Human cells with osteogenic potential in bone tissue research.

Bone regeneration after injury or after surgical bone removal due to disease is a serious medical challenge. A variety of materials are being tested to replace a missing bone or tooth. Regeneration requires cells capable of proliferation and differentiation in bone tissue. Although there are many possible human cell types available for use as a model for each phase of this process, no cell type is ideal for each phase. Osteosarcoma cells are preferred for initial adhesion assays due to their easy cultivation and fast proliferation, but they are not suitable for subsequent differentiation testing due to their cancer origin and genetic differences from normal bone tissue. Mesenchymal stem cells are more suitable for biocompatibility testing, because they mimic natural conditions in healthy bone, but they proliferate more slowly, soon undergo senescence, and some subpopulations may exhibit weak osteodifferentiation. Primary human osteoblasts provide relevant results in evaluating the effect of biomaterials on cellular activity; however, their resources are limited for the same reasons, like for mesenchymal stem cells. This review article provides an overview of cell models for biocompatibility testing of materials used in bone tissue research.

Selective inhibition of acylpeptide hydrolase in SAOS-2 osteosarcoma cells: is this enzyme a viable anticancer target?

Serine hydrolases play crucial roles in many physiological and pathophysiological processes and a panel of these enzymes are targets of approved drugs. Despite this, most of the human serine hydrolases remain poorly characterized with respect to their biological functions and substrates and only a limited number of in vivo active inhibitors have been so far identified. Acylpeptide hydrolase (APEH) is a member of the prolyl-oligopeptidase class, with a unique substrate specificity, that has been suggested to have a potential oncogenic role. In this study, a set of peptides was rationally designed from the lead compound SsCEI 4 and in vitro screened for APEH inhibition. Out of these molecules, a dodecapeptide named Ala 3 showed the best inhibitory effects and it was chosen as a candidate for investigating the anti-cancer effects induced by inhibition of APEH in SAOS-2 cell lines. The results clearly demonstrated that Ala 3 markedly reduced cell viability via deregulation of the APEH-proteasome system. Furthermore, flow cytometric analysis revealed that Ala 3 anti-proliferative effects were closely related to the activation of a caspase-dependent apoptotic pathway. Our findings provide further evidence that APEH can play a crucial role in the pathogenesis of cancer, shedding new light on the great potential of this enzyme as an attractive target for the diagnosis and the quest for selective cancer therapies.

APEH Inhibition Affects Osteosarcoma Cell Viability via Downregulation of the Proteasome.

The proteasome is a multienzymatic complex that controls the half-life of the majority of intracellular proteins, including those involved in apoptosis and cell-cycle progression. Recently, proteasome inhibition has been shown to be an effective anticancer strategy, although its downregulation is often accompanied by severe undesired side effects. We previously reported that the inhibition of acylpeptide hydrolase (APEH) by the peptide SsCEI 4 can significantly affect the proteasome activity in A375 melanoma or Caco-2 adenocarcinoma cell lines, thus shedding new light on therapeutic strategies based on downstream regulation of proteasome functions. In this work, we investigated the functional correlation between APEH and proteasome in a panel of cancer cell lines, and evaluated the cell proliferation upon SsCEI 4-treatments. Results revealed that SsCEI 4 triggered a proliferative arrest specifically in osteosarcoma U2OS cells via downregulation of the APEH-proteasome system, with the accumulation of the typical hallmarks of proteasome: NF-κB, p21(Waf1), and polyubiquitinylated proteins. We found that the SsCEI 4 anti-proliferative effect involved a senescence-like growth arrest without noticeable cytotoxicity. These findings represent an important step toward understanding the mechanism(s) underlying the APEH-mediated downregulation of proteasome in order to design new molecules able to efficiently regulate the proteasome system for alternative therapeutic strategies.

Synergistic Antitumor Interaction of Risedronate Sodium and Standard Anticancer Agents in Canine (D-17) and Human Osteosarcoma (U-2 OS) Cell Lines.

The study discusses in vitro cytotoxicity of a combination of cytostatic drugs (doxorubicin, cisplatin, carboplatin, etoposide) and risedronate sodium against canine and human osteosarcoma (D-17 and U-2 OS). Standard protocols were used for the preparation of cell cultures and evaluation of their viability and apoptosis. MTT assay assessed the culture viability and EC50, while the apoptotic effect of the drugs was checked with a TUNEL assay. Doxorubicin alone showed the strongest cytotoxicity against D-17 (0.056 ± 0.019 µg/mL) and U-2 OS (0.051 ± 0.003 µg/mL), while the lowest cytotoxicity was observed for carboplatin (D-17, 6.45 ± 0.2 µg/mL and U2-OS, 27.5 ± 2.3 µg/mL). Risedronate sodium at 100, 10 and 1 µg/mL lowered viability in OS cell lines by 53.38 ± 1.46 and 49.56 ± 0.7%, 97.08 ± 3.32 and 74.92 ± 4.01%, and 102.67 ± 3.56 and 94.56 ± 3.52%, respectively. In all analyzed drug combinations, risedronate sodium significantly (* p < 0.05) increased the cytotoxicity against tested osteosarcoma cell lines. The decrease in cell viability caused by the studied compound combinations was weaker in canine than in human cell cultures. A combination of doxorubicin (all concentrations), cisplatin (1 µg/mL) and etoposide (1 µg/mL) with 100 µg/mL of risedronate sodium significantly improved the cytotoxicity of the drugs against canine and human osteosarcoma. Administration of carboplatin (1 µg/mL) and risedronate sodium (100 µg/mL), compared to carboplatin per se, produced no significant differences in cytotoxicity against the D-17 cell culture but significantly enhanced cytotoxicity in the U-2 OS line. The strongest apoptosis in both lines was detected for 0.01 µg/mL doxorubicin combined with 100 µg/mL risedronate sodium or 1 µg/mL cisplatin and 100 µg/mL risedronate sodium. In all combinations, the tested compounds revealed a synergistic mechanism of action.

Anticancer activity of an Artemisia annua L. hydroalcoholic extract on canine osteosarcoma cell lines.

Since ancient times, Artemisia annua (A. annua) has been used as a medicinal plant in Traditional Chinese Medicine. In addition, recent studies have investigated the cytotoxic effects of A. annua extracts towards cancer cells. The leading aim of the present research is to evaluate the cytotoxic effects of an hydroalcoholic extract of A. annua on two canine osteosarcoma (OSA) cell lines, OSCA-8 and OSCA-40, focusing on the possible involvement of ferroptosis. The quantitative determination of artemisinin concentration in the extract, culture medium and OSA cells was carried out through the use of an instrumental analytical method based on liquid chromatography coupled with spectrophotometric detection and tandem mass spectrometry (LC-DAD-MS/MS). OSCA-8 and OSCA-40 were exposed to different dilutions of the extract for the EC50 calculation then the uptake of artemisinin by the cells, the effects on the cell cycle, the intracellular iron level, the cellular morphology and the lipid oxidation state were evaluated. A concentration of artemisinin of 63.8 ± 3.4 µg/mL was detected in the extract. A dose-dependent cytotoxic effect was evidenced. In OSCA-40 alterations of the cell cycle and a significantly higher intracellular iron content were observed. In both cell lines the treatment with the extract was associated with lipid peroxidation and with the appearance of a "ballooning" phenotype suggesting the activation of ferroptosis. In conclusion the A. annua idroalcoholic extract utilized in this study showed anticancer activity on canine OSA cell lines that could be useful in treating drug resistant canine OSAs.

Identification of Conserved Pappalysin 1-Derived Circular RNA-Mediated Competing Endogenous RNA in Osteosarcoma.

The etiology of osteosarcoma (OS) is complex and not fully understood till now. This study aimed to identify the miRNAs, circRNAs, and genes (mRNAs) that are differentially expressed in OS cell lines to investigate the mechanism of circRNA-associated competing endogenous RNAs (ceRNAs) in OS. Microarray datasets reporting mRNA (GSE70414), miRNA (GSE70367), and circRNA changes (GSE96964) in human OS cell lines were downloaded, differentially expressed (DE) RNAs were identified, and DEmRNAs were used for the annotation of Gene Ontology (GO) biological processes (BP), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The mechanisms of DEcircRNA-mediated ceRNAs were identified in a step-by-step process. A total of 326 DEmRNAs, 45 DEmiRNAs, and 110 DEcircRNAs were identified from 3 datasets. The DEmRNAs were associated with GO BP terms, including cholesterol biosynthetic process, angiogenesis, extracellular matrix organization and KEGG pathways, including p53 signaling pathway and biosynthesis of antibiotics. The final ceRNA network consisted of 8 DEcircRNAs, including 5 pappalysin (PAPPA) 1-derived DEcircRNAs (hsa_circ_0005456, hsa_circ_0088209, hsa_circ_0002052, hsa_circ_0088214 and has_circ_0008792, all downregulated), 3 DEmiRNAs (hsa-miR-760, hsa-miR-4665-5p and hsa-miR-4539, all upregulated), and downregulated genes (including MMP13 and HMOX1). The ceRNA regulation network of OS was built, which played important roles in the pathogenesis of OS and might be of great importance in therapy.

Potential Antimetastatic Effect of Timosaponin AIII against Human Osteosarcoma Cells through Regulating the Integrin/FAK/Cofilin Axis.

Timosaponin AIII (TSAIII) is a steroidal saponin which demonstrates anti-tumour activities. However, the effect of TSAIII on human osteosarcoma cells remains largely unknown. In this study, we demonstrated that TSAIII exerted a significant inhibitory effect on the distribution of cytoskeletal F-actin and cytoskeletal-related proteins, which contributed to the suppression of cell migration and invasion, without inhibiting cell growth or apoptosis. In the synergistic inhibitory analysis, cotreatment of TSAIII with αVß3 integrin inhibitor [Cyclo(RGDyK)] or focal adhesion kinase (FAK) inhibitor (PF-573228) exerted greater synergistic inhibitory effects on the expression of Intergin αVß3/FAK/cofilin axis, thus inhibiting the migration and invasion capacities of human osteosarcoma cells. TSAIII was demonstrated to significantly inhibit the pulmonary metastasis formation of human osteosarcoma cells in vivo in metastasis animal models. These findings reveal the inhibitory effects of TSAIII on the metastasis progression of human osteosarcoma cells and the regulation of integrin-αVß3-FAK-Src and TESK1/p-cofilin mediated cytoskeletal F-actin pathway. Therefore, TSAIII might represent a novel strategy for the auxiliary treatment of human osteosarcoma cells.

miR-106B-25 Cluster expression: a comparative human and canine osteosarcoma study.

BACKGROUND: Osteosarcoma (OS) is the most common primary malignant bone tumour in dogs and human beings, characterised by similar genetic and clinical features. With the aim to define similarities and differences in the biological aspects involved in OS progression, a comparative study was performed to create a model to improve patient outcome. METHODS: First, the expression of microRNAs (miRNAs) belonging to the cluster miR-106b-25 (miR-106b, miR-25 and miR-93-5p) in human and canine OS tissue was compared. RESULTS: miR-25 and miR-106b presented a variable expression not significantly different from the corresponding normal bone, while miR-93-5p expression was increased in all OS specimens, with higher levels in the canine subset compared with human. Accordingly, its target p21 presented a weaker and less homogeneous immunostaining distribution in the canine group. Given the high expression of miR-93-5p in all OS specimens, the functional response of human 143B and canine DAN OS cells to miRNA inhibition was evaluated. Although p21 expression increased after miR-93-5p inhibition both at mRNA and protein level, a more significant cell response in terms of proliferation and apoptosis was seen in canine OS cells. CONCLUSIONS: In conclusion, canine OS tissue and cell line presented higher expression levels of miR-93-5p than human OS. In addition, the introduction of miR-93-5p inhibitor caused a cell response in 143B and DAN that differed for the more intense functional impact in the canine OS cell line.

In vitro effects of the activity of novel platinum (II) complex in canine and human cell lines.

The anticancer activity of novel platinum derivative, a complex of platinum with tris(2-carboxyethyl)phosphine (Pt-TCEP), has been evaluated in canine (D-17) and human osteosarcoma (U2-OS) cell lines. Viability of cells after incubation for 24 or 72 hours with increasing concentrations (0.625, 1.25, 2.50, 5, 10 and 20 µM) of Pt-TCEP was tested in an MTT assay and compared to effect of cisplatin. Longer-term effect of Pt-TCEP was evaluated in the colony-forming unit assay after 24 hours exposure to the Pt-TCEP (2 and 3 µM) and subsequent incubation for 2 weeks. The influence of the compound on the cell cycle was measured after 24 hours treatment with Pt-TCEP (3 µM). Its pro-apoptotic activity was examined after 24 hours treatment with Pt-TCEP (1.25, 2.50, 5, 10 and 20 µM) using flow cytometry. Expression of main proteins involved in apoptosis was measured after exposure for 24 hours to 3 or 5 µM Pt-TCEP in Western Blot. The compound much more effectively decreased cell viability than cisplatin in case of both cell lines. IC50 of Pt-TCEP was 5.93 ± 0.12 in D-17 and 3.45 ± 0.14 in U2-OS cell lines after 24 hours, and 1.77 ± 0.14 in D-17 and 1.53 ± 0.11 in U2-OS after 72 hours (P < .05). The compound arrested cells in the G2/M phase and inhibited the ability of cells to form colonies. Pt-TCEP induced caspase-dependent apoptosis. The expression of the anti-apoptotic Bcl-XL protein was decreased after Pt-TCEP treatment in both cell lines. The results confirmed anti-cancer activity of Pt-TCEP against canine and human osteosarcoma cell lines.

Multitype Network-Guided Target Controllability in Phenotypically Characterized Osteosarcoma: Role of Tumor Microenvironment.

This study highlights the relevance of network-guided controllability analysis as a precision oncology tool. Target controllability through networks is potentially relevant to cancer research for the identification of therapeutic targets. With reference to a recent study on multiple phenotypes from 22 osteosarcoma (OS) cell lines characterized both in vitro and in vivo, we found that a variety of critical proteins in OS regulation circuits were in part phenotype specific and in part shared. To generalize our inference approach and match cancer phenotypic heterogeneity, we employed multitype networks and identified targets in correspondence with protein sub-complexes. Therefore, we established the relevance for diagnostic and therapeutic purposes of inspecting interactive targets, namely those enriched by significant connectivity patterns in protein sub-complexes. Emerging targets appeared with reference to the OS microenvironment, and relatively to small leucine-rich proteoglycan members and D-type cyclins, among other collagen, laminin, and keratin proteins. These described were evidences shared across all phenotypes; instead, specific evidences were provided by critical proteins including IGFBP7 and PDGFRA in the invasive phenotype, and FGFR3 and THBS1 in the colony forming phenotype.

Evaluation of Apoptosis and Autophagy Inducing Potential of Berberis aristata, Azadirachta indica, and Their Synergistic Combinations in Parental and Resistant Human Osteosarcoma Cells.

Cancer is a multifactorial disease and hence can be effectively overcome by a multi-constituently therapeutic strategy. Medicinal plant extracts represent a perfect example of such stratagem. However, minimal studies have been done till date that portray the effect of extraction techniques on the phyto-constituent profile of plant extracts and its impact on anticancer activity. In the present study, we have evaluated the anticancer potential of methanolic extracts of Berberis aristata root and Azadirachta indica seeds prepared by various extraction techniques in human osteosarcoma (HOS) cells. Soxhlation extract of B. aristata (BAM-SX) and sonication extract of A. indica (AIM-SO) were most effective in inducing apoptosis in parental drug sensitive, as well as resistant cell type developed by repeated drug exposure. Generation of reactive oxygen species and cell cycle arrest preceded caspase-mediated apoptosis in HOS cells. Interestingly, inhibition of autophagy enhanced cell death suggesting the cytoprotective role of autophagy. Combination studies of different methanolic extracts of BAM and AIM were performed, among which, the combination of BAM-SO and AIM-SO (BAAISO) was found to show synergism (IC50 10.27 µg/ml) followed by combination of BAM-MC and AIM-MC (BAAIMC) with respect to other combinations in the ratio of 1:1. BAAISO also showed synergism when it was added to cisplatin-resistant HOS cells (HCR). Chromatographic profiling of BAM-SX and AIM-SO by high performance thin layer chromatography resulted in identification of berberine (Rf 0.55), palmitine (Rf 0.50) in BAM-SX and azadirachtin A (Rf 0.36), azadirachtin B (Rf 0.56), nimbin (Rf 0.80), and nimbolide (Rf 0.43) in AIM-SO. The cytotoxic sensitivity obtained can be attributed to the above compounds. Our results highlight the importance of extraction technique and subsequent mechanism of action of multi-constituential B. aristata and A. indica against both sensitive and drug refractory HOS cells.

Chimaphilin inhibits proliferation and induces apoptosis in multidrug resistant osteosarcoma cell lines through insulin-like growth factor-I receptor (IGF-IR) signaling.

Chimaphilin, an active compound separated from pyrola, possesses the highly efficient antitumor activities. Insulin-like growth factor-I receptor (IGF-IR) plays an important role in tumor cell survival. To look for effective strategies for interrupting IGF-IR signaling pathway, we found that chimaphilin can inhibit the receptor tyrosine kinase activity of IGF-IR. Chimaphilin inhibited the growth of both drug-sensitive and drug-resistant osteosarcoma cell lines in a time and dose-dependent manner; however, it showed relatively little toxicity in normal osteoblast cell lines. Chimaphilin can increase the sensitivity of doxorubicin in doxorubicin-resistant osteosarcoma cell lines. Additionally, small interfering RNA downregulation of IGF-IR expression in drug-resistant cell lines also caused resensitization to doxorubicin. Above all, we conclude that chimaphilin represents a valuable natural source and may potentially be applicable for reversing the drug-resistant phenotype in osteosarcoma therapy.

Mechanisms of methotrexate resistance in osteosarcoma cell lines and strategies for overcoming this resistance.

The aim of the present study was to investigate the underlying mechanisms of methotrexate (MTX) resistance in the human osteosarcoma cell line, Saos-2/MTX4.4, and to evaluate various methods of overcoming the resistance to this chemotherapeutic agent. MMT assays were performed to determine the resistance of the primary (Saos-2) and resistant (Saos-2/MTX4.4) cell lines to MTX, cisplatin [cis-diamminedichloroplatinum II (DDP)], ifosfamide (IFO), Adriamycin (ADM), epirubicin (EPI) and theprubicin (THP). The Saos-2/MTX4.4 cells exhibited a low resistance to IFO, ADM, EPI and THP; however, no resistance to DDP was identified. Overall, the Saos-2/MTX4.4 cells exhibited a greater resistance to all the chemotherapeutic agents investigated compared with the Saos-2 cells. Rhodamine 123 (R123) fluorescence was measured in the Saos-2/MTX4.4 and Saos-2 cells 30 and 60 min after the addition of R123, and R123 plus verapamil (VER). VER administration increased the intracellular accumulation of R123. In addition, reverse transcription-quantitative polymerase chain reaction was performed to determine the mRNA expression levels of multidrug resistance gene 1 (MDR1) in the two cell lines. Although the Saos-2/MTX4.4 cells were more resistant to the chemotherapeutic agents than the Saos-2 cells, no significant difference was identified between the relative mRNA expression levels of MDR1 in the Saos-2/MTX4.4 and Saos-2 cells (0.4350±0.0354 vs. 0.3886±0.0456; P>0.05).