Anlotinib destabilizes PAX3-FOXO1 to induce rhabdomyosarcoma cell death via upregulating NEK2.

BACKGROUND: Rhabdomyosarcoma (RMS) is one of the most common soft tissue sarcomas in children and adolescents, in which PAX3-FOXO1 fusion gene positive patients have very poor prognosis. PAX3-FOXO1 has been identified as an independent prognostic predictor in RMS, with no currently available targeted therapeutic intervention. The novel tyrosine kinase inhibitor anlotinib exhibits a wide range of anticancer effects in multiple types of cancers; however, there have been no relevant studies regarding its application in RMS. MATERIALS AND METHODS: We investigated the effects of PAX3-FOXO1 on the therapeutic efficacy of anlotinib using the CCK-8 assay, flow cytometry, invasion assay, wound healing assay, western blotting, quantitative polymerase chain reaction(qPCR), and xenograft experiments. RNA-seq and co-immunoprecipitation assays were conducted to determine the specific mechanism by which anlotinib regulates PAX3-FOXO1 expression. RESULTS: Anlotinib effectively inhibited RMS cell proliferation and promoted apoptosis and G2/M phase arrest while impeding tumor growth in vivo. Downregulation of PAX3-FOXO1 enhances the antitumor effects of anlotinib. Anlotinib upregulates protein kinase NEK2 and increases the degradation of PAX3-FOXO1 via the ubiquitin-proteasome pathway, leading to a reduction in PAX3-FOXO1 protein levels. CONCLUSION: Anlotinib effectively inhibited the malignant progression of RMS and promoted degradation of the fusion protein PAX3-FOXO1. Anlotinib could be a targeted therapeutic approach to treat PAX3-FOXO1 fusion-positive RMS.

Photo-sonodynamic therapy mediated with OLI_NPs to induce HPV16E7-specific immune response and inhibit cervical cancer in a Tc-1-grafted murine model.

Cervical carcinoma is the fourth most common gynecological cancer. Here we reported the synthesis of oxygen-carried and lipopolysaccharide (LPS)/ indocyanine green (ICG)-loaded nanoparticles (OLI_NPs) for photo-sonodynamic therapy (PSDT) mediated combination therapy to induce systemic antitumor immune responses. We effectively built a new nanoparticle system, a multifunctional nanoagent that integrated the ability of dual-model imaging and therapy for tumors. In this study, we confirmed that OLI_NPs can act as a multifunctional platform that enables not only to diagnose tumors conveniently but also to efficiently provide treatment of in situ tumors, permitting simultaneous dual-mode imaging and localization of the therapy in combination with PSDT-mediated drug release. Furthermore, our combined strategy could effectively depress the tumor development and extend mouse life by the combination of inducing immunogenic cell death (ICD) with encapsulated LPS. In conclusion, combining therapy of OLI_NPs plus PSDT can induce anti-tumor immune responses and tumor antigen-specific immunity in a common TC-1 graft tumor model. Therefore, this combination therapy is a viable technique for cervical cancer treatment.

Photoacoustic mediated multifunctional tumor antigen trapping nanoparticles inhibit the recurrence and metastasis of ovarian cancer by enhancing tumor immunogenicity.

The hypoimmunogenicity of tumors is one of the main bottlenecks of cancer immunotherapy. Enhancing tumor immunogenicity can improve the efficacy of tumor immunotherapy by increasing antigen exposure and presentation, and establishing an inflammatory microenvironment. Here, a multifunctional antigen trapping nanoparticle with indocyanine green (ICG), aluminum hydroxide (Al(OH)3) and oxaliplatin (OXA) (PPIAO) has been developed for tumor photoacoustic/ultrasound dual-modality imaging and therapy. The combination of photothermal/photodynamic therapy and chemotherapy induced tumor antigen exposure and release through immunogenic death of tumor cells. A timely capture and storage of antigens by aluminum hydroxide enabled dendritic cells to recognize and present those antigens spatiotemporally. In an ovarian tumor model, the photoacoustic-mediated PPIAO NPs combination therapy achieved a transition from "cold tumor" to "hot tumor" that promoted more CD8+ T lymphocytes activation in vivo and intratumoral infiltration, and successfully inhibited the growth of primary and metastatic tumors. An in situ tumor vaccine effect was produced from the treated tumor tissue, assisting mice against the recurrence of tumor cells. This study provided a simple and effective personalized tumor vaccine strategy for better treatment of metastatic and recurrent tumors. The developed multifunctional tumor antigen trapping nanoparticles may be a promising nanoplatform for integrating multimodal imaging monitoring, tumor treatment, and tumor vaccine immunotherapy.

Revisiting bone morphogenetic protein-2 knuckle epitope and redesigning the epitope-derived peptides.

The bone morphogenetic protein-2 (BMP2) plays a crucial role in bone formation, growth and regeneration, which adopts a conformational wrist epitope and a linear knuckle epitope to interact with its type-I (BRI) and type-II (BRII) receptors, respectively. In this study, we systematically examine the BRII-recognition site of BMP2 at structural, energetic and dynamic levels and accurately locate hotspots of the recognition at BMP2-BRII complex interface. It is revealed that the traditional knuckle epitope (BMP2 residue range 73-92) do fully match the identified hotspots; the BMP2-recognition site includes the C-terminal region of traditional knuckle epitope as well as its flanked ß-strands. In addition, the protein context of full-length BMP2 is also responsible for the recognition by addressing conformational constraint on the native epitope segment. Therefore, we herein redefine the knuckle epitope to BMP2 residue range 84-102, which has a similar sequence length but is slid along the protein sequence by ~10 residues as compared to traditional knuckle epitope. The redefined one is also a linear epitope that is natively a double-stranded ß-sheet with two asymmetric arms as compared to the natively single ß-strand of the traditional version, although their sequences are partially overlapped to each other. It is revealed that the redefined epitope-derived peptide LN84-102 exhibits an improved affinity by >3-fold relative to the traditional epitope-derived peptide KL73-92 . Even so, the LN84-102 peptide still cannot fully represent the BMP2 recognition event by BRII that has been reported to have a nanomolar affinity. We further introduce a disulfide bond across the two arms of double-stranded ß-sheet to constrain the free LN84-102 peptide conformation, which mimics the conformational constraint addressed by protein context. Consequently, several cyclic peptides are redesigned, in which the LN84-102 (cyc89-101) is determined to exhibit a sub-micromolar affinity; this value is ~5-fold higher than its linear counterpart. Structural analysis also reveals that the cyclic peptide can interact with BRII in a similar binding mode with the redefined knuckle epitope region in full-length BMP2 protein.

Downregulated Long Non-Coding RNA MSC-AS1 Inhibits Osteosarcoma Progression and Increases Sensitivity to Cisplatin by Binding to MicroRNA-142.

BACKGROUND Osteosarcoma (OS) is the most prevalent malignant primary bone tumor, resulting from severe transformation of primitive mesenchymal cells, which induces osteogenesis. Long non-coding RNA (lncRNA) MSC-AS1 triggers osteogenic differentiation by sponging microRNA (miR)-140-5p. The present study assessed the mechanism of lncRNA MSC-AS1 in OS biological features and sensitivity to cisplatin (DDP) by binding to miR-142. MATERIAL AND METHODS Firstly, lncRNA MSC-AS1 expression in OS tissues and cells was analyzed. OS cells were transfected with silenced MSC-AS1 to determine its role in OS biological behaviors, and we also assessed the effect of MSC-AS1 on OS sensitivity to DDP. Then, website prediction and dual-luciferase reporter gene assay were utilized for verification of the binding site between MSC-AS1 and miR-142. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were performed to determine the effect of MSC-AS1 on expression of miR-142, cyclin-dependent kinase 6 (CDK6), and the PI3K/AKT signaling pathway. Xenograft transplantation was also applied to confirm the in vitro experiments. RESULTS Overexpressed MSC-AS1 was associated with poor prognosis of OS patients. OS cell proliferation, invasion, and migration were reduced after silencing MSC-AS1, while cell apoptosis was enhanced. Moreover, silencing MSC-AS1 made OS cells more sensitive to DDP. Interestingly, MSC-AS1 knockdown induced miR-142 expression and reduced CDK6 levels, thereby decreasing the protein expression of p-PI3K/t-PI3K and p-AKT/t-AKT. Silencing MSC-AS1 repressed OS progression in vivo. CONCLUSIONS Our study demonstrated that silencing MSC-AS1 inhibited OS biological behaviors by enhancing miR-142 to decrease CDK6 and inactivating the PI3K/AKT axis. Our results may provide new insights for OS treatment.

Lunasin abrogates the expression of matrix metalloproteinases and reduction of type II collagen.

Impairment of type II collagen caused by MMPs in response to overproduction of IL-1ß is an important step in the pathological progression of osteoarthritis (OA). Lunasin, a well-known peptide present in the soybean, has displayed a positive impact on numerous physiological functions. Little information in the effects of lunasin on cartilage degradation has been sought in clinical research before. Here, we report that lunasin suppressed the increase in MMP-3 and MMP-13 caused by IL-1ß. In addition, we found that lunasin could prevent the decrease in TIMP-1 and TIMP-2 expressions caused by IL-1ß. Notably, lunasin suppressed reduction of type II collagen, the basis for articular cartilage. Lunasin also attenuated activation of the JAK2/STAT1/IRF-1 pathway. These effects of lunasin suggest that it might become a promising therapeutic agent for chondro-protective therapy.

Bufalin attenuates cancer-induced pain and bone destruction in a model of bone cancer.

Bufalin is a natural anti-inflammatory small molecule. Given the close relationship between inflammation and cancer, many scholars have studied the effect of bufalin on cancer in vitro, but in vivo research is still lacking. A murine bone cancer model was used in this study. We conducted pain sensitive test on mice with bone cancer, by nocifensive behavior, mechanical allodynia, and thermal hyperalgesia. Serum levels of bone loss markers with bufalin treatment were measured by ELISA. Expressions of osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) were analyzed in bufalin-treated mice by real-time PCR and Western blot. Cannabinoid 2 receptor (CB2) inverse agonist AM630 was administrated to mice with bone cancer together with bufalin. Bufalin relieved cancer-induced pain and bone destruction in the murine bone cancer model. Serum levels of bone loss markers after bufalin treatment were reduced. Bufalin upregulated OPG and downregulated RANKL. The CB2 receptor inverse agonist, AM630, reduced the pain relief of bufalin treatment in the mouse bone cancer model. This study demonstrates that bufalin relieves cancer-induced pain and bone destruction, which is mediated through the CB2 receptor.

Effects of platelet-derived growth factor on chondrocyte proliferation, migration and apoptosis via regulation of GIT1 expression.

The formation of fibrocartilage, cartilaginous and bony calluses is vital for bone healing following a fracture. Fibroblasts, chondrocytes and osteoblasts are critical functional cells that are involved in these three processes, respectively. Platelet­derived growth factor (PDGF), a growth factor that is released from platelet particles and appears during the early stages at the site of fractures, is essential in bone healing via regulation of cell proliferation and differentiation. However, the effects of PDGF on the chondrocytes remain unclear. In the present study, PDGF promoted phosphorylation of Src and upregulated the expression level of G­protein­coupled receptor kinase interacting protein­1 (GIT1) according to the results of the cell culture of chondrocytes in vitro and western blotting. However, the effect of PDGF on the upregulation of GIT1 expression was mostly inhibited by the Src inhibitor, PP2. After knocking down GIT1 expression using siRNA, the phosphorylation of Src continued to be induced by PDGF, although the expression of GIT1 was inhibited. Furthermore, the results indicated that PDGF promoted chondrocyte proliferation and migration, however, the effect on cell apoptosis induction was suppressed after adding the Src inhibitor, PP2. Additionally, when knocking down GIT1 using siRNA, the expression level of GIT1 decreased, which is similar to the effect of the Src inhibitor, PP2. The current study demonstrates that PDGF may initially activate the phosphorylation of Src, and subsequently induce GIT1 expression to promote chondrocyte proliferation and migration, but suppress cell apoptosis.

MicroRNA-128 inhibits EMT of human osteosarcoma cells by directly targeting integrin α2.

Deregulated expression of miRNAs contributes to the development of osteosarcoma. The present study was to evaluate the level of miR-128 and integrin α2 (ITGA2) in osteosarcoma tissues and cells. We further investigated the molecular mechanisms of miR-128 and ITGA2 in osteosarcoma cell lines. In the present study, we found that miR-128 expression was down-regulated in osteosarcoma tissues and MG-63, U2OS, and SAOS-2 cells (all p < 0.001). By contrast, ITGA2 was up-regulated. Furthermore, we found that miR-128 overexpression suppressed cell migration and invasion of MG-63 cells. Mechanically, miR-128 overexpression inhibited epithelial-mesenchymal transition (EMT) of MG-63 cells. Importantly, we identified that the 3'-untranslated region (3'-UTR) of ITGA2 was a direct target of miR-128. Luciferase reporter assays confirmed that miR-128 binding to the 3'-UTR regions of ITGA2 inhibited the expression of ITGA2 in MG-63 cells. At the same time, overexpressed ITGA2 also reversed EMT inhibited by miR-128. In conclusion, this study suggested that high miR-128 expression suppressed osteosarcoma cell migration, invasion, and EMT development through targeting ITGA2, which may be recommended as a therapeutic target for osteosarcoma.

Integrin-ß1 regulates chondrocyte proliferation and apoptosis through the upregulation of GIT1 expression.

Chondrocytes play a critical role in the repair process of osteoarthritis, which is also known as degenerative arthritis. Integrins, as the key family of cell surface receptors, are responsible for the regulation of chondrocyte proliferation, differentiation, survival and apoptosis through the recruitment and activation of downstream adaptor proteins. Moreover, G-protein-coupled receptor kinase interacting protein-1 (GIT1) exerts its effects on cell proliferation and migration through interaction with various cytokines. It has been previously suggested that GIT1 acts as a vital protein downstream of the integrin-mediated pathway. In the present study, we investigated the effects of integrin-ß1 on cell proliferation and apoptosis, as well as the underlying mechanisms in chondrocytes in vitro. Following transfection with a vector expressing integrin-ß1, our results revealed that the overexpression of integrin-ß1 enhanced GIT1 expression, whereas the knockdown of integrin-ß1 by siRNA suppressed GIT1 expression. However, no significant effect was observed on integrin-ß1 expression following the enforced overexpression of GIT1, which suggests that GIT1 is localized downstream of integrin-ß1. In other words, integrin-ß1 regulates the expression of GIT1. Furthermore, this study demonstrated that integrin-ß1 and GIT1 increased the expression levels of aggrecan and type II collagen, thus promoting chondrocyte proliferation; however, they inhibited chondrocyte apoptosis. Taken together, our data demonstrate that integrin-ß1 plays a vital role in chondrocyte proliferation, differentiation and apoptosis. GIT1 exerts effects similar to those of integrin-ß1 and is a downstream target of integrin-ß1.

Dimethyl fumarate inhibits the expression and function of hypoxia-inducible factor-1α (HIF-1α).

Osteocyte hypoxia has been induced by skeletal unloading and fracture. Hypoxia-dependent regulation of gene expression is mediated by hypoxia-sensitive transcription factors such as hypoxia-inducible factor-1α (HIF-1α). Dimethyl fumarate (DMF) is a recently approved first-line therapy for multiple sclerosis. However, the role of DMF in regulating HIF-1α expression and function has not been evaluated. In this study, we found that DMF inhibited hypoxia-induced expression of HIF-1α and its target genes such as interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF) in MC3T3 E1 cells. Mechanistically, DMF promoted HIF-1α degradation in a proteasome-dependent but von Hippel-Lindau (VHL) protein-independent manner. Importantly, we found that DMF disrupted the interaction between HIF-1α and its chaperone heat shock protein 90 (Hsp90) but promoted the interaction between HIF-1α and the receptor of activated protein kinase C (RACK1). These data suggest that DMF might promote degradation of HIF-1α by affecting its folding and maturation. Based on these observations, we conclude that DMF is a novel inhibitor of HIF-1α.