Osteogenic Activities of Trifolirhizin as a Bioactive Compound for the Differentiation of Osteogenic Cells.

Plant extracts are widely used as traditional medicines. Sophora flavescens Aiton-derived natural compounds exert various beneficial effects, such as anti-inflammatory, anticancer, antioxidant, and antiregenerative activities, through their bioactive compounds, including flavonoids and alkaloids. In the present study, we investigated the biological effects of an S. flavescens-derived flavonoid, trifolirhizin (trifol), on the stimulation of osteogenic processes during osteoblast differentiation. Trifol (>98% purity) was successfully isolated from the root of S. flavescens and characterized. Trifol did not exhibit cellular toxicity in osteogenic cells, but promoted alkaline phosphatase (ALP) staining and activity, with enhanced expression of the osteoblast differentiation markers, including Alp, ColI, and Bsp. Trifol induced nuclear runt-related transcription factor 2 (RUNX2) expression during the differentiation of osteogenic cells, and concomitantly stimulated the major osteogenic signaling proteins, including GSK3ß, ß-catenin, and Smad1/5/8. Among the mitogen-activated protein kinases (MAPKs), Trifol activated JNK, but not ERK1/2 and p38. Trifol also increased the osteoblast-mediated bone-forming phenotypes, including transmigration, F-actin polymerization, and mineral apposition, during osteoblast differentiation. Overall, trifol exhibits bioactive activities related to osteogenic processes via differentiation, migration, and mineralization. Collectively, these results suggest that trifol may serve as an effective phytomedicine for bone diseases such as osteoporosis.

The OsERF115/AP2EREBP110 Transcription Factor Is Involved in the Multiple Stress Tolerance to Heat and Drought in Rice Plants.

The AP2/EREBP family transcription factors play important roles in a wide range of stress tolerance and hormone signaling. In this study, a heat-inducible rice ERF gene was isolated and functionally characterized. The OsERF115/AP2EREBP110 was categorized to Group-IIIc of the rice AP2/EREBP family and strongly induced by heat and drought treatment. The OsERF115/AP2EREBP110 protein targeted to nuclei and suppressed the ABA-induced transcriptional activation of Rab16A promoter in rice protoplasts. Overexpression of OsERF115/AP2EREBP110 enhanced thermotolerance of seeds and vegetative growth stage plants. The OsERF115/AP2EREBP110 overexpressing (OE) plants exhibited higher proline level and increased expression of a proline biosynthesis P5CS1 gene. Phenotyping of water use dynamics of the individual plant indicates that the OsERF115/AP2EREBP110-OE plant exhibited better water saving traits under heat and drought combined stress. Our combined results suggest the potential use of OsERF115/AP2EREBP110 as a candidate gene for genetic engineering approaches to develop heat and drought stress-tolerant crops.

The Rice Abscisic Acid-Responsive RING Finger E3 Ligase OsRF1 Targets OsPP2C09 for Degradation and Confers Drought and Salinity Tolerance in Rice.

Drought and salinity are major important factors that restrain growth and productivity of rice. In plants, many really interesting new gene (RING) finger proteins have been reported to enhance drought and salt tolerance. However, their mode of action and interacting substrates are largely unknown. Here, we identified a new small RING-H2 type E3 ligase OsRF1, which is involved in the ABA and stress responses of rice. OsRF1 transcripts were highly induced by ABA, salt, or drought treatment. Upregulation of OsRF1 in transgenic rice conferred drought and salt tolerance and increased endogenous ABA levels. Consistent with this, faster transcriptional activation of key ABA biosynthetic genes, ZEP, NCED3, and ABA4, was observed in OsRF1-OE plants compared with wild type in response to drought stress. Yeast two-hybrid assay, BiFC, and co-immunoprecipitation analysis identified clade A PP2C proteins as direct interacting partners with OsRF1. In vitro ubiquitination assay indicated that OsRF1 exhibited E3 ligase activity, and that it targeted OsPP2C09 protein for ubiquitination and degradation. Cell-free degradation assay further showed that the OsPP2C09 protein is more rapidly degraded by ABA in the OsRF1-OE rice than in the wild type. The combined results suggested that OsRF1 is a positive player of stress responses by modulating protein stability of clade A PP2C proteins, negative regulators of ABA signaling.

Hydroxychloroquine-loaded hollow mesoporous silica nanoparticles for enhanced autophagy inhibition and radiation therapy.

Radiotherapy (RT) is a major modality for cancer treatment, along with surgery and chemotherapy. Despite its therapeutic effect, the recurrence and metastasis of tumors due to the acquired resistance of cancer cells to RT remain significant clinical problems. Therefore, it is imperative to overcome radioresistance and improve radiosensitivity in cancer patients. Here, we synthesized hydroxychloroquine (HCQ)-loaded hollow mesoporous silica nanoparticles (HMSNs) to enable effective inhibition of radiation-induced cytoprotective autophagy and enhance the therapeutic efficacy of RT. HCQ-HMSN-treated HCT116 colon cancer cells showed a 200-fold higher intracellular uptake of HCQ than that of free HCQ-treated cells, thereby effectively inhibiting the radiation-induced autophagy of cancer cells. In vivo imaging and therapy studies of a tumor xenograft model showed preferential accumulation of HCQ-HMSNs in tumor tissues and significant enhancement of RT by inhibiting autophagy in the tumor sites. Histopathology analyses of major organs, blood chemistry profiles, and changes in body weights of mice confirmed the good biocompatibility of HCQ-HMSNs.

Fucoidan-Based Theranostic Nanogel for Enhancing Imaging and Photodynamic Therapy of Cancer.

In this study, a fucoidan-based theranostic nanogel (CFN-gel) consisting of a fucoidan backbone, redox-responsive cleavable linker and photosensitizer is developed to achieve activatable near-infrared fluorescence imaging of tumor sites and an enhanced photodynamic therapy (PDT) to induce the complete death of cancer cells. A CFN-gel has nanomolar affinity for P-selectin, which is overexpressed on the surface of tumor neovascular endothelial cells as well as many other cancer cells. Therefore, a CFN-gel can enhance tumor accumulation through P-selectin targeting and the enhanced permeation and retention effect. Moreover, a CFN-gel is non-fluorescent and non-phototoxic upon its systemic administration due to the aggregation-induced self-quenching in its fluorescence and singlet oxygen generation. After internalization into cancer cells and tumor neovascular endothelial cells, its photoactivity is recovered in response to the intracellular redox potential, thereby enabling selective near-infrared fluorescence imaging and an enhanced PDT of tumors. Since a CFN-gel also shows nanomolar affinity for the vascular endothelial growth factor, it also provides a significant anti-tumor effect in the absence of light treatment in vivo. Our study indicates that a fucoidan-based theranostic nanogel is a new theranostic material for imaging and treating cancer with high efficacy and specificity.

Targeted, Stimuli-Responsive, and Theranostic 19F Magnetic Resonance Imaging Probes.

Unlike conventional 1H magnetic resonance imaging (MRI), 19F MRI features unambiguous detection of fluorine spins due to negligible background signals. Therefore, it is considered a promising noninvasive and selective imaging method for the diagnosis of cancers and other diseases. For 19F MRI, fluorine-rich molecules such as perfluorocarbons (PFC) have been formulated into nanoemulsions and used as its tracer agent. Along with advancements in other types of nanoparticles as targeted theranostics and stimuli-triggered probes and combined with the advantages of 19F MRI, PFC nanoemulsions are being empowered with these additional functionalities and becoming a promising theranostic platform. In this Review, we provide an overview of fluorine-based materials for sensitive 19F MRI of biological and pathological conditions. In particular, we describe designs and applications of recently reported stimuli-responsive and theranostic 19F MRI probes. Finally, challenges and future perspectives regarding the further development of 19F MRI probes for their clinical applications are described.

Zwitterionic near-infrared fluorophore-conjugated epidermal growth factor for fast, real-time, and target-cell-specific cancer imaging.

Epidermal growth factor receptor (EGFR) is overexpressed in many types of cancers, which is associated with metastatic potential and poor prognosis in cancer patients. Therefore, development of EGFR-targeted sensitive imaging probes has been a challenge in tumor targeting, image-guided cancer surgery, patient-selective anti-EGFR therapy, and efficient targeted therapies. Methods: We synthesized a zwitterionic near-infrared fluorophore (ATTO655)-conjugated epidermal growth factor (EGF) as a novel activatable molecular probe. Fluorescence OFF/ON property and EGFR-targeting specificity of EGF-ATTO655 as well as its utility in real-time near-infrared (NIR) fluorescence imaging of EGFR-positive cancers were evaluated using in vitro and in vivo studies. Results: When conjugated to EGF, the fluorescence of ATTO655 quenched efficiently by photo-induced electron transfer (PET) mechanism between the conjugated dyes and nearby amino acid quenchers (tryptophan/tyrosine residues), which was stably maintained at physiological pH and in the presence of serum for at least 17 h. The fluorescence of EGF-ATTO655 turned on by receptor-mediated endocytosis and subsequent disintegration of EGF in EGFR-positive A431 cancer cells, thereby enabling specific and real-time fluorescence imaging of EGFR-positive cancer cells. Consequently, EGFR-positive tumors could be clearly visualized 3 h post-injection with a significantly high tumor-to-background ratio (TBR = 6.37). Conclusion: This PET mechanism-based OFF/ON type of EGF probe showed great potential for rapid, real-time, and target-cell-specific imaging of EGFR-overexpressing cancers in vitro and in vivo.

Design of Magnetically Labeled Cells (Mag-Cells) for in Vivo Control of Stem Cell Migration and Differentiation.

Cell-based therapies are attractive for treating various degenerative disorders and cancer but delivering functional cells to the region of interest in vivo remains difficult. The problem is exacerbated in dense biological matrices such as solid tissues because these environments impose significant steric hindrances for cell movement. Here, we show that neural stem cells transfected with zinc-doped ferrite magnetic nanoparticles (ZnMNPs) can be pulled by an external magnet to migrate to the desired location in the brain. These magnetically labeled cells (Mag-Cells) can migrate because ZnMNPs generate sufficiently strong mechanical forces to overcome steric hindrances in the brain tissues. Once at the site of lesion, Mag-Cells show enhanced neuronal differentiation and greater secretion of neurotrophic factors than unlabeled control stem cells. Our study shows that ZnMNPs activate zinc-mediated Wnt signaling to facilitate neuronal differentiation. When implemented in a rodent brain stroke model, Mag-Cells led to significant recovery of locomotor performance in the impaired limbs of the animals. Our findings provide a simple magnetic method for controlling migration of stem cells with high therapeutic functions, offering a valuable tool for other cell-based therapies.

Theranostic Nanosystems for Targeted Cancer Therapy.

Nanomaterials have revolutionized cancer imaging, diagnosis, and treatment. Multifunctional nanoparticles in particular have been designed for targeted cancer therapy by modulating their physicochemical properties to be delivered to the target and activated by internal and/or external stimuli. This review will focus on the fundamental "chemical" design considerations of stimuli-responsive nanosystems to achieve favorable tumor targeting beyond biological barriers and, furthermore, enhance targeted cancer therapy. In addition, we will summarize innovative smart nanosystems responsive to external stimuli (e.g., light, magnetic field, ultrasound, and electric field) and internal stimuli in the tumor microenvironment (e.g., pH, enzyme, redox potential, and oxidative stress).

Redox-Responsive Manganese Dioxide Nanoparticles for Enhanced MR Imaging and Radiotherapy of Lung Cancer.

In this study, we synthesized manganese dioxide nanoparticles (MnO2 NPs) stabilized with biocompatible polymers (polyvinylpyrrolidone and polyacrylic acid) and analyzed their effect on non-small cell lung cancer (NSCLC) cells with or without gefitinib resistance in vitro. MnO2 NPs showed glutathione (GSH)-responsive dissolution and subsequent enhancement in magnetic resonance (MR) imaging. Of note, treatment with MnO2 NPs induced significant cytotoxic effects on NSCLC cells, and additional dose-dependent therapeutic effects were obtained upon X-ray irradiation. Normal cells treated with MnO2 NPs were viable at the tested concentrations. In addition, increased therapeutic efficacy could be achieved when the cells were treated with MnO2 NPs in hypoxic conditions. Therefore, we conclude that the use of MnO2 NPs in MR imaging and combination radiotherapy may be an efficient strategy for the imaging and therapy of NSCLC.

Magnetic Tandem Apoptosis for Overcoming Multidrug-Resistant Cancer.

Multidrug resistance (MDR) is a leading cause of failure in current chemotherapy treatment and constitutes a formidable challenge in therapeutics. Here, we demonstrate that a nanoscale magnetic tandem apoptosis trigger (m-TAT), which consists of a magnetic nanoparticle and chemodrug (e.g., doxorubicin), can completely remove MDR cancer cells in both in vitro and in vivo systems. m-TAT simultaneously activates extrinsic and intrinsic apoptosis signals in a synergistic fashion and downregulates the drug efflux pump (e.g., P-glycoprotein) which is one of the main causes of MDR. The tandem apoptosis strategy uses low level of chemodrug (in the nanomolar (nM) range) to eliminate MDR cancer cells. We further demonstrate that apoptosis of MDR cancer cells can be achieved in a spatially selective manner with single-cell level precision. Our study indicates that nanoscale tandem activation of convergent signaling pathways is a new platform concept to overcome MDR with high efficacy and specificity.

A magnetic switch for the control of cell death signalling in in vitro and in vivo systems.

The regulation of cellular activities in a controlled manner is one of the most challenging issues in fields ranging from cell biology to biomedicine. Nanoparticles have the potential of becoming useful tools for controlling cell signalling pathways in a space and time selective fashion. Here, we have developed magnetic nanoparticles that turn on apoptosis cell signalling by using a magnetic field in a remote and non-invasive manner. The magnetic switch consists of zinc-doped iron oxide magnetic nanoparticles (Zn(0.4)Fe(2.6)O(4)), conjugated with a targeting antibody for death receptor 4 (DR4) of DLD-1 colon cancer cells. The magnetic switch, in its On mode when a magnetic field is applied to aggregate magnetic nanoparticle-bound DR4s, promotes apoptosis signalling pathways. We have also demonstrated that the magnetic switch is operable at the micrometre scale and that it can be applied in an in vivo system where apoptotic morphological changes of zebrafish are successfully induced.

Noninvasive remote-controlled release of drug molecules in vitro using magnetic actuation of mechanized nanoparticles.

Mesoporous silica nanoparticles are useful nanomaterials that have demonstrated the ability to contain and release cargos with mediation by gatekeepers. Magnetic nanocrystals have the ability to exhibit hyperthermic effects when placed in an oscillating magnetic field. In a system combining these two materials and a thermally sensitive gatekeeper, a unique drug delivery system can be produced. A novel material that incorporates zinc-doped iron oxide nanocrystals within a mesoporous silica framework that has been surface-modified with pseudorotaxanes is described. Upon application of an AC magnetic field, the nanocrystals generate local internal heating, causing the molecular machines to disassemble and allowing the cargos (drugs) to be released. When breast cancer cells (MDA-MB-231) were treated with doxorubicin-loaded particles and exposed to an AC field, cell death occurred. This material promises to be a noninvasive, externally controlled drug delivery system with cancer-killing properties.