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Abstract Purpose This study aims to investigate the effect of Niraparib in combination with an Anti-GD2 Antibody on osteosarcoma cells. Methods Scratch test was utilized to assess cell migration capacity, while the Transwell experiment was utilized to evaluate cell invasion potential. Cell proliferation was measured using the CCK8 experiment. The affinity between the anti-GD2 antibody and its antigen was determined via ELISA. Tumor growth was evaluated through animal experiments. Western blotting, QRT-PCR, and histological analysis were conducted to examine the expression of relevant proteins and mRNAs. Results MG63 cell line was used for an example. The scratch test showed that the migration rate of osteosarcoma cells in Niraparib + Anti-GD2 group was 1.07 ± 0.04 after 48 h, and 0.34 ± 0.04 in the Control group. Transwell experiment showed that the invasion ability of osteosarcoma cells in Niraparib + Anti-GD2 group was 21.0 ± 1.5, and that in Control group was 87.7 ± 2.9. CCK8 experiment showed that the absorbance value of Niraparib + Anti-GD2 group was 0.16 ± 0.10 on day 5, and that of the Control group was 0.76 ± 0.09. Western blotting showed that the expression levels of BALP and CICP in Niraparib + Anti-GD2 group were 0.751 ± 0.135 and 1.086 ± 0.115, respectively, and those in Control group were 1.025 ± 0.143 and 1.216 ± 0.168, respectively. QRT-PCR results showed that the absorbance values of Niraparib + Anti-GD2 group were 0.173 ± 0.065 and 0.170 ± 0.078 on day 14. The results of animal experiments showed that on day 5, the tumor volume of the Control group was 2433 ± 391, and that of the Niraparib + Anti-GD2 group was 1137 ± 148. Histological analysis showed that the mean density values of Niraparib + Anti-GD2 group were 0.19 ± 0.08 and 0.22 ± 0.07, and those of Control group were 0.26 ± 0.09 and 0.29 ± 0.10. Conclusion The combination of Niraparib and Anti-GD2 antibody significantly inhibits Osteosarcoma cells.

Purpose: This study aims to investigate the effect of Niraparib in combination with an Anti-GD2 Antibody on osteosarcoma cells. Methods: Scratch test was utilized to assess cell migration capacity, while the Transwell experiment was utilized to evaluate cell invasion potential. Cell proliferation was measured using the CCK8 experiment. The affinity between the anti-GD2 antibody and its antigen was determined via ELISA. Tumor growth was evaluated through animal experiments. Western blotting, QRT-PCR, and histological analysis were conducted to examine the expression of relevant proteins and mRNAs. Results: MG63 cell line was used for an example. The scratch test showed that the migration rate of osteosarcoma cells in Niraparib + Anti-GD2 group was 1.07 ± 0.04 after 48 h, and 0.34 ± 0.04 in the Control group. Transwell experiment showed that the invasion ability of osteosarcoma cells in Niraparib + Anti-GD2 group was 21.0 ± 1.5, and that in Control group was 87.7 ± 2.9. CCK8 experiment showed that the absorbance value of Niraparib + Anti-GD2 group was 0.16 ± 0.10 on day 5, and that of the Control group was 0.76 ± 0.09. Western blotting showed that the expression levels of BALP and CICP in Niraparib + Anti-GD2 group were 0.751 ± 0.135 and 1.086 ± 0.115, respectively, and those in Control group were 1.025 ± 0.143 and 1.216 ± 0.168, respectively. QRT-PCR results showed that the absorbance values of Niraparib + Anti-GD2 group were 0.173 ± 0.065 and 0.170 ± 0.078 on day 14. The results of animal experiments showed that on day 5, the tumor volume of the Control group was 2433 ± 391, and that of the Niraparib + Anti-GD2 group was 1137 ± 148. Histological analysis showed that the mean density values of Niraparib + Anti-GD2 group were 0.19 ± 0.08 and 0.22 ± 0.07, and those of Control group were 0.26 ± 0.09 and 0.29 ± 0.10. Conclusion: The combination of Niraparib and Anti-GD2 antibody significantly inhibits Osteosarcoma cells. [ABSTRACT FROM AUTHOR]

We investigated the angiogenesis‐modulating ability of noscapine in vitro using osteosarcoma cell line (MG‐63) and in vivo using a zebrafish model. MTT assay and the scratch wound healing assay were performed on the osteosarcoma cell line (MG‐63) to analyze the cytotoxic effect and antimigrative ability of noscapine, respectively. We also observed the antiangiogenic ability of noscapine on zebrafish embryos by analyzing the blood vessels namely the dorsal aorta, and intersegmental vessels development at 24, 48, and 72 h postfertilization. Real‐time polymerase chain reaction was used to analyze the hypoxia signaling molecules' gene expression in MG‐63 cells and zebrafish embryos. The findings from the scratch wound healing demonstrated that noscapine stopped MG‐63 cancer cells from migrating under both hypoxia and normoxia. Blood vessel development and the heart rate in zebrafish embryos were significantly reduced by noscapine under both hypoxia and normoxia which showed the hemodynamics impact of noscapine. Noscapine also downregulated the cobalt chloride (CoCl2) induced hypoxic signaling molecules' gene expression in MG‐63 cells and zebrafish embryos. Therefore, noscapine may prevent MG‐63 cancer cells from proliferating and migrating, as well as decrease the formation of new vessels and the production of growth factors linked to angiogenesis in vivo under both normoxic and hypoxic conditions. [ABSTRACT FROM AUTHOR]

Aims: Osteosarcoma is the most common primary bone malignancy among children and adolescents. We investigated whether benzamil, an amiloride analogue and sodium-calcium exchange blocker, may exhibit therapeutic potential for osteosarcoma in vitro. Methods: MG63 and U2OS cells were treated with benzamil for 24 hours. Cell viability was evaluated with the MTS/PMS assay, colony formation assay, and flow cytometry (forward/side scatter). Chromosome condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, cleavage of poly-ADP ribose polymerase (PARP) and caspase-7, and FITC annexin V/PI double staining were monitored as indicators of apoptosis. Intracellular calcium was detected by flow cytometry with Fluo-4 AM. The phosphorylation and activation of focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3) were measured by western blot. The expression levels of X-linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), SOD1, and SOD2 were also assessed by western blot. Mitochondrial status was assessed with tetramethylrhodamine, ethyl ester (TMRE), and intracellular adenosine triphosphate (ATP) was measured with BioTracker ATP-Red Live Cell Dye. Total cellular integrin levels were evaluated by western blot, and the expression of cell surface integrins was assessed using fluorescent-labelled antibodies and flow cytometry. Results: Benzamil suppressed growth of osteosarcoma cells by inducing apoptosis. Benzamil reduced the expression of cell surface integrins α5, αV, and β1 in MG63 cells, while it only reduced the expression of αV in U2OS cells. Benzamil suppressed the phosphorylation and activation of FAK and STAT3. In addition, mitochondrial function and ATP production were compromised by benzamil. The levels of anti-apoptotic proteins XIAP, Bcl-2, and Bcl-xL were reduced by benzamil. Correspondingly, benzamil potentiated cisplatin- and methotrexate-induced apoptosis in osteosarcoma cells. Conclusion: Benzamil exerts anti-osteosarcoma activity by inducing apoptosis. In terms of mechanism, benzamil appears to inhibit integrin/FAK/STAT3 signalling, which triggers mitochondrial dysfunction and ATP depletion. Cite this article: Bone Joint Res 2024;13(4):157–168.

Abstract The treatment of osteosarcoma (OS) is still mainly surgery combined with systematic chemotherapy, and gene therapy is expected to improve the survival rate of patients. This study aimed to explore the effect of DEP domain 1 protein (DEPDC1) and kinesin super-family protein 4A (KIF4A) in OS and understand its mechanism. Th expression of DEPDC1 and KIF4A in OS cells was detected by RT-PCR and western blot. The viability, proliferation, invasion and migration of OS cells and tube formation of human umbilical vein endothelial cells (HUVECs) after indicated treatment were in turn detected by CCK-8 assay, EdU staining, wound healing assay, transwell assay and tube formation assay. The interaction between DEPDC1 and KIF4A was predicted by STRING and confirmed by co-immunoprecipitation. The expression of epithelial-mesenchymal transition (EMT)-related proteins, tube formation-related proteins and Hippo signaling pathway proteins was detected by western blot. As a result, the expression of DEPDC1 and KIF4A was all increased in U2OS cells. Down-regulation of DEPDC1 suppressed the viability, proliferation, invasion and migration of U2OS cells and tube formation of HUVECs, accompanied by the increased expression of E-cadherin and decreased expression of N-cadherin, Vimentin and VEGF. DEPDC1 was confirmed to be interacted with KIF4A. Upregulation of KIF4A partially reversed the effect of DEPDC1 interference on the above biological behaviors of U2OS cells. Down-regulation of DEPDC1 promoted the expression of p-LATS1 and p-YAP in Hippo signaling pathway, which was reversed by upregulation of KIF4A. In conclusion, down-regulation of DEPDC1 inhibited the malignant biological behavior of OS cells through the activation of Hippo signaling pathway, which could be reversed by upregulation of KIF4A.

AimsAcridine orange (AO) demonstrates several biological activities. When exposed to low doses of X-ray radiation, AO increases the production of reactive radicals (radiodynamic therapy (AO-RDT)). We elucidated the efficacy of AO-RDT in breast and prostate cancer cell lines, which are likely to develop bone metastases.MethodsWe used the mouse osteosarcoma cell line LM8, the human breast cancer cell line MDA-MB-231, and the human prostate cancer cell line PC-3. Cultured cells were exposed to AO and radiation at various concentrations followed by various doses of irradiation. The cell viability was then measured. In vivo, each cell was inoculated subcutaneously into the backs of mice. In the AO-RDT group, AO (1.0 μg) was locally administered subcutaneously around the tumour followed by 5 Gy of irradiation. In the radiation group, 5 Gy of irradiation alone was administered after macroscopic tumour formation. The mice were killed on the 14th day after treatment. The change in tumour volume by AO-RDT was primarily evaluated.ResultsThe viability of LM8, MDA-MB-231, and PC-3 cells strongly decreased at AO concentration of 1.0 μg/ml and a radiation dose of 5 Gy. In xenograft mouse model, the AO-RDT also showed a strong cytocidal effect on tumour at the backside in osteosarcoma, breast cancer, and prostate cancer. AO-RDT treatment was more effective for tumour control than radiotherapy in breast cancer.ConclusionAO-RDT was effective in preventing the proliferation of osteosarcoma, breast cancer, and prostate cancer cell lines in vitro. The reduction in tumour volume by AO-RDT was also confirmed in vivo.Cite this article: Bone Joint Res 2022;11(10):715–722.

Due to the unique functional properties of shape memory alloys (SMAs) and current scientific interest in Cu-containing biomaterials, a continuously cast Cu-Al-Ni alloy in the form of rods has been investigated as a potential candidate for biomedical application. Additionally, the fact that Cu- complexes have an antitumour effect served as a cornerstone to develop more efficient drugs based on trace element complexes. In line with that, our study aimed to analyse the basic properties of the Cu-Al-Ni alloy, along with its anticancer properties. The detailed chemical analysis of the Cu-Al-Ni alloy was performed using XRF and SEM/EDX analyses. Furthermore, a microstructural and structure investigation was carried out, combined with hardness measurements using the static Vickers method. Observations have shown that the Cu-Al-Ni microstructure is homogeneous, with the presence of typical martensitic laths. XRD analysis confirmed the presence of two phases, β′ (monoclinic) and γ′ (orthorhombic). The viability of osteosarcoma cells in contact with the Cu-Al-Ni alloy was evaluated using epifluorescence microscopy, while their morphology and attachment pattern were observed and analysed using a high-resolution SEM microscope. Biocompatibility testing showed that the Cu-Al-Ni alloy exerted a considerable antineoplastic effect. [ABSTRACT FROM AUTHOR]

Photodynamic therapy (PDT) is a cutting-edge cancer treatment that utilizes both light and photosensitizers (PSs) to attack cancer cells. Methylene blue (MB) has emerged as a highly promising photosensitizer (PS) in PDT therapy due to its exceptional ability to produce singlet oxygen, which is attributed to its high quantum yield. However, the main challenge in utilizing MB in photodynamic therapy is its effective delivery to the target tissue. This challenge can be addressed by utilizing silica nanoparticles (SiNPs) as a drug delivery agent. Silica nanoparticles encapsulate MB and prevent its leakage, offering a novel approach to improving PDT therapy by reducing the toxicity of MB and increasing its bioavailability at the target cell. In this study, an extensive analysis of the size and shape evolution of the synthesized silica nanoparticles loaded with MB was conducted using TEM. Various encapsulated and bare MB concentrations were tested for cytotoxicity against osteosarcoma cells. Moreover, the optimal concentration and exposure time under light (with an intensity of approximately 8.9 mW/cm2 in the visible range) were determined to achieve maximum cell elimination. The results revealed that encapsulated MB in SiNPs exhibited a higher efficacy compared to naked MB, with a 50% increase in concentration effectiveness and a 90% increase in exposure time efficacy. This confirms that encapsulated MB in SiNPs is more effective in killing osteosarcoma cells than bare MB, thereby enhancing photodynamic therapy through increased bioavailability of MB in target cells. The enhanced bioavailability of MB in target cells as a result of its encapsulation in SiNPs makes it a highly promising drug delivery candidate for significantly enhancing the efficacy of photodynamic therapy against osteosarcomas. [ABSTRACT FROM AUTHOR]

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G protein coupled free fatty acid receptors (FFARs) are involved in the pathogenesis of several human diseases. FFAR2 and FFAR3 are activated by the binding of short-chain fatty acids (SCFAs). This study aimed to evaluate the roles of FFAR2 in the regulation of cellular functions in osteosarcoma HOS cells, using acetic acid and propanoic acid as FFAR2 and FFAR3 agonists. FFAR2 and FFAR3 genes were expressed in HOS cells. The cell motile activity of HOS cells was significantly stimulated by acetic acid and propanoic acid. In contrast, acetic acid and propanoic acid had no impact on the activation of matrix metalloproteinase-2 (MMP-2) and MMP-9. In cell survival assay, the cell survival rate to cisplatin (CDDP) of HOS cells was elevated by acetic acid and propanoic acid. To assess the effects of FFAR2 on cellular functions, FFAR2 knockdown (HOS-FFAR2) cells were generated from HOS cells. The cell motile activity of HOS-FFAR2 cells was enhanced by acetic acid and propanoic acid. In the presence of acetic acid and propanoic acid, MMP-2 and MMP-9 activities were reduced in HOS-FFAR2 cells, compared with control cells. When cells were treated with acetic acid and propanoic acid, the cell survival rate to CDDP of HOS-FFAR2 cells was significantly lower than that of control cells. These results suggest that activation of FFAR2-mediated signaling is involved in the modulation of cellular functions in HOS cells. • FFAR2 and FFAR3 genes were expressed in osteosarcoma HOS cells. • The cell motility and survival to CDDP were stimulated by FFAR2 and FFAR3 agonists in HOS cells. • HOS cell motility was enhanced by FFAR2 knockdown. • The cell survival to CDDP of HOS cells was inhibited by FFAR2 knockdown. • FFAR2-mediated signaling is involved in the regulation of cellular functions in HOS cells. [ABSTRACT FROM AUTHOR]

Background: The use of cold atmospheric plasma (CAP) in oncology has been intensively investigated over the past 15 years as it inhibits the growth of many tumor cells. It is known that reactive oxidative species (ROS) produced in CAP are responsible for this effect. However, to translate the use of CAP into medical practice, it is essential to know how CAP treatment affects non-malignant cells. Thus, the current in vitro study deals with the effect of CAP on human bone cancer cells and human osteoblasts. Here, identical CAP treatment regimens were applied to the malignant and non-malignant bone cells and their impact was compared. Methods: Two different human bone cancer cell types, U2-OS (osteosarcoma) and A673 (Ewing's sarcoma), and non-malignant primary osteoblasts (HOB) were used. The CAP treatment was performed with the clinically approved kINPen MED. After CAP treatment, growth kinetics and a viability assay were performed. For detecting apoptosis, a caspase-3/7 assay and a TUNEL assay were used. Accumulated ROS was measured in cell culture medium and intracellular. To investigate the influence of CAP on cell motility, a scratch assay was carried out. Results: The CAP treatment showed strong inhibition of cell growth and viability in bone cancer cells. Apoptotic processes were enhanced in the malignant cells. Osteoblasts showed a higher potential for ROS resistance in comparison to malignant cells. There was no difference in cell motility between benign and malignant cells following CAP treatment. Conclusions: Osteoblasts show better tolerance to CAP treatment, indicated by less affected viability compared to CAP-treated bone cancer cells. This points toward the selective effect of CAP on sarcoma cells and represents a further step toward the clinical application of CAP. [ABSTRACT FROM AUTHOR]

By using Raman microspectroscopy, it aims to elucidate the cellular variations caused by the combination drug of γ‐secretase inhibitor (DAPT) and cisplatin in osteosarcoma (OS) cells. Illustrated by the obtained results of spectral analysis, the intracellular composition significantly changed after combined drug actions compared to the solo DAPT treatment, indicating the synergistic effect of DAPT combined with cisplatin on OS cells. Meanwhile, multivariate curve resolution‐alternating least squares (MCR‐ALS) algorithm was utilized to address the biochemical constitution changes in all investigated groups including the untreated (UT), DAPT (40D) and combined drug (40D + 20C) treated cells. K‐means cluster and univariate imaging were both utilized to visualize the changes in subcellular morphology and biochemical distribution. The presented study provides a unique understanding on the cellular responses to DAPT combined with cisplatin from the natural biochemical perspectives, and laids an experimental foundation for exploring the therapeutic strategies of other combined anticancer drugs in cancer cell model. [ABSTRACT FROM AUTHOR]

Herein, we show intranuclear nanoribbons formed upon dephosphorylation of leucine‐rich L‐ or D‐phosphopeptide catalyzed by alkaline phosphatase (ALP) to selectively kill osteosarcoma cells. Being dephosphorylated by ALP, the peptides are first transformed into micelles and then converted into nanoribbons. The peptides/assemblies first aggregate on cell membranes, then enter cells via endocytosis, and finally accumulate in nuclei (mainly in nucleoli). Proteomics analysis suggests that the assemblies interact with histone proteins. The peptides kill osteosarcoma cells rapidly and are nontoxic to normal cells. Moreover, the repeated stimulation of the osteosarcoma cells by the peptides sensitizes the cancer cells rather than inducing resistance. This work not only illustrates a novel mechanism for nucleus targeting, but may also pave a new way for selectively killing osteosarcoma cells and minimizing drug resistance. [ABSTRACT FROM AUTHOR]

Cite this article: Bone Joint Res 2023;12(5):311–312.

Several semisynthetic, low-cardiotoxicity doxorubicin (DOXO) conjugated have been extensively described, considering the risk of cytotoxicity loss against resistant tumor cells, which mainly present drug efflux capacity. Doxorubicin 14-[4-(4-phenyl-5-thioxo-5H-[1,2]dithiol-3-yl)]-benzoate (H2S-DOXO) was synthetized and tested for its ability to overcome drug resistance with good intracellular accumulation. In this paper, we present a formulation study aimed to develop naked and decorated H2S-DOXO-loaded lipid nanoparticles (NPs). NPs prepared by the "cold dilution of microemulsion" method were decorated with hyaluronic acid (HA) to obtain active targeting and characterized for their physicochemical properties, drug entrapment efficiency, long-term stability, and in vitro drug release. Best formulations were tested in vitro on human-sensitive (MCF7) and human/mouse DOXO-resistant (MDA-MDB -231 and JC) breast cancer cells, on human (U-2OS) osteosarcoma cells and DOXO-resistant human/mouse osteosarcoma cells (U-2OS/DX580/K7M2). HA-decoration by HA-cetyltrimethyl ammonium bromide electrostatic interaction on NPs surface was confirmed by Zeta potential and elemental analysis at TEM. NPs had mean diameters lower than 300 nm, 70% H2S-DOXO entrapment efficiency, and were stable for almost 28 days. HA-decorated NPs accumulated H2S-DOXO in Pgp-expressing cells reducing cell viability. HA-decorated NPs result in the best formulation to increase the inter-cellular H2S-DOXO delivery and kill resistant cells, and therefore, as a future perspective, they will be taken into account for further in vivo experiments on tumor animal model. [ABSTRACT FROM AUTHOR]

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA1 to LPA6) exhibits a variety of malignant properties in cancer cells. Intracellular ATP depletion leads to the development of necrosis and apoptosis. The present study aimed to evaluate the effects of LPA receptor-mediated signaling on the regulation of cancer cell functions associated with ATP reduction. Long-term ethidium bromide (EtBr) treated (MG63-EtBr) cells were established from osteosarcoma MG-63 cells. The intracellular ATP levels of MG63-EtBr cells were significantly lower than that of MG-63 cells. LPAR2, LPAR3, LPAR4 and LPAR6 gene expressions were elevated in MG63-EtBr cells. The cell motile and invasive activities of MG63-EtBr cells were markedly higher than those of MG-63 cells. The cell motile activity of MG-63 cells was increased by LPA4 and LPA6 knockdowns. In cell survival assay, cells were treated with cisplatin (CDDP) every 24 h for 3 days. The cell survival to CDDP of MG63-EtBr cells was lower than that of MG-63 cells. LPA2 knockdown decreased the cell survival to CDDP of MG-63 cells. The cell survival to CDDP of MG-63 cells was inhibited by (2 S)-OMPT (LPA3 agonist). Moreover, the cell survival to CDDP of MG-63 cells was enhanced by LPA4 and LPA6 knockdowns. These results indicate that LPA signaling via LPA receptors is involved in the regulation of cellular functions associated with ATP reduction in MG-63 cells treated with EtBr. [ABSTRACT FROM AUTHOR]

Yan Huang,1 Yong-Qiang Xu,2 Si-Yin Feng,2 Xiang Zhang,2 Jiang-Dong Ni1 1Department of Orthopaedics, The Second Xiangya Hospital of Central South University, Changsha, Hunan Province, People’s Republic of China; 2Department of Orthopaedics, Hunan Provincial People’s Hospital, Changsha, Hunan Province, People’s Republic of ChinaCorrespondence: Jiang-Dong NiDepartment of Orthopaedics, The Second Xiangya Hospital of Central South University, No. 139, Renmin Middle Road, Furong District, Changsha, Hunan Province, People’s Republic of ChinaTel +86-73185295999Email nijiangdong001@csu.edu.cnObjective: This study aimed to investigate the effect of long non-coding TDRG1 on proliferation and migration of osteosarcoma cells through PI3K/AKT signaling pathway.Materials and Methods: Altogether 87 cases of osteosarcoma tissues and adjacent tissues were collected, and osteosarcoma cells and osteoblasts were purchased. The expression of LncRNA TDRG1 in tissues and cells was detected by RT-PCR. Si-NC, si-TDRG1, and Sh-TDRG1 were transfected into osteosarcoma cells. L740Y-P (activator of PI3K/AKT pathway) and LY294002 (inhibitor of PI3k/AKT pathway) were used to interfere with PI3k/Akt signaling pathway in osteosarcoma cells. qRT-PCR was used to detect the expression of TDRG1 in osteosarcoma tissues and cells. WB was used to detect the expression of p-PI3K, p-AKT, N-cadherin, E-Cadherin, vimentin, Bax, Caspase-3, and Bcl-2 in cells. CCK-8, Transwell and cell scratch tests were used to detect cell proliferation, invasion and migration, and flow cytometry was used to detect cell apoptosis.Results: TDRG1 was highly expressed in osteosarcoma, and the levels of p-PI3K and p-AKT were also up-regulated. Cell experiments showed that inhibiting the expression of TDRG1 could inhibit the proliferation, invasion, migration and EMT of osteosarcoma cells, promote the apoptosis of cells, and up-regulating the expression of TDRG1 could promote the proliferation, invasion, migration and EMT of osteosarcoma cells and inhibit the apoptosis of cells. The 740Y-P intervention could reverse the inhibition of Si-TDRG1 on osteosarcoma cell proliferation, invasion, migration and EMT and the promotion of cell apoptosis. LY294002 intervention could reverse the promotion of Sh-TDRG1 on osteosarcoma cell proliferation, invasion, migration and EMT and the inhibition of cell apoptosis.Conclusion: TDRG1 is highly expressed in osteosarcoma tissue. Silencing the expression of osteosarcoma can inhibit the proliferation, invasion, migration and EMT of osteosarcoma cells by inhibiting PI3K/AKT signaling pathway, which may be a new target for diagnosis and treatment of osteosarcoma.Keywords: LncRNA TDRG1, PI3K/AKT, osteosarcoma cells, proliferation, migration

Background: The use of cold atmospheric plasma (CAP) in oncology has been intensively investigated over the past 15 years as it inhibits the growth of many tumor cells. It is known that reactive oxidative species (ROS) produced in CAP are responsible for this effect. However, to translate the use of CAP into medical practice, it is essential to know how CAP treatment affects non-malignant cells. Thus, the current in vitro study deals with the effect of CAP on human bone cancer cells and human osteoblasts. Here, identical CAP treatment regimens were applied to the malignant and non-malignant bone cells and their impact was compared. Methods: Two different human bone cancer cell types, U2-OS (osteosarcoma) and A673 (Ewing’s sarcoma), and non-malignant primary osteoblasts (HOB) were used. The CAP treatment was performed with the clinically approved kINPen MED. After CAP treatment, growth kinetics and a viability assay were performed. For detecting apoptosis, a caspase-3/7 assay and a TUNEL assay were used. Accumulated ROS was measured in cell culture medium and intracellular. To investigate the influence of CAP on cell motility, a scratch assay was carried out. Results: The CAP treatment showed strong inhibition of cell growth and viability in bone cancer cells. Apoptotic processes were enhanced in the malignant cells. Osteoblasts showed a higher potential for ROS resistance in comparison to malignant cells. There was no difference in cell motility between benign and malignant cells following CAP treatment. Conclusions: Osteoblasts show better tolerance to CAP treatment, indicated by less affected viability compared to CAP-treated bone cancer cells. This points toward the selective effect of CAP on sarcoma cells and represents a further step toward the clinical application of CAP.

Introduction. Psoralen is a main active component of Psoralea corylifolia Linn. (Leguminosae). Psoralen has been reported to show antitumor effects and activity to accelerate osteoblastic proliferation. Nevertheless, the antitumor mechanism of psoralen in osteosarcoma has never been elucidated. The current study is aimed to investigate the therapeutic function of psoralen in human osteosarcoma cells and its potential regulatory mechanism. Material and methods. Effects of psoralen (0–70 μg/mL) on the viability of two osteosarcoma cell lines cultured for 48 h was evaluated by MTT assays. The concentration of IC10 (8 μg/mL for MG-63 cells and 9 μg/mL for U2OS cells) was regarded to be a non-cytotoxic dose selected as the working concentration in the subsequent experiments. Effects of psoralen on cell proliferation for 48 h was assessed by colony formation assays. Flow cytometry analyses were performed to measure cell cycle and apoptosis. RT-qPCR and Western blotting were carried out to assess RNA expression and protein levels of endoplasmic reticulum (ER) stress associated factors. Results. Psoralen inhibited osteosarcoma cell viability (IC50 25 μg/mL for MG-63 cells and IC50 40 μg/mL for U2OS cells) in a dose-dependent manner and growth inhibition rate reached the highest level when cells were treated with 70 μg/mL psoralen. Psoralen induced cell cycle arrest in the G0/G1 phase and promoted apoptosis of both MG-63 and U2OS cells. The treatment of psoralen resulted in an increase in ATF-6 and CHOP protein levels as well as a decrease in Bcl-2 protein level, indicating that cell apoptosis induced by psoralen was associated with ER stress. Treatment with 4-PBA, the ER stress inhibitor, attenuated the ability of psoralen to promote apoptosis of MG-63 and U2OS cells. Conclusions. Psoralen showed growth-inhibitory effects in osteosarcoma cells, and induced apoptosis via the ER stress pathway, which might be a potential drug to suppress the development of osteosarcoma. [ABSTRACT FROM AUTHOR]

Bioactive Polycaprolactone/Collagen nanofibers containing gehlenite Nano-powder were fabricated using electrospinning for bone tissue engineering. The physicochemical properties of scaffolds measured using scanning electron microscopy, tensile test, FTIR spectroscopy, and water contact angle measurement test. A slight enhancement (∼20 nm) in fibers diameter was observed by adding gehlenite nanoparticles to the scaffolds, but the fiber diameter distributions for both scaffolds were within the normal range of extracellular matrix (50–500 nm). In vitro experiments demonstrated well attachment and higher proliferation of MG-63 cells on gehlenite nanoparticles containing scaffolds. Besides, the stimulatory cell differentiation effects of Polycaprolactone/Collagen/gehlenite scaffolds were demonstrated by Alizarin red staining. This study represents for advanced of the natural and synthetic polymers for bone tissue engineering. [ABSTRACT FROM AUTHOR]

MicroRNAs play a crucial role in the progression of various cancers, and microRNA-221 (miR-221) has been observed to be significantly overexpressed in osteosarcoma (OS) cells. FBXW11, a vital F-box protein of the ubiquitin-proteasome system, mediates the proliferation and survival of cancer cells by targeting multiple substrates for degradation. FBXW11 inhibits OS growth and metastasis by antagonizing the β-catenin/Wnt signaling pathway. Therefore, we hypothesized that miR-221 targets FBXW11 to mediate Wnt signaling and promote OS proliferation. In this study, we demonstrated the increased expression of miR-221 and FBXW11 in OS tissues and cell lines by real-time polymerase chain reaction (RT-PCR). Moreover, to elucidate the regulatory mechanism(s) of miR-221 and FBXW11 in progression, cell viability and apoptosis were analyzed by the MTT assay and flow cytometry, respectively. The results showed that the overexpression of miR-221 in OS cells dramatically promoted cell growth and cell cycle progression, and inhibited apoptosis, whereas miR-221 inhibitors conversely inhibited proliferation and promoted apoptosis in OS cells. The data also showed that FBXW11 directly targeted miR-221 and miR-221 regulated OS cell proliferation and apoptosis by binding to FBXW11. We further confirmed that miR-221 targeted FBXW11 to promote proliferation and inhibit apoptosis in OS cell lines by inhibiting Wnt signaling. Overall, our study revealed a functional mechanism for miR-221 in OS. Further studies will elucidate its role in the progression of OS and inhibiting miR-221 may represent a useful treatment strategy. [ABSTRACT FROM AUTHOR]

Osteosarcoma (OS) is a primary malignant tumor of bone tissue. Effective chemotherapy may improve the survival of patients with OS. MicroRNAs (miRs) serve significant roles in the regulatory function of tumorigenesis and chemosensitivity of different types of cancer. miR-22 has been revealed to inhibit the proliferation and migration of OS cells, as well as increasing their sensitivity to cisplatin (CDDP). The mechanisms of action behind the functions of miR-22 in OS drug resistance require investigation. Therefore, in the present study, the human OS cell lines (MG-63, U2OS, Saos2 and OS9901) and a drug-resistant cell line (MG-63/CDDP) were cultured. Cell proliferation, apoptosis and autophagy assays were performed to investigate the proliferation, apoptosis and autophagy of cell lines transfected with miR-22 mimic. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were performed to investigate the expression levels of associated genes. The results revealed that miR-22 inhibited the proliferation of MG-63 cells and MG-63/CDDP cells, and enhanced the anti-proliferative ability of CDDP. miR-22 induced apoptosis and inhibited autophagy of MG-63 cells and MG-63/CDDP cells. Apoptosis-related genes, including caspase-3 and Bcl-2-associated X protein were upregulated, while B-cell lymphoma-2 was downregulated in both cell lines transfected with the miR-22 mimic. Autophagy protein 5, beclin1 and microtubules-associated protein 1 light chain 3 were downregulated in both cell lines transfected with miR-22 mimic. Furthermore, the in vitro and in vivo expression levels of metadherin (MTDH) in the OS/OS-CDDP-resistant models were downregulated following transfection with the miR-22 mimic. Therefore, the results of the present study suggested that miR-22 promoted CDDP sensitivity by inhibiting autophagy and inducing apoptosis in OS cells, while MTDH may serve a positive role in inducing CDDP resistance of OS cells. [ABSTRACT FROM AUTHOR]

The present study aimed at investigating how long-chain non-coding RNA (lncRNA) tumor suppressor candidate 7 (TUSC7) regulates the malignant biological behavior of osteosarcoma cells. Tumor tissues and adjacent tissues of 30 patients with osteosarcoma were collected, and the expression levels of lncRNA TUSC7 and miR-375 were detected by RT-qPCR. lncRNA TUSC7 mimic and miR-375 mimic transfection models were established in MG63 osteosarcoma cells, and Transwell assays were used to detect the migration ability of MG63 cells. An MTT assay was used to assess the proliferation ability of MG63 cells. lncRNA TUSC7 in osteosarcoma tissue was significantly lower than that of adjacent tissues, while miR-375 levels were significantly higher than that of adjacent tissues; the two levels have a negative correlation. lncRNA TUSC7 mimic inhibited MG63 proliferation and migration abilities. miR-375 mimic promoted MG63 proliferation and migration abilities. The lncRNA TUSC7 mimic and miR-375 mimic co-transfection system could partially rescue the inhibition of lncRNA TUSC7 mimic on MG63 cells. In conclusion, lncRNA TUSC7 inhibited the proliferation and migration of MG63 osteosarcoma cells by regulating miR-375. [ABSTRACT FROM AUTHOR]

Using confocal Raman micro‐spectroscopy, this study aims to elucidate the cellular responses of the γ‐secretase inhibitor, N‐[N‐(3,5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT), in osteosarcoma (OS) cells in a dose‐ and time‐dependent manner. The K7M2 murine OS cell line was treated with different DAPT doses (0, 10, 20, and 40 μM) for 24 and 48 hours before investigations. Significant compositional changes (nucleic acids, protein and lipid) after DAPT treatment were addressed, which testified inhibitory effect of DAPT on the growth of OS cells. Moreover, both partial least squares‐discriminant analysis (PLS‐DA) and principal component analysis‐linear discriminant analysis (PCA‐LDA) analyses revealed governing composition variations among groups by distinguishing their spectral characteristics. Furthermore, by adopting leave‐one‐out cross validation method, it is shown that PLS‐DA exhibited more classification capacity than PCA‐LDA algorithm. Hence, by understanding the DAPT‐based cellular variations, the achieved results provided an experimental foundation to establish new DAPT‐based anticancer therapeutic strategies, and preclinical Raman analytical methodologies on drug‐cell interactions. [ABSTRACT FROM AUTHOR]

The objective of the current in‐vitro study was to evaluate the biocompatibility of a new type of CAD/CAM scaffold for bone tissue engineering by using human cells. Porous lightweight titanium scaffolds and Bio‐Oss® scaffolds as well as their eluates were used for incubation with human osteoblasts, fibroblasts and osteosarcoma cells. The cell viability was assessed by using fluorescein diazo‐acetate propidium iodide staining. Cell proliferation and metabolism was examined by using MTT‐, WST‐Test and BrdU‐ELISA tests. Scanning electron microscope was used for investigation of the cell adhesion behaviour. The number of devitalised cells in all treatment groups did not significantly deviate from the control group. According to MTT and WST results, the number of metabolically active cells was decreased by the eluates of both test groups with a more pronounced impact of the eluate from Bio‐Oss®. The proliferation of the cells was inhibited by the addition of the eluates. Both scaffolds showed a partial surface coverage after 1 week and an extensive to complete coverage after 3 weeks. The CAD/CAM titanium scaffolds showed favourable biocompatibility compared to Bio‐Oss® scaffolds in vitro. The opportunity of a defect‐specific design and rapid prototyping by selective laser melting are relevant advantages in the field of bone tissue engineering and regenerative medicine. [ABSTRACT FROM AUTHOR]

Vitamin C and iron are both important nutrients for humans and involved in several physiological processes. The biological activities of vitamin C and iron are based on their abilities to accept or donate electrons. Although vitamin C is well known as an excellent electron donor in physiological conditions, it also has pro-oxidant properties, especially with catalytic metal iron. Cancer cells have a higher iron requirement than normal cells, which allows pharmacological ascorbate to kill cancer cells selectively. In this study, we demonstrated that the levels of H2O2 in cells were significantly raised after treated with pharmacological ascorbate, and intracellular labile iron could increase pharmacological ascorbate-mediated oxidative stress by Fenton reaction. Catalytic metal iron plays opposite roles in and outside cells. Intracellular excess labile iron improved ascorbate-induced toxicity, while the excess labile iron in the medium abolished ascorbate-induced toxicity. Fe3+ and Fe2+ have the same effect on ascorbate-induced toxicity, but Fe3+ chelator deferoxamine (DFO) has a profound inhibition effect than Fe2+ chelator 2,2′-bipyridyl (BIP) on ascorbate-induced toxicity. The influence of intracellular labile iron and ascorbate on the ferritin expression may cause selective sensitivity in osteosarcoma cell lines on pharmacological ascorbate. High iron requirement of many cancer cells facilitates pharmacological ascorbate on cancer treatment. In addition, increasing iron content in tumour tissue may be effective strategies to improve the effects of pharmacological ascorbate. [ABSTRACT FROM AUTHOR]

Free fatty acid receptor 1 (FFA1) and FFA4 belong to a family of free fatty acid (FFA) receptors. FFA1- and FFA4-mediated signaling regulates a variety of malignant properties in cancer cells. It is known that stromal cells in the tumor microenvironment promote tumor progression. In the present study, to assess the roles of FFA1 and FFA4 in cellular functions modulated by endothelial cells, highly migratory MG63-CR7(F2) cells were generated from osteosarcoma MG-63 cells, using endothelial F2 cell supernatants. Expression levels of FFAR1 and FFAR4 genes in MG63-CR7(F2) cells were significantly higher than those of MG-63 cells. In cell survival assay, cells were treated with cisplatin (CDDP) every 24 h for 2 days. The cell survival rate of MG-63 cells was significantly elevated by an FFA1 agonist TUG-770 as well as an FFA4 agonist TUG-891. Moreover, the cell survival rate of MG63-CR7(F2) cells was higher than that of MG-63 cells in the presence of TUG-770 or TUG-891, correlating with FFAR1 and FFAR4 expression levels. To validate the effects of FFA1 and FFA4 on cell survival to CDDP, FFA1 and FFA4 knockdown cell were generated from MG-63 cells. The cell survival rate of MG-63 cells was markedly inhibited by FFA1 or FFA4 knockdown. These results suggest that FFA1 and FFA4 may play an important role in the modulation of cellular functions by endothelial cells in osteosarcoma cells. [ABSTRACT FROM AUTHOR]

The study investigates the anticancer activity of mefenamic acid against osteosarcoma, shedding light on its underlying mechanisms and therapeutic potential. Mefenamic acid exhibited robust inhibitory effects on the proliferation of MG-63, HOS, and H2OS osteosarcoma cells in a dose-dependent manner. Moreover, mefenamic acid induced cellular toxicity in MG63 cells, as evidenced by LDH leakage, reflecting its cytotoxic impact. Furthermore, mefenamic acid effectively suppressed the migration and invasion of MG-63 cells. Mechanistically, mefenamic acid induced apoptosis in MG-63 cells through mitochondrial depolarization, activation of caspase-dependent pathways, and modulation of the Bcl-2/Bax axis. Additionally, mefenamic acid promoted autophagy and inhibited the PI3K/Akt/mTOR pathway, further contributing to its antitumor effects. The molecular docking studies provide compelling evidence that mefenamic acid interacts specifically and strongly with key proteins in the PI3K/AKT/mTOR pathway, suggesting a novel mechanism by which mefenamic acid could exert anti-osteosarcoma effects. In vivo studies using a xenograft mouse model demonstrated significant inhibition of MG-63 tumor growth without adverse effects, supporting the translational potential of mefenamic acid as a safe and effective therapeutic agent against osteosarcoma. Immunohistochemistry staining corroborated the in vivo findings, highlighting mefenamic acid's ability to suppress tumor proliferation and inhibit the PI3K/AKT/mTOR pathway within the tumor microenvironment. Collectively, these results underscore the promising therapeutic implications of mefenamic acid in combating osteosarcoma, warranting further investigation for clinical translation and development. [Display omitted] • This study is the first to reveal mefenamic acid's antitumor activity against osteosarcoma. • Mefenamic acid demonstrated a dose-dependent reduction in viability and clonogenic activity of human osteosarcoma MG-63 cells. • Mefenamic acid impede the cells' migratory and invasive behavior, crucial factors in cancer progression. • Mefenamic acid induced cell death in MG-63 cells through the activation of the Bcl-2/Bax axis in mitochondrial pathways. • Mefenamic acid exhibited antitumor effects by suppressing the PI3K/Akt/mTOR signaling pathway. [ABSTRACT FROM AUTHOR]

[Display omitted] Mastering new and efficient ways to obtain successful drug delivery systems (DDS) with controlled release became a paramount quest in the scientific community. Increase of malignant bone tumors and the necessity to optimize an approach of localized drug delivery require research to be even more intensified. Octacalcium phosphate (OCP), with a number of advantages over current counterparts is extensively used in bone engineering. The aim of the present research was to synthesize bioactive and biocompatible doxorubicin (DOX) containing OCP particles. DOX-OCP was successfully obtained in situ in an exhaustive range of added drug (1–20 wt%, theoretical loading). Based on XRD, above 10 wt% of DOX, OCP formation was inhibited and the obtained product was low crystalline α-TCP. In-vitro drug release was performed in pH 7.4 and 6.0. In both pH environments DOX had a continuous release over six weeks. However, the initial drug burst for pH 7.4, in the first 24 h, ranged from 15.9 ± 1.3 % to 33.5 ± 12 % and for pH 6.0 23.7 ± 1.5 % to 36.2 ± 12 %.The DOX-OCP exhibited an inhibitory effect on viability of osteosarcoma cell lines MG63, U2OS and HOS. In contrast, MC3T3-E1 cells (IC50 > 0.062 µM) displayed increased viability and proliferation from 3rd to 7th day. Testing of the DDS on ferroptotic markers (CHAC1, ACSL4 and PTGS2) showed that OCP-DOX does not induce ferroptotic cell death. Moreover, the evaluation of protein levels of cleaved PARP, by western blotting analysis, corroborated that apoptosis is the main pathway of programmed cell death in osteosarcoma cells induced by DOX-OCP. [ABSTRACT FROM AUTHOR]

Therapeutic treatments with Artemisia annua have a long-established tradition in various diseases due to its antibacterial, antioxidant, antiviral, anti-malaria and anti-cancer effects. However, in relation to the latter, virtually all reports focused on toxic effects of A. annua extracts were obtained mostly through conventional maceration methods. In the present study, an innovative extraction procedure from A. annua, based on pressurised cyclic solid–liquid (PCSL) extraction, resulted in the production of a new phytocomplex with enhanced anti-cancer properties. This extraction procedure generated a pressure gradient due to compressions and following decompressions, allowing to directly perform the extraction without any maceration. The toxic effects of A. annua PCSL extract were tested on different cells, including three cancer cell lines. The results of this study clearly indicate that the exposure of human, murine and canine cancer cells to serial dilutions of PCSL extract resulted in higher toxicity and stronger propensity to induce apoptosis than that detected by subjecting the same cells to Artemisia extracts obtained through canonical extraction by maceration. Collected data suggest that PCSL extract of A. annua could be a promising and economic new therapeutic tool to treat human and animal tumours.

OBJECTIVE: To investigate the influences of rapamycin on proliferation and apoptosis of human osteosarcoma MG-63 cells and the mechanisms of action. MATERIALS AND METHODS: The human osteosarcoma MG-63 cells were randomly divided into Control group, Rapamycin group, and Rapamycin + Beclin-1 plasmid transfection group. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was adopted to detect the viability of MG-63 cells in each group, and the 5-Ethynyl-2'-deoxyuridine (EdU) staining and Hoechst staining were applied to determine the proliferation and apoptosis, respectively, of MG-63 cells in each group. The levels of B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) were measured using enzyme-linked immunosorbent assay (ELISA) kits, and the protein expression levels of Beclin-1 and Vps34 in each group of MG-63 cells were tested using the Western blotting. RESULTS: Compared with the Control group, Rapamycin group, and Rapamycin + Beclin-1 plasmid transfection group had markedly weakened the viability of MG-63 cells, inhibited cell proliferation, remarkably increased cell apoptosis rate, elevated Bax level, notably declined Bcl-2 level, and significantly raised the levels of Beclin-1 and Vps34 proteins in MG-63 cells. Besides, the effects in Beclin-1 plasmid transfection group were stronger. CONCLUSIONS: Rapamycin may decrease the viability, inhibit the proliferation, and promote the apoptosis of MG-63 cells by activating autophagy. [ABSTRACT FROM AUTHOR]

The differential expression of miRNAs plays an important role in the study of the occurrence, development, metastasis and prognosis of osteosarcoma. This differential expression also provides a new direction for drug targeted therapy of osteosarcoma. The purpose of this study is to investigate the effect of microrna-x expression on the proliferation, invasion and migration of osteosarcoma cell lines, and to provide theoretical basis for molecular targeted therapy of osteosarcoma. In this paper, whether mir-29c can regulate the invasion, metastasis and proliferation of osteosarcoma cell lines was studied. The effects of mir-29c on the proliferation and metastasis of osteosarcoma cell lines were verified by the methods of Lipofectamine 2000 cell transient transfection, Transwell cell invasion and migration experiment, MTT cell proliferation experiment and other experimental methods. The results showed that the average cell number of F4 / 29cinhibitor was 471.4 ± 87.5, the average cell number of F4 / NC and F4 / blank were 227.4 ± 76.2 and 189.1 ± 36.8, respectively. Mir-29c can inhibit the invasion, migration and proliferation of osteosarcoma cell line sosp9607. [ABSTRACT FROM AUTHOR]

Early diagnosis of cancer often allows for a more vast choice of therapy opportunities. After a cancer diagnosis, staging provides essential information about the extent of disease in the body and the expected response to a particular treatment. The leading importance of classifying cancer patients at the early stage into high or low-risk groups has led many research teams, both from the biomedical and bioinformatics field, to study the application of Deep Learning (DL) methods. The ability of DL to detect critical features from complex datasets is a significant achievement in early diagnosis and cell cancer progression. In this paper, we focus the attention on osteosarcoma. Osteosarcoma is one of the primary malignant bone tumors which usually afflicts people in adolescence. Our contribution to classification of osteosarcoma cells is made as follows: a DL approach is applied to discriminate human Mesenchymal Stromal Cells (MSCs) from osteosarcoma cells and to classify the different cell populations under investigation. Glass slides of different cell populations were cultured including MSCs, differentiated in healthy bone cells (osteoblasts) and osteosarcoma cells, both single cell populations or mixed. Images of such samples of isolated cells (single-type of mixed) are recorded with traditional optical microscopy. DL is then applied to identify and classify single cells. Proper data augmentation techniques and cross-fold validation are used to appreciate the capabilities of a convolutional neural network to address the cell detection and classification problem. Based on the results obtained on individual cells, and to the versatility and scalability of our DL approach, the next step will be its application to discriminate and classify healthy or cancer tissues to advance digital pathology. [ABSTRACT FROM AUTHOR]

Objective To establish the lentivirus RNA interference system targeting adrenomedullin (ADM) of human osteosarcoma cells to efficiently inhibit the expression of ADM gene. Methods We constructed lentivirus vector and designed three shRNA sequences ( shRNA-1, 2, 3) for ADM gene sequence. When the target ADM shRNA was connected to the lentivirus vector, LV-ADM-shRNA positive clones were selected for PCR identification and sequencing. After the oli-gonucleotide insertion sites were confirmed to be correct by sequencing, the target plasmids ( LV-ADM-shRNA 1, 2, 3) were extracted. The lentiviruses were packaged in 293T cells, and the titer of lentivirus was determined by porous dilution method. Result Three lentiviral vectors targeting ADM-shRNA were constructed and lentiviral particles with higher titers were obtained. Conclusion We successful established the RNA interference system with lentivirus-mediated adrenomedullin in human osteosarcoma cells. [ABSTRACT FROM AUTHOR]

Background: PLK1, a typical PLK protein, is the main driver of cancer cell growth and proliferation. It is an inhibitor of the protein kinases that is currently being investigated in clinical studies. It is often used as a tumor marker, as high PLK1 expression correlates with poor prognosis in cancer. Overexpression of MYC is a hallmark of many human cancers. MYC modulates the transcription of thousands of genes that required to coordinate a series of cellular processes, including those essential for growth, proliferation, differentiation, self-renewal and apoptosis. To date, functions of PLK1 and MYC on tumor are mostly studied in separate researches, and studies on their mutual crosstalk are lacking. Purpose: To investigate the mechanism of PLK1 and MYC in regulating progress of osteosarcoma. Methods: Protein level was examined using Western blot. Animal experiments were performed with female FOX CHASE severe combined immunodeficient mice. Mice were randomly divided into experimental or control groups. Results: PLK1 or MYC promoted the proliferation of osteosarcoma cells through the autophagy pathway. PLK1 contributed to MYC protein stabilization. PLK1 inhibition enhanced MYC degradation in osteosarcoma cells. PLK1 inhibition led to a marked decline in MYC protein abundance. The representative MYC target genes were deregulated by PLK1 inhibitors. BI2536 treatment caused a significant delay in xenograft tumor growth in mice injected with U-2 OS cells subcutaneously, with lower mean tumor weight compared to the control group. Conclusion: PLK1 is crucial for MYC stabilization. It promotes cell proliferation by autophagy pathway in osteosarcoma cells. Data validate PLK1 as a potential therapeutic target in osteosarcoma caused by MYC-amplified. [ABSTRACT FROM AUTHOR]

Sterculia foetida seeds are rich in various secondary metabolites such as fatty acids, alkaloids, flavonoids, and saponins, and possess multidisciplinary anti-inflammatory, anti-microbial, anti-diabetic, and anti-obesity pharmacological activities. The present study described the phytochemical compounds along with, anti-microbial, anti-oxidant, and first time in vitro anti-osteosarcoma effects from these seeds. The seeds were collected, authenticated, dried, ground into crude powder, and the secondary metabolites extracted with ethanol. Further, gas chromatography and mass spectrophotometry (GC-MS) analysis was carried out to determine the presence of SFE phytochemical compounds. Anti-microbial activity was confirmed against selected bacterial strains by agar well diffusion and anti-oxidant activity was investigated through 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging method. The anti-proliferative and apoptotic activity was analyzed by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) colorimetric assay, reactive oxygen species (ROS) generation, decrease in the mitochondrial membrane potential (MMP), and nuclear fragmentation assay. The results showed that SFE contained 35 active phytochemical and the secondary metabolites, alkaloids, glycosides, flavonoids, phenols, saponins, terpenoids, and tannins, which demonstrated significant anti-microbial and anti-oxidant potential. The results for SFE anti-cancerous activity on MG-63 osteosarcoma cells were reduced cell viability, generated excessive ROS, induced nuclear fragmentation and decreased MMP in a concentration-dependent manner. It was concluded that SFE had potent anti-oxidant, anti-microbial, and anti-cancerous activity on the MG-63 cells. However, further studies are needed to validate SFE efficacy for prevention and management of osteosarcoma. [ABSTRACT FROM AUTHOR]

Cell migration is closely involved in cancer cell invasion into surrounding tissue and metastasis to the distant organs. It is crucial for understanding the molecular mechanisms that regulate cell migration in cancer cells. The aim of this study is to establish a rapid induction method of highly migratory cells from cancer cells. Osteosarcoma MG-63 and colon cancer DLD1 cells were seeded at 1 × 105 cells in 6-well plates. After 10 min, unattached cells were washed off three times with PBS. The cells which remained attached on the bottom of plates were cultured in DMEM containing 10% FBS. When the cells reached approximately 80% confluence, cells were harvested using trypsin/EDTA. The harvested cells were seeded in other 6-well plates and incubated for 10 min. The unattached cells were washed off and attached cells were further cultured. By repeating this procedure 11–12 times for 2 months, highly migratory MG63-A12 and DLD-A11 cells were obtained from MG-63 and DLD1 cells, respectively. In cell motility assay, the cell motile activities of MG63-A12 and DLD-A11 cells was 10.3 and 13.7 times higher than those of the parental cells, respectively. This procedure is useful to generate highly migratory cells for investigating cellular functions during tumor progression in cancer cells. [ABSTRACT FROM AUTHOR]

Survival rates of osteosarcoma patients could not be significantly improved by conventional chemotherapeutic treatment regimens since the introduction of high-dose chemotherapy 35 years ago. Therefore, there is a strong clinical need for new therapeutic targets and personalized treatment strategies, requiring reliable in vivo model systems for the identification and testing of potential new treatment approaches. Conventional in vivo rodent experiments face ethical issues, are time consuming and costly, being of particular relevance in orphan diseases like osteosarcoma. An attractive alternative to such animal experiments is the chicken chorioallantoic membrane (CAM) assay. The CAM is a highly vascularized, non-innervated extra-embryonic membrane that is perfectly suited for the engraftment of tumor cells. However, only few reports are available for osteosarcoma and reported data are inconsistent. Therefore, the aim of this study was the adaptation and optimization of the CAM assay for its application in osteosarcoma research. Tumor take rates and volumes of osteosarcoma that developed on the CAM were analyzed after modification of several experimental parameters, including egg windowing, CAM pretreatment, inoculation technique and many more. Eight osteosarcoma cell lines were investigated. Our optimized OS-CAM-assay was finally validated against a rat animal xenograft model. Using the cell line MNNG HOS as reference we could improve the tumor take rates from 51% to 94%, the viability of the embryos from initially 40% to >80% and achieved a threefold increase of the tumor volumes. We were able to generate solid tumors from all eight osteosarcoma cell lines used in this study and could reproduce results that were obtained using an osteosarcoma rat animal model. The CAM assay can bridge the gap between in vitro cell culture and in vivo animal experiments. As reliable in vivo model for osteosarcoma research the optimized CAM assay may speed up preclinical data collection and simplifies research on potential new agents towards personalized treatment strategies. Further, in accordance with Russell’s and Burch’s “Principles of Humane Experimental Technique” the reasonable use of this model provides a refinement by minimizing pain and suffering of animals and supports a considerable reduction and/or replacement of animal experiments. [ABSTRACT FROM AUTHOR]

Background and Aim: Osteosarcoma is a devastating tumor of bone, primarily affecting adolescents. Parthenolide, a naturally occurring small molecule that interferes with NF-κB signaling, has recently attracted considerable attention because of its pharmacological action involving anti-cancer effects. However, the mechanism of the cytotoxic effect exerted by parthenolide on tumor cells is not clearly defined today. Methods: In this study, the effects of parthenolide were evaluated and characterized in human osteosarcoma cancer cell. Cell viability was assessed by CCK-8. Apoptosis was assessed by Annexin V-FITC/PI Flow cytometry assay. Relative quantitative real-time PCR and western blot were used to determine the expressions of genes and proteins. Results: Our results suggest that parthenolide did not cause caspase-dependent cell death in osteosarcoma cancer cells, as indicated by the absence of significant early apoptosis as well as caspase-3 cleavage. Instead, parthenolide increased the autophagy and mitophagy, as characterized by increased PINK1 and Parkin translocation to mitochondria and enhanced autophagy proteins. The induction of autophagy by parthenolide was associated with the increase of reactive oxygen species (ROS). ROS antioxidants N-acetylcysteine (NAC) attenuated parthenolide-induced autophagy activity. Conclusions: Our findings unveil a novel mechanism of drug action by parthenolide in osteosarcoma cancer cells and suggest a potential value of treating osteosarcoma cancer through a caspase-independent autophagic cell death by ROS activation.

Purpose: Drug resistance has been recognized to be a major obstacle to the chemotherapy for osteosarcoma. And the potential importance of hypoxia as a target to reverse drug resistance in osteosarcoma has been indicated, though the mechanism underlining such role is not clarified. The present study aims to investigate the role of hypoxia in the drug resistance in osteosarcoma cells via activating AMP-activated protein kinase (AMPK) signaling. Experimental design: We investigated the promotion of the resistance to doxorubicin of osteosarcoma MG-63 and U2-os cells in vitro, and then determined the role of hypoxia-inducible factor-1 (HIF-1)α and HIF-1β, the activation and regulatory role of AMPK in the osteosarcoma U2-os cells which were treated with doxorubicin under hypoxia. Results: It was demonstrated that hypoxia significantly reduced the sensitivity of MG-63 and U2-os cells to doxorubicin, indicating an inhibited viability reduction and a reduced apoptosis promotion. And such reduced sensitivity was not associated with HIF-1α, though it was promoted by hypoxia in U2-os cells. Interestingly, the AMPK signaling was significantly promoted by hypoxia in the doxorubicin-treated U2-os cells, with a marked upregulation of phosphorylated AMPK (Thr 172) and phosphorylated acetyl-CoA carboxylase (ACC) (Ser 79), which were sensitive to the AMPK activator, AICAR and the AMPK inhibitor, Compound C. Moreover, the promoted AMPK activity by AICAR or the downregulated AMPK activity by Compound C significantly reduced or promoted the sensitivity of U2-os cells to doxorubicin. Conclusion: The present study confirmed the AMPK signaling activation in the doxorubicin-treated osteosarcoma cells, in response to hypoxia, and the chemical upregulation or downregulation of AMPK signaling reduced or increased the chemo-sensitivity of osteosarcoma U2-os cells in vitro. Our study implies that AMPK inhibition might be a effective strategy to sensitize osteocarcoma cells to chemotherapy.

Our results showed that the expression of calcium‐sensing receptor (CaSR) and the concentration of intracellular calcium were enhanced in the osteosarcoma cells MG‐63 compared with in the normal osteoblasts hFOB1.19. GdCl3 (CaSR agonist) significantly increased the proliferation rate of MG‐63 cells, the expression of PCNA and Cyclin D1 and decreased the expression of p21Cip/WAF‐1. The effect of NPS2390 (CaSR antagonist) on the above indicators was opposite to GdCl3. In addition, GdCl3 up‐regulated the phosphorylation of ERK1/2, PI3K and Akt and the proliferation rate of MG‐63 cells. However, these effects of GdCl3 were cancelled by LY294002 (a PI3K inhibitor) or PD98059 (an ERK1/2 inhibitor). Our results demonstrate that activation of CaSR promotes osteosarcoma cell multiplication by up‐regulating ERK1/2 and PI3K‐Akt pathways. [ABSTRACT FROM AUTHOR]

Abstract Magnetic nanoparticles (MNPs) functionalized with different therapeutics delivered by mesenchymal stem cells represent a promising approach to improve the typical drug delivery methods. This innovative method, based on the "Trojan horse" principle, faces however important challenges related to the viability of the MNPs-loaded cells and drug stability. In the present study we report about an in vitro model of adipose-derived stem cells (ADSCs) loaded with palmitate-coated MNPs (MNPsPA) as antitumor drug carriers targeting a 3D tissue-like osteosarcoma cells. Cell viability, MNPsPA-drug loading capacity, cell speed, drug release rate, magnetization and zeta potential were determined and analysed. The results revealed that ADSCs loaded with MNPsPA-drug complexes retained their viability at relatively high drug concentrations (up to 1.22 pg antitumor drug/cell for 100% cell viability) and displayed higher speed compared to the targeted tumor cells in vitro. The magnetization of the sterilized MNPsPA complexes was 67 emu/g within a magnetic field corresponding to induction values of clinical MRI devices. ADSCs payload was around 9 pg magnetic material/cell, with an uptake rate of 6.25 fg magnetic material/min/cell. The presented model is a proof-of-concept platform for stem cells-mediated MNPs-drug delivery to solid tumors that could be further correlated with MRI tracking and magnetic hyperthermia for theranostic applications. Highlights • tumor targeting via magnetic nanoparticle-loaded stem cells as delivery agents was identified as a promising approach for cancer chemotherapy • ADSCs loaded with MNPs coated with palmitic acid and mitoxantrone retain their viability and gain clinically relevant magnetization • mitoxantrone-functionalized magnetic nanoparticles carried by ADSC are shown to target osteosarcoma tissue like structures in vitro [ABSTRACT FROM AUTHOR]

Cultured cells easily develop resistance to kinesin-5 inhibitors (K5Is) often by overexpressing a related motor protein, kinesin-12/KIF15, or by acquiring mutations in the N-terminal motor domain of kinesin-5/KIF11 itself. We aimed to identify novel mechanisms responsible for resistance to S-trityl L-cysteine (STLC), one of the K5Is, using human osteosarcoma cell lines. Among six lines examined, U-2OS and HOS survived chronic STLC treatment and gave rise to resistant cells with IC50s at least 10-fold higher than those of the respective parental lines. Depletion of KIF15 largely eliminated the acquired K5I resistance in both cases, consistent with the proposed notion that KIF15 is indispensable for it. In contrast to the KIF11-independent property of the cells derived from HOS, those derived from U-2OS still required KIF11 for their growth and, intriguingly, expressed a C-terminal truncated variant of KIF11 resulting from a frame shift mutation (S1017fs). All of the isolated clones harbored the same mutation, suggesting its clonal expansion in the cell population due to the growth advantage during chronic STLC treatment. Transgenic expression of KIF11S1017fs in the parental U-2OS cells, as well as in HeLa cells, conferred a moderate but reproducible STLC resistance, probably owing to STLC-resistant localization of the mutant KIF11 on mitotic spindle. Our observations indicate that both KIF15 and the C-terminal-truncated KIF11 contributes to the STLC resistance of the U-2OS derived cells. [ABSTRACT FROM AUTHOR]

Dogs diagnosed with appendicular osteosarcoma typically succumb to metastatic disease within a year of diagnosis. The current standard of care for curative intent, amputation followed by adjuvant chemotherapy, increases survival time but chemoresistance is a major contributor to mortality. Unfortunately, the mechanisms driving the progression of metastatic disease and the development of chemoresistance are unknown. One theory is that autophagy may contribute to chemoresistance by providing neoplastic cells with a mechanism to survive chemotherapy treatment. Our objective was to evaluate the effect of combining an autophagy inhibitor with a standard chemotherapeutic drug on response to chemotherapy in canine appendicular osteosarcoma cells. We hypothesized that combining the autophagy inhibitor spautin-1 with doxorubicin treatment would enhance chemoresponsiveness. Using commercial (D17) and primary cell lines derived from 1° and 2° sites of osteosarcoma, we showed that this combination treatment enhances cell killing and inhibits colony formation. Our findings support the theory that autophagy contributes to chemoresistance in canine appendicular osteosarcoma and indicate that adding an autophagy inhibitor to the standard of care has the potential to improve outcome. [ABSTRACT FROM AUTHOR]

Resveratrol shows potent anti-tumor therapeutic properties in various tumors. However, the exact effect of resveratrol on osteosarcoma cells, especially cancer stem cells, remains unclear. In this study, we examined the effect of resveratrol on osteosarcoma stem cells and explored the underlying molecular mechanisms. Resveratrol inhibited cell viability, self-renewal ability and tumorigenesis of osteosarcoma cells, whereas showed no significant inhibition effects to normal osteoblast cells. Mechanically, resveratrol treatment decreased cytokines synthesis and inhibited JAK2/STAT3 signaling, which was consistent with the decline of cancer stem cells marker, CD133. Exogenous STAT3 activation attenuated the cancer stem cell elimination effects of resveratrol treatment. Our results demonstrated that resveratrol inhibited osteosarcoma cell proliferation and tumorigenesis ability, which was correlated with cytokines inhibition related JAK2/STAT3 signaling blockage. Resveratrol may be a promising therapeutic agent for osteosarcoma management. [ABSTRACT FROM AUTHOR]

Background: Osteosarcoma (OS) is a primary malignant bone tumor with a high fatality rate. Many circRNAs have been proved to play important roles in the pathogenesis of some diseases. However, the occurrence of circRNAs in OS remains little known. Methods: The circular RNA (circRNA) expression file GSE96964 dataset, which included seven osteosarcoma cell lines and one control sample (osteoblast cell line), was downloaded from the Gene Expression Omnibus (GEO) database to explore the potential function of circRNAs in osteosarcoma by competing endogenous RNA (ceRNA) analysis. Three gene expression profiles of OS were downloaded from GEO database and then used for the pathway enrichment analysis, Venn analysis and protein-protein interaction (PPI) network analysis. Real-time qPCR validation and RNA interference were conducted to verify our prediction. Results: Differentially expressed circRNAs between OS and control, including 8 up-regulated and 102 down-regulated circRNAs, were generated and ceRNA analysis for 5 most up-regulated or 5 most down-regulated circRNAs in OS were then performed. The pathway enrichment analysis of gene expression profiles indicated differentially expressed genes (DEGs) of three gene profiles significantly enriched in cell cycle pathway, cell adhesion molecules (CAMs) pathway, oxidative phosphorylation pathway, cytokine-cytokine receptor interaction pathway, p53 signaling pathway and proteoglycans in cancer pathway, which were critical important pathways in the pathogenesis of OS. The Venn analysis showed that 2 (one is a pseudogene) up-regulated and 39 down-regulated DEGs were co-expressed in all three gene profiles. Then PPI networks of 41 co-expressed DEGs (up- and down-regulated DEGs) were constructed to predict their functions using the GeneMANIA. The expression levels of these related RNAs also matched our predictions really well. Conclusion: Ultimately, we found cell adhesion molecule 1 (CADM1) gene was not only a co-expression mRNA of the three mRNA expression profiles of OS, but also are predicted to be regulated by hsa_circ_0032462, hsa_circ_0028173, hsa_circ_0005909 by functioning as miRNAs ‘Sponge’ in human osteosarcoma. These over-expressed circRNAs may result in the over expression of CADM1 which promote the development of OS. We envision this discovery of these important moleculars, incuding hsa_circ_0032462, hsa_circ_0028173, hsa_circ_0005909 and CADM1 may lead to further development of new concepts, thus allowing for more opportunities in diagnosis and therapy of OS. [ABSTRACT FROM AUTHOR]