Apoptosis Of Osteosarcoma Cells Research Articles

Arnicolide D Inhibits Proliferation and Induces Apoptosis of Osteosarcoma Cells through PI3K/Akt/mTOR Pathway.

Osteosarcoma is considered as the most prevalent form of primary malignant bone cancer, prompting a pressing need for novel therapeutic options. Arnicolide D, a sesquiterpene lactone derived from the traditional Chinese herbal medicine Centipeda minima (known as E Bu Shi Cao in Chinese), showed anticancer efficacy against several kinds of cancers. However, its effect on osteosarcoma remains unclear. This study aimed to investigate the anticancer activity of arnicolide D and the underlying molecular mechanism of its action in osteosarcoma cells, MG63 and U2OS. Cell viability and proliferation were evaluated through MTT assay and colony formation assay following 24 h and 48 h treatment with different concentrations of arnicolide D. Flow cytometry was employed to examine cell cycle progression and apoptosis after 24 h treatment of arnicolide D. Western blotting was performed to determine the expression of the PI3k, Akt and m-TOR and their phosphorylated forms. Our findings revealed that arnicolide D treatment resulted in a significant reduction in cell viability, the inhibition of proliferation, and the induction of apoptosis and cell cycle arrest in the G2/M phase. Furthermore, arnicolide D could inhibit the activation of PI3K/Akt/mTOR pathway in osteosarcoma cells. Based on our results, arnicolide D demonstrated significant anti-osteosarcoma activity and held the potential to be considered as a therapeutic candidate for osteosarcoma in the future.

A novel prognostic signature related to programmed cell death in osteosarcoma.

Osteosarcoma primarily affects children and adolescents, with current clinical treatments often resulting in poor prognosis. There has been growing evidence linking programmed cell death (PCD) to the occurrence and progression of tumors. This study aims to enhance the accuracy of OS prognosis assessment by identifying PCD-related prognostic risk genes, constructing a PCD-based OS prognostic risk model, and characterizing the function of genes within this model. We retrieved osteosarcoma patient samples from TARGET and GEO databases, and manually curated literature to summarize 15 forms of programmed cell death. We collated 1621 PCD genes from literature sources as well as databases such as KEGG and GSEA. To construct our model, we integrated ten machine learning methods including Enet, Ridge, RSF, CoxBoost, plsRcox, survivalSVM, Lasso, SuperPC, StepCox, and GBM. The optimal model was chosen based on the average C-index, and named Osteosarcoma Programmed Cell Death Score (OS-PCDS). To validate the predictive performance of our model across different datasets, we employed three independent GEO validation sets. Moreover, we assessed mRNA and protein expression levels of the genes included in our model, and investigated their impact on proliferation, migration, and apoptosis of osteosarcoma cells by gene knockdown experiments. In our extensive analysis, we identified 30 prognostic risk genes associated with programmed cell death (PCD) in osteosarcoma (OS). To assess the predictive power of these genes, we computed the C-index for various combinations. The model that employed the random survival forest (RSF) algorithm demonstrated superior predictive performance, significantly outperforming traditional approaches. This optimal model included five key genes: MTM1, MLH1, CLTCL1, EDIL3, and SQLE. To validate the relevance of these genes, we analyzed their mRNA and protein expression levels, revealing significant disparities between osteosarcoma cells and normal tissue cells. Specifically, the expression levels of these genes were markedly altered in OS cells, suggesting their critical role in tumor progression. Further functional validation was performed through gene knockdown experiments in U2OS cells. Knockdown of three of these genes-CLTCL1, EDIL3, and SQLE-resulted in substantial changes in proliferation rate, migration capacity, and apoptosis rate of osteosarcoma cells. These findings underscore the pivotal roles of these genes in the pathophysiology of osteosarcoma and highlight their potential as therapeutic targets. The five genes constituting the OS-PCDS model-CLTCL1, MTM1, MLH1, EDIL3, and SQLE-were found to significantly impact the proliferation, migration, and apoptosis of osteosarcoma cells, highlighting their potential as key prognostic markers and therapeutic targets. OS-PCDS enables accurate evaluation of the prognosis in patients with osteosarcoma.

System analysis based on Anoikis-related genes identifies MAPK1 as a novel therapy target for osteosarcoma with neoadjuvant chemotherapy

BackgroundOsteosarcoma (OS) is the most common bone malignant tumor in children, and its prognosis is often poor. Anoikis is a unique mode of cell death.However, the effects of Anoikis in OS remain unexplored.MethodDifferential analysis of Anoikis-related genes was performed based on the metastatic and non-metastatic groups. Then LASSO logistic regression and SVM-RFE algorithms were applied to screen out the characteristic genes. Later, Univariate and multivariate Cox regression was conducted to identify prognostic genes and further develop the Anoikis-based risk score. In addition, correlation analysis was performed to analyze the relationship between tumor microenvironment, drug sensitivity, and prognostic models.ResultsWe established novel Anoikis-related subgroups and developed a prognostic model based on three Anoikis-related genes (MAPK1, MYC, and EDIL3). The survival and ROC analysis results showed that the prognostic model was reliable. Besides, the results of single-cell sequencing analysis suggested that the three prognostic genes were closely related to immune cell infiltration. Subsequently, aberrant expression of two prognostic genes was identified in osteosarcoma cells. Nilotinib can promote the apoptosis of osteosarcoma cells and down-regulate the expression of MAPK1.ConclusionsWe developed a novel Anoikis-related risk score model, which can assist clinicians in evaluating the prognosis of osteosarcoma patients in clinical practice. Analysis of the tumor immune microenvironment and chemotherapeutic drug sensitivity can provide necessary insights into subsequent mechanisms. MAPK1 may be a valuable therapeutic target for neoadjuvant chemotherapy in osteosarcoma.

Clemastine and hyperthermia enhance sensitization of osteosarcoma cells for apoptosis

ABSTRACT Clemastine is an antagonist of histamine H1 receptor may provide benefits in the treatment of osteosarcoma (OS). In the current study, we used hyperthermia approach to sensitize OS cells to clemastine-mediated cell death. Osteosarcoma U-2 OS and Saos-2 cells were treated with clemastine at 37°C, followed by 42°C for 2 h, and released at 37°C for 6 h. The impact of clemastine and hyperthermia on OS cell survival and autophagy-mediated cell death was investigated. Exposure of U-2 OS and Saos-2 cells to clemastine and hyperthermia (42°C) inhibited dose-dependent clemastine-mediated cell survival by increasing cell apoptosis. Hyperthermia and clemastine exposure modulated inflammatory and unfolded protein response (UPR) signaling differentially in U-2 OS and Saos-2 cells. Exposure of U-2 OS and Saos-2 cells to hyperthermia and clemastine inhibited AKT/mTOR and induced expression of the autophagy biomarkers LC3B II and LC3-positive puncta formation. The inhibition of autophagy by 3-methyladenine blocked hyperthermia and clemastine-mediated induction of LC3B II, LC3-positive puncta formation, and OS cell apoptosis. These results indicate that clemastine and hyperthermia sensitize OS cell lines by inducing increased autophagic cell death. Collectively, our data suggest that hyperthermia along with antihistamine therapy may provide an improved approach for the treatment of OS.

High expression of SRSF1 facilitates osteosarcoma progression and unveils its potential mechanisms

BackgroundSRSF1, a member of Serine/Arginine-Rich Splicing Factors (SRSFs), has been observed to significantly influence cancer progression. However, the precise role of SRSF1 in osteosarcoma (OS) remains unclear. This study aims to investigate the functions of SRSF1 and its underlying mechanism in OS.MethodsSRSF1 expression level in OS was evaluated on the TCGA dataset, TAGET-OS database. qRT-PCR and Western blotting were employed to assess SRSF1 expression in human OS cell lines as well as the interfered ectopic expression states. The effect of SRSF1 on cell migration, invasion, proliferation, and apoptosis of OS cells were measured by transwell assay and flow cytometry. RNA sequence and bioinformatic analyses were conducted to elucidate the targeted genes, relevant biological pathways, and alternative splicing (AS) events regulated by SRSF1.ResultsSRSF1 expression was consistently upregulated in both OS samples and OS cell lines. Diminishing SRSF1 resulted in reduced proliferation, migration, and invasion and increased apoptosis in OS cells while overexpressing SRSF1 led to enhanced growth, migration, invasion, and decreased apoptosis. Mechanistically, Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and Gene Set Enrichment Analysis (GSEA) revealed that the biological functions of SRSF1 were closely associated with the dysregulation of the protein targeting processes, location of the cytosolic ribosome, extracellular matrix (ECM), and proteinaceous extracellular matrix, along with the PI3K-AKT pathway, Wnt pathway, and HIPPO pathway. Transcriptome analysis identified AS events modulated by SRSF1, especially (Skipped Exon) SE events and (Mutually exclusive Exons) MXE events, revealing potential roles of targeted molecules in mRNA surveillance, RNA degradation, and RNA transport during OS development. qRT-PCR confirmed that SRSF1 knockdown resulted in the occurrence of alternative splicing of SRRM2, DMKN, and SCAT1 in OS.ConclusionsOur results highlight the oncogenic role of high SRSF1 expression in promoting OS progression, and further explore the potential mechanisms of action. The significant involvement of SRSF1 in OS development suggests its potential utility as a therapeutic target in OS.

Bioactive Glasses Modulate Anticancer Activity and Other Polyphenol-Related Properties of Polyphenol-Loaded PCL/Bioactive Glass Composites.

In this work, bioactive glass (BG) particles obtained by three different methods (melt-quenching, sol-gel, and sol-gel-EISA) were used as modifiers of polyphenol-loaded PCL-based composites. The composites were loaded with polyphenolic compounds (PPh) extracted from sage (Salvia officinalis L.). It was hypothesized that BG particles, due to their different textural properties (porosity, surface area) and surface chemistry (content of silanol groups), would act as an agent to control the release of polyphenols from PCL/BG composite films and other significant properties associated with and affected by the presence of PPh. The polyphenols improved the hydrophilicity, apatite-forming ability, and mechanical properties of the composites and provided antioxidant and anticancer activity. As the BG particles had different polyphenol-binding capacities, they modulated the kinetics of polyphenol release from the composites and the aforementioned properties to a great extent. Importantly, the PPh-loaded materials exhibited multifaceted and selective anticancer activity, including ROS-mediated cell cycle arrest and apoptosis of osteosarcoma (OS) cells (Saos-2) via Cdk2-, GADD45G-, and caspase-3/7-dependent pathways. The materials showed a cytotoxic and antiproliferative effect on cancerous osteoblasts but not on normal human osteoblasts. These results suggest that the composites have great potential as biomaterials for treating bone defects, particularly following surgical removal of OS tumors.

Tumor mitochondrial oxidative phosphorylation stimulated by the nuclear receptor RORγ represents an effective therapeutic opportunity in osteosarcoma

Osteosarcoma (OS) is the most common malignant bone tumor with a poor prognosis. Here, we show that the nuclear receptor RORγ may serve as a potential therapeutic target in OS. OS exhibits a hyperactivated oxidative phosphorylation (OXPHOS) program, which fuels the carbon source to promote tumor progression. We found that RORγ is overexpressed in OS tumors and is linked to hyperactivated OXPHOS. RORγ induces the expression of PGC-1β and physically interacts with it to activate the OXPHOS program by upregulating the expression of respiratory chain component genes. Inhibition of RORγ strongly inhibits OXPHOS activation, downregulates mitochondrial functions, and increases ROS production, which results in OS cell apoptosis and ferroptosis. RORγ inverse agonists strongly suppressed OS tumor growth and progression and sensitized OS tumors to chemotherapy. Taken together, our results indicate that RORγ is a critical regulator of the OXPHOS program in OS and provides an effective therapeutic strategy for this deadly disease.

Reversine inhibits proliferation and induces apoptosis of human osteosarcoma cells through targeting MEK1

Reversine, or 2-(4-morpholinoanilino)-6-cyclohexylaminopurine, is a 2,6-disubstituted purine derivative. This small molecule shows anti-tumor potential by playing a central role in the inhibition of several kinases related to cell cycle regulation and cytokinesis. In this study, systematic review demonstrated the feasibility and pharmacological mechanism of anti-tumor effect of reversine. Firstly, we grafted MNNG/HOS, U-2 OS, MG-63 osteosarcoma cell aggregates onto chicken embryonic chorioallantoic membrane (CAM) to examine the tumor volume of these grafts after reversine treatment. Following culture, reversine inhibited the growth of osteosarcoma cell aggregates on CAM significantly. In vitro experiment, reversine suppressed osteosarcoma cell viability, colony formation, proliferation, and induced apoptosis and cell cycle arrest at G0-G1 phase. Scratch wound assay demonstrated that reversine restrained cell migration. Reversine increased the protein expression of E-cadherin. The mRNA expression of Rac1, RhoA, CDC42, PTK2, PXN, N-cadherin, Vimentin in MNNG/HOS, U-2 OS and MG-63 cells were suppressed and PTEN increased after reversine treatment. Network pharmacology prediction, molecular docking and systematic review revealed MEK1 can be used as an effective target for reversine to inhibit osteosarcoma. Western blot results show the regulation of MEK1 and ERK1/2 by reversine was not consistent in different osteosarcoma cell lines, but we found that reversine significantly inhibited the protein expression of MEK1 in MNNG/HOS, U-2 OS and MG-63. All these suggested that reversine can exert its anti-tumor effect by targeting the expression of MEK1.

Osteosarcoma cell death induced by innovative scaffolds doped with chemotherapeutics.

Osteosarcoma (OS) cancer treatments include systemic chemotherapy and surgical resection. In the last years, novel treatment approaches have been proposed, which employ a drug-delivery system to prevent offside effects and improves treatment efficacy. Locally delivering anticancer compounds improves on high local concentrations with more efficient tumour-killing effect, reduced drugs resistance and confined systemic effects. Here, the synthesis of injectable strontium-doped calcium phosphate (SrCPC) scaffold was proposed as drug delivery system to combine bone tissue regeneration and anticancer treatment by controlled release of methotrexate (MTX) and doxorubicin (DOX), coded as SrCPC-MTX and SrCPC-DOX, respectively. The drug-loaded cements were tested in an in vitro model of human OS cell line SAOS-2, engineered OS cell line (SAOS-2-eGFP) and U2-OS. The ability of doped scaffolds to induce OS cell death and apoptosis was assessed analysing cell proliferation and Caspase-3/7 activities, respectively. To determine if OS cells grown on doped-scaffolds change their migratory ability and invasiveness, a wound-healing assay was performed. In addition, the osteogenic potential of SrCPC material was evaluated using human adipose derived-mesenchymal stem cells. Osteogenic markers such as (i) the mineral matrix deposition was analysed by alizarin red staining; (ii) the osteocalcin (OCN) protein expression was investigated by enzyme-linked immunosorbent assay test, and (iii) the osteogenic process was studied by real-time polymerase chain reaction array. The delivery system induced cell-killing cytotoxic effects and apoptosis in OS cell lines up to Day 7. SrCPC demonstrates a good cytocompatibility and it induced upregulation of osteogenic genes involved in the skeletal development pathway, together with OCN protein expression and mineral matrix deposition. The proposed approach, based on the local, sustained release of anticancer drugs from nanostructured biomimetic drug-loaded cements is promising for future therapies aiming to combine bone regeneration and anticancer local therapy.

Repurposing the diuretic benzamil as an anti-osteosarcoma agent that acts by suppressing integrin/FAK/STAT3 signalling and compromising mitochondrial function.

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. 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. 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. 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.

Paucatalinone A from Paulownia Catalpifolia Gong Tong Elicits mitochondrial-mediated cancer cell death to combat osteosarcoma.

As the global cancer burden escalates, the search for alternative therapies becomes increasingly vital. Natural products, particularly plant-derived compounds, have emerged as promising alternatives to conventional cancer treatments due to their diverse bioactivities and favorable biosafety profiles. Here, we investigate Paucatalinone A, a newly discovered geranylated flavanone derived from the fruit of Paulownia Catalpifolia Gong Tong, notable for its significant anti-cancer properties. We revealed the capability of Paucatalinone A to induce apoptosis in osteosarcoma cells and deciphered its underlying mechanisms. Our findings demonstrate that Paucatalinone A substantially augments apoptosis, inhibits cell proliferation, and demonstrates a pronounced anti-tumor effect in a murine model of osteosarcoma. Mechanistically, Paucatalinone A disrupts calcium homeostasis and exacerbates intracellular reactive oxygen species accumulation, leading to mitochondrial impairment, cytoskeletal collapse, and caspase-dependent apoptotic cell death. This study underscores the potential of Paucatalinone A in initiating apoptosis in cancer cells and highlights the therapeutic efficacy of plant-derived agents in treating osteosarcoma, offering a viable approach for managing other intractable cancers.

METTL3 Promotes Osteosarcoma Metastasis via an m6A-dependent Epigenetic Activity of CBX4.

Osteosarcoma cells are prone to metastasis, and the mechanism of N6-methyladenosine (m6A) methylation modification in this process is still unclear. Methylation modification of m6A plays an important role in the development of osteosarcoma, which is mainly due to abnormal expression of enzymes related to methylation modification of m6A, which in turn leads to changes in the methylation level of downstream target genes messenger RNA (mRNA) leading to tumor development. We analyzed the expression levels of m6A methylation modification-related enzyme genes in GSE12865 whole-genome sequencing data. And we used shRNA (short hairpin RNA) lentiviral interference to interfere with METTL3 (Methyltransferase 3) expression in osteosarcoma cells. We studied the cytological function of METTL3 by Cell Counting Kit-8 (CCK8), flow cytometry, migration and other experiments, and the molecular mechanism of METTL3 by RIP (RNA binding protein immunoprecipitation), Western blot and other experiments. We found that METTL3 is abnormally highly expressed in osteosarcoma and interferes with METTL3 expression in osteosarcoma cells to inhibit metastasis, proliferation, and apoptosis of osteosarcoma cells. We subsequently found that METTL3 binds to the mRNA of CBX4 (chromobox homolog 4), a very important regulatory protein in osteosarcoma metastasis, and METTL3 regulates the mRNA and protein expression of CBX4. Further studies revealed that METTL3 inhibited metastasis of osteosarcoma cells by regulating CBX4. METTL3 has been found to be involved in osteosarcoma cells metastasis by CBX4 affecting the protein expression of matrix metalloproteinase 2 (MMP2), MMP9, E-Cadherin and N-Cadherin associated with osteosarcoma cells metastasis. These results suggest that the combined action of METTL3 and CBX4 plays an important role in the regulation of metastasis of osteosarcoma, and therefore, the METTL3-CBX4 axis pathway may be a new potential therapeutic target for osteosarcoma.

MTORC1 accelerates osteosarcoma progression via m6A-dependent stabilization of USP7 mRNA

Osteosarcoma (OS) is considered a sex steroid hormone-dependent bone tumor. The development and progression of OS are regulated by 17β-estradiol (E2). However, the detailed mechanisms of E2-modulated OS progression remained to be elucidated. Here, we found that E2-activated mammalian target of rapamycin (mTOR) signaling promoted N6-methyladenosine (m6A) modification through regulating WTAP. Inhibition of mTOR complex 1 (mTORC1) reversed E2-activated WTAP expression. Meanwhile, inhibition of mTORC1 suppressed OS cell proliferation and migration. Deficiency of TSC2 activated mTORC1 signaling and enhanced OS cell proliferation and migration, while abrogated by Rapamycin. Interestingly, mTOMC1 promoted mRNA stability of ubiquitin-specific protease 7 (USP7) through m6A modification. Loss of USP7 suppressed the proliferation, migration, and ASC specks, while promoted apoptosis of OS cells. USP7 interacted with NLRP3 and deubiquitinated NLRP3 through K48-ubiquitination. USP7 was upregulated and positive correlation with NLRP3 in OS patients with high level of E2. Loss of USP7 suppressed the progression of OS via inhibiting NLRP3 inflammasome signaling pathway. Our results demonstrated that E2-activtated mTORC1 promoted USP7 stability, which promoted OS cell proliferation and migration via upregulating NLRP3 expression and enhancing NLRP3 inflammasome signaling pathway. These results discover a novel mechanism of E2 regulating OS progression and provide a promising therapeutic target for OS progression.

Mefenamic acid exhibits antitumor activity against osteosarcoma by impeding cell growth and prompting apoptosis in human osteosarcoma cells and xenograft mice model

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.

Synthesized tin oxide nanoparticles promote apoptosis in human osteosarcoma cells

Complicated protocols have been used to tune the size of tin oxide nanoparticles (SnO2 NPs) in conventional chemical synthesis approaches. In this study, we addressed this issue through the combination of polarity solvent reduction and sonication as a potential strategy for optimizing the size of SnO2 NPs for anticancer applications. Then the anticancer activity and safety of synthesized SnO2 NPs on U-2OS human osteosarcoma cells and normal human osteoblast, NHOst, respectively, were assessed by several assays. SEM and TEM analyses revealed that synthesized SnO2 NPs had a spherical-shaped morphology with an average particle size of about 10 nm. FTIR result showed a broad absorption peak at 649 cm– 1, corresponding to the stretching vibration of the O–Sn–O bond. XRD analysis exhibited eight main peaks, revealing a tetragonal system of rutile-like structure. DLS study showed that synthesized SnO2 NPs had an average hydrodynamic size of 43.93 nm with a zeta potential value of −31.00 mV. Cellular assays displayed that SnO2 NPs mitigate the growth of U-2OS human osteosarcoma cells (IC50: 15.50 µg/mL) with partial cytotoxicity on normal osteoblasts. It was then discovered that SnO2 NPs triggered stress effects by raising the levels of LDH release, ROS, and MDA and lowering those of SOD, CAT activity, and GSH content. Also, it was discovered that SnO2 NPs activated both intrinsic and extrinsic signaling pathways mediated by MMP collapse, elevation of caspase-3, −9, −8 activities and corresponding mRNA, and upregulation of Bax/Bcl-2 mRNA ratio. Overall, this study found that extrinsic and mitochondrial apoptotic pathways can both be activated by synthesized SnO2 NPs to induce apoptosis in human osteosarcoma cells. This finding calls for additional research in the future.

3D-Printed Magnesium Peroxide-Incorporated Scaffolds with Sustained Oxygen Release and Enhanced Photothermal Performance for Osteosarcoma Multimodal Treatments.

The hypoxic microenvironment in osteosarcoma inevitably compromises the antitumor effect and local bone defect repair, suggesting an urgent need for sustained oxygenation in the tumor. The currently reported oxygen-releasing materials have short oxygen-releasing cycles, harmful products, and limited antitumor effects simply by improving hypoxia. Therefore, the PCL/nHA/MgO2/PDA-integrated oxygen-releasing scaffold with a good photothermal therapy effect was innovatively constructed in this work to achieve tumor cell killing and bone regeneration functions simultaneously. The material distributes MgO2 powder evenly on the scaffold material through 3D printing technology and achieves the effect of continuous oxygen release (more than 3 weeks) through its slow reaction with water. The in vitro and in vivo results also indicate that the scaffold has good biocompatibility and sustained-release oxygen properties, which can effectively induce the proliferation and osteogenic differentiation of bone mesenchymal stem cells, achieving excellent bone defect repair. At the same time, in vitro cell experiments and subcutaneous tumorigenesis experiments also confirmed that local oxygen supply can promote osteosarcoma cell apoptosis, inhibit proliferation, and reduce the expression of heat shock protein 60, thereby enhancing the photothermal therapy effect of polydopamine and efficiently eliminating osteosarcoma. Taken together, this integrated functional scaffold provides a unique and efficient approach for antitumor and tumor-based bone defect repair for osteosarcoma treatment.

Investigating the synergy of Shikonin and Valproic acid in inducing apoptosis of osteosarcoma cells via ROS-mediated EGR1 expression

BackgroundOsteosarcoma is the most prevalent malignant bone tumour with a poor prognosis. Shikonin (SHK) is derived from the traditional Chinese medicine Lithospermum that has been extensively studied for its notable anti-tumour effects, including for osteosarcoma. However, its application has certain limitations. Valproic acid (VPA) is a histone deacetylase inhibitor (HDACI) that has recently been employed as an adjunctive therapeutic agent that allows chromatin to assume a more relaxed state, thereby enhancing anti-tumour efficacy. PurposeThis study was aimed to investigate the synergistic anti-tumour efficacy of SHK in combination with VPA and elucidate its underlying mechanism. Methods/Study designCCK-8 assays were utilized to calculate the combination index. Additional assays, including colony formation, acridine orange/ethidium bromide double fluorescent staining, and flow cytometry, were employed to evaluate the effects on osteosarcoma cells. Wound healing and transwell assays were utilized to assess cell mobility. RNA sequencing, PCR, and Western blot analyses were conducted to uncover the underlying mechanism. Rescue experiments were performed to validate the mechanism of apoptotic induction. The impact of SHK and VPA combination treatment on primary osteosarcoma cells was also assessed. Finally, in vivo experiments were conducted to validate its anti-tumour effects and mechanism. ResultsThe combination of SHK and VPA synergistically inhibited the proliferation and migration of osteosarcoma cells in vitro and induced apoptosis in these cells. Through a comprehensive analysis involving RNA sequencing, PCR, Western blot, and rescue experiments, we have substantiated our hypothesis that the combination of SHK and VPA induced apoptosis via the ROS-EGR1-Bax axis. Importantly, our in vivo experiments corroborated these findings, demonstrating the potential of the SHK and VPA combination as a promising therapeutic approach for osteosarcoma. ConclusionThe combination of SHK and VPA exerted an anti-tumour effect by inducing apoptosis through the ROS-EGR1-Bax pathway. Repurposing the old drug VPA demonstrated its effectiveness as an adjunctive therapeutic agent for SHK, enhancing its anti-tumour efficacy and revealing its potential value. Furthermore, our study expanded the application of natural compounds in the anti-tumour field and overcame some of their limitations through combination therapy. Finally, we enhanced the understanding of the mechanistic pathways linking reactive oxygen species (ROS) accumulation and apoptosis in osteosarcoma cells. Additionally, we elucidated the role of EGR1 in osteosarcoma cells, offering novel strategies and concepts for the treatment of osteosarcoma.

LncRNA RPARP-AS1 promotes the progression of osteosarcoma cells through regulating lipid metabolism.

Osteosarcoma (OS) is a highly malignant tumor, and its dysregulated lipid metabolism is associated with tumorigenesis and unfavorable prognosis. Interestingly, long noncoding RNAs (lncRNAs) have emerged as pivotal regulators of lipid metabolism, exerting notable impacts on tumor proliferation. Nevertheless, the involvement of RPARP-AS1, a novel lipid metabolism-associated lncRNA, remains unexplored in the context of OS. This study aims to identify functionally relevant lncRNAs impacting OS proliferation and lipid metabolism and seeks to shed light on the upstream regulatory mechanisms governing lipogenic enzyme activity. Based on comprehensive bioinformatic analysis and the establishment of a risk model, we identified seven lncRNAs significantly associated with clinical characteristics and lipid metabolism-related genes in patients with OS. Among these, RPARP-AS1 was selected for in-depth investigation regarding its roles in OS proliferation and lipid metabolism. Experimental techniques including RT-qPCR, Western blot, cell viability assay, assessment, and quantification of free fatty acids (FFAs) and triglycerides (TGs) were utilized to elucidate the functional significance of RPARP-AS1 in OS cells and validate its effects on lipid metabolism. Manipulation of RPARP-AS1 expression via ectopic expression or siRNA-mediated knockdown led to alterations in epithelial-mesenchymal transition (EMT) and expression of apoptosis-associated proteins, thereby influencing OS cell proliferation and apoptosis. Mechanistically, RPARP-AS1 was found to augment the expression of key lipogenic enzymes (FABP4, MAGL, and SCD1) and potentially modulate the Akt/mTOR pathway, thereby contributing to lipid metabolism (involving alterations in FFA and TG levels) in OS cells. Collectively, our findings establish RPARP-AS1 as a novel oncogene in OS cells and suggest its role in fostering tumor growth through the enhancement of lipid metabolism.

Dipsacus Asperoides-Derived Exosomes-Like Nanoparticles Inhibit the Progression of Osteosarcoma via Activating P38/JNK Signaling Pathway.

Osteosarcoma is a prevalent and highly malignant primary bone tumor. However, current clinical therapeutic drugs for osteosarcoma are not suitable for long-term use due to significant side effects. Therefore, there is an urgent need to develop new drugs with fewer side effects. Dipsacus asperoides C. Y. Cheng et T. M. Ai, a traditional Chinese medicine, is commonly used for its anti-inflammatory, anti-pain, bone fracture healing, and anti-tumor effects. In this study, we investigated the effects of exosome-like nanoparticles derived from Dipsacus asperoides (DAELNs) on osteosarcoma cells in vitro and in vivo. DAELNs were isolated and purified from Dipsacus asperoides and their physical and chemical properties were characterized using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The cellular uptake of DAELNs in osteosarcoma cells was analyzed by PKH26 staining. The proliferation, invasion, migration, and apoptosis of osteosarcoma cells were assessed using CCK8 assay, EdU assay, colony-formation assay, transwell assay, wound healing assay, and mitochondrial membrane potential measurement, respectively. The regulatory mechanism of DAELNs inhibiting the progression of osteosarcoma via activating P38/JNK signaling pathway was investigated using Western blotting and immunohistochemistry. Moreover, the therapeutic effects of DAELNs were evaluated using in vivo small animal imaging assay, HE staining, and immunohistochemistry. Our results showed that DAELNs inhibited the proliferation, invasion, migration, and fostered the apoptosis of osteosarcoma cells in vitro and suppressed the tumor growth of osteosarcoma cells in a xenograft nude mouse model. Furthermore, the bio-distribution of DiD-labeled DAELNs showed preferential targeting of osteosarcoma tumors and excellent biosafety in histological analysis of the liver and kidney. Mechanistically, DAELNs activated the P38/JNK signaling pathway-induced apoptosis. Taken together, DAELNs are novel, natural, and osteosarcoma-targeted agents that can serve as safe and effective therapeutic approaches for the treatment of osteosarcoma.

Silencing FUT4 Inhibits the Progression of Osteosarcoma through Activation of FOXO1.

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. To explore the biological significance of the connection between FUT4 and FOXO1 in osteosarcoma growth. 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. 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. 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.