Saos-2 Cells Research Articles

Circ_KIAA0922 regulates Saos-2 cell proliferation and osteogenic differentiation by regulating the miR-148a-3p/SMAD5 axis and activating the TGF-β signaling pathway.

Circular RNAs (circRNAs) are emerging as important regulators in human disease, but their function in osteoporosis (OP) is not sufficiently known. The aim of this study was to identify the possible molecular mechanism of circ_KIAA0922 in osteogenic differentiation of Saos-2 cells in vitro and the interactions among circ_KIAA0922, miR-148a-3p, and SMAD family member 5 (SMAD5). Circ_ KIAA0922, miR-148a-3p, and SMAD5 were overexpressed by transient transfection. Dual-luciferase reporter assay system was used to analyze the combination among circ_KIAA0922, miR-148a-3p, and SMAD5. In addition, the levels of circ_KIAA0922, miR-148a-3p, SMAD5, osteocalcin (OCN), and runt-related transcription factor 2 (RUNX2) were detected using RT-qPCR or western blot analysis. Alizarin red staining was performed to analyze the degree of osteogenic differentiation under the control of circ_KIAA0922, miR-148a-3p, and SMAD5. We found that circ_KIAA0922 knockdown inhibited the proliferation and osteogenic differentiation of Saos-2 cells. Circ_KIAA0922 directly targeted miR-148a-3p, and miR-148a-3p inhibition reversed the effects of circ_KIAA0922 knockdown on the proliferation and osteogenic differentiation of Saos-2 cells. Overexpression of SMAD5 promoted the proliferation and osteogenic differentiation of Saos-2 cells and attenuated the inhibitory effect of miR-148a-3p on cell proliferation and osteogenic differentiation. In conclusion, circ_ KIAA0922 facilitated Saos-2 cell proliferation and osteogenic differentiation via the circ_KIAA0922/ miR-148a-3p/ SMAD5 axes in vitro, thus providing insights into the mechanism of osteogenic differentiation by circ_ KIAA0922.

Investigation of the efficacy of epidermal growth factor, boric acid and their combination in cartilage injury in rats: An experimental study.

In this study, we aimed to determine the bioefficacy of epidermal growth factor (EGF), boric acid (BA), and their combination on cartilage injury in rats. In in vitro setting, the cytotoxic effects of BA, EGF, and their combinations using mouse fibroblast cell (L929), human bone osteosarcoma cell (Saos-2), and human adipose derived mesenchymal stem cells (hAD-MSCs) were determined by applying MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] test. In in vivo setting, 72 rats were randomly divided into four groups. A standard chondral defect was created and microfracture was performed in all groups. Group A was determined as the control group. In addition to the standard procedure, Group B received 100 ng/mL of EGF, Group C received a combination of 100 ng/mL of EGF and 10 µg/mL of BA combination, and Group D 20 µg/mL of BA. The cytotoxic effect of the combinations of EGF dilutions (1, 5, 10, 25, 50, 100, 200 ng/mL) with BA (100, 300, 500 µg/mL) was observed only in the 72-h application period and in Saos-2. The cytotoxic effect of BA was reduced when combined with EGF. There was no significant difference in the histopathological scores among the groups (p=0.13). Our study showed that EGF and low-dose BA application had a positive effect on cartilage healing in rats. Significant decreases in recovery scores were observed in the other groups. The combination of EGF and BA promoted osteoblast growth. Detection of lytic lesions in the group treated with 20 µg/mL of BA indicates that BA may have a cytotoxic effect.

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.

RPS19 and RPL5 Contribute to DNA Double-Strand Break Repair

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome most often due to germline pathogenic variants (PVs) in ribosomal protein (RP) genes, with RPS19 and RPL5 being the most frequently mutated. In addition to hypoplastic anemia, individuals have a moderately increased risk of myelodysplastic syndrome, acute myeloid leukemia, and solid cancers, most commonly colorectal cancer and osteosarcoma. A causal link between RP deficiency and cancer predisposition has yet to be firmly established. Our work aims to examine the impact of deficiencies in RPS19 and RPL5 on DNA double-strand break (DSB) repair with the overarching hypothesis that disruptions in DSB repair could increase mutagenesis and ultimately contribute to cancer development in DBA. We found that DBA patient-derived lymphoblastoid cells were hypersensitive to ionizing radiation (IR) and had delayed resolution of the DNA damage marker, γ-H2AX, at IR-induced foci. Additionally, we found that both RPS19- and RPL5-knocked down (KD) bone marrow-derived CD34+ cells had delayed resolution of DSBs after IR in neutral comet assays. These data suggest that RPS19 and RPL5 deficiencies lead to impaired DSB repair. We, therefore, assessed the effect of RPS19- and RPL5-KD on the four main DSB repair pathways using well-established U2OS (osteosarcoma) DSB reporter cell lines. We found decreased homologous recombination (HR) and single-strand annealing (SSA) upon RPS19-KD, whereas the efficiencies of nonhomologous end-joining (NHEJ) and alternative end-joining (alt-EJ) were unchanged. Consistent with an HR defect, RPS19-KD cells were hypersensitive to PARP inhibition. In contrast, RPL5-KD cells had increased NHEJ and alt-EJ, whereas the efficiencies of HR and SSA were unchanged. Together, these results suggest that shifts in DSB repair pathway choice, with less high-fidelity HR, or more error-prone NHEJ or mutagenic alt-EJ, could contribute to mutagenesis in the context of RPS19 or RPL5 PVs. To investigate the mechanism(s) underlying these effects, we examined cell cycle profiles and found decreased G1 and increased G2/M cells following both RPS19- and RPL5-KD, which suggests altered cell cycle profiles are not responsible for DSB repair effects since NHEJ occurs principally in G1 and HR is restricted to G2. Given the essential roles of RPS19 and RPL5 in translation, we also examined levels of crucial proteins in each DSB repair pathway. Surprisingly, we found increased levels of NHEJ proteins DNA ligase IV (LIG4) and XRCC4 in both RPS19 and RPL5-KD cells. However, restoring LIG4/XRCC4 to normal levels via siRNA KD did not normalize the DSB repair efficiencies. Since HR and SSA require extensive resection of the 5' DNA strand at DSBs, we examined whether RPS19-KD impaired this key step by assessing the accumulation of nuclear RPA2, which associates with end-resected DNA. We found nuclear RPA2 levels were not reduced but rather increased following IR compared to the control, indicating the reduction in HR and SSA efficiency in these cells was not due to impaired end resection and may, instead, involve a downstream step in HR that we are currently investigating. To assess if RPS19 and RPL5 might directly contribute to repair, we monitored the recruitment of fluorescently tagged RPS19 and RPL5 to DSBs. We found that these proteins were rapidly recruited to sites of laser microirradiation and sites of chromatin associated Fok1-nuclease (Figure 1), both of which induce DSBs. The recruitment of both proteins was hindered by PARP inhibition and enhanced for RPS19 with PARG inhibition, suggesting RPS19, in particular, may be parylated or interact with parylated proteins. Similar studies were performed in Saos2 cells, which, in contrast to U2OS cells, are p53 null. Whereas RPS19 was recruited to sites of laser microirradiation in Saos2 cells, RPL5 was not. Interestingly, a transcriptionally hyperactive p53 (∆CTD), but not a wild-type or a dominant-negative form of p53 (p.R248Q), restored RPL5 recruitment in Saos2 cells, suggesting a p53 downstream target is required for its recruitment. We are currently investigating proteins that mediate RPS19 and RPL5 DSB recruitment. Together, our results support a model whereby altered DNA DSB repair in RPS19- and RPL5-deficient cells may contribute to cancer development in DBA.

Anticancer Potential of Farnesol Against Human Osteosarcoma Saos-2 Cells and Human Colorectal Carcinoma HCT-116 Cells.

Increased colorectal carcinoma (CRC) and osteosarcomaprevalence, low survival rate, poor prognosis, and the limitations of existing anticancer therapies like side effects of drugs, non-specificity, short half-life, etc., pose a need for novel anticancer drugs.Farnesol, an organic sesquiterpene compound, found in the essential oils of various plants has been shown to possess antioxidant, anti-inflammatory, and anticancer properties. However, the anticancer effect of farnesol againstCRCand osteosarcoma has not yet been adequately elucidated. The aim of the study was to analyze the anticancer effects of farnesol against human osteosarcoma and CRC cell lines. Human osteosarcoma (Saos-2) and colorectal carcinoma (HCT-116) cell lines were procured and cultured at37oC and 5% CO2. The cells were treated with 10, 20, 40, 60, 80, and 100 µM/ml and 20, 40, 60, 80, 100, and 120 µM/ml of farnesol for 24 hours, respectively.3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromideassay was performed to assess the cytotoxicity of farnesol on Saos-2 and HCT-116 cells. Acridine orange/ethidium bromidestaining was carried out to analyze apoptosis.4',6-diamidino-2-phenylindolestaining was done to observe the nuclear changes.Dichloro-dihydro-fluorescein diacetatestaining was performed to assess the farnesol-induced reactive oxygen species (ROS)-mediated cell death. Farnesol reduced the viability and proliferation of Saos-2 and HCT-116 cells in a dose-dependent manner. Farnesol was able to alter the cellular and nuclear morphology of Saos-2 and HCT-116 cells, promoting cell death. Farnesol-induced apoptosis inhuman osteosarcoma and colorectal carcinoma cell lines. Early apoptosis was observed in farnesol-treated HCT-116 cells. Additionally, ROS-mediated apoptotic cell death was reported in Saos-2 cells. Farnesol has the potential to induce cytotoxicity against human osteosarcoma and CRC cell lines.

Single-nucleotide polymorphism profiling by multimodal-targeted next-generation sequencing in methotrexate-resistant and -sensitive human osteosarcoma cell lines

Introduction: Methotrexate (MTX) is one of the most important drugs included in the first-line protocols to treat high-grade osteosarcoma (HGOS). Although several polymorphisms have been reported to be associated with drug response or MTX-related toxicity in pharmacogenetic studies, their role in the development of MTX resistance in HGOS is still unclear.Methods: Therefore, in this study, 22 single nucleotide polymorphisms (SNPs) in 4 genes of the folate metabolism, 7 MTX transporter genes, and 2 SNPs of the tumor protein p53 (TP53) gene were investigated using a custom multimodal-targeted next-generation sequencing (mmNGS) approach in 8 MTX-resistant and 12 MTX-sensitive human HGOS cell lines. The panel was validated by TaqMan genotyping assays.Results: High instability of TP53 rs1642785 was observed in all U-2OS/MTX variants. Allele changes of the solute carrier family 19 member 1/replication factor C subunit 1 (SLC19A1, previously known as RFC1) and rs1051266 were identified in all Saos-2/MTX-resistant variants in both DNA- and RNA- derived libraries compared to the parental Saos-2 cell line. Allele changes of methylenetetrahydrofolate reductase (MTHFR) rs1801133 were identified only in the RNA-derived libraries of the two U2OS variants with the highest MTX resistance level. Significantly upregulated gene expression associated with the development of MTX resistance was revealed for dihydrofolate reductase (DHFR) whereas SLC19A1 was downregulated. In addition, a fusion transcript of DHFR (ex4) and MutS Homolog 3 (MSH3) (ex9) was identified in the RNA libraries derived from the two U-2OS variants with the highest MTX resistance level.Conclusion: This innovative mmNGS approach enabled the simultaneous exploration of SNPs at DNA and RNA levels in human HGOS cell lines, providing evidence of the functional involvement of allele changes associated with the development of MTX resistance.

Modeling phenotypic heterogeneity towards evolutionarily inspired osteosarcoma therapy

Osteosarcoma is the most common bone sarcoma in children and young adults. While universally delivered, chemotherapy only benefits roughly half of patients with localized disease. Increasingly, intratumoral heterogeneity is recognized as a source of therapeutic resistance. In this study, we develop and evaluate an in vitro model of osteosarcoma heterogeneity based on phenotype and genotype. Cancer cell populations vary in their environment-specific growth rates and in their sensitivity to chemotherapy. We present the genotypic and phenotypic characterization of an osteosarcoma cell line panel with a focus on co-cultures of the most phenotypically divergent cell lines, 143B and SAOS2. Modest environmental (pH, glutamine) or chemical perturbations dramatically shift the success and composition of cell lines. We demonstrate that in nutrient rich culture conditions 143B outcompetes SAOS2. But, under nutrient deprivation or conventional chemotherapy, SAOS2 growth can be favored in spheroids. Importantly, when the simplest heterogeneity state is evaluated, a two-cell line coculture, perturbations that affect the faster growing cell line have only a modest effect on final spheroid size. Thus the only evaluated therapies to eliminate the spheroids were by switching therapies from a first strike to a second strike. This extensively characterized, widely available system, can be modeled and scaled to allow for improved strategies to anticipate resistance in osteosarcoma due to heterogeneity.

In vitro and in vivo killing effects of methionine enkephalin on osteosarcoma

ObjectiveThis study aimed to investigate the underlying regulatory effects of methionine enkephalin (MENK) on osteosarcoma. MethodsThe Cell Counting Kit-8 assay, clone formation, wound healing, transwell assay, and flow cytometry were performed to measure the effects of MENK on the proliferation, migration, invasion, and apoptosis of MG-63 and Saos-2 cells. Opiate growth factor receptor expression (OGFr) in cells was stably knocked down using siRNA. A tumor model was established by inoculating MG-63 cells into mice. Flow cytometry was performed to identify alterations in mice bone marrow, spleen, and tumor tissue immune cells. The phenotype of tumor-associated macrophages was determined using immunohistochemistry. After OGFr knockdown or/and treatment with MENK, Bax, Bcl-2, caspase 3, caspase 9, and PARP expression levels were characterized using qRT-PCR, western blot, and WES, respectively. ResultsMENK could significantly inhibit the proliferation, invasion, and migration of MG-63 and Saos-2, arrest the cell cycle in the G0/G1 phase, upregulate Bax, caspase 3, caspase 9, and PARP expression, and downregulate Bcl-2 expression. Tumor size and weight were lower in the MENK group than those in the control group. MENK-treated mice exhibited a reduced ratio of CD11b + Gr-1 + myeloid-derived suppressor cells. MENK increased the ratio of M1-type macrophages and decreased the proportion of M2-type macrophages in tumor tissue. Furthermore, the level of TNF-α significantly increased while that of IL-10 decreased in MENK-treated mice. The effect of MENK could be partly reversed by OGFr knockdown. ConclusionMENK reduces the abundance of myeloid-derived suppressor cells, induces M1 polarization of macrophages, and exhibits an inhibitory effect on osteosarcoma.

Osseointegration of functionally graded Ti6Al4V porous implants: Histology of the pore network

The additive manufacturing of titanium into porous geometries offers a means to generate low-stiffness endosseous implants with a greater surface area available for osseointegration. In this work, selective laser melting was used to produce gyroid-based scaffolds with a uniform pore size of 300 μm or functionally graded pore size from 600 μm to 300 μm. Initial in vitro assessment with Saos-2 cells showed favourable cell proliferation at pore sizes of 300 and 600 μm. Following implantation into rabbit tibiae, early histological observations at four weeks indicated some residual inflammation alongside neovessel infiltration into the scaffold interior and some early apposition of mineralized bone tissue. At twelve weeks, both scaffolds were filled with a mixture of adipocyte-rich marrow, micro-capillaries, and mineralized bone tissue. X-ray microcomputed tomography showed a higher bone volume fraction (BV/TV) and percentage of bone-implant contact (BIC) in the implants with 300 μm pores than in the functionally graded specimens. In functionally graded specimens, localized BV/TV measurement was observed to be higher in the innermost region containing smaller pores (estimated at 300–400 μm) than in larger pores at the implant exterior. The unit cell topology of the porous implant was also observed to guide the direction of bone ingrowth by conducting along the implant struts. These results suggest that in vivo experimentation is necessary alongside parametric optimization of functionally graded porous implants to predict short-term and long-term bone apposition.

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.

Boldine Treatment Induces Cytotoxicity in Human Colorectal Carcinoma and Osteosarcoma Cells.

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.

Evaluation of the Biological Effect of Non-UV-Activated Bergapten on Selected Human Tumor Cells and the Insight into the Molecular Mechanism of Its Action.

There is some evidence that non-photoactivated psoralens may be active against breast and colon tumor cells. Therefore, we evaluated the antiproliferative, proapoptotic, and anti-migrative effect of 5-methoxypsoralen (5-MOP) isolated from Peucedanum tauricum MB fruits in human colorectal adenocarcinoma (HT-29 and SW620), osteosarcoma (Saos-2 and HOS), and multiple myeloma (RPMI8226 and U266). Dose- and cell-line-dependent effects of 5-MOP on viability and proliferation were observed, with the strongest inhibitory effect against Saos-2 and a moderate effect against the HOS, HT-29, and SW620 cells. Multiple myeloma showed low sensitivity. The high viability of human normal cell cultures (HSF and hFOB) in a wide range of 5-MOP concentrations tested (6.25-100 µM) was confirmed. Moreover, the migration of treated Saos-2, SW620, and HT-29 cell lines was impaired, as indicated via a wound healing assay. Flow cytometry analysis conducted on Saos-2 cells revealed the ability of 5-MOP to block the cell cycle in the G2 phase and trigger apoptosis, which was accompanied by a loss of mitochondrial membrane potential, caspases (-9 and -3) activation, the altered expression of the Bax and Bcl-2 proteins, and decreased AKT phosphorylation. This is the first report evaluating the antiproliferative and antimigratory impact of non-UV-activated bergapten on the abovementioned (except for HT-29) tumor cells, which provides new data on the potential role of 5-MOP in inhibiting the growth of various types of therapeutic-resistant cancers.

Nelumbo nucifera Leaf Extract Induces Cytotoxicity in Osteosarcoma Saos-2 Cells.

Background Osteosarcoma is the eighth most common cancer and its prevalence in children makes it a global concern. Existing medications and treatments like high-dose methotrexate possess harmful side effects. Therefore, novel herbal drugs like Nelumbo nucifera are of utmost importance. Aim To analyze a novel anticancer herbal drug, Nelumbo nucifera leaf extractfor its cytotoxic potential against osteosarcoma. Materials and method Nelumbo nucifera leaf extract was prepared. Saos-2 Cells (human osteosarcoma cell line) were treated with Nelumbo nucifera leaf extract (25, 50, 75, 100, 125, and 150 µg/ml) for 24 hours which were then subjected to MTT assay, morphological analysis and DAPI staining. Results The results suggested that Nelumbo nucifera leaf extract had a concentration-dependent cytotoxic effect onSaos-2 cell line. The extract significantly reduced the number of viable cells, inhibited proliferation and induced morphological changes in Saos-2 cells. Conclusion Nelumbo nucifera has the potential to induce cytotoxicity against osteosarcoma cell lines and hence, this study provides a novel therapeutic regimen for the treatment of osteosarcoma.

Chemical characterization of the crude extract of Sauromatum venosum (voodoo lily) and docking study with 12-O-acetylingol 8-tiglate for cytotoxicity testing in SaOS2 (osteoblastic osteosarcoma cells).

The current study was carried out to investigate the anticancer potential of Sauromatum venosum (SV) tuber by gas chromatography with high-resolution mass spectrometry (GC-HRMS) analysis of ethanolic (eSV), hydroalcoholic (hSV), and aqueous extracts (wSV), and in silico study were performed to investigate the main targets of 12-O-acetylingol 8-tiglate by computational docking. The GC-HRMS analysis of three plant samples was carried out on a system equipped with a high-resolution mass spectrometer. The major compounds were identified in all crude extracts. Computation docking analysis was performed for the prediction of the main target of the cancer proliferation of active compound of the Sauromatum venosum tuber extract in cancer therapy. A total of 45 phytocompounds were detected including diterpenoids, esters of fatty acid, hydrocarbons, and alkanes in the tuber of SV. Among all the crude samples tested, eSV showed the lowest IC50 value treated with SaOS2 cells. 12-O-acetylingol 8-tiglate is one of the phytocompounds identified in eSV extract and has been found to exhibit cytotoxic effects against various cancer cells, as reported in the research. It shows the optimum binding affinity with -8.59kcal/mol binding energy with a molecular target protein TNF-α (PDB ID: 7PKA). The observed interactions strongly support the anticancer activity of 12-O-acetylingol 8-tiglate and its role in the medicinal efficacy of the plant. These findings highlight the potential of the compound as a valuable source for the development of a therapeutic agent aimed at combating cancer. However, it is important to note that additional in vitro and in vivo studies are required to validate these findings and establish the therapeutic potential.

Mutant p53 Gain-of-Function Induces Migration and Invasion through Overexpression of miR-182-5p in Cancer Cells.

The master-key TP53 gene is a tumor suppressor that is mutated in more than 50% of human cancers. Some p53 mutants lose their tumor suppressor activity and acquire new oncogenic functions, known as a gain of function (GOF). Recent studies have shown that p53 mutants can exert oncogenic effects through specific miRNAs. We identified the differentially expressed miRNA profiles of the three most frequent p53 mutants (p53R273C, p53R248Q, and p53R175H) after their transfection into the Saos-2 cell line (null p53) as compared with p53WT transfected cells. The associations between these miRNAs and the signaling pathways in which they might participate were identified with miRPath Software V3.0. QRT-PCR was employed to validate the miRNA profiles. We observed that p53 mutants have an overall negative effect on miRNA expression. In the global expression profile of the human miRNome regulated by the p53R273C mutant, 72 miRNAs were underexpressed and 35 overexpressed; in the p53R175H miRNAs profile, our results showed the downregulation of 93 and upregulation of 10 miRNAs; and in the miRNAs expression profile regulated by the p53R248Q mutant, we found 167 decreased and 6 increased miRNAs compared with p53WT. However, we found overexpression of some miRNAs, like miR-182-5p, in association with processes such as cell migration and invasion. In addition, we explored whether the induction of cell migration and invasion by the p53R48Q mutant was dependent on miR-182-5p because we found overexpression of miR-182-5p, which is associated with processes such as cell migration and invasion. Inhibition of mutant p53R248Q and miR-182-5p increased FOXF2-MTSS1 levels and decreased cell migration and invasion. In summary, our results suggest that p53 mutants increase the expression of miR-182-5p, and this miRNA is necessary for the p53R248Q mutant to induce cell migration and invasion in a cancer cell model.

In Vitro Characterization of Doped Bioglass 45S5/HAp Coatings Obtained by CoBlastTM Deposition

Bone replacement is one of the major medical procedures in the oral surgery field due to the progressive ageing population and to illness or trauma in younger age groups. The use of implants without biological activity and effective osseointegration increases the chances of implant failure. This work aims to improve the interaction between implants and bone by using Bioglass 45S5 (BG)/hydroxyapatite (HAp) mixtures, including copper-, zinc-, and cerium-doped BG, as well as co-doping by the mentioned metals, as coatings produced by the CoBlastTM technique. All coatings present a uniform coverage of the Ti-6Al-4V substrate. Furthermore, in vitro testing using human osteosarcoma Saos-2 cells indicated that BG/HAp coatings have no cytotoxic effect, and the used of doping agents did not alter cell adhesion, proliferation, or alkaline phosphatase (ALP) expression when compared to undoped coating. These results demonstrate that BG/HAp by CoBlastTM can be a solution to improve implants’ osseointegration.

Synthesis, characterization, and in vitro cytotoxic evaluation of hydroxyapatite submicron particles functionalized with 2-chlorobenzoylthiourea as a drug delivery system

In this study, hydroxyapatite (HAp) nanoparticles (NPs) were synthesized via hydrothermal synthesis method. The surfaces of HAp NPs were functionalized with (3-aminopropyl)triethoxysilane (APTES) and 2-chlorobenzoylthiourea (BT) to obtain HAp–APTES–BT NPs. The structural characterization of HAp submicron particles was made via FTIR and X-ray diffraction techniques. HAp–APTES and HAp–APTES–BT submicron particles were characterized via FTIR. The particle size of HAp submicron particles was determined to be 1281, 1484, and 1718 nm, and their zeta potentials were determined to be −3.38, −3.25, and −3.18. Zoledronic acid (ZA) was loaded onto the HAp–APTES–BT submicron particles under supercritical CO2 conditions (25 °C, 200 bar, 1 h). Drug release profiles of HAp–APTES–BT–ZA submicron particles were investigated in phosphate buffered saline solution (pH 7.4, 37 °C) for 72 h. The cytotoxic effects of HAp–APTES–BT–ZA submicron particles on Saos-2 cells were determined by 3-(4,5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide test.

Eugenol: In Vitro and In Ovo Assessment to Explore Cytotoxic Effects on Osteosarcoma and Oropharyngeal Cancer Cells.

Cancer is a significant health problem worldwide; consequently, new therapeutic alternatives are being investigated, including those found in the vegetable kingdom. Eugenol (Eug) has attracted attention for its therapeutic properties, especially in stomatology. The purpose of this study was to investigate the cytotoxicity of Eug, in vitro, on osteosarcoma (SAOS-2) and oropharyngeal squamous cancer (Detroit-562) cells, as well as its potential irritant effect in ovo at the level of the chorioallantoic membrane (CAM). The data obtained following a 72 h Eug treatment highlighted the reduction in cell viability up to 41% in SAOS-2 cells and up to 37% in Detroit-562 cells, respectively. The apoptotic-like effect of Eug was indicated by the changes in cell morphology and nuclear aspect; the increase in caspase-3/7, -8 and -9 activity; the elevated expression of Bax and Bad genes; and the increase in luminescence signal (indicating phosphatidylserine externalization) that preceded the increase in fluorescence signal (indicating the compromise of membrane integrity). Regarding the vascular effects, slight signs of coagulation and vascular lysis were observed, with an irritation score of 1.69 for Eug 1 mM. Based on these results, the efficiency of Eug in cancer treatment is yet to be clarified.

Calcium hypochlorite cytotoxicity mechanism in fibroblasts and effect on osteoblast mineralization.

To determine the cytotoxicity mechanism of 2.5% calcium hypochlorite [Ca(OCl)2 ] in L929 fibroblasts and the effect of this solution on human osteoblast-like cells (Saos-2) mineralization, compared to that of 2.5% sodium hypochlorite (NaOCl). L929 fibroblasts were exposed to Ca(OCl)2 and NaOCl at different dilutions for 10 min. Cell metabolism was assessed by methyl-thiazole-tetrazolium (MTT); lysosome integrity, by neutral red (NR) assay; type of cell death, by flow cytometry (apoptosis/necrosis); cytoskeleton, by actin and α-tubulin fluorescence and cell ultrastructure, by transmission electron microscopy (TEM). The alkaline phosphatase (ALP) activity and mineralized nodule formation were determined in Saos-2 by thymolphthalein release and alizarin red staining (ARS), respectively. The data were analysed by two-way anova and Bonferroni's post-test (α = .05). Ca(OCl)2 promoted higher cell viability and a lower percentage of apoptosis and necrosis than NaOCl (p < .05). Ca(OCl)2 and NaOCl decreased cell metabolism and lysosome integrity, induced the breakdown of microtubules and actin filaments, promoted alterations of rough endoplasmic reticulum and disruption of mitochondrial cristae. Additionally, Ca(OCl)2 did not induce ALP activity and had no effect on mineralized nodules formation. Although Ca(OCl)2 and NaOCl promoted the same cytotoxicity mechanism, Ca(OCl)2 was less cytotoxic than NaOCl. As for ALP activity, no differences were observed between NaOCl and Ca(OCl)2 . The production of mineralized nodules induced by Ca(OCl)2 was lower than those induced by NaOCl, but was not different from those induced by the control group.

Effects of surface micro-deformation on the cellular response to metallic implant materials

In the current study, it is aimed to get a comprehensive understanding of how cellular response to metallic biomaterial surfaces can be controlled via surface parameter specifics, as well as microstructural modifications. For this purpose, a surface micro-deformation procedure was designed where patterns with different parameters, specifically different indent size and repeating frequencies, were created on 316 L stainless steel sample surfaces by using a Vicker's microhardness testing device. The effects of the changes in surface properties following the formation of the different patterns were characterized via scanning electron microscopy (SEM), optical microscopy and profilometer. It was observed that surface roughness significantly increased as a result of the surface micro-deformation process and additionally, dislocation activities of different levels were triggered depending on the specifics of the formed micro-deformation pattern. The effects of the surface micro-deformation process on cellular response were investigated through in vitro experiments where Saos-2 cell line was used. Significant improvements were obtained in terms of cellular viability, proliferation, attachment and cell differentiation behaviors on the samples with surface micro-deformation. Correlating the effects surface parameters such as surface roughness, dislocation density close to the surface and topographical feature specifics on cellular response, it has been observed that by manipulating the metal microstructure and optimizing the surface micro-deformation patterning parameters, cellular response can be significantly enhanced. It was shown that specific surface patterns with smaller indent depth and narrower spacings, as well as optimum dislocation density levels and surface roughness yields superior cellular response. Overall, the obtained information about the specifics of surface features of metallic biomaterials on cellular response can be used for the design of novel implant surface modification methods to improve osseointegration.