HCT-116 and Saos-2 cell lines were subjected to different concentrations of boldine treatment (5, 10, 20, 30, 40, and 50 μg/mL) and (10, 20, 40, 60, and 80 µg/mL), respectively, for 24 hours. The cytotoxicity was analyzed by MTT assay, AO/EBstaining, DCFH-DA assay, and scratch assay. Results The MTT assay, microscopic analysis, and staining showed that boldine had dose-dependent cytotoxic effects against HCT-116 and Saos-2 cell lines by inhibiting their proliferation, viability, and migration, and inducing ROS-mediated apoptosis. Conclusion The study concluded that boldine had a concentration-dependent cytotoxic effect on human CRC and osteosarcoma cell lines.

BackgroundOsteosarcoma is the most common primary tumor of bone. Interleukin-33 (IL-33) is a pro-inflammatory cytokine that also participates in tumor progression. This study aimed to investigate the role of IL-33 in human osteosarcoma cell viability, proliferation, apoptosis, and epithelial-mesenchymal transition (EMT) in vitro and the molecular mechanisms involved.Material/MethodsThe normal osteoblast cell line, hFOB 1.19, and the human osteosarcoma cell lines SOSP-9607, SAOS2, MG63, and U2OS were studied. The expression of IL-33 mRNA and protein in human osteosarcoma cell lines were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. The effects of IL-33 on human osteosarcoma cell viability, apoptosis, EMT, and the signaling pathways were studied using the MTT assay, flow cytometry, qRT-PCR, and Western blot.ResultsIL-33 was upregulated in human osteosarcoma cell lines, including U2OS cells. The use of an IL-33 gene plasmid promoted osteosarcoma cell viability, inhibited cell apoptosis, increased the expression of Bcl-2, and reduced the expression of Bax. IL-33 reduced the level of E-cadherin and increased the levels of N-cadherin and matrix metalloproteinase-9 (MMP-9) in osteosarcoma cells at the mRNA and protein level. The use of the IL-33 plasmid increased the protein expression levels of p-AKT and the p-AKT/AKT ratio in osteosarcoma cells, and IL-33 siRNA reversed these findings.ConclusionsIL-33 was highly expressed in human osteosarcoma cells. Down-regulation of IL-33 reduced cell viability and EMT of osteosarcoma cells, and induced cell apoptosis through activation of the PI3K/AKT signaling pathway.

Transcriptional regulation of cell- and stage-specific genes is a crucial process in the development of mesenchymal tissues. Here we have investigated the regulatory mechanism of the expression of the chondromodulin-I (ChM-I) gene, one of the chondrocyte-specific genes, in osteogenic cells using osteosarcoma (OS) cells as a model. Methylation-specific sequence analyses revealed that the extent of methylation in the core-promoter region of the ChM-I gene was correlated inversely with the expression of the ChM-I gene in OS primary tumors and cell lines. 5-Aza-deoxycytidine treatment induced the expression of the ChM-I gene in ChM-I-negative OS cell lines, and the induction of expression was associated tightly with the demethylation of cytosine at -52 (C(-52)) in the middle of an Sp1/3 binding site to which the Sp3, but not Sp1, bound. The replacement of C(-52) with methyl-cytosine or thymine abrogated Sp3 binding and also the transcription activity of the genomic fragment including C(-52). The inhibition of Sp3 expression by small interfering RNA reduced the expression of the ChM-I gene in ChM-I-positive normal chondrocytes, indicating Sp3 as a physiological transcriptional activator of the ChM-I gene. These results suggest that the methylation status of the core-promoter region is one of the mechanisms to determine the cell-specific expression of the ChM-I gene through the regulation of the binding of Sp3.

Matrine, a natural product, has been demonstrated to be a promising chemotherapeutic drug for some cancers. Using flow cytometric analysis of the cell cycle and apoptosis, we found that matrine inhibited the proliferation and induced apoptosis in the human osteosarcoma (OS) cell lines MG63, HOS, U2OS, and SAOS2 in vitro in a dose-dependent manner. We therefore assessed the role of the serine/threonine kinase Akt in the regulation of matrine-mediated cell growth inhibition and apoptosis induction in human OS cell lines. After treatment for 48 h, matrine induced G0/G1-stage cell cycle arrest in MG63, U2OS, and SAOS2 cells associated with an increase in the expression of p27(Kip1) and a decrease in the expression of Akt, glycogen synthase kinase 3 (GSK3)-β (Ser9), and cyclin D1. Furthermore, the pro-apoptotic factor Bax was upregulated. Overall, our findings suggest that matrine may be an effective anti-osteosarcoma drug due to its ability to inhibit proliferation and induce apoptosis in OS cells, possibly through the involvement of Akt signaling.

PurposeCD81 is a member of the tetraspanin family of membrane proteins. Recently, it has been shown that CD81 may be involved in cancer cell proliferation and metastasis. As yet, however, there have been few reports on the expression and role of CD81 in osteosarcoma.MethodsThe expression of CD81 was investigated in human osteoblast cell line hFOB1.19 and in human osteosarcoma cell lines Saos2, MG63 and 143B. The expression of CD81 was inhibited in osteosarcoma cells using siRNA after which cell proliferation, migration and invasion were assessed. We also used Western blotting to investigate the phosphorylation status of Akt, Erk, JNK and p38, and measured the expression of MMP-2, MMP-9 and MT1-MMP. In addition, we used a CRISPR/Cas9 system to stably knock out CD81 expression in 143B cells, transplanted the cells into mice, and assessed tumor formation and lung metastasis in these mice compared to those in the control group.ResultsWe found that CD81 was expressed in the human osteoblast cell line and in all osteosarcoma cell lines tested. The osteosarcoma cell line 143B exhibited a particularly high level of expression. In addition, we found that osteosarcoma cell proliferation, migration and invasion were decreased after CD81 inhibition, and that the phosphorylation of Akt and Erk was suppressed. Also, the expression levels of MMP-2, MMP-9 and MT1-MMP were found to be suppressed, with MMP-9 showing the greatest suppression. In vivo, we found that mice transplanted with CD81 knockout 143B cells exhibited significantly less tumor formation and lung metastasis than mice in the control group.ConclusionBased on our findings we conclude that inhibition of CD81 suppresses intracellular signaling and reduces tumorigenesis and lung metastasis in osteosarcoma cells.

Objective: To study the effect of long non-coding RNA LINC-PINT on proliferation and apoptosis of osteosarcoma cells. Methods: Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expressions of LINC-PINT and miR-524-5p in normal osteoblast hFOB and human osteosarcoma cell lines HOS, MG63 and SAOS2 cells. The pcDNA plasmid, pcDNA-LINC-PINT plasmid, negative control siRNA (si-NC), si-LINC-PINT, negative control mimics (miR-NC), miR-524-5p mimics (miR-524-5p), pcDNA-LINC-PINT combined with miR-NC, pcDNA-LINC-PINT combined with miR-524-5p were transfected into HOS cells with liposome, respectively. The protein expressions of PCNA and cleaved-caspase-3 in the cells were detected by western blot. Cell proliferation ability was detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. The apoptosis was detected by flow cytometry. The transcriptional activity was detected by double luciferase reporter assay. Results: Compared with normal osteoblast hFOB cell (1.00±0.08 vs 1.00±0.06), the expressions of LINC-PINT were down-regulated (0.18±0.01; 0.33±0.01; 0.42±0.01), while the expressions of miR-524-5p were up-regulated (2.65±0.23; 1.68±0.14; 1.51±0.13) in human osteosarcoma cell lines HOS, MG63 and SAOS2 cells, respectively. Overexpression of LINC-PINT significantly inhibited the proliferation (0.41±0.05 vs. 0.62±0.05 for 48 h; 0.57±0.05 vs. 1.06±0.09 for 72 h, both P<0.05) while promoted the apoptosis (25.28±2.15 vs. 9.01±0.17, P<0.01) of HOS cells. Knockdown of LINC-PINT or overexpression of miR-524-5p can significantly promote the proliferation and inhibit apoptosis of HOS cells. Moreover, miR-524-5p inhibited the fluorescence activity of wild-type LINC-PINT (0.31±0.03) in HOS cells when comparred with miR-NC (1.00±0.03) and was negatively regulated by LINC-PINT. Overexpression of miR-524-5p reversed the proliferation inhibition and apoptosis-promotion effects of LINC-PINT in HOS cells. Conclusions: Long non-coding RNA LINC-PINT can inhibit the proliferation and promote apoptosis of osteosarcoma cells through targeting miR-524-5p, which will provide a new target for the treatment of osteosarcoma.

Skeletal alkaline phosphatase specific activity is an index of the osteoblastic phenotype in subpopulations of the human osteosarcoma cell line SaOS-2

Insulin-like growth factors I (IGF-I) and II (IGF-II) are anabolic for osteoblastic cells. Although expression of IGF-I and IGF-II mRNA has been demonstrated in rodent osteoblastic cells, little is known about IGF gene expression in human osteoblastic cell models. In this study we characterized IGF-I and -II mRNA expression in (1) normal human osteoblast-like (hOB) cells, (2) a simian virus 40 immortalized hOB (HOBIT) cell line, and (3) human osteosarcoma cell lines SaOS-2, TE-85, MG-63, and U-2. Since cross-hybridization of IGF cDNA probes with ribosomal RNA obscures detection of some of the multiple IGF transcripts in human cells, we replaced Northern analysis with the more specific ribonuclease protection assay (RPA). We also used the reverse transcriptase-polymerase chain reaction (RT-PCR) to assess whether mRNAs were present at trace levels. IGF-I mRNA expression was consistently observed in normal hOB cells only and by both RT-PCR and RPA. Among IGF-I transcript variants, Ea IGF-I mRNA was more abundant than the Eb mRNA in normal hOB cells. Trace levels of IGF-I mRNA were variably detected in SaOS-2 and U-2 osteosarcoma cells when RT-PCR was performed, but we found no IGF-I mRNA in HOBIT, TE-85, or MG-63 cells. IGF-II mRNA was expressed in normal hOB, HOBIT, TE-85, and U-2 cells as assessed by either method.(ABSTRACT TRUNCATED AT 250 WORDS)

The Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) is a hematopoietic growth factor that regulates the in vitro and in vivo proliferation and differentiation of hematopoietic cells through the interaction with a specific heterodimeric receptor complex (GM-CSFR), consisting of an alpha and a beta chain with molecular weights of 80 and 120 KDa, respectively. We have studied the expression of the GM-CSFR (alpha chain) on the surface of the human osteosarcoma cell line SaOS-2 and the in vitro effects of different concentrations (10, 100, and 200 ng/ml) of GM-CSF on GM-CSFR expression and the biological activity of SaOS-2 cells. Our data show that SaOS-2 cells express GM-CSFR and that GM-CSF can down-regulate the expression of its own receptor on these cells. Furthermore, to evaluate the biological effects of GM-CSF on SaOS-2 cells, we have investigated cell proliferation and differentiation of these cells treated with different doses of the growth factor through: (1) a morphological analysis of typical osteoblast differentiation markers such as osteopontin and BSP-II; (2) measurement of alkaline phosphatase (ALP) activity; (3) production of bone ECM components (collagen I, fibronectin, tenascin, and laminin); (4) production of interleukin-6 (IL-6) and osteocalcin in the culture medium. The results show that the in vitro treatment of SaOS-2 cells with recombinant human GM-CSF causes a decreased cell proliferation and an increased production of osteopontin, BSP-II, ALP, IL-6, and most but not all ECM components. These findings suggest that GM-CSF can regulate proliferation and differentiation of osteoblast-like SaOS-2 cells and could also play an unexpected role in the maturation of bone tissue.

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.

Background: Orento, a traditional Japanese medicine, is known as Kampo medicine in Japan. We investigated the possible efficacy of Kampo medicine for periodontal disease. In this study, we examined the in vitro effects of orento on the proliferation of the inflammatory cytokines interleukin (IL)-6 and IL-8, the production of type 1 collagen, and the secretion of alkaline phosphatase (ALP) in the human osteosarcoma cell line Saos-2 (Saos-2 cells). Methods: The proliferation of Saos-2 cells was assessed by MTT assay. IL-6 and IL-8 levels, type 1 collagen production and ALP secretion were evaluated using enzyme-linked immunosorbent assay and ALP assays. Saos-2 cells were treated with or without 0.1, 1, 10, 100 and 1000 μg/mL of orento for 24 h. Results: Orento (10 μg/mL) significantly induced the proliferation of Saos-2 cells. At this concentration, orento suppressed IL-6 and IL-8 and enhanced type 1 collagen production and ALP secretion. Conclusions: These results indicate that orento controls the IL-6 and IL-8 secretion and cellular metabolism of osteoblasts, resulting in the secretion of early bone-related biomarkers.

Supplementary Table 1 from Prostate-Specific Antigen Modulates Genes Involved in Bone Remodeling and Induces Osteoblast Differentiation of Human Osteosarcoma Cell Line SaOS-2