Identification of therapeutic targets for osteosarcoma by integrating single-cell RNA sequencing and network pharmacology.

Background: Osteosarcoma (OS) is a common primary tumor with extensive heterogeneity. In this study, we used single-cell RNA sequencing (scRNA-seq) and network pharmacology to analyze effective targets for Osteosarcoma treatment. Methods: The cell heterogeneity of the Osteosarcoma single-cell dataset GSE162454 was analyzed using the Seurat package. The bulk-RNA transcriptome dataset GSE36001 was downloaded and analyzed using the CIBERSORT algorithm. The key targets for OS therapy were determined using Pearson's correlation analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed on key targets. The DeepDR algorithm was used to predict potential drugs for Osteosarcoma treatment. Molecular docking analysis was performed to verify the binding abilities of the predicted drugs and key targets. qRT-PCR assay was used to detect the expression of key targets in osteoblasts and OS cells. Results: A total of 21 cell clusters were obtained based on the GSE162454 dataset, which were labeled as eight cell types by marker gene tagging. Four cell types (B cells, cancer-associated fibroblasts (CAFs), endothelial cells, and plasmocytes) were identified in Osteosarcoma and normal tissues, based on differences in cell abundance. In total, 17 key targets were identified by Pearson's correlation analysis. GO and KEGG analysis showed that these 17 genes were associated with immune regulation pathways. Molecular docking analysis showed that RUNX2, OMD, and CD4 all bound well to vincristine, dexamethasone, and vinblastine. The expression of CD4, OMD, and JUN was decreased in Osteosarcoma cells compared with osteoblasts, whereas RUNX2 and COL9A3 expression was increased. Conclusion: We identified five key targets (CD4, RUNX2, OMD, COL9A3, and JUN) that are associated with Osteosarcoma progression. Vincristine, dexamethasone, and vinblastine may form a promising drug-target pair with RUNX2, OMD, and CD4 for Osteosarcoma treatment.

B16 Membrane-Coated Vesicles for Combined Photodynamic Therapy and Immunotherapy Shift Immune Microenvironment of Melanoma.

Introduction: Coating of nanomedicine with cell membranes has attracted increasing attention as it can boost biocompatibility and improve the efficiency of treatment. Herein, we prepared innovative tumor cell-membrane-coated vesicles based on photodynamic therapy (PDT) drug indocyanine green (ICG) and explore the effect on melanoma in vitro and in vivo. Methods: ICG was coated with B16 cell membranes (I@BM NVs) by sonication and extrusion, and the morphological characteristics of I@BM NVs were evaluated by transmission electron microscopy (TEM) and NP-tracking analysis. Homologous cellular uptake was evaluated by flow cytometry (FCM) after staining by DiD dye. Cellular cytotoxicity was evaluated by cell counting kit-8 assay and the anti-tumor effect in vitro was assessed by FCM and western blotting. The anti-tumor effect in vivo was evaluated in a B16 xenograft model in mice. The tumor micro-environment was investigated by FCM and real-time PCR. Results: The vesicles are stable and uniform in nature, and show strong homologous targeting in vivo and in vitro. The vesicles can generate reactive oxygen species to induce apoptosis of B16 cells under near-infrared irradiation. Furthermore, the I@BM NVs induce a significant anti-tumor response in vivo, and perform better with respect to both tumor growth inhibition and lifespan extension. Analysis of immunocytes in the tumor microenvironment showed significant reductions in numbers of myeloid-derived suppressor cells and tumor-associated M2 macrophages in mice in the I@BM NVs group. This was accompanied by significant increases in numbers of M1 macrophages and proliferative CD4+/CD8+ T cells. Expression levels of IFN-γ and IL-2 increased in the I@BM NVs group, while expression of TGF-ß and IL-10 decreased. Conclusion: The results show that the I@BM NVs are feasible drugs for the treatment of melanoma by inducing cell apoptosis under NIR and shifting the immunosuppressive tumor microenvironment in vivo.

Retraction: NF-κB inhibition promotes apoptosis in androgen-independent prostate cancer cells by the photothermal effect via the IκBα/AR signaling pathway.

Retraction of 'NF-κB inhibition promotes apoptosis in androgen-independent prostate cancer cells by the photothermal effect via the IκBα/AR signaling pathway' by Chenfei Kong et al., Biomat. Sci., 2019, 7, 2559-2570, https://doi.org/10.1039/C8BM01007B.

Microplastics in sediments from an interconnected river-estuary region.

Microplastics are an emerging pollutant of international concern due to its wide distribution through various pathways. Estuary is an important pathway for land microplastics to enter into the oceans by rivers. In this study, we hypothesized that microplastics would sink into estuary sediment during diffusion and transmission from river before entering into the sea, which results in higher accumulation of microplastics in proximity to river-estuary than in the oceans. In order to demonstrate this hypothesis, sediment samples were collected from an estuary and its two main inputting rivers and the microplastics in these samples were analyzed. In the collected sediment samples, 19 types of polymers, including the three most common polymers (polyethylene, polyethylene terephthalate, and poly(propylene:ethylene)), were identified and confirmed by FT-IR. Eight types of polymers were consistently detected in all samples, while 11 types of polymers were occasionally found in some samples. These microplastics exhibited four shapes and their percentages followed the high-to-low order of film, fragment, fiber and pellet. A relatively lower abundance of microplastics was found in river sediments from Shuangtaizi River with an average of 170 ± 96 particles/kg d.w., compared to that from Daliao River with an average of 237 ± 129 particles/kg d.w., but it was higher than that from Liaohe Estuary with an average of 120 ± 46 particles/kg d.w. Furthermore, the highest concentration of microplastics was found at the mouth of rivers, showing high accumulation where the freshwater and saltwater meet. Results from this study, including the abundance, characteristics and spatial distribution of microplastic pollution in sediments from an interconnected river-estuary system, revealed the fate and distribution of microplastics in the river and estuary environment.

Investigation of the microplastics profile in sludge from China's largest Water reclamation plant using a feasible isolation device.

Sewage sludge, which is widely applied to land as a fertilizer, is a key source of microplastics in the environment. We sought to develop a feasible device for isolation of microplastic from sewage sludge for further understanding their fates in the environment. In the present study, an effective isolation device, consisting of a fritted glass funnel and a glass filtration apparatus, was constructed to extract microplastics from sludge with nearly 100% recovery efficiency. Then, a high abundance of microplastics was detected in sludge sampled from China's largest sewage treatment plant. Among the 25 types of microplastic polymers confirmed by Fourier transform infrared spectroscopy, poly(11-bromoundecyl acrylate) (PBA) and poly(11-bromoundecyl methacrylate) (PBMA) accounted for 23.63% of total microplastics detected. Rayon, polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), and copolymers, such as PP/PE and poly(styrene:acrylonitrile:butadiene) (ABS), were also detected. The shapes of these microplastics consisted of pellets, fragments, films, and microfibers. Characterization of the isolated microplastics revealed that domestic applications and vehicle products were the major sources of microplastic in sewage treatment sludge. Some priority recommendations were issued based on these results. In conclusion, the present study demonstrate that the device is effective for the isolation of microplastics from sludge.

Identification of differentially expressed genes in MG63 osteosarcoma cells with drug­resistance by microarray analysis.

Osteosarcoma is the most common type of primary malignant bone tumor, with extremely poor prognosis in patients with metastatic disease and resistance to therapy, such as multidrug regimens. The mechanisms of drug resistance are quite complex and have not been fully elucidated; thus, novel therapeutic targets should be identified to alleviate drug resistance in osteosarcoma. In the present study, the transcriptomes of the human osteosarcoma cell line MG63 and vincristine (VCR)­resistant MG63 cells were compared by microarray analysis. A total of 1,300 genes (602 upregulated and 698 downregulated) were reported to be differentially expressed in MG63/VCR compared with MG63 cells. Bioinformatics analysis predicted that the differentially expressed genes were mainly enriched in the B cell receptor, UVA­induced mitogen­activated protein kinases and receptor tyrosine kinase 2/3 signaling pathways. In the present study, 10 of the dysregulated genes, including roundabout homolog 1, death­associated protein kinase 1 and A­kinase anchor protein 12 were further evaluated by reverse transcription­quantitative polymerase chain reaction. These results may aid the validation of candidate biomarkers for the treatment and prognosis of osteosarcoma, and provide novel insight into the molecular mechanisms underlying the drug resistance of osteosarcoma cells.

NF-κB inhibition promotes apoptosis in androgen-independent prostate cancer cells by the photothermal effect via the IκBα/AR signaling pathway.

The photothermal response of nanomaterials provides a basis for many biomedical applications, including diagnosis (e.g., biosensor and photoacoustic imaging) and treatment (e.g., drug delivery and photothermal therapy). The use of nano-materials for cancer phototherapy (solid tumor ablation) can cause cell necrosis and apoptosis. However, photothermal effects using the same material can differ among tumor cell types, and the molecular mechanisms underlying these differences are not clear. We used polydopamine (PDA)-coated branched Au-Ag nanoparticles (Au-Ag@PDA NPs) for the photothermal treatment of two prostate cancer cell lines. The therapeutic effect was evaluated by CCK8, flow cytometry, and expression analyses of related genes by western blotting. Photothermal therapy resulted in oxidative stress in prostate cancer cells and activated the mitochondrial-related apoptosis pathway, increasing the Bax expression. In addition, we observed a greater photothermal treatment effect on the androgen-dependent cells LNCaP than the androgen-independent cells DU145. Pretreatment with an inhibitor of the NF-κB signaling pathway (BAY 11-7082) enhanced the expression of BAX in the DU145 cells and increased the sensitivity of the cells to the heat treatment of Au-Ag@PDA NPs both in vitro and in vivo. Our findings explain the differences in the observed effects of photothermal therapy and provide the direction for further improvements to this strategy.

Inhibition of Autophagy Promoted Apoptosis and Suppressed Growth of Hepatocellular Carcinoma Upon Photothermal Exposure.

Photothermal therapy (PTT) is a recently developed and promising strategy for the treatment of hepatocellular carcinoma (HCC). However, apoptosis has been extensively investigated as the mechanism of the underlying effect of PTT on cancer to date. Here, we explored alternative mechanisms of these therapeutic effects, including the activation of cell-cycle arrest and autophagy during PTT in addition to apoptosis under mild temperature. We treated the HCC cell line HepG2 with polydopamine (PDA)-coated branched Au-Ag nanoparticles at various concentrations along with PTT using an 808-nm laser. Apoptosis was evaluated based on flow cytometry, western blot analysis of apoptosis related proteins (BAX, BCL2, caspase 3), Hoechst staining, and TUNEL staining. To explore the role of autophagy, we treated cells with the autophagy inhibitor chloroquine diphosphate. Enhancement of apoptosis by PTT with nanoparticle treatment was observed after autophagy was inhibited. Moreover, inhibition of autophagy markedly enhance the suppression of tumor growth in vivo in a HepG2 mouse xenograft model. These results suggest that further exploration of the mechanism of PTT can help guide its clinical application, and that autophagy inhibition combined with PTT could be a promising strategy for HCC treatment.

Polydopamine-coated Au-Ag nanoparticle-guided photothermal colorectal cancer therapy through multiple cell death pathways.

Nanoparticles are emerging as a new therapeutic modality due to their high stability, precise targeting, and high biocompatibility. Branched Au-Ag nanoparticles with polydopamine coating (Au-Ag@PDA) have strong near-infrared absorbance and no cytotoxicity but high photothermal conversion efficiency. However, the photothermal activity of Au-Ag@PDA in vivo and in vitro has not been reported yet, and the mechanism underlying the effects of photothermal nanomaterials is not clear. Therefore, in this study, the colorectal cancer cell line HCT-116 and nude mice xenografts were used to observe the photothermal effects of Au-Ag@PDA in vivo and in vitro. The results suggest that Au-Ag@PDA NPs significantly inhibited cell proliferation and induced apoptosis in colorectal cancer cells. Moreover, Au-Ag@PDA NP-mediated photothermal therapy inhibited the growth of tumors at doses of 50 and 100 µg in vivo. The mechanisms through which Au-Ag@PDA NPs induced colorectal cancer cell death involved multiple pathways, including caspase-dependent and -independent apoptosis, mitochondrial damage, lysosomal membrane permeability, and autophagy. Thus, our findings suggest that Au-Ag@PDA NPs could be used as potential antitumor agents for photothermal ablation of colorectal cancer cells.

Photothermal exposure of polydopamine-coated branched Au-Ag nanoparticles induces cell cycle arrest, apoptosis, and autophagy in human bladder cancer cells.

PURPOSE: Polydopamine-coated branched Au-Ag nanoparticles (Au-Ag@PDA NPs) exhibit good structural stability, biocompatibility, and photothermal performance, along with potential anticancer efficacy. Here, we investigated the cytotoxicity of Au-Ag@PDA NPs against human bladder cancer cells (T24 cells) in vitro and in vivo, as well as the underlying molecular mechanisms of photothermal therapy-induced T24 cell death. MATERIALS AND METHODS: T24 cells were treated with different doses of Au-Ag@PDA NPs followed by 808 nm laser irradiation, and the effects on cell proliferation, cell cycle, apoptosis, and autophagy were analyzed. To confirm the mechanisms of inhibition, real-time PCR and Western blot analysis were used to evaluate markers of cell cycle, apoptosis, autophagy, and the AKT/ERK signaling pathway. Moreover, we evaluated the effects of the treatment on mitochondrial membrane potential and ROS generation to confirm the underlying mechanisms of inhibition. Finally, we tested the T24 tumor inhibitory effects of Au-Ag@PDA NPs plus laser irradiation in vivo using a xenograft mouse model. RESULTS: Au-Ag@PDA NPs, with appropriate laser irradiation, dramatically inhibited the proliferation of T24 cells, altered the cell cycle distribution by increasing the proportion of cells in the S phase, induced cell apoptosis by activating the mitochondria-mediated intrinsic pathway, and triggered a robust autophagy response in T24 cells. Moreover, Au-Ag@PDA NPs decreased the expression of phosphorylated AKT and ERK and promoted the production of ROS that function upstream of apoptosis and autophagy. In addition, Au-Ag@PDA NP-mediated photothermolysis also significantly suppressed tumor growth in vivo. CONCLUSION: This preclinical study can provide a mechanistic basis for Au-Ag@PDA NP-mediated photothermal therapy toward promotion of this method in the clinical treatment of bladder cancer.

LIM kinase 1 serves an important role in the multidrug resistance of osteosarcoma cells.

Multidrug resistance (MDR) is a major challenge for the management of the majority of cancers. The precise molecular mechanisms of MDR remain elusive. In a previous study, a multidrug resistant osteosarcoma model [MG63/vincristine (VCR)] was established by intermittent exposure of MG63 cells to gradually increasing concentrations of VCR. These cells exhibited cross-resistance to multiple structurally and mechanistically unrelated chemotherapeutic agents. The development of MDR was associated with increased expression of LIM kinase 1 (LIMK1). Compared with that in normal human fetal osteoblasts (hFOB) 1.19, the messenger RNA and protein expression of LIMK1 was significantly elevated both in MG63 and U2OS osteosarcoma cells. To observe the expression pattern of LIMK1 in osteosarcoma, immunohistochemical analyses were performed on specimens derived from 6 patients. The results indicated that LIMK1 was expressed to a greater extent in the tumor parenchyma than in the mesenchyme. The role of LIMK1 in MDR was confirmed by transfecting plasmids coding LIMK1-small interfering RNA (siRNA), wild-type-LIMK1 or empty vector into MG63/VCR cells, and measuring the expression of LIMK1 and multidrug resistance protein 1 (MDR1), also known as P-gycoprotein (P-gp). The results demonstrated that the level of MDR1/P-gp was positively correlated with the level of LIMK1. This correlation was also shown with the doxorubicin efflux assay and by measuring apoptosis. Specifically, after 6 h of incubation with VCR, 25.6% of the cells transfected with the LIMK1-siRNA plasmid were apoptotic compared with 6.2% in the empty vector group and 1.3% in the group of cells transfected with the wild-type-LIMK1 plasmid. Thus, it was concluded that LIMK1 serves a key role in the MDR of osteosarcoma and functions through MDR1.

Hypoxia regulates IL-17A secretion from nasal polyp epithelial cells.

Hypoxia creates a microenvironment conducive to polypogenesis by regulating immune responses of the nasal polyp (NP) epithelium. We explored the immunocompetence of NP and control epithelial cells in response to hypoxia, to investigate potential relationships with polypogenesis. Three groups of tissue samples were collected: inferior turbinate (IT)and NP from individuals with chronic rhinosinusitis with NPs (CRSwNP), and control IT. A positive relationship was detected between HIF1α, HIF2α protein expression in epithelial cells and endoscope score in NP samples, while there was a negative correlation between HIF1α expression and degree of eosinophil infiltration. Epithelial IL-17A expression was lower in NPs than in IT samples from either controls or patients with CRSwNP. Primary human nasal epithelial cells were cultured under hypoxic or normoxic conditions. Enzyme-linked immunosorbent assays demonstrated decreased IL-17A expression upon prolonged exposure to hypoxia in both IT and NP samples from patients with CRSwNP, while IL-17A increased in control IT epithelial cells; correlation and time-dependency were observed between HIF1α and IL-17A expression in both IT and NP samples from patients with CRSwNP. These observations suggest that hypoxia is involved in the pathogenesis of NPs through regulation of IL-17A secretion and HIF1α and HIF2α expression in the NP epithelium.

Characterization of multidrug­resistant osteosarcoma sublines and the molecular mechanisms of resistance.

Multidrug resistance (MDR) is a challenge for the treatment of cancer and the underlying molecular mechanisms remain elusive. The current study exposed MG63 osteosarcoma cells to increasing concentrations of vincristine (VCR) to establish four VCR­resistant MG63/VCR cell sublines (MG63/VCR1, 2, 3 and 4). The drug resistance indices (RI) of these sublines was detected with the CCK­8 assay and determined to be163, 476, 1,247, and 2,707­fold higher than that of parental cells, respectively. These sublines also exhibited cross­resistance to doxorubicin, paclitaxel and pirarubicin. With increased RI, the proliferative capacity of these sublines was gradually reduced and cell morphology was also altered, characterized by increased formation of pseudopodia and long cytoplasmic processes at opposite poles. However, the migration capacity and expression of certain drug resistance­associated genes were not in accordance with the increased RI; multidrug resistance protein 1 (MDR1) expression was significantly increased in these sublines compared with parental cells. However, in the highly resistant MG63/VCR3 and MG63/VCR4 cells, MDR­associated protein 1, topoisomerase II and LIM domain kinase 1 levels were significantly reduced compared with the moderately resistant MG63/VCR2 cells. Expression of glutathione S­transferase­π mRNA was determined using reverse transcription­quantitative polymerase chain reaction and determined that it was not changed between MG63 and MG63/VCR cells. The data of the present study demonstrated that the molecular alterations of drug resistance may change with the degree of drug resistance. Taking cell morphology into consideration, the intratumor clonal and phenotypic heterogeneity may be responsible for drug resistance. These MG63/VCR sublines may be a valuable tool to assess drug resistance and the underlying mechanisms, and to identify novel drug resistance­associated genes or strategies to overcome MDR in human osteosarcoma.