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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., (© 2024. The Author(s).)

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 (CoCl 2 ) 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., (© 2024 Wiley Periodicals LLC.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: It has been reported that inhibition of Fucosyltransferase4 (FUT4) to activate Forkhead box O1 (FOXO1) can lead to apoptosis of cancer cells, however, the mechanism in osteosarcoma is still unclear., Objective: To explore the biological significance of the connection between FUT4 and FOXO1 in osteosarcoma growth., Methods: In vitro tests were conducted using the human osteoblast cell line and the osteosarcoma cell lines. QRT-PCR assay as well as western blot assay were used to ascertain the relative expression levels of FUT4 and FOXO1 in the cells. By using the CCK-8 assay, colony assay, EDU assay, wound healing assay and Transwell assay, osteosarcoma cells' ability to proliferate, migrate and invade were examined in relation to si- FUT4. TUNEL test was used to evaluate Si-impact FUT4's on KHOS and U2OS apoptosis in osteosarcoma cells. Western blot assay was used to identify the expression of proliferative, migrating and apoptosis-related protein markers in osteosarcoma cells KHOS and U2OS and the expression of important proteins in the Wnt/ β-catenin signaling pathway., Results: In comparison with osteoblasts, osteosarcoma cells expressed more FUT4. The osteosarcoma cells' capacities to proliferate, invade, and migrate were markedly inhibited by the inhibition of FUT4 expression, which also increased osteosarcoma cell apoptosis. The Wnt/β-catenin signaling pathway was blocked by upregulating FOXO1 expression, which was in turn inhibited by inhibiting FUT4 expression., Conclusion: Osteosarcoma cells express more FUT4. The Wnt/β-catenin signaling pathway has a significant effect on osteosarcoma cell death, and inhibition of FUT4 expression may target FOXO1 activation to decrease osteosarcoma cells' ability to proliferate, invade, and migrate., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

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.

The current design scheme on anti-cancer materials is mainly through tuning the mechanical properties of the materials to induce apoptosis in cancer cells, with the involvement of Rho/ROCK signaling pathway. We hypothesize that tuning the motility is another potential important approach to modifying the tumor microenvironment and inducing tumor apoptosis. To this aim, we have prepared RGD-modified substrates to regulate cell motility through modification of RGD with different concentrations, and systematically examined the effect of motility on the apoptosis of tumor cells, and the potential involvement of Wnt signaling pathway. Our studies indicated that RGD modification could be readily used to tune the motility of cancer cells. High RGD concentration significantly suppressed the migration of cancer cells, leading to significantly increased apoptosis rate, about three times of that of the unmodified samples. Western-blot analysis also showed that cell with low motility expressed more caspase-3 and PARP proteins. Further RNA sequence study strongly suggested that low motility inhibited the canonical Wnt signaling pathway, which in turn led to the activation of the mitochondria-associated caspase signaling pathway, and ultimately to the apoptosis of osteosarcoma cells. Activation of the Wnt-β-catenin pathway through HLY78 significantly suppressed the apoptosis of MG-63 cells, further suggesting the critical role of Wnt pathway in motility-regulated-apoptosis of tumor cells. Our findings shed insights to understand the underlying mechanisms that induced the tumor cell apoptosis, and might provide new strategy for designing the novel anti-tumor materials., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., (© 2023. The Author(s).)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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.

Objective: To investigate the inhibitory effect of miR-125b-5p on proliferation and migration of osteosarcoma and the role of RAB3D in mediating this effect., Methods: The expression level of miR-125b-5p was detected by qRT-PCR in a normal bone cell line (hFOB1.19) and in two osteosarcoma OS cell lines (MG63 and HOS). A miR-125b-5p mimic or inhibitor was transfected in the osteosarcoma cell lines via liposome and the changes in cell proliferation and migration were detected with EDU and Transwell experiments. Bioinformatic analysis was conducted for predicting the target gene of miR-125b-5p, and the expression level of RAB3D in hFOB1.19, MG63, and HOS cells was detected by Western blotting. In the two osteosarcoma cell lines transfected with miR-125b-5p mimic or inhibitor, the expression levels of RAB3D mRNA and protein in osteosarcoma cells were examined with qRT-PCR and Western blotting. The effects of RAB3D overexpression, RAB3D knockdown, or overexpression of both miR-125b-5p and RAB3D on the proliferation and migration of cells were assessed using EDU and Transwell experiments., Results: The two osteosarcoma cell lines had significantly lower expression levels of miR-125b-5p ( P < 0.05). Bioinformatic analysis predicted that RAB3D was a possible target gene regulated by miR-125b-5p. In osteosarcoma cells, overexpression of miR-125b-5p significantly lowered the expression of RAB3D protein ( P < 0.05); inhibiting miR-125b-5p expression significantly decreased RAB3D expression only at the protein level ( P < 0.05) without obviously affecting its mRNA level. Modulation of miR-125b-5p and RAB3D levels produced opposite effects on proliferation and migration of osteosarcoma cells, and in cells with overexpression of both miR-125b-5p and RAB3D, the effect of RAB3D on cell proliferation and migration was blocked by miR-125b-5p overexpression ( P < 0.05)., Conclusion: Overexpression of miR-125b-5p inhibits the proliferation and migration of osteosarcoma cells by regulating the expression of RAB3D at the post-transcriptional level.

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., (© 2022 Wiley-VCH GmbH.)

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., (© 2022 Wiley-VCH GmbH.)

Aims: Acridine 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., Methods: We 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., Results: The 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., Conclusion: AO-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):685-692.

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 (H 2 S-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 H 2 S-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% H 2 S-DOXO entrapment efficiency, and were stable for almost 28 days. HA-decorated NPs accumulated H 2 S-DOXO in Pgp-expressing cells reducing cell viability. HA-decorated NPs result in the best formulation to increase the inter-cellular H 2 S-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.

Many studies indicated that static magnetic fields (SMFs) have anti-cancer effects. However, effect of SMFs on cancer cells with strength exceeding 12 T are rarely reported. The intracellular iron could participate in the reactive oxygen species (ROS) production and affect cell proliferation. This study aimed to investigate the effect of 12 T high static magnetic field (HiSMF) on osteosarcoma cells and the relationship with intracellular iron. The 12 T HiSMF was generated by a superconducting magnet. The proliferation was evaluated by CCK-8 assays and cell counting. The apoptosis, cell cycle distribution, and ROS were evaluated by flow cytometry. Intracellular iron status was evaluated by atomic absorption spectroscopy and Calcein-AM/2,2'-bipyridyl. The expression of cell cycle and iron metabolism-related genes were analyzed by Western Blot. The result showed that 12 T HiSMF exposure suppressed the proliferation of osteosarcoma cell lines MNNG/HOS, U-2 OS, and MG63 via cell cycle arrest in S and G2/M. Meanwhile, 12 T HiSMF increasing intracellular ROS, and its antitumor effect was reduced by antioxidant. Furthermore, the intracellular total and free iron levels, the expression of FTH1 and DMT1 were increased by 12 HiSMF. The iron chelator (DFO) could reduce the cytotoxicity of 12 T HiSMF on osteosarcoma cells. Moreover, 12 T HiSMF could enhance the cytotoxicity of cisplatin and sorafenib in osteosarcoma cells. In Conclusion, 12 T HiSMF could suppress osteosarcoma cells proliferation via intracellular iron and ROS related cell cycle arrest, and have application potential in osteosarcoma therapy combined with sorafenib and cisplatin., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background/aims: Silica nanoparticles (SiNPs) have been promising vehicles for drug delivery. Cichorium Pumilum (CP), a natural photosensitizer (PS), has been reported to have many useful effects in cancer treatment. However, the poor water solubility and its low bioavailability have confined its use as a suitable photosensitizer for photodynamic therapy. Therefore, a subtle approach is required to overcome these drawbacks., Materials and Methods: We have synthesized a silica nanoparticles loaded with Cichorium Pumilum. The nanoparticles structural morphologies have been charectrized by Transmission Electron Microscopy (TEM). The cytotoxicity for different concentrations of naked and encapsulated CP was evaluated. Moreover, the optimal concentration of naked and encapsulated CP with exposure time to a light (Maximum intensity at 350nm ∼0.27mW/cm 2 ) required to eliminate the used cells (Osteosarcoma cells) were also measured., Results: The results showed that encapsulated CP in SiNPs exhibited relatively higher efficacy than the naked CP by + 157.14 % of exposure time efficacy and + 49.45% of concentration efficacy, and encapsulated CP was also confirmed to be effective in eradicating osteosarcoma cells., Conclusion: The engineered silica nanoparticles loaded with CP enhanced the photodynamic therapy by increasing the CP bioavailability., (Copyright © 2022. Published by Elsevier B.V.)

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. Convallatoxin, a natural cardiac glycoside, exhibits potent anti-tumor activities. Literature has confirmed that PTHR1 is highly expressed in OS tissues and cells and downregulation of PTHR1 could decrease the invasion and growth of OS cells and increase tumor differentiation. In addition, PTHR1 could activate Wnt signaling pathway to promote the malignant functions of OS. In the present study, MG63 and U2OS cells were treated with 0, 12.5, 25, and 50 nM convallatoxin in order to elucidate the precise function of convallatox on the malignant behaviors of OS cells. Moreover, MG63 and U2OS cells treated with convallatoxin were transfected with Ov-PTHR1 or sh-DKK1, aiming to explore whether convallatoxin impeded the malignant progression of OS by modulating PTHR1 and Wnt/β-catenin pathway. CCK-8, wound healing and transwell assays were employed to assess the proliferation, migration, and invasion of OS cells. Differentiation markers (collagen 1, osteopontin, RANKL, Runx2, osteocalcin) were measured to evaluate OS cell differentiation. Results illuminated that convallatoxin suppressed proliferation, migration, and invasion as well as promoted osteogenic differentiation of OS cells. Besides, convallatoxin inhibited PTHR1 expression and inactivated Wnt/β-catenin pathway and PTHR1 overexpression activated Wnt/β-catenin pathway. Furthermore, PTHR1 overexpression or DKK1 knockdown reversed the suppressing effects of convallatoxin on OS cell proliferation, migration, and invasion, as well as the enhancing effect of convallatoxin on OS cell osteogenic differentiation. Collectively, convallatoxin may repress the malignant progression of OS by blocking PTHR1 and Wnt/β-catenin pathway.

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA 1 to LPA 6 ) 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 LPA 4 and LPA 6 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. LPA 2 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 (LPA 3 agonist). Moreover, the cell survival to CDDP of MG-63 cells was enhanced by LPA 4 and LPA 6 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., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Incomplete removal of tumor cells and insufficient osseointegration are the main causes of bone tumor recurrence and implantation failure. In the present study, a multifunctional titanium-based bioactive implant for near-infrared-triggered synergy therapy to overcome these hurdles is engineered, composed of titanium dioxide (TiO 2 ) nanoparticles doped with fluorine (F)/dopamine (PDA)/collagen. The TiO 2 nanoparticles designed in this work can simultaneously exhibit excellent near-infrared-activated photothermal and photocatalytic properties. Besides, the layer designed in this work show excellent anti-tumor activity under irradiation with 808 nm light due to the synergetic effect of hyperthermia and reactive oxygen species (ROS), and Saos-2 cells can be eradicated within 10 min. Moreover, modification of PDA and collagen endue the Ti alloy excellent osteogenic activity., (Copyright © 2022. Published by Elsevier B.V.)

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.

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.

Osteosarcoma (OS) is the most common primary malignant tumor of bone. It is a common phenomenon that osteosarcoma cells have a hypoxic microenvironment. Hypoxia can dedifferentiate cells of several malignant tumor types into stem cell-like phenotypes. However, the role of hypoxia in stemness induction and the expression of cancer stem cell (CSC) markers in human osteosarcoma cells has not been reported. The present study examined the effects of hypoxia on stem-like cells in the human osteosarcoma MNNG/HOS cells. Under the incubation with 1% oxygen, the expression of CSCs markers (Oct-4, Nanog and CD133) in MNNG/HOS cells were increased. Moreover, MNNG/HOS cells cultured under hypoxic conditions were more likely to proliferate into spheres and resulted in larger xenograft tumor. Hypoxia also increased the mRNA and protein levels of hypoxia-inducible factor (HIF)-1α. Then rapamycin was used, which has been shown to lower HIF-1α protein level, to inhibit the hypoxic response. Rapamycin suppressed the expression of HIF-1α protein and CSCs markers (Oct4, Nanog and CD133) in MNNG/HOS cells. In addition, pretreatment with rapamycin reduced the efficiency of MNNG/HOS cells in forming spheres and xenograft tumors. The results demonstrated that hypoxia (1% oxygen) can dedifferentiate some of the MNNG/HOS cells into stem cell-like phenotypes, and that the mTOR signaling pathway participates in this process via regulating the expression of HIF-1α protein., (Copyright: © Lin et al.)

Background and Objectives: 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., Methods: 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., Results: 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., Interpretation and Conclusions: 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., (Copyright © 2021 IMSS. Published by Elsevier Inc. All rights reserved.)

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA 1 to LPA 6 ) exhibits a variety of biological responses. In tumor microenvironment, endothelial cells promote cancer cell functions. In this study, we investigated the roles of endothelial cells in the regulation of cell motile activity via LPA 2 and LPA 3 in human osteosarcoma MG-63 cells. In cell motility assay, the cell motile activity of MG-63 cells was markedly increased by the supernatants of endothelial F2 cells. MG-63 cell motility elevated by the supernatants was enhanced by GRI-977143 (LPA 2 agonist) and reduced by (2S)-OMPT (LPA 3 agonist). LPAR2 and LPAR3 expressions were increased in highly migratory MG63-CR7(F2) cells, which were generated from MG-63 cells by co-culture with F2 cell supernatants. MG63-CR7(F2) cell motility was stimulated by LPA treatment. In the presence of F2 cell supernatants, MG63-CR7(F2) cell motility was markedly enhanced by GRI-977143 and suppressed by (2S)-OMPT. Autotaxin (ATX) enzymatically converts lysophosphatidylcholine (LPC) to LPA. ATX expression was higher in MG63-CR(F2) cells than in MG-63 cells. MG63-CR7(F2) cell motility was markedly increased by LPC in comparison with MG-63 cells. In addition, MG63-CR(F2) cell motility was significantly stimulated by the supernatants of LPC treated F2 cells. The present results suggest that the activation of LPA signaling via LPA 2 and LPA 3 by endothelial cells is involved in the modulation of cell motile activity of MG-63 cells., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Carbon ion radiotherapy (CIRT) is more effective than conventional photon beam radiotherapy in treating osteosarcoma (OSA); however, the outcomes of CIRT alone are still unsatisfactory. In this study, we aimed to investigate whether miR-29b acts as a radiosensitizer for CIRT. The OSA cell lines U2OS and KHOS were treated with carbon ion beam alone, γ-ray irradiation alone, or in combination with an miR-29b mimic. OSA cell death as well as invasive and migratory abilities were analyzed through viability, colony formation, Transwell, and apoptosis assays. miR-29 expression was downregulated in OSA tissues compared to that in normal tissues and was associated with metastasis and relapse in patients with OSA. Further, miR-29b was found to directly target the transcription factor Sp1 and suppress the activation of the phosphatase and tensin homolog (PTEN)-AKT pathway. Conversely, Sp1 was found to attenuate the inhibitory effects of miR-29b in OSA cells. When used in combination with miR-29b mimic, carbon ion beam markedly inhibited invasion, migration, and proliferation of OSA cells and promoted apoptosis by inhibiting AKT phosphorylation in a Sp1/PTEN-mediated manner. Taken together, miR-29b mimic improved the radiosensitivity of OSA cells via the PTEN-AKT-Sp1 signaling pathway, presenting a novel strategy for the development of carbon ion beam combination therapy., Competing Interests: None., (AJCR Copyright © 2020.)

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., (© 2020 Wiley-VCH GmbH.)

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., (Copyright: © Wang et al.)

Confocal Raman microspectroscopy (CRM) analysis provides subcellular compositional and morphology related information. In this study, we used CRM in conjunction with multivariate statistical analysis to elucidate the time-dependent impact of the γ-secretase inhibitor, DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) of osteosarcoma (OS) cells. The interactions of DAPT (20 μM) with a murine OS cell line K7M2 at 24 and 48 h were monitored. The spectral characteristics of drug action were identified to illustrate the cellular compositional alterations, showing that DAPT induced apoptosis by reducing the protein, lipid and nucleic acid content and structural changes. Multivariate algorithms, principal component analysis (PCA) and linear discriminant analysis (LDA) revealed a clear separation among cells in the untreated control (UT), 24H (DAPT-treated for 24 h), and 48H (DAPT-treated for 48 h) groups, achieving sensitivities of 100%, 96%, 100% and specificities of 98%, 100%, 100%, respectively. After point-scanned spectral imaging, K-means clustering analysis (KCA) was further used to visualize sub-cellular morphological changes and the underlying spectral characteristics in a temporal sequence. Compared with the UT group, Raman imaging results exhibited gradually increased nuclear division of OS cells with DAPT treatment duration extension, along with changes in the physiology of other organelles within the cell. By providing a unique perspective for understanding the temporary cellular responses to DAPT at molecular level, the achieved results form the foundation of strategies for the application of CRM and other Raman-based techniques for studying the therapeutic responses of other anticancer agents in cancer model systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: 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., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Huang et al.)

Search for natural substances in association with conventional chemotherapeutic drugs with a chemiosensitizing action easily accessible to the tumor mass has encouraged our studies on voacamine (VOA) and its monomeric units, voacangine and vobasine. Our previous results showed that VOA sensitized multidrug resistant (MDR) osteosarcoma cells (U-2 OS/DX) to doxorubicin (DOX) cytotoxicity. VOA, extracted by Peschiera fuchsiaefolia plant, is a bisindole alkaloid consisting of an Iboga skeleton (voacangine) directly linked to a 2-acyl indole unit (vobasine). High-performance thin-layer chromatography densitometry demonstrated the purity of VOA, voacangine and vobasine samples. Flow cytometry analysis showed that VOA, voacangine and vobasine enhanced DOX accumulation of U-2 OS/DX cells, in equally way, whereas VOA reduced more efficiently DOX efflux. Optical microscopy and clonogenic assay confirmed that VOA was more effective than voacangine and vobasine in enhancing DOX cytotoxic effect. These results showed that monomers linked together are necessary to modulate resistant phenotype of osteosarcoma cells. To complete the study, we evaluated the effect of three compounds on microtubules by confocal microscopy, suggesting that only the whole molecule depolymerizes the microtubules blocking so DOX efflux-mediated by vesicles., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 H 2 O 2 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. Fe 3+ and Fe 2+ have the same effect on ascorbate-induced toxicity, but Fe 3+ chelator deferoxamine (DFO) has a profound inhibition effect than Fe 2+ 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.

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.

Osteosarcoma (OSA) is the most common malignant bone tumor in children and adolescents. The overall five-year survival rate for all bone cancers is below 70%; however, when the cancer has spread beyond the bone, it is about 15-30%. Herein, we evaluated the effects of carbon-ion beam irradiation alone or in combination with zoledronic acid (ZOL) on OSA cells. Carbon-ion beam irradiation in combination with ZOL significantly inhibited OSA cell proliferation by arresting cell cycle progression and initiating KHOS and U2OS cell apoptosis, compared to treatments with carbon-ion beam irradiation, X-ray irradiation, and ZOL alone. Moreover, we observed that this combination greatly inhibited OSA cell motility and invasion, accompanied by the suppression of the Pi3K/Akt and MAPK signaling pathways, which are related to cell proliferation and survival, compared to individual treatments with carbon-ion beam or X-ray irradiation, or ZOL. Furthermore, ZOL treatment upregulated microRNA (miR)-29b expression; the combination with a miR-29b mimic further decreased OSA cell viability via activation of the caspase 3 pathway. Thus, ZOL-mediated enhancement of carbon-ion beam radiosensitivity may occur via miR-29b upregulation; co-treatment with the miR-29b mimic further decreased OSA cell survival. These findings suggest that the carbon-ion beam irradiation in combination with ZOL has high potential to increase OSA cell death.

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA 1 to LPA 6 ) regulates a variety of malignant properties of cancer cells. It is known that endothelial cells promote tumor progression and chemoresistance. The present study aimed to investigate the roles of LPA 5 in cellular functions modulated by endothelial cells and anticancer drug in osteosarcoma cells. Human osteosarcoma MG-63 cells were maintained in endothelial F2 cell supernatants. After culturing for 3 months, MG63-F2 cells were established. LPAR5 expression level in MG63-F2 cells was significantly elevated, compared with MG-63 cells. The cell motile activity of MG63-F2 cells was markedly higher than that of MG-63 cells. To validate the effects of LPA 5 on cell motile activity, LPA 5 knockdown cells were generated from MG-63 cells. The cell motile activity of MG-63 cells was inhibited by LPA 5 knockdown. The cell survival to cisplatin (CDDP) was reduced in MG-63 cells treated with LPA. In the presence of LPA, the cell survival rate was significantly lower in MG63-F2 cells than MG-63 cells, correlating with LPAR5 expression. LPA 5 knockdown cells indicated the high cell survival rate to CDDP. Moreover, LPAR5 expression level was increased in the long-term CDDP treated MG63-C cells. The cell survival to CDDP of MG63-C cells was enhanced by LPA 5 knockdown. These results suggest that cellular functions are regulated through LPA 5 -mediatd signaling induced by endothelial cells and CDDP in MG-63 cells., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Confocal Raman microspectral imaging was adopted to elucidate the cellular drug responses of osteosarcoma cells (OC) to N-[N-(3, 5-difluorophenyl acetyl)-L-alanyl]-sphenylglycine butyl ester (DAPT), a γ-secretase inhibitor, by identifying the drug induced subcellular compositional and structural changes. Methods : Spectral information were acquired from cultured osteosarcoma cells treated with 0 (Untreated Group, UT), 10 (10 μM DAPT treated, 10T), 20 μM (20 μM DAPT treated, 20T) DAPT for 24 hours. A one-way ANOVA and Tukey's honest significant difference (HSD) post hoc multiple test were sequentially applied to address spectral features among three groups. Multivariate algorithms such as K-means clustering analysis (KCA) and Principal component analysis (PCA) were used to highlight the structural and compositional differences, while, univariate imaging was applied to illustrate the distribution pattern of certain cellular components after drug treatment. Results : Major biochemical changes in DAPT-induced apoptosis came from changes in the content and structure of proteins, lipids, and nucleic acids. By adopted multivariate algorithms, the drug induced cellular changes was identified by the morphology and spectral characteristics between untreated cells and treated cells, testified that DAPT mainly acted in the nuclear region. With the increase of the drug concentration, the content of main subcellular compositions, such nucleic acid, protein, and lipid decreased. In an addition, DAPT-induced nuclear fragmentation and apoptosis was depicted by the univariate Raman image of major cellular components (nucleic acids, proteins and lipids). Conclusions : The achieved Raman spectral and imaging results illustrated detailed DAPT-induced subcellular compositional and structural variations as a function of drug dose. Such observations can not only explain drug therapeutic mechanisms of OC DAPT treatment, and also provide new insights for accessing the medicine curative efficacy and predicting prognosis., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Licochalcone A (LicA) is isolated from the roots of Glycyrrhiza glabra and possesses antitumor and anti-invasive activities against several tumor cells. However, the antitumor effects of LicA on human osteosarcoma cells have yet to be demonstrated either in vitro or in vivo., Methods: Cell viability was measured by MTT assay. Apoptosis and mitochondrial dysfunction were detected with Annexin V/PI staining and JC-1 staining by flow cytometry. The expressions of caspase- or mitochondrial-related proteins were demonstrated by western blotting. Antitumor effect of LicA on 143B xenograft mice in vivo., Results: LicA could inhibit cell proliferation and induce apoptosis in human osteosarcoma cells, as evidenced by a decrease in cell viability, loss of mitochondrial membrane potentials, and activation of caspases. LicA treatment substantially reduced the expression of Bcl-2 and Mcl-1 and increased the expression of cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP, and Bax in HOS and U2OS cells. Moreover, mitochondrial membrane potential and apoptosis suppression mediated by Z-VAD or tauroursodeoxycholic acid significantly reduced LicA-induced mitochondria-dependent apoptosis. The study also determined that LicA treatment induced p38MAPK phosphorylation, but siRNA-p38 or BIRB796 substantially reversed cell viability through the inhibition of mitochondria-dependent apoptosis pathways. Finally, an in vivo study revealed that LicA significantly inhibited 143B xenograft tumor growth., Conclusions: These findings demonstrate that LicA has antitumor activities against human osteosarcoma cells through p38MAPK regulation of mitochondria-mediated intrinsic apoptotic pathways in vitro and in vivo., Competing Interests: The authors declare that they have no competing interests

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., Competing Interests: The authors report no conflicts of interest in this work., (© 2019 Mo et al.)

The development of advanced techniques of fabrication of three-dimensional (3D) microenvironments for the study of cell growth and proliferation has become one of the major motivations of material scientists and bioengineers in the past decade. Here, we present a novel residueless 3D structuration technique of poly(dimethylsiloxane) (PDMS) by water-in-PDMS emulsion casting and subsequent curing process in temperature-/pressure-controlled environment. Scanning electron microscopy and X-ray microcomputed tomography allowed us to investigate the impact of those parameters on the microarchitecture of the porous structure. We demonstrated that the optimized emulsion casting process gives rise to large-scale and highly interconnected network with pore size ranging from 500 μm to 1.5 mm that turned out to be nicely adapted to 3D cell culture. Experimental cell culture validations were performed using SaOS-2 (osteosarcoma) cell lines. Epifluorescence and deep penetration imaging techniques as two-photon confocal microscopy unveiled information about cell morphology and confirmed a homogeneous cell proliferation and spatial distribution in the 3D porous structure within an available volume larger than 1 cm 3 . These results open alternative scenarios for the fabrication and integration of porous scaffolds for the development of 3D cell culture platforms.

Osteosarcoma (OS), the most common malignant bone tumor, is the main cause of cancer-related deaths in children and young adults. Despite the combination of surgery and multi-agent chemotherapy, patients with OS who develop resistance to chemotherapy or experience recurrence have a dismal prognosis. MicroRNAs (miRNAs) are a class of small noncoding RNAs that repress their targets by binding to the 3'-UTR and/or coding sequences, leading to the inhibition of gene expression. miR-221 is found to be up-regulated in tumors when compared with their matched normal osteoblast tissues. We also observed significant miR-221 up-regulation in the OS cell lines, MG-63, SaoS-2, and U2OS, when compared with the normal osteoblast cell line, HOb. Overexpression of miR-221 promoted OS cell invasion, migration, proliferation, and cisplatin resistance. MG-63 and SaoS-2 cells transfected with miR-221 mimics were more resistant to cisplatin. The IC 50 of MG-63 cells transfected with control mimics was 1.24 μM. However, the IC 50 of MG-63 cells overexpressing miR-221 increased to 7.65 μM. Similar results were found in SaoS-2 cells, where the IC 50 for cisplatin increased from 3.65 to 8.73 μM. Thus, we report that miR-221 directly targets PP2A subunit B (PPP2R2A) in OS by binding to the 3'-UTR of the PPP2R2A mRNA. Restoration of PPP2R2A in miR-221-overexpressing OS cells recovers the cisplatin sensitivity of OS cells. Therefore, the present study suggests a new therapeutic approach by inhibiting miR-221 for anti-chemoresistance in OS., (© 2019 The Author(s).)

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 × 10 5 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.

Down-regulation of p16INK4a and miR-146b-5p contributes to tumorigenesis in osteosarcoma (OS). However, the correlation between p16INK4a and miR-146b-5p in OS proliferation remains largely unknown. In the present study, we demonstrated that miR-146b-5p expression was positively correlated with p16INK4a in OS, but inversely correlated with TNF receptor associated factor 6 (TRAF6) expression. Overexpression of miR-146b-5p dramatically suppressed OS cell proliferation. Mechanistically, we validated TRAF6 as a direct functional target of miR-146b-5p and found that miR-146b-5p overexpression significantly decreased the level of phosphorylated PI3k and Akt, which are the pivotal downstream effectors of TRAF6. Moreover, TRAF6 expression was positively correlated with Ki-67 but inversely correlated with miR-146b-5p expression. In OS cells, silencing of TRAF6 mimicked the anti-tumor effects of miR-146b-5p. p16INK4a is an important tumor suppressor gene frequently down-regulated in OS. We found that this inhibitory effect is associated with the suppression of the miR-146b-5p, and is mediated via up-regulating TRAF6 expression. Our findings identified p16INK4a and miR-146b-5p as tumor suppressors, and suggested p16INK4a, miR-146b-5p and TRAF6 as potential therapeutic candidates for malignant OS., (© 2019 The Author(s).)

Background: The 12-hydroxy-14-dehydroandrographolide (DP) is a predominant component of the traditional herbal medicine Andrographis paniculata (Burm. f.) Nees (Acanthaceae). Recent studies have shown that DP exhibits potent anti-cancer effects against oral and colon cancer cells., Objective: This investigation examined the potential effects of DP against osteosarcoma cell., Methods: A cell analyzer was used to measure cell viability. The cell growth and proliferation were performed by Flow cytometry and BrdU incorporation assay. The cell migration and invasion were determined by wound healing and transwell assay. The expression of EMT related proteins was examined by Western blot analysis., Results: In this study, we found that DP treatment repressed osteosarcoma (OS) cell growth in a dose-dependent manner. DP treatment significantly inhibited OS cell proliferation by arresting the cell cycle at G2/M phase. In addition, DP treatment effectively inhibited the migration and invasion abilities of OS cells through wound healing and Transwell tests. Mechanistic studies revealed that DP treatment effectively rescued the epithelialmesenchymal transition (EMT), while forced expression of SATB2 in OS cells markedly reversed the pharmacological effect of DP on EMT., Conclusion: Our data demonstrated that DP repressed OS cell growth through inhibition of proliferation and cell cycle arrest; DP also inhibited metastatic capability of OS cells through a reversal of EMT by targeting SATB2. These findings demonstrate DP's potential as a therapeutic drug for OS treatment., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors mediates various biological effects in cancer cells. This study aimed to investigate the roles of LPA receptors in the regulation of cellular functions during tumor progression in osteosarcoma cells. Long-term cisplatin (CDDP)-treated MG63-C and MG63-R7-C cells were generated from osteosarcoma MG-63 and highly-migratory MG63-R7 cells, respectively. LPAR2 and LPAR3 expression levels were significantly higher in MG63-C cells than in MG-63 cells, while LPAR1 expression was reduced. MG63-C cells were highly motile, compared with MG-63 cells. MG63-C cell motility was suppressed by LPA 2 knockdown and enhanced by the LPA 1 /LPA 3 antagonist, dioctanoylglycerol pyrophosphate. LPAR2 and LPAR3 expression levels were significantly elevated in MG63-R7-C cells in comparison with MG63-R7 cells. MG63-R7-C cells were found to be highly invasive, correlating with metalloproteinase-2 activation. MG63-R7-C cells formed large colonies, whereas colony formation was absent from MG63-R7 cells. Notably, MG63-R7-C cell activities were inhibited by LPA 2 knockdown. These results suggest that LPA signaling via LPA 2 plays an important role in the acquisition of malignant properties during tumor progression in MG-63 cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Achieved Silica Nanoparticles (SiNPs) to encapsulate the photosensitizer [Protoporphyrin IX (PpIX)] in photodynamic therapy (PDT) application was reported in this research., Materials and Methods: Cytotoxicity for five different concentrations of encapsulated and naked PpIX was measured. Optimum concentration and optimum exposure time of encapsulated and naked PpIX that needed to destroy the cells (Osteosarcoma cells) was measured., Results: The results showed that the encapsulated PpIX has more efficacy compared to the naked PpIX and the applicability of the encapsulated PpIX-SiNPs was proved on osteosarcoma cells., Conclusion: The results established the important in-vitro photodynamic effectiveness of PpIX-SiNP, which may open a new application for PpIX in its clinical and in-vitro studies.

Sclareol (sclarcol) is an organic compound extracted from sage clary plants. Recent study has showed its anti-tumor effects against breast cancer, gastric carcinoma, osteosarcoma and colorectal cancer. However, its exact mechanisms in inhibiting tumor growth remain unknown. This study thus observed the effect of sclareol on the proliferation of osteosarcoma cells, in an attempt to investigate the role of sclarcol on osteosarcoma growth. MG63 osteosarcoma cell was treated with different concentrations of sclarcol. CCK8 assay was used to test its effect on cell proliferation. LC50 value was then determined to obtain optimal treatment dose. MG63 cells were then divided into control and drug treated group, for measuring apoptosis and mitochondrial membrane potential by flow cytometry, and the expression of cytochrome c, Bax and Bcl-2 by western blot. CCK8 analysis showed that sclareol inhibited MG63 cell proliferation, with an LC50 value at 11.0 μM. Flow cytometry results showed the apoptotic cell ratio was 13.8%, 24.1% and 37.3% after treated with 2.0 μM, 4.0 μM and 8.0 μM sclareol respectively. Compared to control group, sclareol significantly depressed mitochondrial membrane potential of MG63 cells, increased the expression of cytochrome c and Bax, but decreased Bcl-2 expression. In conclusion, Sclareol can inhibit the proliferation of MG63 cells, and induce cell apoptosis and decrease mitochondrial membrane potential suggesting the inhibition of osteosarcoma cells by sclareol might be via both apoptosis induction and decreasing mitochondrial membrane potential.

Butyrate, an essential factor for colonocytes and regulator in the development of colon cancer, is partially absorbed by the gut. It influences the proliferation and differentiation of several cell types including osteoblasts. We evaluated the effects of different doses of butyrate on differentiation and functionality of osteosarcoma cells in vitro and the expression of a pro-inflammatory phenotype in a normal or inflammatory environment. SaOS-2 osteosarcoma cells were induced to differentiate and contemporarily treated for 24 h, 48 h, or 7 days with sodium butyrate 10 -4 , 5 × 10 -4 , or 10 -3  M in the presence or absence of tumor necrosis factor alpha (TNFα) 1 ng/mL, a pro-inflammatory stimulus. Despite the mild effects on proliferation and alkaline phosphatase activity, butyrate dose- and time-dependently induced the expression of a differentiated phenotype (RUNX2, COL1A1 gene expression, and osteopontin gene and protein expression). This was associated with a partial inhibition of nuclear factor kappa B (NF-κB) activation and the induction of histone deacetylase 1 expression. The net effect was the expression of an anti-inflammatory phenotype and the increase in the osteoprotegerin-to-receptor activator of nuclear factor kappa-B ligand (RANKL) ratio. Moreover, butyrate, especially at the highest dose, counteracted the effects of the pro-inflammatory stimulus of TNFα 1 ng/mL. Butyrate affects osteosarcoma cell metabolism by anticipating the expression of a differentiated phenotype and by inducing the expression of anti-inflammatory mediators.