In Vivo Albumin Traps Photosensitizer Monomers from Self-Assembled Phthalocyanine Nanovesicles: A Facile and Switchable Theranostic Approach.

Albumin is a promising candidate as a biomarker for potential disease diagnostics and has been extensively used as a drug delivery carrier for decades. In these two directions, many albumin-detecting probes and exogenous albumin-based nanocomposite delivery systems have been developed. However, there are only a few cases demonstrating the specific interactions of exogenous probes with albumin in vivo, and nanocomposite delivery systems usually suffer from tedious fabrication processes and potential toxicity of the complexes. Herein, we demonstrate a facile "one-for-all" switchable nanotheranostic (NanoPcS) for both albumin detection and cancer treatment. In particular, the in vivo specific binding between albumin and PcS, arising from the disassembly of injected NanoPcS, is confirmed using an inducible transgenic mouse system. Fluorescence imaging and antitumor tests on different tumor models suggest that NanoPcS has superior tumor-targeting ability and the potential for time-modulated, activatable photodynamic therapy.

Expression of LRIG1, a Negative Regulator of EGFR, Is Dynamically Altered during Different Stages of Gastric Carcinogenesis.

Leucine-rich repeats and immunoglobulin-like domains (LRIG)-1 is a transmembrane protein that antagonizes epidermal growth factor receptor signaling in epithelial tissues. LRIG1 is down-regulated in various epithelial cancers, including bladder, breast, and colorectal cancer, suggesting that it functions as a tumor suppressor. However, its role in gastric carcinogenesis is not well understood. Here, we investigated the changes in LRIG1 expression during the stages of gastric cancer. We used a DMP-777-induced spasmolytic polypeptide-expressing metaplasia mouse model and a tissue array of human gastric cancer lesions. The effects of LRIG1 knockdown were also assessed using the human gastric cancer cell line SNU638 in a xenograft model. LRIG1 expression varied over the course of gastric carcinogenesis, increasing in spasmolytic polypeptide-expressing metaplasia lesions but disappearing in intestinal metaplasia and cancer lesions, and the increase was concurrent with the up-regulation of epidermal growth factor receptor. In addition, LRIG1 knockdown promoted the tumorigenic potential in vitro, which was manifested as increased proliferation, invasiveness, and migration as well as increased tumor size in vivo in the xenograft model. Furthermore, LRIG1 expression was determined to be a positive prognostic biomarker for the survival of gastric cancer patients. Collectively, our findings indicate that LRIG1 expression is closely related wto gastric carcinogenesis and may play a vital role as a tumor suppressor through the modulation of epidermal growth factor receptor activity.

The mRNA and Protein Levels of Tubulin and ß-Actin Are Greatly Reduced in the Proximal Duodenum of Mice Relative to the Rest of the Small Intestines.

To accurately quantify mRNA and protein levels, it is critical to choose appropriate internal standards. As the expression of housekeeping genes is assumed to remain constant, they are often employed to normalize signals to correct for sample-to-sample variations. However, recent studies have documented that ß-actin and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression levels change in response to various stimuli during proliferation, activation, and differentiation. We investigated levels of α-, ß-, γ-tubulin, ß-actin, and GAPDH vary across the gastrointestinal tract of mice. We found that different regions of the small intestines had dramatically different expression profiles, as measured by western blot, quantitative Reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemical staining. These results revealed that the expression levels of tubulins and ß-actin were dramatically lower in the proximal duodenum, relative to the rest of the small intestines. These varying levels of housekeeping genes may reflect differences in the activities of specialized tissues and suggest unique requirements for tubulins in these tissue types. We conclude that the use of a single housekeeping gene to normalize gene expression in the gastrointestinal tracts of mice may introduce errors, as measured differences in gene expression may reflect regulation of the internal control rather than the mRNA or protein under investigation.

Microscopic nodes and ducts inside lymphatics and on the surface of internal organs are rich in granulocytes and secretory granules.

The blood and lymphatic systems are the two well-established circulatory systems. The existence of a third circulatory system representing acupuncture meridians was claimed in the 1960s. The very existence and function of the system, however, remained uncertain. We have found that microscopic nodes and ducts inside lymphatics, as well as on the surface of internal organs of the rat. The nodes and ducts are covered by a layer of EMP-3-positive spindle-shaped epithelium with, below, a layer of vWF-positive but CD31-negative endothelium. The nodes contain a variety of immune cells, usually enriched with mast cells, eosinophils, neutrophils and histiocytes, as well as chromaffin cells, other granule-containing cells. Secretory granules originating from the mast cells in the nodes appear to pass along ductules, two or more of which make up a duct. Our results reveal a potential circulatory system whose anatomical structure and cellular content differ from the blood and lymph systems, and which may be involved in the transport of secretory granules.

The position of the target site for engineered nucleases improves the aberrant mRNA clearance in in vivo genome editing.

Engineered nucleases are widely used for creating frameshift or nonsense mutations in the target genes to eliminate gene functions. The resulting mRNAs carrying premature termination codons can be eliminated by nonsense-mediated mRNA decay. However, it is unclear how effective this process would be in vivo. Here, we found that the nonsense-mediated decay was unable to remove the mutant mRNAs in twelve out of sixteen homozygous mutant mice with frameshift mutations generated using engineered nucleases, which is far beyond what we expected. The frameshift mutant proteins translated by a single nucleotide deletion within the coding region were also detected in the p53 mutant mice. Furthermore, we showed that targeting the exons present downstream of the exons with a start codon or distant from ATG is relatively effective for eliminating mutant mRNAs in vivo, whereas the exons with a start codon are targeted to express the mutant mRNAs. Of the sixteen mutant mice generated, only four mutant mice targeting the downstream exons exhibited over 80% clearance of mutant mRNAs. Since the abnormal products, either mutant RNAs or mutant proteins, expressed by the target alleles might obscure the outcome of genome editing, these findings will provide insights in the improved performance of engineered nucleases when they are applied in vivo.

RAE1 mediated ZEB1 expression promotes epithelial-mesenchymal transition in breast cancer.

Breast cancer metastasis accounts for most of the deaths from breast cancer. Since epithelial-mesenchymal transition (EMT) plays an important role in promoting metastasis of cancer, many mechanisms regarding EMT have been studied. We previously showed that Ribonucleic acid export 1 (RAE1) is dysregulated in breast cancer and its overexpression leads to aggressive breast cancer phenotypes by inducing EMT. Here, we evaluated the functional capacity of RAE1 in breast cancer metastasis by using a three-dimensional (3D) culture system and xenograft models. Furthermore, to investigate the mechanisms of RAE1-driven EMT, in vitro studies were carried out. The induction of EMT with RAE1-overexpression was confirmed under the 3D culture system and in vivo system. Importantly, RAE1 mediates upregulation of an EMT marker ZEB1, by binding to the promoter region of ZEB1. Knockdown of ZEB1 in RAE1-overexpressing cells suppressed invasive and migratory behaviors, accompanied by an increase in epithelial and a decrease in mesenchymal markers. Taken together, these data demonstrate that RAE1 contributes to breast cancer metastasis by regulating a key EMT-inducing factor ZEB1 expression, suggesting its potential as a therapeutic target.

Sequential Protein-Responsive Nanophotosensitizer Complex for Enhancing Tumor-Specific Therapy.

A major challenge in cancer treatment is the development of effective tumor-specific therapeutic methods that have minimal side effects. Recently, a photodynamic therapy (PDT) technique using activatable photosensitizers (aPSs) has shown great potential for cancer-specific treatment. Here, we develop a sequential protein-responsive aPS (PcC4-MSN-O1) that is based on zinc(II) phthalocyanine derivative (PcC4)-entrapped mesoporous silica nanoparticles (MSNs) and a wrapping DNA (O1) as a biogate. Inside the nanostructure of PcC4-MSN-O1, PcC4 shows self-quenching photoactivity. However, when PcC4-MSN-O1 sequentially reacts with telomerase and albumin, its photoactivity is dramatically turned on. Therefore, PcC4-MSN-O1 displays selective phototoxicity against cancer cells ( e.g., HeLa) over normal cells ( e.g., HEK-293). Following systemic PcC4-MSN-O1 administration, there is an obvious accumulation in HeLa tumors of xenograft-bearing mice, and laser irradiation clearly induces the inhibition of tumor growth. Moreover, the time-modulated activation process in tumors and the relatively fast excretion of PcC4-MSN-O1 indicate its advantages in reducing potential side effects.

Facile Supramolecular Approach to Nucleic-Acid-Driven Activatable Nanotheranostics That Overcome Drawbacks of Photodynamic Therapy.

Supramolecular chemistry provides a "bottom-up" method to fabricate nanostructures for biomedical applications. Herein, we report a facile strategy to directly assemble a phthalocyanine photosensitizer (PcS) with an anticancer drug mitoxantrone (MA) to form uniform nanostructures (PcS-MA), which not only display nanoscale optical properties but also have the capability of undergoing nucleic-acid-responsive disassembly. These supramolecular assemblies possess activatable fluorescence emission and singlet oxygen generation associated with the formation of free PcS, mild photothermal heating, and a concomitant chemotherapeutic effect associated with the formation of free MA. In vivo evaluations indicate that PcS-MA nanostructures have a high level of accumulation in tumor tissues, are capable of being used for cancer imaging, and have significantly improved anticancer effect compared to that of PcS. This study demonstrates an attractive strategy for overcoming the limitations of photodynamic cancer therapy.

In vivo near-infrared imaging and phototherapy of tumors using a cathepsin B-activated fluorescent probe.

The development of multifunctional reagents for simultaneous specific near-infrared (NIR) imaging and phototherapy of tumors is of great significance. This work describes the design of a cathepsin B-activated fluorescent probe (CyA-P-CyB) and its applications as an NIR imaging probe for tumor cells and as a phototherapy reagent for tumors. In vitro experiments demonstrated that CyA-P-CyB was activated via the cleavage of a peptide linker by cathepsin B in tumor cells to produce fluorescence in the NIR region based on a FRET mechanism. MTT assays showed that the phototoxicity of CyA-P-CyB toward cells depended on the activity of cathepsin B, and the probe exhibited specific phototoxicity toward tumor cells. CyA-P-CyB was also successfully applied to the in vivo imaging and phototherapy of tumors. Histological analysis indicated that CyA-P-CyB had no cytotoxic effects on seven mouse tissues (lung, liver, heart, kidney, pancreas, spleen and brain) after the CyA-P-CyB treatment and laser irradiation.

Wnt-3a regulates chondrocyte differentiation via c-Jun/AP-1 pathway.

Our previous study indicated that interleukin (IL)-1beta induces expression of several Wnt proteins in chondrocytes and causes chondrocyte dedifferentiation via the c-Jun/activator protein-1 (AP-1) pathway. This study examined whether Wnt-3a causes chondrocyte dedifferentiation via the c-Jun/AP-1 pathway. Wnt-3a inhibited chondrogenesis of mesenchymal cells by stabilizing cell-cell adhesion in a manner independent of beta-catenin transcriptional activity. Wnt-3a also induced dedifferentiation of articular chondrocytes by stimulating the transcriptional activity of beta-catenin-T cell-factor/lymphoid-enhancer-factor (Tcf/Lef) complex. In chondrocytes, Wnt-3a caused the expression of c-Jun and its phosphorylation by c-Jun N-terminal kinase (JNK), resulting in activation of AP-1. AP-1 activation suppressed the expression of Sox-9, a major transcription factor regulating type II collagen expression. Collectively, our results suggest that Wnt-3a inhibits chondrogenesis by stabilizing cell-cell adhesion and that it causes dedifferentiation of chondrocytes by activating of beta-catenin-Tcf/Lef transcriptional complex and the c-Jun/AP-1 pathway.

Mouse models of gastric carcinogenesis.

Gastric cancer is one of the most common cancers in the world. Animal models have been used to elucidate the details of the molecular mechanisms of various cancers. However, most inbred strains of mice have resistance to gastric carcinogenesis. Helicobacter infection and carcinogen treatment have been used to establish mouse models that exhibit phenotypes similar to those of human gastric cancer. A large number of transgenic and knockout mouse models of gastric cancer have been developed using genetic engineering. A combination of carcinogens and gene manipulation has been applied to facilitate development of advanced gastric cancer; however, it is rare for mouse models of gastric cancer to show aggressive, metastatic phenotypes required for preclinical studies. Here, we review current mouse models of gastric carcinogenesis and provide our perspectives on future developments in this field.

Role of MyD88 in TLR agonist-induced functional alterations of human adipose tissue-derived mesenchymal stem cells.

Toll-like receptors (TLRs) sense microorganism components and are critical host mediators of inflammation during infection. Recently, TLRs have been reported to be involved in cell proliferation and differentiation. We previously reported that TLR agonists might affect proliferation and differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs). In this study, we sought to determine whether TLR signaling is dependent on MyD88 in hASCs. The hASCs were downregulated using LV-GFP-miR-MyD88, a lentiviral construct inserted siRNA against human MyD88 that significantly inhibited cell proliferation. MyD88 downregulation reduced NF-kappaB activation and enhancement of osteogenic differentiation induced by peptidoglycan (PGN) more significantly than that induced by lipopolysaccharide (LPS). Although LPS- and PGN-induced cytokine secretions were decreased greatly by MyD88 downregulation, IFN-gamma-induced protein-10 (IP10) and IFNbeta expression were enhanced by LPS irrespective of the downregulation of MyD88. These results suggest that TLR signaling is mediated via MyD88-independent pathways as well as MyD88-dependent pathways in hASCs and that MyD88 contributes to the regulation of cell proliferation and differentiation in hASCs.

c-Jun/activator protein-1 mediates interleukin-1beta-induced dedifferentiation but not cyclooxygenase-2 expression in articular chondrocytes.

Interleukin (IL)-1beta is a major catabolic pro-inflammatory cytokine involved in cartilage destruction-associated processes, such as loss of the differentiated chondrocyte phenotype (dedifferentiation) and inflammation. Here, we investigated the role of c-Jun and activator protein-1 (AP-1) in IL-1beta-induced dedifferentiation and cyclooxygenase (COX)-2 expression in primary cultured chondrocytes. IL-1beta induced expression and transient phosphorylation of c-Jun in primary cultured chondrocytes. Ectopic expression of c-Jun was sufficient to cause dedifferentiation, whereas expression of dominant negative c-Jun blocked IL-1beta-induced dedifferentiation. Interestingly, modulation of c-Jun expression did not affect IL-1beta-induced COX-2 expression. Further experiments revealed that c-Jun phosphorylation was mediated by c-Jun N-terminal kinase and was required for IL-1beta-induced dedifferentiation but not COX-2 expression. Consistent with its ability to induce phosphorylation of c-Jun, IL-1beta caused transient activation of AP-1, which is necessary for IL-1beta-induced dedifferentiation. IL-1beta treatment suppressed expression of Sox-9, a major transcription factor that regulates type II collagen expression. Inhibition of c-Jun N-terminal kinase or AP-1 reversed IL-1beta-induced suppression of Sox-9, and ectopic expression of c-Jun was sufficient to cause suppression of Sox-9. Our results collectively suggest that IL-1beta suppresses type II collagen expression in articular chondrocytes by inducing expression and phosphorylation of c-Jun, AP-1 activation, and subsequent suppression of Sox-9.

Regulation of beta-catenin signaling and maintenance of chondrocyte differentiation by ubiquitin-independent proteasomal degradation of alpha-catenin.

Accumulation of beta-catenin and subsequent stimulation of beta-catenin-T cell-factor (Tcf)/lymphoid-enhancerfactor (Lef) transcriptional activity causes dedifferentiation of articular chondrocytes, which is characterized by decreased type II collagen expression and initiation of type I collagen expression. This study examined the mechanisms of alpha-catenin degradation, the role of alpha-catenin in beta-catenin signaling, and the physiological significance of alpha-catenin regulation of beta-catenin signaling in articular chondrocytes. We found that both alpha- and beta-catenin accumulated during dedifferentiation of chondrocytes by escaping from proteasomal degradation. Beta-catenin degradation was ubiquitination-dependent, whereas alpha-catenin was proteasomally degraded in a ubiquitination-independent fashion. The accumulated alpha- and beta-catenin existed as complexes in the cytosol and nucleus. The complex formation between alpha- and beta-catenin blocked proteasomal degradation of alpha-catenin and also inhibited beta-catenin-Tcf/Lef transcriptional activity and the suppression of type II collagen expression associated with ectopic expression of beta-catenin, the inhibition of proteasome, or Wnt signaling. Collectively, our results indicate that ubiquitin-independent degradation of alpha-catenin regulates beta-catenin signaling and maintenance of the differentiated phenotype of articular chondrocytes.