Osteosarcoma Cell Proliferation Research Articles

IL-13Rα2 Is Involved in Resistance to Doxorubicin and Survival of Osteosarcoma Patients

Background/Objectives: Interleukin 13 receptor alpha 2 (IL-13Rα2) is a receptor with a high affinity for IL-13 and is involved in the progression of human cancers. However, studies on the role of IL-13Rα2 in osteosarcoma are limited. Therefore, this study aimed to investigate the expression and roles of IL-13Rα2 in the progression of osteosarcoma. Methods: This study evaluated the roles of IL-13Rα2 in osteosarcomas by evaluating tumor tissues from 37 human osteosarcomas and osteosarcoma cells. Results: Immunohistochemical positivity of IL-13Rα2 was an independent indicator of shorter overall survival and relapse-free survival of 37 osteosarcoma patients and 26 subpopulations of patients who received adjuvant chemotherapy with multivariate analysis. In U2OS and KHOS/NP osteosarcoma cells, overexpression of IL-13Rα2 significantly increased proliferation, migration, and invasion of cells, all of which decreased with knockdown of IL-13Rα2. Overexpression of IL-13Rα2 increased expression of TGF-β, snail, cyclin D1, and BCL2 but decreased BAX, and knockdown of IL-13Rα2 caused a decrease in expression of these molecules. In addition, both in vitro and in vivo, proliferation of osteosarcoma cells increased, and apoptosis decreased with overexpression of IL-13Rα2 under treatment with doxorubicin. Knockdown of IL-13Rα2 sensitized osteosarcoma cells to the cytotoxic effect of doxorubicin. Conclusions: The results of this study suggest that the expression of IL13Rα2 might be used as a potential prognostic indicator in osteosarcoma patients. Furthermore, it is observed that IL13Rα2 influences the resistance to the chemotherapeutic agent doxorubicin. Therefore, a therapeutic trial targeting IL13Rα2 might be a new therapeutic strategy for osteosarcoma, especially those highly expressing IL13Rα2.

LncRNA GClnc1 promotes osteosarcoma progression by stabilizing NONO and blocking FBXW7-mediated ubiquitination

BackgroundLong non-coding RNA (lncRNA) plays a vital role in the occurrence and development of varieties of tumors. Previous studies have shown that lncRNA GClnc1 is highly expressed in osteosarcoma (OS). However, the mechanism of lncRNA GClnc1 in osteosarcoma has not been fully elucidated. In this study, we investigated the biological roles of lncRNA GClnc1 in osteosarcoma and unveiled its underlying mechanisms.MethodsThe expression of lncRNA GClnc1 in OS cells was detected by real-time quantitative PCR (qRT-PCR). The functional roles of lncRNA GClnc1 were examined by CCK8, trans-well, scratch wound healing assay, colony formation, and apoptosis assays in osteosarcoma cells upon silencing or overexpressing GClnc1. Western blot analysis, qRT-PCR, and RNA co-immunoprecipitation (RIP) assays were used to detect the interaction between lncRNA GClnc1 and NONO.ResultsThe expression of lncRNA GClnc1 was up-regulated in osteosarcoma cell lines. Knockdown of lncRNA GClnc1 suppressed the cell growth, migration, and invasion of OS cells, whereas the over-expression of GClnc1 improved the proliferation, migration, and invasion of OS cells. Mechanistically, we identified that lncRNA GClnc1 regulates the stability of NONO by blocking FBXW7-mediated ubiquitination degradation. Additionally, overexpression of NONO can reverse GClnc1 silencing exerted suppression of the cell proliferation, migration, and invasion, and vice versa.ConclusionsOur study elucidated that lncRNA GClnc1 participates in the progression of OS by regulating the NONO signal pathway. Targeting GClnc1 provides a potential target for future clinical treatment of OS.

RBM15-mediated the m6A modification of MAT2A promotes osteosarcoma cell proliferation, metastasis and suppresses ferroptosis.

Methionine adenosyltransferase 2A (MAT2A) has been found to mediate osteosarcoma (OS) progression. Therefore, more roles and mechanisms of MAT2A in the development of OS deserve further exploration. The mRNA and protein levels of MAT2A and RNA binding motif protein 15 (RBM15) were tested by quantitative real-time PCR and western blot (WB). Cell proliferation and metastasis were examined using EdU assay and transwell assay. The protein levels of metastasis-related markers and ferroptosis-related marker were measured by WB. Cell ferroptosis was assessed via testing GSH, ROS, and Fe2+levels. Mice xenograft model was constructed to explore the roles of MAT2A and RBM15 in vivo. RBM15 and MAT2A interaction was assessed by MeRIP assay and dual-luciferase reporter assay. High expression of MAT2A was observed in OS tumor tissues and cells. MAT2A knockdown reduced OS cell proliferation, migration, invasion and enhanced ferroptosis. Silencing of MAT2A inhibited OS tumor growth in vivo. RBM15 was upregulated in OS tumor tissues and cells, which could promote MAT2A expression by N6-methyladenosine (m6A) modification. Downregulation of RBM15 repressed OS cell behaviors and tumorigenesis by decreasing MAT2A expression. In conclusion, MAT2A, regulated by RBM15-mediated m6A modification, accelerated OS malignant progression by increasing cell proliferation, metastasis and decreasing ferroptosis.

SLC38A5 suppresses ferroptosis through glutamine-mediated activation of the PI3K/AKT/mTOR signaling in osteosarcoma

BackgroundSolute carrier family 38 member 5 (SLC38A5) is an amino acid transporter that plays a significant role in various cellular biological processes and may be involved in regulating the progression of tumors However, its function and underlying mechanism in osteosarcoma remain unexplored.MethodsUtilizing various database analyses and experiments, we have explored the dysregulation of SLC38A5 in osteosarcoma and its prognostic value. A series of in vitro functional experiments, including CCK-8, colony formation, wound healing, and transwell invasion assays, were conducted to evaluate the effects of SLC38A5 on the proliferation, migration, and invasion of osteosarcoma cells. Downstream pathways of SLC38A5 were explored through methods such as western blot and metabolic assays, followed by a series of validations. Finally, we constructed a subcutaneous xenograft tumor model in nude mice to explore SLC38A5 function in vivo.ResultsSLC38A5 is upregulated in osteosarcoma and is associated with poor prognosis in patients. Upregulation of SLC38A5 promotes proliferation, migration, and invasion of osteosarcoma cells, while the PI3K inhibitor BKM120 can counteract these effects. Additionally, silencing of SLC38A5 inhibits tumor growth in vivo. Mechanistically, SLC38A5 mediates the activation of the PI3K/AKT/mTOR signaling pathway by transporting glutamine, which subsequently enhances the SREBP1/SCD-1 signaling pathway, thereby suppressing ferroptosis in osteosarcoma cells.ConclusionSLC38A5 promotes osteosarcoma cell proliferation, migration, and invasion via the glutamine-mediated PI3K/AKT/mTOR signaling pathway and inhibits ferroptosis. Targeting SLC38A5 and the PI3K/AKT signaling axis may provide a meaningful therapeutic strategy for the future treatment of osteosarcoma.Graphical abstracts

Tanshinone II A Facilitates Chemosensitivity of Osteosarcoma Cells to Cisplatin via Activation of p38 MAPK Pathway.

To examine the mechanism of action of tanshinone II A (Tan II A) in promoting chemosensitization of osteosarcoma cells to cisplatin (DDP). The effects of different concentrations of Tan II A (0-80 µ mol/L) and DDP (0-2 µ mol/L) on the proliferation of osteosarcoma cell lines (U2R, U2OS, 143B, and HOS) at different times were examined using the cell counting kit-8 and colony formation assays. Migration and invasion of U2R and U2OS cells were detected after 24 h treatment with 30 µ mol/L Tan II A, 0.5 µ mol/L DDP alone, and a combination of 10 µ mol/L Tan II A and 0.25 µ mol/L DDP using the transwell assay. After 48 h of treatment of U2R and U2OS cells with predetermined concentrations of each group of drugs, the cell cycle was analyzed using a cell cycle detection kit and flow cytometry. After 48 h treatment, apoptosis of U2R and U2OS cells was detected using annexin V-FITC apoptosis detection kit and flow cytometry. U2R cells were inoculated into the unilateral axilla of nude mice and then the mice were randomly divided into 4 groups of 6 nude mice each. The 4 groups were treated with equal volume of Tan II A (15 mg/kg), DDP (3 mg/kg), Tan II A (7.5 mg/kg) + DDP (1.5 mg/kg), and normal saline, respectively. The body weight of the nude mice was weighed, and the tumor volume and weight were measured. Cell-related gene and signaling pathway expression were detected by RNA sequencing and Kyoto Encyclopedia of Genes and Genomes pathway analysis. p38 MAPK signaling pathway proteins and apoptotic protein expressions were detected by Western blot. In vitro studies have shown that Tan II A, DDP and the combination of Tan II A and DDP inhibit the proliferation, migration and invasion of osteosarcoma cells. The inhibitory effect was more pronounced in the Tan II A and DDP combined treatment group (P<0.05 or P<0.01). Osteosarcoma cells underwent significantly cell-cycle arrest and cell apoptosis by Tan II A-DDP combination treatment (P<0.05 or P<0.01). In vivo studies demonstrated that the Tan II A-DD combination treatment group significantly inhibited tumor growth compared to the Tan II A and DDP single drug group (P<0.01). Additionally, we found that the combination of Tan II A and DDP treatment enhanced the p38 MAPK signaling pathway. Western blot assays showed higher p-p38, cleaved caspase-3, and Bax and lower caspase-3, and Bcl-2 expressions with the combination of Tan II A and DDP treatment compared to the single drug treatment (P<0.01). Tan II A synergizes with DDP by activating the p38/MAPK pathway to upregulate cleaved caspase-3 and Bax pro-apoptotic gene expressions, and downregulate caspase-3 and Bcl-2 inhibitory apoptotic gene expressions, thereby enhancing the chemosensitivity of osteosarcoma cells to DDP.

UBE2C, targeted by miR-140-3p, promotes the progression of osteosarcoma via PI3K/AKT signaling pathway.

UBE2C was reported to play carcinogenic effects in diverse cancers. However, the role of UBE2C in osteosarcoma was poorly understood, and its functional mechanisms were not fully clarified. RT-qPCR was used to assess the expression of UBE2C mRNA and miR-140-3p, and western blot technique was used to examine the UBE2C protein and PI3K/AKT pathway-associated proteins. CCK-8 test was applied to measure cell proliferation, and wound healing assay were used to measure migration. Using animal studies, the function of UBE2C invivo was evaluated. Dual-luciferase reporter assay was used to confirm the potential interaction among UBE2C and miR-140-3p. In osteosarcoma cells as well as tumor samples, UBE2C was strongly expressed. Osteosarcoma cell proliferation as well as cell migration were inhibited by UBE2C knockdown, and PI3K/AKT signaling activity was diminished. In addition, UBE2C knockdown impeded tumor growth in animal models. UBE2C expression was lessened by miR-140-3p as miR-140-3p targets it. UBE2C is overexpressed which promote osteosarcoma proliferation as well as migration, and strengthened the PI3K/AKT signaling activity, while forced miR-140-3p expression partially abolished these effects. UBE2C, targeted by miR-140-3p, drove carcinogenic effects in osteosarcoma through activating the PI3K/AKT pathway.

METTL3/YTHDF1 stabilizes CORO6 expression promoting osteosarcoma progression through glycolysis

This study investigates the role of CORO6 (Coronin 6) in the development of osteosarcoma. Osteosarcoma is a common malignant bone tumor in children and adolescents, characterized by rapid and irregular bone growth and a high risk of distant lung metastasis. CORO6 is a member of the Coronin family, known for its conserved WD40 repeat domain. This structure allows CORO6 to inhibit actin dynamics through interactions with F-actin and Arp2/3, thereby affecting the organization of the cytoskeleton. Our research found that in osteosarcoma patients, the levels of CORO6 are significantly elevated. Experimental observations showed that reducing the expression of CORO6 significantly inhibits the growth, migration, and invasion abilities of osteosarcoma cells. Moreover, in vivo experiments demonstrated that the absence of CORO6 effectively inhibits the growth of osteosarcoma in animal models. We also discovered that CORO6 promotes the proliferation, migration and invasion capabilities of osteosarcoma cells by activating the Wnt/β-catenin signaling pathway. Moreover, CORO6 play a critical important role in glycosis of osteosarcoma cells. Mechanically, we found that METTL3/YTHDF1 induced m6A modification of CORO6 mRNA promoted the expression of CORO6 by enhancing its stability. These findings offer new directions for the treatment of osteosarcoma, suggesting that CORO6 could be a novel prognostic biomarker and an effective therapeutic target for patients.In summary, CORO6, as an oncogene, plays a key role in the development of osteosarcoma, providing a crucial theoretical basis for the development of new osteosarcoma treatment strategies.

Inhibition of mitochondrial OMA1 ameliorates osteosarcoma tumorigenesis

OMA1 is an ATP-independent zinc metalloprotease essential for maintaining mitochondrial homeostasis and plays a vital role in tumorigenesis. Depending on the type of cancer, a decrease in OMA1 expression has been linked to a varying prognosis for patients. The role of OMA1 in human osteosarcoma (OS), one of the most prevalent malignant bone tumors, remains elusive. Here, we observed elevated OMA1 expression in OS tumor tissues from four patients with advanced OS. Knockout of OMA1 in OS cells significantly reduces OS tumor weight and size, and lung metastatic nodules in BALB/c nude mice. Immunohistochemistry analysis showed a significant decrease in Ki67 and an increase in Cleaved-caspase 3 in OMA1 knockout tumor samples. Mechanistically, we found that OMA1 deficiency increases the levels of PINK1 and Parkin and consequently induces excessive mitophagy, leading to increased apoptosis and reduced cell proliferation and invasion in OS cells. Specifically, OMA1 deficiency reduces the amount of cytosolic p53 and p53-associated cytosolic Parkin but increases mitochondrial p53, which may lead to enhanced apoptosis. Regarding the effect on cell proliferation and invasion, loss of OMA1 reduces mitochondrial ROS levels and increases cytosolic glycogen synthase kinase 3β (GSK3β) levels, thereby increasing interaction between GSK3β and β-catenin and then reducing cytosolic and nuclear β-catenin. This contributes to reduced cell proliferation and migration in OMA1-deficient cells. Moreover, we found that ciclopirox (CPX), an antifungal drug, induces OMA1 self-cleavage and L-OMA1 degradation in cultured OS cells. CPX also reduces tumor development of control OS cells but not OMA1-deficient OS cells in mice. These findings strongly support the important role of OMA1 in OS tumorigenesis and suggest that OMA1 may be a valuable prognostic marker and a promising therapeutic target for OS.

Trifluoperazine exerts anti-osteosarcoma effect by inducing mitochondria-dependent apoptosis via AKT/TXNIP signaling pathway

The survival rates for patients with osteosarcoma (OS) have stagnated over the past few decades. It is essential to find new therapies and drugs. A licensed antipsychotic medication called trifluoperazine (TFP) significantly reduces the growth of several cancers. However, the exact molecular pathways of TFP in OS remain to be discovered. Our research revealed that TFP greatly reduced OS cell migration and growth and caused the arrest of G0/G1 cell cycle. Combined with RNA-Seq data and further research, we confirmed that TFP promoted reactive oxygen species (ROS) production by elevating thioredoxin binding protein (TXNIP) expression to induce mitochondria-dependent apoptosis. Interestingly, we first demonstrated that AKT was an upstream regulatory target of TXNIP in OS cells. Dephosphorylation of AKT led to an increase in TXNIP expression, further elucidating the anticancer mechanism of TFP. In vivo, TFP inhibited subcutaneous OS cell proliferation and induced OS cell apoptosis without noticeable side effects. In conclusion, our findings imply that TFP is a potential treatment for OS.

Curcumin and Methotrexate: A Promising Combination for Osteosarcoma Treatment via Hedgehog Pathway Inhibition

Osteosarcoma (OS) is the most severe bone tumor in children. A chemotherapy regimen includes a combination of high-dose Methotrexate (MTX), doxorubicin, and cisplatin. These drugs cause acute and chronic side effects, such as infections, thrombocytopenia, neutropenia, DNA damage, and inflammation. Therefore, to identify new therapeutic strategies, effective and with a safety profile, is necessary. The Hedgehog (Hh) signaling pathway involved in tumorigenesis is active in OS. Hh components Patched receptor 1 (PTCH1), Smoothened (SMO), and glioma-associated oncogene homolog transcription factors (GLI1 and GLI2) are overexpressed in OS cell lines and patient samples. Curcumin (CUR)—with antioxidant and anti-cancer properties—downregulates Hh components in cancer, inhibiting progression. This study investigates CUR effects on the MG-63 OS cell line, alone and combined with MTX, to propose a novel therapeutic approach. Our study suggests CUR as a novel therapeutic agent in OS, particularly when combined with MTX. Targeting the Hh signaling pathway, CUR and MTX showed significant pro-apoptotic effects, increasing the BAX/Bcl-2 ratio and total apoptotic cell percentage. They reduced the expression of Hh pathway components (PTCH1, SMO, GLI1, and GLI2), inhibiting OS cell proliferation, survival, and invasion. CUR and MTX combined determined a β-Catenin decrease and a trend toward reducing NF-kB and matrix metalloproteinases (MMP-2 and MMP-9). Our findings suggest CUR as a support to OS treatment, improving outcomes and reducing the adverse effects of current therapies.

Retraction of: miR-627-3p inhibits osteosarcoma cell proliferation and metastasis by targeting PTN.

Retraction of: miR-627-3p inhibits osteosarcoma cell proliferation and metastasis by targeting PTN.

Deoxypodophyllotoxin Mediates Autophagy Death through Inhibition of GRP78 in Human Osteosarcoma.

Glucose-regulated protein 78 (GRP78), as a chaperone protein, can protect the endoplasmic reticulum of cells and is expressed to influence chemoresistance and prognosis in cancer. Deoxypodophyllotoxin (DPT) is a compound with antitumor effects on cancers. DPT inhibits the proliferation of osteosarcoma by inducing apoptosis, necrosis, or cell cycle arrest. This study was performed to demonstrate the molecular mechanism by which DPT attenuates osteosarcoma progression through GRP78. Natural compound libraries and western blot (WB) were used to screen the inhibitors of osteosarcoma GRP78. The expression of mitochondria-related genes in cancer cells of the treatment group was detected by quantitative real-time PCR (qPCR) and WB. 3-(4,5)- Dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) and 5-ethynyl-2'- deoxyuridine (EDU) were used to discover the activity and proliferation of osteosarcoma cells treated with DPT. We constructed an in vivo mouse model of DPT drug therapy and carried out immunohistochemical detection of xenografts. The treated osteosarcoma cells were analyzed using bioinformatics and electron microscopy. The data were analyzed finally. DPT inhibited osteosarcoma cell survival and the growth of tumor xenografts. It promoted up-regulation of BCL2-associated X protein (Bax) and B-cell CLL/lymphoma 2 (Bcl-2), which serves to mediate and attenuate, respectively, the killing activities of DPT through mitochondria dysfunction. The effect of DPT against cancer cells could be attenuated by the overexpression of GRP78, characterized by the inactivation of the caspase cascade. The loss of GRP78 in osteosarcoma cells negatively mediated the basal level of autophagyassociated genes. DPT stimulated autophagy via the phosphoinositide 3-kinase (PI3K)-v-akt murine thymoma viral oncogene homolog (AKT), a mechanistic target of rapamycin (mTOR) axis. The autophagy caused by DPT played an active role in the osteosarcoma of humans and blocked the apoptotic cascade. Combination treatment with the GRP78 inhibitor DPT and pharmacological autophagy inhibitors will be a meaningful method of obviating osteosarcoma cells.

Deubiquitinase USP10 promotes osteosarcoma autophagy and progression through regulating GSK3β-ULK1 axis

BackgroundDeubiquitinating enzymes (DUBs) are pivotal in maintaining cell homeostasis by regulating substrate protein ubiquitination in both healthy and cancer cells. Ubiquitin-specific protease 10 (USP10) belongs to the DUB family. In this study, we investigated the clinical and pathological significance of USP10 and Unc-51-like autophagy activating kinase 1 (ULK1) in osteosarcoma (OS), as well as the mechanism of USP10 action in ULK1-mediated autophagy and disease progression.ResultsThe analysis of OS and adjacent normal tissues demonstrated that USP10 and ULK1 were significantly overexpressed in OS, and a positive association between their expression and malignant properties was observed. USP10 knockdown in OS cells reduced ULK1 mRNA and protein expression, whereas USP10 overexpression increased ULK1 mRNA and protein expression. In vitro experiments showed that USP10 induced autophagy, cell proliferation, and invasion by enhancing ULK1 expression in OS cell lines. Furthermore, we found that the regulation of ULK1-mediated autophagy, cell proliferation, and invasion in OS by USP10 was dependent on glycogen synthase kinase 3β (GSK3β) activity. Mechanistically, USP10 promoted ULK1 transcription by interacting with and stabilising GSK3β through deubiquitination, which, in turn, increased the activity of the ULK1 promoter, thereby accelerating OS progression. Using a xenograft mouse model, we showed that Spautin-1, a small-molecule inhibitor targeting USP10, significantly reduced OS development, with its anti-tumour activity significantly enhanced when combined with the chemotherapeutic agent cisplatin.ConclusionCollectively, we demonstrated that the USP10-GSK3β-ULK1 axis promoted autophagy, cell proliferation, and invasion in OS. The findings imply that targeting USP10 may offer a promising therapeutic avenue for treating OS.

The sEVs miR-487a/Notch2/GATA3 axis promotes osteosarcoma lung metastasis by inducing macrophage polarization toward the M2-subtype

Small extracellular vesicles (sEVs) are important mediators of intercellular communication between tumor cells and their surrounding environment. Furthermore, the mechanisms by which miRNAs carried in tumor sEVs regulate macrophage polarization remain largely unknown. To concentrate sEVs, we used the traditional ultracentrifugation method. Western blot, NanoSight, and transmission electron microscopy were used to identify sEVs. To determine the function of sEVs-miR-487a, we conducted in vivo and in vitro investigations. The intercellular communication mechanism between osteosarcoma cells and M2 macrophages, mediated by sEVs carrying miR-487a, was validated using luciferase reporter assays, transwell assays, and Western blot analysis. In vitro, sEVs enriched in miR-487a and delivered miR-487a to macrophages, promoting macrophage polarization toward an M2-like type, which promotes proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of osteosarcoma cells. In vivo, sEVs enriched in miR-487a facilitate lung metastasis of osteosarcoma. Moreover, plasma miR-487a in sEVs was shown to be a potential biomarker applicable for osteosarcoma diagnosis. In summary, miR-487a derived from osteosarcoma cells can be transferred to macrophages via sEVs, then promote macrophage polarization towards an M2-like type by targeting Notch2 and activating the GATA3 pathway. In a feedback loop, the activation of macrophages accelerates epithelial-mesenchymal transition (EMT), which in turn promotes the migration, invasion, and lung metastasis of osteosarcoma cells. This reciprocal interaction between activated macrophages and osteosarcoma cells contributes to the progression of the disease. Our data demonstrate a new mechanism that osteosarcoma tumor cells derived exosomal-miR-487a which is involved in osteosarcoma development by regulating macrophage polarization in tumor microenvironment (TME).

Growth inhibitory potential of quinone-rich fraction of Plumbago zeylanica L. Against MG-63 osteosarcoma cells via Bax/Bcl-2 modulation

ObjectiveThe objectives of this study were to explore the cytotoxic potential of different fractions (Hexane, chloroform, ethyl acetate and methanol) of Plumbago zeylanica L. root powder. MethodologyIn this study, PzMH, PzMC, PzME, and PzMM fractions from the root powder of P. zeylanica were obtained through the cold maceration technique. The cytotoxic properties of these fractions on MG-63 cells were assessed using the MTT assay. The most potent fraction was further analyzed to understand its cytotoxic mechanism. To explore its apoptosis-inducing potential, microscopic and flow cytometric studies were conducted. Additionally, western blot analysis was used to examine the expression levels of proteins associated with apoptotic pathways. ResultsPzMH showed the highest cytotoxicity against MG-63 osteosarcoma cells, with a GI50 value of 53.07 µg/ml. Flow cytometric studies revealed an upsurge in reactive oxygen species and disruption of mitochondrial membrane potential. The highest concentration of PzMH fraction (183.48 µg/ml) led to 62 % cell population arrest at G0/G1 stage. The anti-apoptotic protein Bcl2 was downregulated, with p53, cleaved-caspase 9, cleaved-caspase3, Bad and Bax expression upregulated. HPLC analysis revealed the presence of lawsone, p-coumaric acid, plumbagin, and 4-hydroxy benzaldehyde, indicating that the quinone-rich fraction of Plumbago zeylanica L. attenuates the proliferation of MG-63 osteosarcoma cells through the Bax/Bcl-2 pathway. ConclusionTo the best of our current knowledge, this marks the first documented case where the hexane fraction enriched with quinones extracted from the roots of P. zeylanica demonstrates its ability to induce apoptosis by influencing the Bax/Bcl2 pathway.

The expression and clinical significance of ARHGAP25 in osteosarcoma based on bioinformatics analysis

ARHGAP25, a member of the ARHGAP family, encodes a negative regulator of Rho-GTPase that is important for actin remodeling, cell polarity, and cell migration. ARHGAP25 is down-regulated in a variety of solid tumors and promotes cancer cell growth, migration, and invasion. However, nothing is understood about ARHGAP25’s biological function in osteosarcoma. This work used qPCR and WB to confirm the expression of ARHGAP25 in osteosarcoma following the initial analysis of its expression in pan-cancer. For GO and KEGG analysis, we have chosen 300 genes from the TARGET osteosarcoma data that had the strongest positive correlation with ARHGAP25, and we created nomogram and calibration charts. We simultaneously overexpressed ARHGAP25 in osteosarcoma cells to examine its impact on apoptosis and proliferation. By using MSP, we determined their methylation status in osteosarcoma cells and normal bone cells. We observed that ARHGAP25 was significantly downregulated in a range of malignancies, including osteosarcoma, and was associated with poor patient outcomes. The decrease of ARHGAP25 expression in osteosarcoma is related to DNA methylation. Overexpression of ARHGAP25 induced apoptosis and inhibited the proliferation of osteosarcoma cells in vitro. In addition, ARHGAP25 is also associated with immune-related pathways in osteosarcoma. These findings suggest that ARHGAP25 is a valuable prognostic biomarker in osteosarcoma patients.

Effect of ac4C acetyltransferase NAT10 on tumor progression via ATF4/ASNS mediated asparagine biosynthesis in osteosarcoma.

234 Background: Osteosarcoma is the most common primary malignant bone tumor in children and adolescents, characterized by high malignancy, high mortality, and high disability rates. Investigating the mechanism of osteosarcoma progression, identifying new targets, and developing effective therapy are of great significance for improving patient clinical outcomes. NAT10-mediated ac4C modification is widely present in mRNA and plays important roles in multiple biological processes of mRNA. NAT10 and ac4C modification also play a crucial role in the occurrence and development of malignant tumors such as bladder cancer, esophageal cancer, and breast cancer. Methods: Through RNA-seq and functional screening, NAT10 was identified as the potential therapeutic target. The biological functions of NAT10 and ac4C modification in osteosarcoma were explored in vivo and in vitro. Downstream target of NAT10 was identified through acRIP-seq combined with RNA-seq. The ac4C modification of downstream target gene were detected using RIP-qPCR and its regulatory effect on mRNA stability was explored using RNA half-life analysis. Drug screening was performed using FDA-approved drugs and a small molecule compound library to identify NAT10 inhibitors. The efficacy of inhibitors in osteosarcoma treatment is validated through in vivo, in vitro experiments and patient-derived tumor xenograft (PDX) models. Results: Functional screening targeting RNA modification regulatory factors demonstrated that inhibiting NAT10 significantly reduced the proliferation, migration and invasion of osteosarcoma cells. NAT10 knockout significantly inhibited the proliferation and metastasis of osteosarcoma cells, as well as the malignant progression in vivo. The acRIP-seq and RNA-seq revealed that ATF4 is a downstream target gene regulated by NAT10 in osteosarcoma. NAT10 promoted ac4C modification of ATF4 mRNA, enhanced the stability of ATF4 mRNA, and ATF4 promoted the transcription and expression of ASNS. ASNS catalyzes the synthesis of asparagine (Asn), thereby promoting the malignant progression of osteosarcoma. Drug screening identified Paliperidone and AG-401 as potential inhibitors of NAT10. Through cell functional experiments, orthotopic osteosarcoma models, organoids model, and PDX models, it was demonstrated that Paliperidone and AG-401 can inhibit the malignant progression of osteosarcoma. Conclusions: NAT10 was highly expressed in osteosarcoma and associated with patient prognosis. Through ac4C modification, NAT10 regulated the synthesis of asparagine mediated by ATF4/ASNS, providing materials for protein and nucleotide synthesis in tumor cells, thus promoting the malignant progression of osteosarcoma. Paliperidone and AG-401 were identified as potential NAT10 inhibitors and targeting NAT10 could be a promosing strategy in osteosarcoma treatment.

MTF2 facilitates the advancement of osteosarcoma through mediating EZH2/SFRP1/Wnt signaling

BackgroundOsteosarcoma is a soft tissue neoplasm with elevated recurrence risk and highly metastatic potential. Metal response element binding transcriptional factor 2 (MTF2) has been revealed to exert multiple activities in human tissues. The present research was conducted to explore the functions and related response mechanism of MTF2 in osteosarcoma which have not been introduced yet.MethodsBioinformatics tools identified the differential MTF2 expression in osteosarcoma tissues. MTF2 expression in osteosarcoma cells was examined with Western blot. Cell Counting Kit-8 (CCK-8) assay, 5-Ethynyl-2’-deoxyuridine (EDU) staining, wound healing as well as transwell assays measured cell proliferation, migration and invasion, respectively. Flow cytometry assay detected the cellular apoptotic level. Western blot also measured the expressions of proteins associated with epithelial mesenchymal transition (EMT), apoptosis and enhancer of zeste homolog 2 (EZH2)/secreted frizzled-related protein 1 (SFRP1)/Wnt signaling. Co-immunoprecipitation (Co-IP) assay confirmed MTF2-EZH2 interaction.ResultsMTF2 expression was increased in osteosarcoma tissues and cells. MTF2 interference effectively inhibited the proliferation, migration and invasion of osteosarcoma cells and promoted the cellular apoptotic rate. MTF2 directly bound to EZH2 and MTF2 silence reduced EZH2 expression, activated SFRP1 expression and blocked Wnt signaling in osteosarcoma cells. EZH2 upregulation or SFRP1 antagonist WAY-316606 partly counteracted the impacts of MTF2 down-regulation on the SFRP1/Wnt signaling and the biological phenotypes of osteosarcoma cells.ConclusionsMTF2 might down-regulate SFRP1 to activate Wnt signaling and drive the progression of osteosarcoma via interaction with EZH2 protein.

The use of matrine to inhibit osteosarcoma cell proliferation via the regulation of the MAPK/ERK signaling pathway.

Matrine is an alkaloid extracted from Sophorus beans of the legume family, and it has significant effects and a variety of pharmacological activities. Osteosarcoma(OS) is a common malignant bone tumor that is characterized by high incidence and rapid progression. There have been some preliminary studies on the therapeutic effect of matrine on OS, but the specific mechanism remains unclear. The aim of this study was to investigate the antitumor effect of matrine on HOS cells and the underlying molecular mechanism. The effects of matrine on the proliferation, apoptosis and cell cycle progression of HOS cells were determined by CCK-8 assay, TUNEL assay and flow cytometry in vitro. Wound healing and Transwell invasion assays were used to observe the effect of matrine on the migration and invasion of HOS cells. The mechanism underlying the antitumor effect of matrine on HOS cells was investigated by Western blotting. Matrine significantly inhibited HOS cell proliferation, promoted HOS cell apoptosis, and arrested HOS cells in the G1 phase of the cell cycle. Both wound healing and Transwell invasion assays showed that matrine inhibited HOS cell migration and invasion. Western blotting results showed that matrine inhibited the activation of the MAPK/ERK signaling pathway. We found that matrine also downregulated Bcl-2 expression, which may be related to protein synthesis inhibition. Matrine can inhibit the proliferation of HOS cells, arrest HOS cells in the G1 phase, and promote HOS cell apoptosis through the MAPK/ERK signaling pathway.

Small Nuclear Ribonucleoprotein Polypeptides B and B1 Promote Osteosarcoma Progression via Activating the Ataxia-Telangiectasia Mutated Signaling Pathway through Ribonucleotide Reductase Subunit M2

Osteosarcoma is a malignant bone tumor characterized by high metastatic potential and recurrence rates post-therapy. The small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB), a core component of spliceosome, have been reported to exhibit upregulation across several cancer types. However, the precise role of SNRPB in osteosarcoma progression remains poorly elucidated. Herein, we explored SNRPB expression in human osteosarcoma tissues and normal bone tissues by immunohistochemical staining (IHC) staining, revealing a notable upregulation of SNRPB in osteosarcoma, correlating with diminished survival rates. Moreover, the in vitro loss-of-function experiments showed that SNRPB knockdown significantly suppressed the osteosarcoma cell proliferation and migration, as well as tubule formation of HUVECs, while enhancing osteosarcoma cell apoptosis. Mechanistically, we revealed that SNRPB promoted the transcription of ribonucleotide reductase subunit M2 (RRM2) via E2F transcription factor 1 (E2F1). Further rescue experiments indicated that RRM2 was required for SNRPB-induced malignant behaviors in osteosarcoma. Additionally, we confirmed that the function of SNRPB in osteosarcoma cell growth and apoptosis was associated with ATM signaling pathway activation. In conclusion, our findings provide initial insights into the underlying mechanisms governing SNRPB-induced osteosarcoma progression and proposed for SNRPB as a novel therapeutic target in osteosarcoma management.