Human Osteosarcoma Cell Line Research Articles

Polymeric Nanoformulation of Zoledronic Acid Rescues Osteoblasts From the Harmful Effect of its Native Form: An in-Vitro Investigation of Cytotoxic Potential on Osteoblasts and Osteosarcoma Cells.

Osteosarcoma is a malignant tumor, fatal for pediatric patients who do not respond to chemotherapy, alternative therapies and drugs could provide better outcomes.Zoledronic acid belonging to the class of bisphosphonates (BP) has direct antitumor ability preventing Ras GTPases modification and stimulating apoptosis. Despite advances in maintaining balance in skeletal events and direct anticancer property, Zoledronic acid causes some cytotoxicity to normal healthy pre-osteoblast cells, hampering mineralization and differentiation. The study reports the preparation and evaluation of a nanoformulation that can diminish the existing drawbacks of administering native Zoledronic The cytotoxic effect is evaluated on bone cancer cells and healthy bone cells with 3 different cell lines namely, K7M2 (mouse osteosarcoma cell line), SaOS2 (human osteosarcoma cell line) and MC3T3E1 (healthy cell counterpart). It is observed that Zoledronic acid nanoformulation is uptaken more (95%) in K7M2 whereas in MC3T3-E1 the percent population internalizing NPs is 45%. Zoledronic acid has a sustained release of 15% after 96h from the nanoparticle which leads to a rescuing effect on the normal pre-osteoblast cells. In conclusion, we can state that Zoledronic acid nanoformulation can be used as a good platform for a sustained release system with minimum side effects to normal bone cells. This article is protected by copyright. All rights reserved.

Exploring the Antimicrobial, Anticancer, and Apoptosis Inducing Ability of Biofabricated Silver Nanoparticles Using Lagerstroemia speciosa Flower Buds against the Human Osteosarcoma (MG-63) Cell Line via Flow Cytometry.

Biosynthesized nano-composites, such as silver nanoparticles (AgNPs), can be engineered to function as smart nano-biomedicine platforms for the detection and management of diverse ailments, such as infectious diseases and cancer. This study determined the eco-friendly fabrication of silver nanoparticles using Lagerstroemia speciosa (L.) Pers. flower buds and their efficacy against antimicrobial and anticancer activities. The UV-Visible spectrum was found at 413 nm showing a typical resonance spectrum for L. speciosa flower bud extract-assisted silver nanoparticles (Ls-AgNPs). Fourier transform infrared analysis revealed the presence of amines, halides, and halogen compounds, which were involved in the reduction and capping agent of AgNP formation. X-ray diffraction analysis revealed the face-centered cubic crystals of NPs. Energy dispersive X-ray verified the weight of 39.80% of silver (Ag), TEM analysis revealed the particles were spherical with a 10.27 to 62.5 nm range, and dynamic light scattering recorded the average particle size around 58.5 nm. Zeta potential showed a significant value at -39.4 mV, and finally, thermo-gravimetric analysis reported higher thermal stability of Ls-AgNPs. Further, the obtained Ls-AgNPs displayed good antimicrobial activity against clinical pathogens. In addition, a dose-dependent decrease in the anticancer activity by MTT assay on the osteosarcoma (MG-63) cell line showed a decrease in the cell viability with increasing in the concentration of Ls-AgNPs with an IC50 value of 37.57 µg/mL. Subsequently, an apoptotic/necrosis study was conducted with the help of Annexin-V/PI assay, and the results indicated a significant rise in early and late apoptosis cell populations. Therefore, green synthesized Ls-AgNPs were found to have potent antimicrobial and anticancer properties making them fascinating choices for future bio-medical implementations.

Fabrication of highly ordered willemite/PCL bone scaffolds by 3D printing: Nanostructure effects on compressive strength and in vitro behavior

In this study, first willemite (Zn2SiO4) micro and nano-powders were synthesized by the sol-gel method. X-ray diffraction (XRD), transmission electron microscopy (TEM), and dynamic light scattering (DLS) were applied to characterize the crystalline phases and particle size of powders. Then polycaprolactone (PCL) polymer scaffolds containing 20 wt% willemite were successfully fabricated by the DIW 3D printing (direct ink writing) method. The effects of willemite particle size on compressive strength, elastic modulus, degradation rate, and bioactivity of the composite scaffolds were investigated. The results showed that nanoparticle willemite/PCL (NW/PCL) scaffolds had 33.1% and 58.1% higher compressive strength and the elastic modulus of NW/PCL were 1.14 and 2.45 times better compared to micron size willemite/PCL (MW/PCL) and pure PCL scaffolds, respectively. Scanning electron microscopy (SEM) images and Energy-dispersive X-ray spectroscopy map (EDS map) results indicated that willemite nanoparticles, unlike microparticles, were smoothly embedded in the scaffold struts. In vitro tests also revealed an improvement in bone-like apatite formation ability and an increase in the degradation rate up to 2.17% by decreasing the willemite particle size to 50 nm. In addition, NW/PCL rendered significant enhancement in cell viability and cell attachment during the culture of MG-63 human osteosarcoma cell line. Nanostructure had also a positive effect on ALP activity and biomineralization in vitro.

Prognostic value and mechanism of long non-coding RNA DLEU1 in osteosarcoma

To investigate the prognostic value and mechanism of long non-coding RNA DLEU1(LncRNA DLEU1) in osteosarcoma. The tissue samples and clinical data of 86 patients with osteosarcoma treated by orthopaedic surgery in our hospital from January 2012 to December 2014 were retrospectively collected. The expression of LncRNA DLEU1 in pathological tissues was detected by qRT-PCR, then the patients were divided into high and low expression of LncRNA DLEU1 groups. Osteosarcoma cell line HOS was divided into two groups, down-regulated expression group (si-DLEU1 group) and negative control group (si-NC group). LncRNA DLEU1 siRNA and negative control sequence were transfected by Lipofectamine 3000. Chi-square test was used to analyze the relationship between the expression of LncRNA DLEU1 and the clinicopathological factors of osteosarcoma. Kaplan-Meier method was used to compare the difference of the overall survival rate of osteosarcoma patients between the high and low expression groups of LncRNA DLEU1. The risk factors affecting the overall survival rate of osteosarcoma were analyzed by single factor and multifactor analysis. The number of invasive cells in the two groups was determined and compared by Transwell assay. The expression of LncRNA DLEU1 in osteosarcoma tissue was higher than that in adjacent tissues (P<0.001). The expression of LncRNA DLEU1 in human osteosarcoma cell lines (MG-63, U-2 OS, and HOS) was significantly higher than that in human osteoblast line hFOB 1.19 (P<0.001). The expression of LncRNA DLEU1 was significantly correlated with Enneking stage (P<0.001), distant metastasis (P=0.016), and histological grade (P=0.028). The 1-year overall survival rate of the LncRNA DLEU1 high expression group was significantly higher than that of the low expression group (90.7% vs 60.5%, P<0.001). The 5-year overall survival rate of the LncRNA DLEU1 high expression group was significantly higher than that of the low expression group (32.6% vs 11.6%, P<0.001). Univariate analysis showed that Enneking stage (P<0.001), tumor size (P=0.043), distant metastasis (P<0.001), histological grade (P<0.001), and expression of LncRNA DLEU1 (P<0.001) were risk factors for overall survival of osteosarcoma patients. Multivariate analysis showed that high expression of LncRNA DLEU1 [HR=1.948, 95% CI(1.141, 3.641), P=0.012] and distant metastasis[HR=4.108, 95% CI(2.169, 7.780), P<0.001] were independent risk factors for overall survival of osteosarcoma patients. The number of invasive cells in si-DLEU1 group was significantly lesser than that in si-NC group(139±13 vs 357±31, P<0.001). High expression of LncRNA DLEU1 is a molecular marker affecting the prognosis of osteosarcoma patients. Downregulation of LncRNA DLEU1 can inhibit the invasion of osteosarcoma cells.

Cowpea isoflavones enhance the osteoblast differentiation and antioxidant capacity in synergy with vitamin D and β-carotene: A mechanistic in vitro study.

Background: Osteoporosis (OS) is a pathological condition that makes bones susceptible to fractures by affecting the balance between bone formation and resorption. Recent literature uncovered the possible potential of bioactive compounds with antioxidant mechanisms to counter the issue. Cowpea (CP) isoflavones based on our previous study, vitamin D and natural antioxidant β-carotene for its pleotropic protective effects were assessed alone and in combination. Aim: The study aims to assess the antioxidant and osteoblast differentiation abilities of cowpea isoflavones alone and in combination of vitamin D (VD) and β-carotene (BC) in the human osteosarcoma cell line Saos2. Methods: Saos2 cells were maintained in cell culture conditions and concentrations of CP extract (genistein + daidzein), BC and VD required to increase cell proliferation were estimated using MTT assay. Upon treating cells with the EC50 concentrations, lysates were prepared and levels of alkaline phosphatase (ALP) and osteocalcin were evaluated using ELISA. Oxidative stress parameters and osteoblast differentiation markers were evaluated. Results: CP extract (genistein + daidzein), BC and VD concentrations which enhanced the cell proliferation rate were determined and elevated levels of ALP and osteocalcin upon treatment was observed. Anti-oxidant stress parameters studied showed an increase in cells upon treatment compared to control. Significant alterations in levels of protein involved in osteoblast differentiation are observed upon treatment. Conclusion: Cowpea isoflavones has shown a significant activity against OS by elevating antioxidant parameters and inducing osteoblast differentiation in the present study.

Immunogenic cell death after combined treatment with radiation and ATR inhibitors is dually regulated by apoptotic caspases.

Inhibitors of the ATR kinase act as radiosensitizers through abrogating the G2 checkpoint and reducing DNA repair. Recent studies suggest that ATR inhibitors can also increase radiation-induced antitumor immunity, but the underlying immunomodulating mechanisms remain poorly understood. Moreover, it is poorly known how such immune effects relate to different death pathways such as caspase-dependent apoptosis. Here we address whether ATR inhibition in combination with irradiation may increase the presentation of hallmark factors of immunogenic cell death (ICD), and to what extent caspase activation regulates this response. Human lung cancer and osteosarcoma cell lines (SW900, H1975, H460, U2OS) were treated with X-rays and ATR inhibitors (VE822; AZD6738) in the absence and presence of a pan-caspase inhibitor. The ICD hallmarks HMGB1 release, ATP secretion and calreticulin surface-presentation were assessed by immunoblotting of growth medium, the CellTiter-Glo assay and an optimized live-cell flow cytometry assay, respectively. To obtain accurate measurement of small differences in the calreticulin signal by flow cytometry, we included normalization to a barcoded control sample. Extracellular release of HMGB1 was increased in all the cell lines at 72 hours after the combined treatment with radiation and ATR inhibitors, relative to mock treatment or cells treated with radiation alone. The HMGB1 release correlated largely - but not strictly - with loss of plasma membrane integrity, and was suppressed by addition of the caspase inhibitor. However, one cell line showed HMGB1 release despite caspase inhibition, and in this cell line caspase inhibition induced pMLKL, a marker for necroptosis. ATP secretion occurred already at 48 hours after the co-treatment and did clearly not correlate with loss of plasma membrane integrity. Addition of pan-caspase inhibition further increased the ATP secretion. Surface-presentation of calreticulin was increased at 24-72 hours after irradiation, but not further increased by either ATR or caspase inhibition. These results show that ATR inhibition can increase the presentation of two out of three ICD hallmark factors from irradiated human cancer cells. Moreover, caspase activation distinctly affects each of the hallmark factors, and therefore likely plays a dual role in tumor immunogenicity by promoting both immunostimulatory and -suppressive effects.

Fabrication of a composite 3D-printed titanium alloy combined with controlled in situ drug release to prevent osteosarcoma recurrence

Osteosarcoma is a malignant bone tumor occurring in adolescents. Surgery combined with adjuvant or neoadjuvant chemotherapy is the standard treatment. However, systemic chemotherapy is associated with serious side effects and a high risk of postoperative tumor recurrence, leading to a high amputation rate and mortality in cancer patients. Implant materials that can simultaneously repair large bone defects and prevent osteosarcoma recurrence are in urgent need. Herein, an intelligent system comprising 3D-printed titanium scaffold (TS) and pH-responsive PEGylated paclitaxel prodrugs was fabricated for bone defect reconstruction and recurrence prevention following osteosarcoma surgery. The drug-loaded implants exhibited excellent stability and biocompatibility for supporting the activity of bone stem cells under normal body fluid conditions and the rapid release of drugs in response to faintly acidic environments. An in vitro study demonstrated that five human osteosarcoma cell lines could be efficiently eradicated by paclitaxel released in an acidic microenvironment. Using mice models, we demonstrated that the drug-loaded TS can enable a pH-responsive treatment of postoperative tumors and effectively prevent osteosarcoma recurrence. Therefore, local implantation of this composite scaffold may be a promising topical therapeutic method to prevent osteosarcoma recurrence.

Anticancer effect of crizotinib on osteosarcoma cells by targeting c-Met signaling pathway.

C-Met receptor and its ligand hepatocyte growth factor (HGF) are overexpressed in a variety of osteosarcoma cell lines and osteosarcoma pathological samples. It is suggested that c-Met/HGF plays an important role in the development of osteosarcoma. This study aimed to explore the anticancer effect of the c-Met-targeted drug crizotinib on osteosarcoma (OS) cells. The effects of crizotinib on the proliferation of osteosarcoma cells (SaOS2, MG-63 and MNNG) at different concentrations were detected by CCK8. Human osteosarcoma cell line MG-63 was used as an in vitro model to evaluate the effects of 2.5 μM crizotinib, 5.0 μM crizotinib and DMSO on cell apoptosis, cell cycle, migration and invasion. The expression of the c-Met signaling pathway in osteosarcoma cells was detected by western blot. The results showed that crizotinib inhibited the proliferation of cell lines in a concentration-dependent manner. Crizotinib significantly increased the number of apoptotic cells compared with the control group. Compared with the control group, crizotinib increased G0/G1 phase cells and decreased S phase cells. Compared with the control group, crizotinib inhibited the migration and invasion of osteosarcoma cells and decreased the expression of c-Met/Gab1/STAT5. This study will provide a promising therapeutic target and theoretical basis for the clinical application of crizotinib in osteosarcoma.

Cytotoxic effects of walnut hull extracts against human osteosarcoma cell line (U2OS)

This study aimed to evaluate the total phenolic, total flavonoid content and the antioxidant activity of walnut (Juglans regia L.) hull (JRH) extracts and investigate the cytotoxic effect of different walnut hull extracts on osteosarcoma U2OS cell line and study their impact on apoptosis, autophagy and heat shock protein 90. For this purpose, the dried walnut hulls were extracted by soxhlet apparatus using methanol, ethanol, ethyl acetate and hexane as extraction solvents. The total phenolic content, total flavonoid content and antioxidant activity of each extract were determined by three colorimetric methods: Folin-Ciocalteu method, AlCl3 method and DPPH radical scavenging assay, respectively. In order to investigate the osteosarcoma U2OS cells viability in response to treatment with each JRH extracts, MTT assays were performed. qRT-PCR was used to investigate the cytotoxic effect of the extracts. Caspase-3 and Bcl-2 were checked to determine their role in apoptosis, Beclin 1 for autophagy and HSP90 for viability/proliferation. Overall, the highest extraction yield (41.9 %.), total phenolic content (85.16 ± 5.43 mg GAE/g), total flavonoid content (105.49 ± 6.44 mg RE/g), the antioxidant activity (IC50 = 15.484 μg/mL) and cytotoxic effect on osteosarcoma cell line (IC50 = 180.20 ± 27 μg/mL) were determined in JRH methanol, ethanol, ethyl acetate, ethanol, methanol extracts respectively. The assessments of the present investigation demonstrated that all JRH extracts reduced the viability of osteosarcoma U2OS cells in a dose dependent manner and methanol extract showed the highest anticancer activity by upregulating the gene expression of Caspase-3 and downregulating the gene expressions of Beclin-1, Bcl-2 and HSP-90.

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.

Radiolabeling, Quality Control, and Cell Binding Studies of New 99mTc-Labeled Bisphosphonates: 99mTc-Ibandronate Sodium.

Early detection of bone cancer is critical for treating symptoms, minimizing pain, and increasing overall quality of life. It is critical to develop novel radiopharmaceuticals with high labeling efficiency and stability for the diagnosis of bone cancer. This research aims to design a novel radiopharmaceutical that may be used to diagnose bone cancer. In this study, ibandronate sodium (IBD), a bisphosphonate analog, was radiolabeled with technetium-99m [99mTc] and quality control tests on the newly developed radiopharmaceutical ([99mTc]Tc-IBD) were performed using radioactive thin layer chromatography. After that, the incorporation of [99mTc]Tc-IBD into hydroxyapatite (HA) crystals and a human bone osteosarcoma cell line (U2OS) was tested. According to the results obtained, optimal radiolabeling procedure was obtained for [99mTc]Tc-IBD with 200 μg.mL-1 IBD, 20 μg stannous chloride, and 99mTc with 37 MBq radioactivity. The reaction mixture was adjusted to pH 5.5 and incubated at room temperature for 15 min. The radiochemical purity of [99mTc]Tc-IBD was found to be greater than 95% at room temperature for up to 6 h. Additionally, chromatography analysis showed >95% [99mTc]Tc-IBD complex formation with promising stability for up to 24 h in saline and up to 2 h in cell medium. The percentage binding of IBD to HA was 83.70 ± 3.67 and the logP of [99mTc]Tc-IBD was -1.1014. The radiolabeled complex exhibited a higher rate of cell incorporation to U2OS cells compared to Reduced/Hydrolyzed 99mTcO4 -. The newly produced radiopharmaceutical is very promising according to the results of in vitro cell culture, HA binding, and quality studies, and will be a step forward for further studies in nuclear medicine for bone cancer diagnostics.

Shedding Light on Osteosarcoma Cell Differentiation: Impact on Biomineralization and Mitochondria Morphology.

Osteosarcoma (OS) is the most common primary malignant bone tumor and its etiology has recently been associated with osteogenic differentiation dysfunctions. OS cells keep a capacity for uncontrolled proliferation showing a phenotype similar to undifferentiated osteoprogenitors with abnormal biomineralization. Within this context, both conventional and X-ray synchrotron-based techniques have been exploited to deeply characterize the genesis and evolution of mineral depositions in a human OS cell line (SaOS-2) exposed to an osteogenic cocktail for 4 and 10 days. A partial restoration of the physiological biomineralization, culminating with the formation of hydroxyapatite, was observed at 10 days after treatment together with a mitochondria-driven mechanism for calcium transportation within the cell. Interestingly, during differentiation, mitochondria showed a change in morphology from elongated to rounded, indicating a metabolic reprogramming of OS cells possibly linked to an increase in glycolysis contribution to energy metabolism. These findings add a dowel to the genesis of OS giving new insights on the development of therapeutic strategies able to restore the physiological mineralization in OS cells.

The MMP-2 histone H3 N-terminal tail protease is selectively targeted to the transcription start sites of active genes

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

Targeting CD70 using CAR NK cells to enhance NK cells cytolytic effect against osteosarcoma

Abstract Background: Cure rates for osteosarcoma (OS) have not improved in &amp;gt;30 years and no new effectivetherapies have been identified for metastatic disease. Natural killer (NK) cells have become an attractive tool for cancer immunotherapy. Efficay is improved by direct tumor targeting introducing a specific chimeric antigen receptor (CAR). Emerging knowledge suggests CD70 to be a viable candidate as expression is higher in lung metastases and it has been correlated with tumor escape from immune surveillance. Objective: To assess whether using CD70 CAR-NK cells we can enhance trafficking, homing and proliferation of NK cells and therefore therapeutic efficacy against OS. Methods: We analyzed the cBioportal database to verify CD70 expression in OS. Surface expression of CD70 on human OS cell lines and normal osteoblasts was determined by flow cytometry. CD70 CAR constructs were sequenced, verified and transduced into NK cells. CD70 CAR NK and control NK cells cytolytic activity against OS cells was evaluated by IncuCyte. In vitrocytokine release was measured upon CD70 CAR NK/NK cells exposure to OS cells (IsoPlexis). Results: Database analysis confirmed OS CD70 gene amplification. Flow cytometry showed variable expression of CD70 on OS cells and no expression on human osteoblasts. There was an increase in CD70 CAR NK percent lysis against OS cells at the highest effector:target ratios compared to control NK cells. We found a predominant release in the chemoattractive/stimulatory cytokines upon OS cells exposure to CD70 CAR NK cells. Conclusion: CD70 has immunotherapy potential against OS. CD70 CAR NK cells have increased cytolytic activity against OS cells in vitro. Cytolytic activity may be influenced by the release of specific cytokines.

In Vitro and In Vivo Studies of Antibacterial Coatings on Titanium Alloy Implants for Veterinary Application.

The aim of this work was the evaluation of biological properties of hybrid coatings modified with Ag, Cu, and Zn nanoparticles (NPs) applied on TPLO medical implants by the sol-gel process. The implant coatings enriched with various concentrations of metallic NPs were investigated in the in vitro bactericidal efficacy tests against Gram+ and Gram- bacteria and pathogenic yeast. Next, the designed materials were tested on human osteosarcoma cell lines. The cells adhesion, proliferation, viability, and differentiation were investigated. The cell growth wasevaluated using SEM, and the metallic ion release was measured. The results revealed that the NPs concentration in the hybrid layers decreased with the incubation time. In the last stage, the implants were tested in vivo on six canine patients. Three months after the operation, the radiological evaluation of the performed anastomosis was carried out as well as the histopathological evaluation of tissue regeneration. The strongest bactericidal efficacy was observed for the layers containing AgNPs. Along with an increased concentration of metallic additives, a growing toxic effect was clearly observed. The most pronounced toxic effect was especially evident with the AgNPs concentration exceeding 1 mol %. In all the operated patients, no deviations were found during the follow-up examinations in the postoperative period. The low dose of AgNPs in the hybrid layer facilitated the tissue healing process. It was proven that silver nanoparticles may accelerate the bone healing process. The correct tissue reparation was observed.

Combination of Everolimus and Bortezomib Inhibits the Growth and Metastasis of Bone and Soft Tissue Sarcomas via JNK/p38/ERK MAPK and AKT Pathways.

The combination of the mammalian target of rapamycin and proteasome inhibitors is a new treatment strategy for various tumors. Herein, we investigated the synergistic effect of everolimus and bortezomib on tumor growth and metastasis in bone and soft tissue sarcomas. The antitumor effects of everolimus and bortezomib were assessed in a human fibrosarcoma (FS) cell line (HT1080) and mouse osteosarcoma (OS) cell line (LM8) by MTS assays and Western blotting. The effects of everolimus and bortezomib on HT1080 and LM8 tumor growth in xenograft mouse models were evaluated using tumor volume and the number of metastatic nodes of the resected lungs. Immunohistochemistry was used to evaluate cleaved PARP expression. The combination therapy decreased FS and OS cell proliferation compared with either drug alone. This combination induced more intense p-p38, p-JNK, and p-ERK and activated apoptosis signals, such as caspase-3, compared with single-agent treatment. The combination treatment reduced p-AKT and MYC expression, decreased FS and OS tumor volumes, and suppressed lung metastases of OS. The combination therapy inhibited tumor growth in FS and OS and metastatic progression of OS via the JNK/p38/ERK MAPK and AKT pathways. These results could aid in the development of new therapeutic strategies for sarcomas.

Cytotoxicity of Carbon Nanotubes, Graphene, Fullerenes, and Dots.

The cytotoxicity of carbon nanomaterials is a very important issue for microorganisms, animals, and humans. Here, we discuss the issues of cytotoxicity of carbon nanomaterials, carbon nanotubes, graphene, fullerene, and dots. Cytotoxicity issues, such as cell viability and drug release, are considered. The main part of the review is dedicated to important cell viability issues. They are presented for A549 human melanoma, E. coli, osteosarcoma, U2-OS, SAOS-2, MG63, U87, and U118 cell lines. Then, important drug release issues are discussed. Bioimaging results are shown here to illustrate the use of carbon derivatives as markers in any type of imaging used in vivo/in vitro. Finally, perspectives of the field are presented. The important issue is single-cell viability. It can allow a correlation of the functionality of organelles of single cells with the development of cancer. Such organelles are mitochondria, nuclei, vacuoles, and reticulum. It allows for finding biochemical evidence of cancer prevention in single cells. The development of investigation methods for single-cell level detection of viability stimulates the cytotoxicity investigative field. The development of single-cell microscopy is needed to improve the resolution and accuracy of investigations. The importance of cytotoxicity is drug release. It is important to control the amount of drug that is released. This is performed with pH, temperature, and electric stimulation. Further development of drug loading and bioimaging is important to decrease the cytotoxicity of carbon nanomaterials. We hope that this review is useful for researchers from all disciplines across the world.

COPS6 promotes tumor progression and reduces CD8+ T cell infiltration by repressing IL-6 production to facilitate tumor immune evasion in breast cancer.

Due to poor T cell infiltration, tumors evade immune surveillance. Increased CD8+ T cell infiltration in breast cancer suggests a satisfactory response to immunotherapy. COPS6 has been identified as an oncogene, but its role in regulating antitumor immune responses has not been defined. In this study, we investigated the impact of COPS6 on tumor immune evasion in vivo. Tumor transplantation models were established in C57BL/6 J mice and BALB/c nude mice. Flow cytometry was conducted to identify the role of COPS6 on tumor-infiltrating CD8+ T cells. By analyzing the TCGA and GTEx cohort, we found that COPS6 expression was significantly up-regulated in a variety of cancers. In human osteosarcoma cell line U2OS and non-small cell lung cancer cell line H1299, we showed that p53 negatively regulated COPS6 promoter activity. In human breast cancer MCF-7 cells, COPS6 overexpression stimulated p-AKT expression as well as the proliferation and malignant transformation of tumor cells, whereas knockdown of COPS6 caused opposite effects. Knockdown of COPS6 also significantly suppressed the growth of mouse mammary cancer EMT6 xenografts in BALB/c nude mice. Bioinformatics analysis suggested that COPS6 was a mediator of IL-6 production in the tumor microenvironment and a negative regulator of CD8+ T cell tumor infiltration in breast cancer. In C57BL6 mice bearing EMT6 xenografts, COPS6 knockdown in the EMT6 cells increased the number of tumor-infiltrating CD8+ T cells, while knockdown of IL-6 in COPS6KD EMT6 cells diminished tumor infiltrating CD8+ T cells. We conclude that COPS6 promotes breast cancer progression by reducing CD8+ T cell infiltration and function via the regulation of IL-6 secretion. This study clarifies the role of p53/COPS6/IL-6/CD8+ tumor infiltrating lymphocytes signaling in breast cancer progression and immune evasion, opening a new path for development of COPS6-targeting therapies to enhance tumor immunogenicity and treat immunologically "cold" breast cancer.

Oyster shell-derived nano-hydroxyapatite and proanthocyanidin pretreatment on dentinal tubule occlusion and permeability before and after acid challenge—an in vitro study

This in vitro study evaluated the dentinal tubule occlusion (TO), depth of penetration (DoP), and dentin permeability (DP) of oyster shell-derived nanohydroxyapatite (os-nHAp) with and without 15% proanthocyanidin (PA) pretreatment. os-nHAp was synthesized via the precipitation method and it was characterized. The morphology and particle size of os-nHAp were analyzed using a high-resolution transmission electron microscope (HRTEM). Cytotoxicity of os-nHAp, PA/os-nHAp, and casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) was assessed by (3-(4,5-dimethythiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) assay using human osteosarcoma (MG-63) cell line. One hundred and ninety-seven dentin discs of 3 mm thickness were prepared from the crown portion of extracted human teeth. The dentinal surfaces of the discs were etched for 2 min with 6% citric acid to simulate dentin hypersensitivity. Five discs were randomly selected and the patency of dentinal tubules was confirmed using a scanning electron microscope (SEM). The remaining 192 discs were divided into four groups (n = 48) depending on the type of remineralization as follows: group 1: os-nHAp, group 2: PA/os-nHAp, group 3: CPP-ACP, and group 4: no treatment. The remineralization protocol was followed for 21 days. Out of the 48 dentin discs in each group, 32 discs were used to evaluate dentinal tubule occlusion (TO) and depth of penetration (DoP) using SEM. The remaining 16 discs were subjected to an assessment of dentin permeability (DP) using a hydraulic conductance model. TO, DoP and DP were evaluated after remineralization and acid challenge. Characterization studies confirmed the presence of pure phase apatite. HRTEM confirmed the nanometric particle size of os-nHAp. MTT assay results showed that all the tested materials exhibited >80% cell viability when tested up to a concentration of 100 µg/mL. The results demonstrated a significantly higher mean percentage of TO, DoP, and lesser mean DP after remineralization in groups 1, 2, and 3 (p < 0.05). After the acid challenge, group 3 showed a significant reduction in TO and DoP, and increased DP (p < 0.05). However, no such changes were observed in groups 1 and 2. Within the limitations of this study, it can be concluded that os-nHAp and PA/os-nHAp could serve as potential and durable therapeutic agents in the treatment of dentin hypersensitivity.Graphical

Targeting RNA Polymerase I Transcription Activity in Osteosarcoma: Pre-Clinical Molecular and Animal Treatment Studies

The survival rate of patients with osteosarcoma (OS) has not improved over the last 30 years. Mutations in the genes TP53, RB1 and c-Myc frequently occur in OS and enhance RNA Polymerase I (Pol I) activity, thus supporting uncontrolled cancer cell proliferation. We therefore hypothesised that Pol I inhibition may be an effective therapeutic strategy for this aggressive cancer. The Pol I inhibitor CX-5461 has demonstrated therapeutic efficacy in different cancers in pre-clinical and phase I clinical trials; thus, the effects were determined on ten human OS cell lines. Following characterisation using genome profiling and Western blotting, RNA Pol I activity, cell proliferation and cell cycle progression were evaluated in vitro, and the growth of TP53 wild-type and mutant tumours was measured in a murine allograft model and in two human xenograft OS models. CX-5461 treatment resulted in reduced ribosomal DNA (rDNA) transcription and Growth 2 (G2)-phase cell cycle arrest in all OS cell lines. Additionally, tumour growth in all allograft and xenograft OS models was effectively suppressed without apparent toxicity. Our study demonstrates the efficacy of Pol I inhibition against OS with varying genetic alterations. This study provides pre-clinical evidence to support this novel therapeutic approach in OS.