OBJECTIVE:To investigate the synergistic effects of polyphyllin I(PPI)combined with tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)on the growth of osteosarcoma cells through downregulating the Wnt/β-catenin signaling pathway. METHODS:Cell viability,apoptosis and cell cycle distribution were examined using cell counting kit-8 and flow cytometry assays.The morphology of cancer cells was observed with inverted phase contrast microscope.The migration and invasion abilities were examined by xCELLigence real time cell analysis DP system and transwell assays.The expressions of poly(adenosine diphosphate-ribose)polymerase,C-Myc,Cyclin B1,cyclin-dependent kinases 1,N-cadherin,Vimentin,Active-β-catenin,β-catenin,p-glycogen synthase kinase 3β(GSK-3β)and GSK-3β were determined by Western blotting assay. RESULTS:PPI sensitized TRAIL-induced decrease of viability,migration and invasion,as well as increase of apoptosis and cell cycle arrest of MG-63 and U-2 OS osteosarcoma cells.The synergistic effect of PPI with TRAIL in inhibiting the growth of osteosarcoma cells was at least partially realized through the inactivation of Wnt/β-catenin signaling pathway. CONCLUSION:The combination of PPI and TRAIL is potentially a novel treatment strategy of osteosarcoma.

Background: Osteosarcoma is the most common primary bone cancer in children and adolescents with high metastatic ability. Aim: This study aimed to explore the inhibitory effects of (S)-10-hydroxycamptothecin (HCPT) on osteosarcoma cell growth and metastasis as well as the underlying mechanism. Methods: The osteosarcoma cells of 143B and U-2 OS (U-2), treated with HCPT (20, 100, or 300 nM), underwent detections, such as CCK-8, flow cytometry, Transwell, wound healing, and immunoblotting. EMT-related key proteins, like N-cadherin, Snail, and Vimentin, were found to be down-regulated, while E-cadherin was up-regulated dose-dependently in HCPT-exposed 143B and U-2 cells. Additionally, incubation of 143B and U-2 cells with HCPT for 3 hours dosedependently reduced the expression ratios of p-LATS1/LATS1, p-MST1/MST1, p-YAP/YAP, and p-TAZ/TAZ. Results: Taken together, our study has demonstrated HCPT to inhibit osteosarcoma growth and metastasis potentially by activating the HIPPO signaling pathway and reversing EMT. Conclusion: HCPT might be a candidate agent for the prevention and treatment of osteosarcoma

Osteosarcoma is the most common malignant bone tumor and is frequently diagnosed in juvenile. Cellular senescence is a fundamental hallmark of osteosarcoma and plays a vital role in the initiation and progression of aging and tumorigenesis. Long non-coding RNAs (lncRNAs) are implicated in tumorigenesis. In this study, six cellular senescence-related lncRNAs with independent prognostic significance in juvenile osteosarcoma patients were identified through univariate Cox regression analysis, least absolute shrinkage and selection operator (LASSO) regression analysis, and multivariate Cox regression analysis. Prognostic significance was further confirmed by Kaplan–Meier (KM) survival curves, co-expression interaction networks, and sankey diagrams. A prognostic model of cellular senescence-related genes in juvenile osteosarcoma patients was then constructed using multivariate Cox regression analysis based on these six genes. High- and low-risk groups were identified according to the median risk score calculated by the prognostic model. The favorable prognostic significance of this model was demonstrated through survival curves, receiver operating characteristic (ROC) curves, distribution scatter plots and lncRNA expression heatmaps. Furthermore, cellular senescence-related lncRNAs were validated by enrichment analysis, immunological correlation analysis, m6A correlation analysis, and drug sensitivity correlation analysis. These findings are important for improving the prognosis of juvenile osteosarcoma patients and understanding the mechanisms underlying cellular senescence in juvenile osteosarcoma development.

BackgroundsPD-1 inhibitors and TKIs have been used to treat advanced osteosarcoma, but there is still a lack of intuitive data for the comparison of their efficacy. We conducted a meta-analysis to evaluate their therapeutic benefits.MethodsA systematic methodological search of five primary electronic databases was performed. Studies with a randomized design of any type about PD-1 inhibitors or TKIs for the treatment of advanced osteosarcoma were included. The primary outcomes mainly included CBR, PFS, OS and ORR, The CR, PR, SD and AEs were the secondary outcomes. The survival period (months) of patients was taken as the main analysis data. Random-effects models were used for meta-analysis.ResultsEight immunocheckpoint inhibitors in 327 patients from 10 clinical trials were finally evaluated. For OS, TKIs [11.67 months (95% CI, 9.32-14.01)] show more obvious advantages than PD-1 inhibitors [6.37 months (95% CI, 3.96-8.78)]. For PFS, TKIs [4.79 months (95% CI, 3.33-6.24)] are longer than PD-1 inhibitors [1.46 months (95% CI, 1.23-1.69)]. Although there was no fatal event, attention should still be paid, especially during the combined application of PD-1 inhibitors with TKIs since their obvious AEs.ConclusionsThe findings of this study suggest that patients with advanced osteosarcoma, TKIs may be more beneficial than PD-1 inhibitors. TKIs combined with PD-1 inhibitors has a bright future in the treatment of advanced osteosarcoma, but we should always pay attention to the strong side effects.

The important roles of long non-coding RNAs (lncRNAs) in cancer have been studied, such as regulating the proliferation, epithelial-mesenchymal transition (EMT), migration, infiltration, and autophagy of cancer cells. Localization detection of lncRNAs in cells can provide insight into their functions. By designing the lncRNA-specific antisense chain sequence followed by labeling with fluorescent dyes, RNA fluorescence in situ hybridization (FISH) can be applied to detect the cellular localization of lncRNAs. Together with the development of microscopy, the RNA FISH techniques now even allow for visualization of the poorly expressed lncRNAs. This method can not only detect the localization of lncRNAs alone, but also detect the colocalization of other RNAs, DNA, or proteins by using double-color or multicolor immunofluorescence. Here, we have included the detailed experimental operation procedure and precautions of RNA FISH by using lncRNA small nucleolar RNA host gene 6 (SNHG6) in human osteosarcoma cells (143B) as an example, to provide a reference for researchers who want to perform RNA FISH experiments, especially lncRNA FISH.

Lung adenocarcinoma (LUAD) is the main histological type of lung cancer with an unfavorable survival rate. Metastasis is the leading LUAD-related death with Epithelial-Mesenchymal Transition (EMT) playing an essential role. The anticancer efficacies of the active ingredients in Chonglou have been widely reported in various cancers. However, the potential therapeutic targets of the Chonglou active ingredients in LUAD patients remain unknown. Here, the network pharmacology and bioinformatics were performed to analyze the associations of the clinical characteristics, immune infiltration factors and m6A-related genes with the EMT-related genes associated with LUAD (EMT-LUAD related genes), and the molecular docking, STRING, GO, and KEGG enrichment for the drug targets of Chonglou active ingredients associated with EMT (EMT-LUAD-Chonglou related genes). And, cell viability analysis and cell invasion and infiltration analysis were used to confirm the theoretical basis of this study. A total of 166 EMT-LUAD related genes were identified and a multivariate Cox proportional hazards regression model with a favorable predictive accuracy was constructed. Meanwhile, the immune cell infiltration, immune cell subsets, checkpoint inhibitors and the expression of m6A-related genes were significantly associated with the risk scores for EMT-LUAD related genes with independent significant prognostic value of all included LUAD patients. Furthermore, 12 EMT-LUAD-Chonglou related genes with five core drug targets were identified, which participated in LUAD development through extracellular matrix disassembly, collagen metabolic process, collagen catabolic process, extracellular matrix organization, extracellular structure organization and inflammatory response. Moreover, we found that the active ingredients of Chonglou could indeed inhibit the progression of lung adenocarcinoma cells. These results are oriented towards EMT-related genes to achieve a better understanding of the role of Chonglou and its targets in osteosarcoma development and metastasis, thus guiding future preclinical studies and facilitating clinical translation of LUAD treatment.

The important roles of long non-coding RNAs (lncRNAs) in cancer have been studied, such as regulating the proliferation, epithelial-mesenchymal transition (EMT), migration, infiltration, and autophagy of cancer cells. Localization detection of lncRNAs in cells can provide insight into their functions. By designing the lncRNA-specific antisense chain sequence followed by labeling with fluorescent dyes, RNA fluorescence in situ hybridization (FISH) can be applied to detect the cellular localization of lncRNAs. Together with the development of microscopy, the RNA FISH techniques now even allow for visualization of the poorly expressed lncRNAs. This method can not only detect the localization of lncRNAs alone, but also detect the colocalization of other RNAs, DNA, or proteins by using double-color or multicolor immunofluorescence. Here, we have included the detailed experimental operation procedure and precautions of RNA FISH by using lncRNA small nucleolar RNA host gene 6 (SNHG6) in human osteosarcoma cells (143B) as an example, to provide a reference for researchers who want to perform RNA FISH experiments, especially lncRNA FISH.

骨肉瘤是临床常见原发性恶性骨肿瘤,目前针对骨肉瘤的治疗主要包括化疗和手术治疗等.但长期化疗所产生的耐药问题,严重影响其对部分骨肉瘤患者的疗效,降低患者长期生存率.中药在治疗骨肉瘤方面具有不良反应少、多靶点、多阶段性特色,使其成为骨肉瘤耐药领域研究热点.文章通过综述中药有效单体逆转骨肉瘤耐药研究现状,探讨中药治疗骨肉瘤耐药的作用机制及研究进展,以期为中医药参与临床治疗骨肉瘤化疗耐药提供理论依据.

Targeted therapies of programmed cell death protein-1 (PD-1) inhibitors and protein tyrosinase inhibitors (TKIs) have offered new treatment strategies and promoted the exploration of clinical evidence-based medicine for advanced osteosarcoma. This meta-analysis evaluated the efficacy of PD-1 inhibitors and TKIs in the treatment of advanced osteosarcoma. Trials for advanced osteosarcoma from electronic databases using keywords were comprehensively searched. Stata software was used to compare the overall survival (OS), the progression free survival (PFS), and the serious adverse events (AEs). Eight immunocheckpoint inhibitors in 327 patients from 10 clinical trials were finally evaluated. For OS, TKIs [11.67 months (95% CI, 9.32-14.01)] show more obvious advantages than PD-1 inhibitors [6.37 months (95% CI, 3.96-8.78)]. For PFS, TKIs [4.79 months (95% CI, 3.33-6.24)] are longer than PD-1 inhibitors [1.46 months (95% CI, 1.23-1.69)]. Based on the current evidences, TKIs are identified to be better than PD-1 inhibitors, and PD-1 inhibitors combined with TKIs are promising in the treatment of advanced osteosarcoma. Although there was no fatal event, attention should still be paid, especially during the combined application of PD-1 inhibitors with TKIs since their obvious AEs.Funding: Our current work is supported by grants from (1) National Key R&D Program of China (2020YFE0201600), (2) National Nature Science Foundation (81973877 and 82174408). (3) Research Projects within Budget of Shanghai University of Traditional Chinese Medicine (2021LK047).Declaration of Interest: All authors declare no competing interests.Ethical Approval: MISSING

Increasing studies have revealed significant associations between TOP2A with oncogenesis and prognosis of human cancers; however, pan-cancer analysis has not been reported. Here, we explored the potential carcinogenic function and the association with clinical outcomes of TOP2A in 33 different human cancers. The results showed that TOP2A was amplified in 31 investigated cancers; TOP2A expression was significantly associated with metastasis of six different cancers and significantly associated with the survival of patients in ten different cancers; TOP2A-encoded protein was obviously upregulated in five available cancers; phosphorylated TOP2A protein at S1106 was significantly upregulated in all six available cancers. Moreover, TOP2A expression was found to be associated with the cancer-associated immune cell infiltration, including fibroblasts, Tregs, and macrophages. In addition, the Kyoto encyclopedia of genes and genomes (KEGG) pathway and Gene Ontology (GO) enrichment analyses revealed a most significant association between TOP2A with the Wnt signaling pathway and DNA conformation change. This work provides a comprehensive knowledge of TOP2A in different cancers, including carcinogenic function, prognostic values for metastasis, and clinical outcomes.

As the most common male malignancy, prostate cancer (PC) ranks second in mortality, primarily due to a 65%-75% bone metastasis rate. Therefore, it is essential to understand the process and related mechanisms of prostate cancer bone metastasis for developing new therapeutics. For this, an animal model of bone metastasis is an essential tool. Here, we report detailed procedures to generate a bone metastasis mouse model via intra-cardiac injection of prostate cancer cells. A bioluminescence imaging system can determine whether prostate cancer cells have been accurately injected into the heart and monitor cancer cell metastasis since it has great advantages in monitoring metastatic lesion development. This model replicates the natural development of disseminated cancer cells to form micro-metastases in the bone and imitates the pathological process of prostate cancer bone metastasis. It provides an effective tool for further exploration of the molecular mechanisms and the in vivo therapeutic effects of this disease.

骨肉瘤是一种原发性、侵袭性恶性肿瘤,极易发生早期转移且缺乏有效治疗靶点.手术和放化疗及二者的联合应用是目前主要的治疗手段,但对转移性骨肉瘤患者总体生存率的提高仍未有明显改善.骨肉瘤在中医属于"癌病"范畴,中医药治疗骨肉瘤历史悠久,并且在防治骨肉瘤转移方面能够发挥积极作用,但其机制尚未十分明确.信号通路在骨肉瘤转移的发生过程中具有重要作用,并且是研究骨肉瘤转移的病理机制和治疗药物的作用靶点之一.近年来,针对中药有效成分防治骨肉瘤转移相关信号通路进行了大量研究,现将此做一综述,以期为中医药防治骨肉瘤转移、新药开发和临床诊治提供借鉴.

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

目的 建立具有高肺转移能力的人源骨肉瘤细胞系,为探索骨肉瘤肺转移的发生机制提供研究模型.方法 将GFP和荧光素酶标记的人源骨肉瘤143B细胞重悬到MEM培养基并与Matrigel胶按1:1比例混匀,制成浓度为1×107/mL的细胞悬液.取10μL注射到4周龄BALB/c裸鼠的左侧胫骨内,建立胫骨内原位注射肺转移瘤模型(n=6).收集肺转移结节制成单细胞悬液,在完全培养基中贴壁培养,并用新霉素(G418)抗性筛选后扩增,再次接种于裸鼠胫骨内;采用同样方法在体内循环2次后获得的细胞命名为143B-HLM.采用体外实时细胞迁移实验检测细胞迁移情况,RT-qPCR和Western blot检测迁移浸润关键分子标志物的表达情况,同时观察肺转移瘤模型肺部肿瘤结节的形成情况.结果 与亲本人源骨肉瘤143B细胞相比,建立的143B-HLM细胞体外迁移能力增强,波形蛋白(Vimentin)和锌指转录因子(Slug)的蛋白表达量增加(均P<0.05),Vimentin、Slug、锌指转录抑制因子(Snail)和凋亡抑制基因(Survivin)的mRNA表达水平也升高(均P<0.05).与注射143B细胞的裸鼠相比,裸鼠胫骨内原位注射143B-HLM细胞的荷瘤模型中,原位肿瘤体积减小(P<0.05),肺部肿瘤转移荧光信号显著增强,肺转移的裸鼠数目也更多(P<0.01).结论 骨肉瘤肺转移细胞经体内循环筛选及分离,成功建立了稳定的具有高肺转移能力的骨肉瘤细胞系143B-HLM.

Background: As the leading primary bone cancer in adolescents and children, osteosarcoma patients with metastasis show a five-year-survival-rate of 20-30%, without improvement over the past 30 years.Wnt/β-catenin is important in promoting osteosarcoma development.DKK3 is a Wnt/β-catenin antagonist and predicted to have the specific binding site in 3′-UTR with miR-214-3p.Methods: miR-214-3p and DKK3 levels were investigated in human osteosarcoma tissues and cells by RT-qPCR; the prognostic importance of DKK3 level in osteosarcoma patients was determined with Log-rank test; direct binding between DKK3 with miR-214-3p was identified with targetscan; anti-osteosarcoma mechanism of cantharidin was investigated by miR-214-3p silence/over-expression with or without cantharidin treatment, and nuclear/cytoplasmic protein assay in osteosarcoma cells.Results: Down-regulated DKK3 indicated poor prognosis of osteosarcoma patients.Up-regulated miR-214-3p promoted proliferation and migration, while suppressed apoptosis of osteosarcoma cells by increasing β-catenin nuclear translocation and LEF1 translation via degradation of DKK3.Cantharidin suppressed viabilities, migration and invasion, while promoted cell cycle arrest and apoptosis in 143B and U-2 OS cells via down-regulating miR-214-3p to up-regulate DKK3, thus inhibited p-GSK-3β expression, β-catenin nuclear translocation and LEF1 translation.Meanwhile, cantharidin inhibited tumor growth in xenograft-bearing mice with 143B cell injection in tibia.Conclusion: miR-214-3p mediated Wnt/β-catenin/LEF1 signaling activation by targeting DKK3 to promote oncogenesis of osteosarcoma; cantharidin inhibited proliferation and metastasis of osteosarcoma cells via down-regulating miR-214-3p to up-regulate DKK3 and decrease β-catenin nuclear translocation, indicating that cantharidin may be a prospective candidate for osteosarcoma treatment by targeting miR-214-3p/DKK3/β-catenin signaling.