Apoptosis In Saos-2 Cells Research Articles

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

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. Human osteosarcoma Saos-2 cells were treated with genistein for 24 and 48 hours. Cytotoxicity and apoptosis were measured. 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. Genistein may inhibit proliferation through the up-regulation of p21 and p27 and viability by inducing apoptosis in Saos-2 cells.

Combinatorial anticancer effects of multi metal ion and drug substitute with hydroxyapatite coatings on surgical grade 316LSS stainless steel alloys towards biomedical applications

The present study highlights the metal ions and existing drug ampicillin-loaded nano-hydroxyapatite (nHAP) coated on 316 stainless steel alloy (316LSS) using the electrodeposition method. In addition, the sample phase purity and crystallinity of the HAp composite coatings on 316LSS were analyzed by X-ray diffraction analysis (XRD) and Fourier transform infrared (FT-IR) spectroscopy using the KBr pellet method. The coating microstructure/morphology was characterized using a scanning electron microscope, which showed a flower-like morphology with the attachment of energy-dispersive X-ray spectroscopy (EDX) to confirm the major element groups in the synthesized sample. The TGA results showed that weight loss occurred in the temperature range of up to 800 °C for oxidation and reduction reactions. Additionally, the beat-optimized nanomaterial showed promising in vitro bioactivity against disease-causing microorganisms, displaying a significant zone of inhibition. Furthermore, cytotoxicity assessment using the MTT assay demonstrated that the synthesized samples effectively inhibited cell proliferation in human osteosarcoma cell lines (Saos-2). AO/EB double staining performed under a fluorescence microscope indicated changes in the morphology of the apoptotic cell nuclei. In addition, the relationship between ROS and nanomaterial-induced apoptosis in Saos-2 cells was analyzed by flow cytometry. MMP (ΔѰmt) profiling showed that the HAp composite increased the effective mitochondrial membrane damage or depolarization in the human osteosarcoma cell line. Moreover, the TUNEL assay suggested that treated nanomaterial with Saos-2 cells showed enhanced green fluorescence intensity, indicating terminal DNA damage. The obtained results meet the recommended physiological standard for synthesized 316LSS HAp composites to guide bone tissue regeneration.

Thymoquinone Potentiates Methotrexate Mediated-Apoptosis in Saos-2 Osteosarcoma Cell Line.

Recently, various studies have concentrated on the therapeutic potential of thymoquinone (TQ), a natural polyphenol, in various human malignancies, including osteosarcoma. However, the underlying mechanisms in TQ-mediated anti-cancer effects are not yet fully understood. Therefore, the present study investigated the effect of TQ on methotrexate (MTX)-induced apoptosis in Saos-2 cells. Saos-2 cells were treated with MTX, TQ, and a combination of both, and cell viability was assessed by MTT assay. mRNA expression of apoptotic markers, including Bax, Bcl-2, and caspase-3, was assessed using quantitative real-time polymerase chain reaction (qRT-PCR). MTX resulted in significant inhibition of cell proliferation in a dose-dependent manner. The combination of TQ and MTX inhibited proliferation compared to single treatments (P<0.05). TQ also induced apoptosis by regulating pro-apoptotic markers including Bax and caspase-3 and reducing anti-apoptotic mediators including Bcl-2. In addition, TQ increased MTX-induced apoptosis in Saos-2 cells. The findings of the present study highlight new insights into understanding the role of TQ as a potential therapeutic agent in osteosarcoma by increasing MTX-induced apoptosis.

Quercetin Augments Cisplatin-Induced Apoptosis, DNA Damage Response, and MiR-22 Expression While It Prevents DNA Repair in Osteosarcoma Cells.

Osteosarcoma (OS) patients are commonly treated with chemotherapeutic agents like cisplatin (Cis). Quercetin with fewer side effects can improve the potency of chemotherapy and be used in combinational therapies. Herein, we aimed to evaluate the effects of Cis plus quercetin on DNA damage response (DDR), DNA repair, and apoptosis in Saos-2 cells. The effects of Cis and quercetin single or in combination on Saos-2 cell viability and the cytotoxicity of the drugs were measured by MTT assay. The expression of DDR and repair components including P53, ATM, ATR, RAD51, and H2AX, and also miR-22 were analyzed by real-time PCR. The rate of apoptosis was measured by flow cytometry. Quercetin potentiated the cytotoxic effects of Cis in Saos-2 cells. The IC50 of Cis reduced from 6.12 µM to 4.25 µM. The combination of quercetin and Cis was associated with the up-regulation of miR-22 and DDR components, including P53, ATM, ATR, and H2AX as well as the down-regulation of RAD51. Moreover, this combined regimen significantly induced apoptosis in Saos-2 cells compared to mono drugs. The co-treatment of quercetin and Cis can accelerate DNA damage, DNA damage response, and apoptosis while interfering with the DNA repair process in Saos-2 cells. Moreover, this combination provokes the tumor suppressor miR-22 expression in these cells.

Self-Assembling Imageable Silk Hydrogels for the Focal Treatment of Osteosarcoma.

Background: The standard treatment for osteosarcoma comprises complete surgical resection and neoadjuvant chemotherapy, which may cause serious side effects and partial or total limb loss. Therefore, to avoid the disadvantages of traditional treatment, we developed self-assembling imageable silk hydrogels for osteosarcoma. Methods: We analysed whether iodine induced apoptosis in MG-63 and Saos-2 cells by using CCK-8 and flow cytometry assays and transmission electron microscopy. Western blotting was used to analyse the pathway of iodine-induced apoptosis in osteosarcoma cells. PEG400, silk fibroin solution, polyvinylpyrrolidone iodine (PVP-I), and meglumine diatrizoate (MD) were mixed to produce an imageable hydrogel. A nude mouse model of osteosarcoma was established, and the hydrogel was injected locally into the interior of the osteosarcoma with X-ray guidance. The therapeutic effect and biosafety of the hydrogel were evaluated. Results: Iodine treatment at 18 and 20 µM for 12 h resulted in cell survival rate reduced to 50 ± 2.1% and 50.5 ± 2.7% for MG-63 and Sao-2 cells, respectively (p < 0.01). The proportion of apoptotic cells was significantly higher in the iodine-treatment group than in the control group (p < 0.05), and apoptotic bodies were observed by transmission electron microscopy. Iodine could regulate the death receptor pathway and induce MG-63 and Saos-2 cell apoptosis. The hydrogels were simple to assemble, and gels could be formed within 38 min. A force of less than 50 N was required to inject the gels with a syringe. The hydrogels were readily loaded and led to sustained iodine release over 1 week. The osteosarcoma volume in the PEG-iodine-silk/MD hydrogel group was significantly smaller than that in the other three groups (p < 0.001). Caspase-3 and poly (ADP-ribose) polymerase (PARP) expression levels were significantly higher in the PEG-iodine-silk/MD hydrogel group than in the other three groups (p < 0.001). Haematoxylin and eosin (H&E) staining showed no abnormalities in the heart, liver, spleen, lung, kidney, pancreas or thyroid in any group. Conclusions: Self-assembling imageable silk hydrogels could be injected locally into osteosarcoma tissues with X-ray assistance. With the advantages of good biosafety, low systemic toxicity and minimal invasiveness, self-assembling imageable silk hydrogels provide a promising approach for improving the locoregional control of osteosarcoma.

Design and development of novel 1,2,3-triazole chalcone derivatives as potential anti-osteosarcoma agents via inhibition of PI3K/Akt/mTOR signalling pathway.

Osteosarcoma (OS) is an uncommon tumour that mainly affects bone in children and adolescents. The current treatment options of OS are of limited significance due to their immense side effects. In the present manuscript, we have developed a novel series of 1,2,3-triazole chalcone derivatives as potential agents against OS. The compounds were synthesized and evaluated for their PI3K and mTOR inhibitory activity using luminescent kinase assay, and Lance ultra assay, resp. The entire set of compounds showed significant to moderate inhibition of both kinases in the nanomolar range. The three most active compounds: 4e (N-(4-(3-(1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)acryloyl)phenyl)-4-nitrobenzamide), 4f (N-(4-(3-(1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)acryloyl)phenyl)-4-chlorobenzamide) and 4g (4-bromo-N-(4-(3-(1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)acryloyl)phenyl)benzamide), were evaluated for anticancer activity against human OS cancer cell line (MG-63), liver cancer cell line (HepG2), lung cancer cell line (A549) and cervical cancer (HeLa), using MTT assay. Among the tested series, compound 4e showed a better inhibitory profile than gedatolisib against PI3K and was approximately comparable to that of gedatolisib against mTOR. The most significant inhibitory activity was observed for compound 4e against all cell lines (MG-63, HepG2, A549 and HeLa), still somewhat lower to comparable to that of gedatolisib, but with the highest potency against MG-63 cells. Compound 4e was further tested for anti-cancer activity against other OS cells and showed to be equipo-tent to gedatolisib against U2OS and Saos-2 cells. Moreover, it was also found non-toxic to normal cells (BEAS-2B and MCF 10A). The effect of compound 4e was further determined on apoptosis of Saos-2 cells by Annexin-PI assay, where it significantly amplified the percentage of apoptotic cells. Novel 1,2,3-triazole chalcone derivatives are potential agents against OS.

Necdin, one of the important pathway proteins in the regulation of osteosarcoma progression by microRNA-200c

ABSTRACT MicroRNA-200c (miR-200c) generally acts as a tumor suppressor in multiple cancer types and a promising therapeutic target in tumorigenesis. However, only a few studies have explained the role of miR-200c in the development of osteosarcoma (OS). In this study, we investigated the role of miR-200c in OS progression and identified the regulatory pathway protein NDN involved in inhibiting the occurrence and development of OS. Firstly, we found that miR-200c is downregulated in OS cells and tissues. As well, in vitro and in vivo experiments showed that upregulating miR-200c inhibits the proliferation, invasion, metastasis of Saos-2 cells, promotes the apoptosis of Saos-2 cells and suppresses tumor growth in mice, indicating miR-200c plays a major role in regulating the OS progression. Furthermore, bioinformatics analysis showed that an anti-tumor protein, necdin (NDN), might be a potential target by miR-200c. To verify this hypothesis, we measured the expression level of NDN in OS cells and tissues and found NDN is downregulated, suggesting NDN is functional in OS progression. Moreover, we found that the expression levels of NDN and miR-200c in in vivo and in vitro experiments were positively correlated. However, the results of dual-luciferase reporter gene experiment showed miR-200c does not directly act on the 3ʹ untranslated region (UTR) of NDN gene, indicating that NDN might be an important pathway protein which regulates OS progression in the presence of miR-200c. Therefore, miR-200c/NDN could be potential targets for developing effective treatment against OS.

The Effect of TQ and Cis in OS.

Osteosarcoma (OS) is a primary bone sarcoma with a high recurrence rate and poorer prognosis. The application of natural agents in combinational therapies can increase the efficacy of treatment and decrease the side effects. Herein, we aimed to evaluate the effects of Thymoquinone (TQ) combined with Cisplatin on apoptosis and its underlying mechanisms in the Saos-2 cells. The effects of TQ and Cisplatin on Saos-2 cell viability were measured using an MTT assay. Western blotting was applied for the measurement of γH2AX protein expression. The expression levels of 8-Hydroxy-2'-deoxyguanosine (8-oxo-dG) were evaluated by enzyme-linked immunosorbent assay (ELISA). DCFH-DA fluorescence dye was used to detect reactive oxygen species (ROS) formation. For evaluation of apoptosis, flow cytometry was employed. TQ dramatically promotes the cytotoxic effects of Cisplatin. TQ considerably enhanced the expression levels of 8-oxo-dG and γ-H2AX in Saos-2 cells. After TQ treatment, ROS levels were increased; furthermore, TQ treatment resulted in the potentiation of Cisplatin-induced apoptosis in Saos-2 cells compared to either TQ or Cisplatin treated cells. In general, TQ plus Cisplatin resulted in potentiated cellular cytotoxicity by increasing ROS level and inducing oxidative DNA damage, leading to the potent induction of apoptosis in tumor cells.

Curcumin Ameliorates Palmitic Acid-Induced Saos-2 Cell Apoptosis Via Inhibiting Oxidative Stress and Autophagy.

Objectives We aimed to determine the effects of curcumin on palmitic acid- (PA-) induced human osteoblast-like Saos-2 cell apoptosis and to explore the potential molecular mechanisms in vitro level. Methods Saos-2 cell were cultured with PA with or without curcumin, N-acetylcysteine (NAC, anti-oxidant), 3-methyladenine (3-MA, autophagy inhibitor) AY-22989 (autophagy agonist) or H2O2. Then, the effects of PA alone or combined with curcumin on viability, apoptosis, oxidative stress, and autophagy in were detected by CCK-8, flow cytometry assay and western blot. Results We found that autophagy was induced, oxidative stress was activated, and apoptosis was promoted in PA-induced Saos-2 cells. Curcumin inhibited PA-induced oxidative stress, autophagy, and apoptosis in Saos-2 cells. NAC successfully attenuated oxidative stress and apoptosis, and 3-MA attenuated oxidative stress and apoptosis in palmitate-induced Saos-2 cells. Interestingly, NAC inhibited PA-induced autophagy, but 3-MA had no obvious effects on oxidative stress in PA-treated Saos-2 cells. In addition, curcumin inhibited H2O2 (oxidative stress agonist)-induced oxidative stress, autophagy, and apoptosis, but curcumin had no obvious effect on AY-22989 (autophagy agonist)-induced autophagy and apoptosis. Conclusion The present study demonstrated that oxidative stress is an inducer of autophagy and that curcumin can attenuate excess autophagy and cell apoptosis by inhibiting oxidative stress in PA-induced Saos-2 cells.

Flavonoid compound breviscapine suppresses human osteosarcoma Saos-2 progression property and induces apoptosis by regulating mitochondria-dependent pathway.

This study was aimed to investigate the ability of a flavonoid compound breviscapine (BVP) to suppress growth and elicit apoptosis in human osteosarcoma (OS) Saos‐2 cells. The cells were cultured in vitro and treated with three concentrations of BVP (80, 160, and 320 μg/ml). Moreover, C57 mice were injected with Saos‐2 cells to establish a subcutaneous xenograft model, and they were subsequently treated with three doses of BVP via intraperitoneal injection. The viability of the cells was examined by the Cell Counting Kit‐8 method. The apoptotic cells were assessed by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The tumor volume and weight were monitored from day 3 through day 21 after the last injection. The expression of bax, bcl‐2, and cytochrome c (cyt c) mRNA was detected by a real‐time polymerase chain reaction. The protein levels of bax, bcl‐2, cyt c, caspase 3, and caspase 9 were evaluated by Western blot. The expression and distribution of bcl‐2 and bax in tissues were detected by immunohistochemistry. Compared with the control group, BVP treatment inhibited cell proliferation and induced apoptosis of Saos‐2 cells in vitro. Consistently, treatment of mice bearing transplanted tumors with BVP suppressed the growth of OS tumors and promoted cell apoptosis; it also reduced tumor volume and weight. Mechanistically, BVP‐induced apoptosis was mediated by the mitochondria‐dependent pathway, as evidenced by the increased expression of bax and cyt c and the decreased expression of bcl‐2, as well as activation of caspase 9 and caspase 3 in vitro and in vitro. Collectively, BVP inhibits growth and promotes apoptosis of OS by activating the mitochondrial apoptosis pathway.

CacyBP/SIP promotes tumor progression by regulating apoptosis and arresting the cell cycle in osteosarcoma.

Osteosarcoma (OS) is the most common primary malignant bone tumor in pediatric and adolescent patients. The calcyclin-binding protein/Siah-1-interacting protein (CacyBP/SIP) performs an essential function in cell proliferation and apoptosis. The present study investigated the effect of CacyBP/SIP in OS cell proliferation and apoptosis. CacyBP/SIP mRNA expression levels were evaluated in four OS cell lines by quantitative PCR. CacyBP/SIP expression was downregulated in Saos-2 cells using a lentivirus transfection system and the transfection efficiency was analyzed. The effects of CacyBP/SIP downregulation on Saos-2 cell proliferation and colony-formation ability were evaluated by MTT and colony-formation assays. The effect of CacyBP/SIP knockdown on Saos-2 cell cycle and apoptosis was analyzed by flow cytometry cell sorting. The Cancer Genome Atlas (TCGA) data was analyzed for validation. Human OS cell lines Saos-2, MG-63, HOS and U20S expressed CacyBP/SIP mRNA. CacyBP/SIP knockdown significantly inhibited cell proliferation and colony-formation ability. G1/S phase arrest was induced by CacyBP/SIP downregulation, which also resulted in the downregulation of CDK and cyclins and the upregulation of p21. In addition, CacyBP/SIP downregulation induced Saos-2 cell apoptosis mediated by Bax and Bcl-2. High expression of CacyBP/SIP was significantly associated with poor prognosis in TCGA sarcoma database. Thus, CacyBP/SIP performs important functions in the proliferation and apoptosis of human OS cells.

MicroRNA-708-5p affects proliferation and invasion of osteosarcoma cells by targeting URGCP.

Osteosarcoma is an aggressive cancer of the skeletal system which remains a challenge for the current therapeutic strategies due to unclear etiology and molecular mechanisms of pathogenesis. The current study aimed to determine the expression levels, role and molecular mechanism of microRNA-708-5p (miR-708-5p) in the development of osteosarcoma. The expression level of miR-708-5p was detected using reverse transcription-quantitative polymerase chain reaction. miR-708-5p was overexpressed in SaOS-2 cells using miR-708-5p mimics. Cell viability, apoptosis, migration and invasion were determined using Cell Counting kit-8 assay, flow cytometry, wound healing and transwell assays, respectively. The results indicated that miR-708-5p was significantly downregulated in osteosarcoma tissues and cells, and its overexpression significantly inhibited cell viability, invasion and migration and induced apoptosis of SaOS-2 cells. Furthermore, the present results indicated that miR-708-5p directly targeted the 3′-untranslated region of up-regulator of cell proliferation (URGCP) and negatively regulated its expression in SaOS-2 cells. Taken together, the current study suggested that miR-708-5p may inhibit the growth and invasion of osteosarcoma cells via regulating the URGCP/NF-κB signaling pathway. Further research on these molecules in osteosarcoma may provide novel insights into the target therapy for this disease.

Palmitic acid induces human osteoblast-like Saos-2 cell apoptosis via endoplasmic reticulum stress and autophagy.

Palmitic acid (PA) is the most common saturated long-chain fatty acid in food that causes cell apoptosis. However, little is known about the molecular mechanisms of PA toxicity. In this study, we explore the effects of PA on proliferation and apoptosis in human osteoblast-like Saos-2 cells and uncover the signaling pathways involved in the process. Our study showed that endoplasmic reticulum (ER) stress and autophagy are involved in PA-induced Saos-2 cell apoptosis. We found that PA inhibited the viability of Saos-2 cells in a dose- and time-dependent manner. At the same time, PA induced the expression of ER stress marker genes (glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP)), altered autophagy-related gene expression (microtubule-associated protein 1 light chain 3 (LC3), ATG5, p62, and Beclin), promoted apoptosis-related gene expression (Caspase 3 and BAX), and affected autophagic flux. Inhibiting ER stress with 4-PBA diminished the PA-induced cell apoptosis, activated autophagy, and increased the expression of Caspase 3 and BAX. Inhibiting autophagy with 3-MA attenuated the PA and ER stress-induced cell apoptosis and the apoptosis-related gene expression (Caspase 3 and BAX), but seemed to have no obvious effects on ER stress, although the CHOP expression was downregulated. Taken together, our results suggest that PA-induced Saos-2 cell apoptosis is activated via ER stress and autophagy, and the activation of autophagy depends on the ER stress during this process.

Anti-ganglioside GD2 monoclonal antibody synergizes with cisplatin to induce endoplasmic reticulum-associated apoptosis in osteosarcoma cells.

Cisplatin is an effective chemotherapeutic agent for osteosarcoma (OS) and has been shown to induce endoplasmic reticulum (ER) stress-associated apoptosis in human cancer cells. Ganglioside GD2-specific antibodies can inhibit tumor cell viability without involvement of the immune system. A recent study has shown that antiGD2 monoclonal antibody (mAb) 14G2a effectively inhibits the viability and invasiveness of human OS cells. In this study, we explored the effect of anti-GD2 mAb and cisplatin alone and in combination on ER stress-associated apoptosis in osteosarcoma cells. MG-63 and Saos-2 human OS cells were treated with cisplatin and/or an-GD2 mAb 14G2a for 48 hours. Cisplatin and 14G2a dose-dependently induced apoptosis in MG-63 and Saos-2 cells. They in combination induced 70%-77% of apoptosis in MG-63 cells and 79%-85% of apoptosis in Saos-2 cells, exhibiting a synergistic effect stronger than addition of their individual effects over the control level. Showing no significant effect on the expression of protein kinase RNA-like ER kinase (PERK), cisplatin and 14G2a exhibited a marked synergistic effect on inducing phosphorylation/activation of PERK, phosphorylation/inactivation of eukaryotic translation initiation factor 2α (eIF2α), expression of CHOP, in parallel to inducing the caspase-3 activity and apoptosis in MG-63 and Saos-2 cells. The effects were abolished by lentivirus-mediated knockdown of PERK. Particularly, PERK knockdown abolished 63% and 65% of the combined apoptotic effect of cisplatin and 14G2a on MG-63 and Saos-2 cells, respectively. In conclusion, this study provides the first evidence supporting that cisplatin and 14G2a synergize to induce ER stress-associated apoptosis in human OS cells through activating the PERK ER stress pathway by synergistically inducing phosphorylation/activation of PERK. Our findings add new insights into the pharmacologic effects of anti-GD2 mAb in anticancer treatment and suggest that cisplatin plus anti-GD2 mAb could be a new effective therapeutic strategy for OS.

MicroRNA-21 Inhibits the Apoptosis of Osteosarcoma Cell Line SAOS-2 via Targeting Caspase 8.

Currently, multiple microRNAs (miRNAs) have been found to play vital roles in the pathogenesis of osteosarcoma. This study aimed to investigate the role of miR-21 in osteosarcoma. The level of miR-21 in 20 pairs of osteosarcoma and corresponding adjacent tissues was monitored by qPCR. Human osteosarcoma cell line SAOS-2 was transfected with either miR-21 mimic or miR-21 inhibitor, and then cell viability, survival, and apoptosis were measured by MTT, colony formation assay, and flow cytometry. A target of miR-21 was predicted by the microRNA.org database and verified in vitro by using luciferase reporter, qPCR, and Western blot analyses. Finally, cells were cotransfected with siRNA against caspase 8 and miR-21 inhibitor, and the apoptotic cell rate was determined again. Results showed that the mRNA level of miR-21 was highly expressed in osteosarcoma tissues compared with adjacent tissues. Overexpression of miR-21 improved cell viability and survival but suppressed apoptosis. Caspase 8 was a direct target of miR-21, and it was negatively regulated by miR-21. Moreover, miR-21 suppression attenuated caspase 8 silencing and induced the decrease in apoptosis. In conclusion, overexpression of miR-21 suppressed SAOS-2 cell apoptosis via directly targeting caspase 8.

Tumstatin induces apoptosis and stimulates phosphorylation of p65NF-κB in human osteoblastic osteosarcoma Saos-2 cells.

The present study was aimed to investigate the effect of tumstatin on inhibition of proliferation and induction of apoptosis in Saos-2 human osteosarcoma cells and to understand the mechanism involved. Inhibition of cell proliferation was analyzed by MTT assay and induction of apoptosis through nuclear fragmentation assay. Viability of Saos-2 cells was reduced to 19% on treatment with 25µM concentration of tumstatin after 48h. Presence of characteristic apoptotic nuclei, rounded cell shape and shrunken size were caused by tumstatin treatment at 25µM concentration. The level of mRNA corresponding to PTEN, FasR and FasL was increased significantly in tumstatin treated Saos-2 cells compared to untreated control. Investigation of the mechanism revealed NF-κB activation by phosphorylation on serine 536. The activated NF-κB was translocated into the nucleus from the cytoplasm on treatment with tumstatin. Degradation of the IκBα by tumstatin was found to be much slower compared to that induced by treatment with TNF-α. Thus, tumstatin inhibits proliferation and induces apoptosis in Saos-2 cells through activation of NF-κB and its translocation to the nucleus. Therefore, tumstatin can play an important role in the treatment of osteosarcoma.

Anti-citrullinated protein antibodies promote apoptosis of mature human Saos-2 osteoblasts via cell-surface binding to citrullinated heat shock protein 60

We hypothesized that anti-citrullinated protein antibodies (ACPAs) react with osteoblast surface citrullinated proteins and affect cell function, leading to joint damage in patients with rheumatoid arthritis (RA). First, we purified ACPAs by cyclic citrullinated peptide (CCP)-conjugated affinity column chromatography. The cognate antigens of ACPAs on Saos-2 cells, a sarcoma osteogenic cell line generated from human osteoblasts, were probed by ACPAs, and the reactive bands were analyzed using proteomic analyses. We found that ACPAs bind to Saos-2 cell membrane, and several protein candidates, including HSP60, were identified. We then cloned and purified recombinant heat shock protein 60 (HSP60) and citrullinated HSP60 (citHSP60) and investigated the effect of ACPAs on Saos-2 cell. We confirmed that HSP60 obtained from Saos-2 cell membrane were citrullinated and reacted with ACPAs, which induces Saos-2 cells apoptosis via binding to surface-expressed citHSP60 through Toll-like receptor 4 signaling. ACPAs promoted interleukin (IL)-6 and IL-8 expression in Saos-2 cells. Finally, sera from patients with RA and healthy controls were examined for their titers of anti-HSP60 and anti-citHSP60 antibodies using an enzyme-linked immunosorbent assay. The radiographic change in patients with RA was evaluated using the Genant-modified Sharp scoring system. Patients with RA showed higher sera titers of anti-citHSP60, but not anti-HSP60, antibodies when compared with controls. In addition, the anti-citHSP60 level was positively associated with increased joint damage in patients with RA. In conclusion, Saos-2 cell apoptosis was mediated by ACPAs via binding to cell surface-expressed citHSP60 and the titer of anti-citHSP60 in patients with RA positively associated with joint damage.

GDC-0152 attenuates the malignant progression of osteosarcoma promoted by ANGPTL2 via PI3K/AKT but not p38MAPK signaling pathway.

Angiopoietin-like protein 2 (ANGPTL2) plays an important role in inflammatory carcinogenesis and tumor metastasis. The compound GDC-0152 is a peptidomimetic small molecule antagonist of inhibitor of apoptosis (IAP) proteins with antitumor activity. However, the interaction between ANGPTL2 and GDC-0152 has not been studied. It has been proven that ANGPTL2 promotes metastasis of osteosarcoma. Therefore, in the present study, the effect of GDC-0152 on the malignant progression of osteosarcoma promoted by ANGPTL2 was investigated. Human osteosarcoma cell line SaOS2 cells were pre-treated or non-treated with GDC-0152 and then exposed to recombinant human ANGPTL2. The viability of SaOS2 cells was determined by MTT assay, the migration of SaOS2 cells was analyzed by chamber migration assay kit, and the SaOS2 cell apoptosis was determined by fluorescence-activated cell sorting (FACS) and nuclear staining. Treatment with ANGPTL2 increased SaOS2 cell growth and migration and decreased cell apoptosis. The increased cell growth and decreased cell apoptosis were significantly attenuated in SaOS2 cells receiving GDC-0152. However, the ANGPTL2-increased SaOS2 cell migration was not inhibited by GDC-0152 treatment. Furthermore, western blot analysis showed that the activation of phosphatidyl inositol 3-kinase (PI3K) (p85), PI3K (p110), protein kinase B (Akt) (Ser473), Akt (Thr308) and p38 mitogen-activated protein kinase (p38MAPK) were upregulated by ANGPTL2. Quantitative real-time polymerase chain reaction (qTR-PCR) and gelatin zymography showed that the mRNA expression and activity of matrix metalloproteinase-9 (MMP-9) and matrix metalloproteinase-2 (MMP-2) were also increased by ANGPTL2. The upregulated activation of PI3K and Akt were significantly suppressed by the treatment of GDC-0152. In contrast, GDC-0152 did not suppress ANGPTL2-induced p38MAPK phosphorylation, MMP-9/MMP-2 mRNA expression or MMP-9/MMP-2 activity. Taken together, these data indicate that GDC-0152 attenuates the malignant progression of osteosarcoma promoted by ANGPTL2 via PI3K/AKT but not p38MAPK signaling pathway. The present study indicated a novel therapeutic strategy to inhibit tumor growth by indirectly preventing ANGPTL2 signaling.

MiR-218 expression in osteosarcoma tissues and its effect on cell growth in osteosarcoma cells.

To investigate the expression of miR-218 and its clinical significance in osteosarcoma tissues and explore its effect on proliferation and apoptosis in osteosarcoma cells. miR-218 expression was detected in 76 samples of surgically resected osteosarcoma and matched normal tumor-adjacent tissues using quantitative reverse transcription polymerase chain reaction (qRT-PCR). MiR-218 was over-expressed by exogenous miR-218 plasmids in Saos-2 cells, and then BrdU cell proliferation assay and flow cytometry were used to determine cell proliferation and apoptosis. The expression of miR-218 in osteosarcoma tissues was significantly lower than those in normal tumor-adjacent tissues (t=8.735, P<0.001). MiR-218 expression in tumor tissues was significantly correlated with tumor size (χ(2)=5.380, P=0.020), clinical stage (χ(2)=6.692, P=0.010) and distant metastasis (χ(2)=4.180, P=0.041). MiR-218 was obviously over-expressed by exogenous miR-218 plasmids (t=19.42, P<0.001), and miR-218 overexpression significantly reduced cell proliferation (t=9.045, P<0.001) and induced apoptosis (t=12.38, P<0.001) in Saos-2 cells. The low-expression of miR-218 is correlated with the poor clinicopathological features in osteosarcoma. Moreover, miR-218 overexpression reduces cancer cell proliferation and induces apoptosis in Saos-2 cells, suggesting that miR-218 may play a key role in the progression of human osteosarcoma.

Effect of microRNA-101 on proliferation and apoptosis of human osteosarcoma cells by targeting mTOR.

Studies have proved that microRNA-101 (miR-101) functions as a tumor suppressor and is associated with growth and apoptosis of various human cancers. However, the role of miR-101 in osteosarcoma and the possible mechanism by which miR-101 affects the tumor growth and apoptosis have not been fully elucidated. In this study, we found that the expression of miR-101 was down-regulated in osteosarcoma tissues and Saos-2 cell line as compared with that in adjacent non-neoplastic bone tissues and the osteoblastic cell line. To better characterize the role of miR-101 in osteosarcoma, we used a gain-of-function analysis by transfecting human osteosarcoma cell line Saos-2 with chemically synthesized miR-101 mimics. The results showed that overexpression of miR-101 inhibited the proliferation and promoted the apoptosis of Saos-2 cells. Meanwhile, bioinformatic analysis demonstrated that mTOR gene was a direct target of miR-101. Overexpression of miR-101 significantly decreased the expression of mTOR at both mRNA and protein levels in Saos-2 cells, consequently inhibiting Saos-2 cells proliferation and promoting cells apoptosis in an mTOR-dependent manner. Taken together, these data suggest that miR-101 may act as a tumor suppressor, which is commonly downregulated in both osteosarcoma tissues and cells. mTOR plays an important role in mediating miR-101 dependent biological functions in osteosarcoma. Reintroduction of miR-101 may be a novel therapeutic strategy by down-regulating mTOR expression.