The miR-23b/27b/24-1 Cluster Inhibits Hepatic Fibrosis by Inactivating Hepatic Stellate Cells.

BACKGROUND & AIMS: Hepatic fibrosis is characterized by hepatic stellate cell (HSC) activation and transdifferentiation-mediated extracellular matrix (ECM) deposition, which both contribute to cirrhosis. However, no antifibrotic regimen is available in the clinic. microRNA-23b/27b/24-1 cluster inhibition of transforming growth factor-ß (TGF-ß) signaling during hepatic development prompted us to explore whether this cluster inhibits HSC activation and hepatic fibrosis. METHODS: Experimental fibrosis was studied in carbon tetrachloride (CCl4)-treated C57BL/6 mice. After administration of miR-23b/27b/24-1 lentivirus or vehicle, animals were euthanized for liver histology. In primary rat HSC and HSC-T6, the anti-fibrotic effect of miR-23b/27b/24-1 cluster was furtherly investigated by RNA-sequencing, luciferase reporter assay, western blotting and bioinformatic means. RESULTS: In this study, we showed that increasing the miR-23b/27b/24-1 level through intravenous delivery of miR-23b/27b/24-1 lentivirus ameliorated mouse hepatic fibrosis. Mechanistically, the miR-23b/27b/24-1 cluster directly targeted messenger RNAs, which reduced the protein expression of 5 secretory profibrotic genes (TGF-ß2, Gremlin1, LOX, Itgα2, and Itgα5) in HSCs. Suppression of the TGF-ß signaling pathway by down-regulation of TGF-ß2, Itgα2, and Itgα5, and activation of the bone morphogenetic protein signaling pathway by inhibition of Gremlin1, decreased extracellular matrix secretion of HSCs. Furthermore, down-regulation of LOX expression softened the ECM. Moreover, a reduction in tissue inhibitors of metalloproteinase 1 expression owing to weakened TGF-ß signaling increased ECM degradation. CONCLUSIONS: Hepatic overexpression of the miR-23b/27b/24-1 cluster blocked hepatic fibrosis and may be a novel therapeutic regimen for patients with hepatic fibrosis.

Class C1 decoy oligodeoxynucleotide inhibits profibrotic genes expression in rat hepatic stellate cells.

The aim of the present study was to investigate whether class C1 decoy oligodeoxynucleotides (ODNs) can inhibit the expression of pro­fibrotic genes associated with rat hepatic stellate cell (HSC) activation and hepatic fibrosis. Luciferase reporter assays were performed to test the promoter activities of transforming growth factor (TGF)­ß and its downstream target genes following transfection of decoy ODNs and plasmids into HSC­T6 cells, and western blot assays were performed to measure the protein expression of those genes following decoy ODN transfection. Class C1 decoy ODNs were confirmed to inhibit the promoter activity of TGF­ß and its downstream target genes, such as type 1 collagen (COLI)α1, tissue inhibitor of metalloproteinases (TIMP)1 and α­smooth muscle actin by Gaussia luciferase reporter assay, and to further downregulate the expression of TGF­ß, SMAD3, COLIα1 and TIMP1 by western blotting in activated HSC­T6 cells. In conclusion, class C1 decoy ODNs inhibited pro­fibrotic gene expression in rat HSCS by downregulating TGF­ß signaling.

Mitochondrial-targeted penetrating peptide delivery for cancer therapy.

INTRODUCTION: Mitochondria are promising targeting organelles for anticancer strategies; however, mitochondria are difficult for antineoplastic drugs to recognize and bind. Mitochondria-penetrating peptides (MPPs) are unique tools to gain access to the cell interior and deliver a bioactive cargo into mitochondria. MPPs have combined or delivered a variety of antitumor cargoes and obviously inhibited the tumor growth in vivo and in vitro. MPPs create new opportunities to develop new treatments for cancer. AREAS COVERED: We review the target sites of mitochondria and the target-penetration mechanism of MPPs, different strategies, and various additional strategies decorated MPPs for tumor cell mitochondria targeting, the decorating mattes including metabolism molecules, RNA, DNA, and protein, which exploited considered as therapeutic combined with MPPs and target in human cancer treatment. EXPERT OPINION/COMMENTARY: Therapeutic selectivity that preferentially targets the mitochondrial abnormalities in cancer cells without toxic impact on normal cells still need to be deepen. Moreover, it needs appropriate study designs for a correct evaluation of the target delivery outcome and the degradation rate of the drug in the cell. Generally, it is optimistic that the advances in mitochondrial targeting drug delivery by MPPs plasticity outlined here will ultimately help to the discovery of new approaches for the prevention and treatment of cancers.

Calreticulin is an effective immunologic adjuvant to tumor-associated antigens.

As a key molecule involved in cell recognition, calreticulin (CRT) may be expressed on the surface of (pre-) apoptotic cells and provide the signal that is recognized by dendritic cells (DCs) or other antigen presenting cells (APCs), which results in phagocytosis. Within the APCs, tumor-associated antigens (TAAs) may be subsequently presented to T lymphocytes, which triggers a specific antitumor immune response. It has been hypothesized that CRT is able to act as the immunologic adjuvant and translocate itself and TAAs to the cell surface and induce a specific antitumor immune response. In the present study, CRT was demonstrated to translocate itself and mucin 1 (MUC1), a breast cancer antigen, to the surface of 4T1 cells and the MUC1-CRT-coated cells were able to induce apoptosis in a time-dependent manner. When DCs were infected with adenovirus containing MUC1-CRT, an increase in T cell proliferation and cytokine production was exhibited. These results suggest that CRT may act as an immunologic adjuvant with MUC1 and induce a strong immune response.

Novel peptide MT23 for potent penetrating and selective targeting in mouse melanoma cancer cells.

Cell-penetrating peptides (CPPs) have a great potential for intracellular delivery of cell-impermeable biological macromolecules in clinical therapy. However, their lack of cell and tissue specificity remains the primary limitation for their clinical development as drug delivery vehicles. In this study, based on phage display and an in silico approach, we found a novel CPP-MT23 with mouse melanoma cell specificity, it can only enter B16 melanoma cancer cells and without any cytotoxicity, Moreover, MT23 showed higher penetration efficiency based on fluorescence microcopy and quantitative assay, and it has capability for mediating functional Apoptin into cells in vitro or in vivo. Moreover, MT23-Apoptin can significantly inhibit tumor growth and induce the cell apoptosis in B16 tumor bearing mice. To sum up, all the results implicated that MT23 has the potential to deliver exogenous therapeutic proteins for further use and it also expected to lay the foundation for developing human melanoma cancer cell specific CPP.

Gremlin1 Accelerates Hepatic Stellate Cell Activation Through Upregulation of TGF-Beta Expression.

Gremlin1, the antagonist of bone morphogenetic protein-7 and one of the target genes of transforming growth factor (TGF)-ß signal pathway, plays an important role in embryonic development and its expression decreases along with aging. To explore the expression of gremlin1 in liver fibrosis and the causal link between gremlin1 and hepatic stellate cell (HSC) activation, we detected the expression of gremlin1 in mice with hepatic fibrosis induced by porcine serum using real time quantitative PCR (RT-qPCR) and immunohistochemical staining. The hepatic fibrosis mice were evaluated by the external feature of the liver, histology, hepatic function, collagen deposition, and the expression of fibrosis-related genes (genes COLIα2 and COLIVα2) in the liver. In the HSC-T6, western blotting was used to analyze the expression of α-smooth muscle actin (α-SMA), COL1α, and TGF-ß1 in conditions of overexpression of gremlin1 or gremlin1 being knocked down by specific siRNA, respectively. The results showed that the mRNA expression of the gremlin1 gene was significantly increased consistent with increased expression of COLIα2 and COLIVα2 in the liver tissue of the hepatic fibrosis mice. Increased expression of gremlin1 coincided with the same area of the collagen deposition. Furthermore, the results also showed that the expression of α-SMA, COLIα1, and TGF-ß1 was consistent with the expression of gremlin1 not only in the HSC-T6 overexpressing gremlin1 but also in the HSC-T6 that gremlin1 is knocked down by specific siRNA. The findings suggest that gremlin1 might play an important role in the progression of hepatic fibrosis and that it modulates HSC activation.

[Glycogen synthase kinase3 and prostate cancer: An update].

Glycogen synthase kinase3 (GSK3α and GSK3ß) are serine/threonine protein kinases acting on numerous substrates and involved in the regulation of various cellular functions such as their proliferation, survival, glycogen metabolism, and autophagy. Accumulating evidence indicates that the expression of GSK3α is increased mainly in androgendependent while that of GSK3ß in androgenindependent prostate cancer, and that GSK3ß is also involved in the regulation of the transactivation of the androgen receptor (AR) and growth of prostate cancer. Animal experiments have proved that some GSK3 inhibitors, such as lithium, can significantly suppress tumor growth in different animal models of prostate cancer. The GSK3 inhibitor is promising to be an important agent for the clinical management of prostate cancer.

Suppression of hepatic stellate cell activation through downregulation of gremlin1 expression by the miR-23b/27b cluster.

The imbalance between transforming growth factor ß and bone morphogenetic protein 7 signaling pathways is a critical step in promoting hepatic stellate cell activation during hepatic fibrogenesis. Gremlin1 may impair the balance. Something remains unclear about the regulatory mechanisms of gremlin1 action on hepatic stellate cell activation and hepatic fibrosis. In the current study, gremlin1 overexpression promotes activation of hepatic stellate cells. Knockdown of gremlin1 with siRNAs suppresses hepatic stellate cell activation and attenuates hepatic fibrosis in rat model. Our results also show that miR-23b/27b cluster members bind to 3'-untranslated region of gremlin1 resulting in reduction of transforming growth factor ß, α-smooth muscle actin and collagenI α1/2 gene expression. Our findings suggest that gremlin1 promotes hepatic stellate cell activation and hepatic fibrogenesis through impairment of the balance between transforming growth factor ß and bone morphogenetic protein 7 signaling pathways. The miR-23b/27b cluster suppresses activation of hepatic stellate cells through binding gremlin1 to rectify the imbalance.

Liganded Vitamin D Receptor Through Its Interacting Repressor Inhibits the Expression of Type I Collagen α1.

Hepatic fibrosis is a reversible process involving plenty of transcription factors and pathways. Vitamin D receptor (VDR) as a member of ligand-induced transcription factors can interact with 9-cis retinoid X receptor (RXR) and VDR-interacting repressor (VDIR) to mediate transactivation or transrepression in the absence or in the presence of VDR ligand to regulate the expression of VDR target genes. The active form of vitamin D [1α,25(OH)2D3] can downregulate the expression of type I collagen both α1 and α2 (COLIα1 and COLIα2) in hepatic stellate cells (HSC-T6) in a time-dependent fashion, which provides a new direction for hepatic fibrosis therapy. As one of VDR target genes, rat COLIα1 gene contains 1αnVDRE (E-box1 and E-box2) in its promoter, and unliganded VDR/RXR may bind to 1αnVDRE through VDIR to mediate transactivation, whereas liganded VDR/RXR may bind to 1αnVDRE through VDIR for transrepression. The results suggested a sort of relying on each other relationship between VDR/RXR and VDIR in regulating the expression of COLIα1 gene in HSC-T6 cells, which established VDR as a potential target for blocking and even reversing hepatic fibrosis.

SP1 and UTE1 Decoy ODNs inhibit activation and proliferation of hepatic stellate cells by targeting tissue inhibitors of metalloproteinase 1.

BACKGROUND: The excessive accumulation of extracellular matrix of hepatic fibrosis is positively correlated with tissue inhibitors of metalloproteinase 1 (TIMP1). Here we aimed to investigate whether TIMP1 may be down-regulated by Decoy ODNs strategy to capture transcriptional factor upstream TIMP1 element 1 (UTE1) and specificity protein 1(SP1). RESULTS: By luciferase reporter assays, we confirmed that these Decoy ODNs could influence the promoter activation of TIMP-1, α-SMA and Collagen Iα2 (COLΙα2) genes as well as the enhancer activation of TRE in HSC-T6 cells, and the combination tended to be more effective than SP1 or UTE1 Decoy ODN alone. Western blot analysis also demonstrated down-regulation of the expression of those target genes except for TGF-ß. Furthermore, we observed that the viability of HSC-T6 cells at 72 h was significantly in decline in combination group. CONCLUSION: The combination of SP1 and UTE1 Decoy ODNs treatments inhibit the activation and proliferation of HSCs more effectively than one of the Decoy ODNs through co-regulation of TIMP1 and TGF-ß signal pathway but not the expression of TGF-ß itself.

Efficient therapeutic delivery by a novel cell-permeant peptide derived from KDM4A protein for antitumor and antifibrosis.

Cell-penetrating peptide (CPP) based delivery have provided immense potential for the therapeutic applications, however, most of nonhuman originated CPPs carry the risk of possible cytotoxicity and immunogenicity, thus may restricting to be used. Here, we describe a novel human-derived CPP, denoted hPP10, and hPP10 has cell-penetrating properties evaluated by CellPPD web server, as well as In-Vitro and In-Vivo analysis. In vitro studies showed that hPP10-FITC was able to penetrate into various cells including primary cultured cells, likely through an endocytosis pathway. And functionalized macromolecules, such as green fluorescent protein (GFP), tumor-specific apoptosis inducer Apoptin as well as biological active enzyme GCLC (Glutamate-cysteine ligase, catalytic subunit) can be delivered by hPP10 in vitro and in vivo. Collectively, our results suggest that hPP10 provide a novel and versatile tool to deliver exogenous proteins or drugs for clinical applications as well as reprogrammed cell-based therapy.

Enhancing DPYSL3 gene expression via a promoter-targeted small activating RNA approach suppresses cancer cell motility and metastasis.

To explore a novel strategy in suppressing tumor metastasis, we took the advantage of a recent RNA activation (RNAa) theory and used small double-strand RNA molecules, termed as small activating RNAs (saRNA) that are complimentary to target gene promoter, to enhance transcription of metastasis suppressor gene. The target gene in this study is Dihydro-pyrimidinase-like 3 (DPYSL3, protein name CRMP4), which was identified as a metastatic suppressor in prostate cancers. There are two transcriptional variants of DPYSL3 gene in human genome, of which the variant 2 is the dominant transcript (DPYSL3v2, CRMP4a) but is also significantly down-regulated in primary prostate cancers. A total of 8 saRNAs for DPYSL3v1 and 14 saRNAs for DPYSL3v2 were tested in multiple prostate cancer cell lines. While none of the saRNAs significantly altered DPYSL3v1 expression, 4 saRNAs showed a strong enhancing effect on DPYSL3v2 expression, resulting in reduced cell mobility in vitro. To achieve a prostate cancer-specific delivery for in vivo testing, we conjugated the most potent saV2-9 RNA molecule with the prostate-specific membrane antigen (PSMA)-targeting aptamer A10-3.2. The conjugates successful increased DPYSL3v2 gene expression in PSMA-positive but not PSMA-negative prostate cancer cells. In nude mice bearing orthotopic xenograft of prostate cancer, a 10-day consecutive treatment with the saV2-9 conjugates significantly suppress distal metastasis compared to the control saRNAs. Analysis of xenograft tissues revealed that DPYSL3v2 expression was largely increased in saV2-9 conjugate-treated group compared to the control group. In conclusion, DPYSL3v2 promoter-targeted saRNA molecules might be used as an adjunctive therapy to suppress prostate cancer metastasis.

Relationship between Structure and Conformational Change of the Vitamin D Receptor Ligand Binding Domain in 1α,25-Dihydroxyvitamin D3 Signaling.

Vitamin D Receptor (VDR) belongs to the nuclear receptor (NR) superfamily. Whereas the structure of the ligand binding domain (LBD) of VDR has been determined in great detail, the role of its amino acid residues in stabilizing the structure and ligand triggering conformational change is still under debate. There are 13 α-helices and one ß-sheet in the VDR LBD and they form a three-layer sandwich structure stabilized by 10 residues. Thirty-six amino acid residues line the ligand binding pocket (LBP) and six of these residues have hydrogen-bonds linking with the ligand. In 1α,25-dihydroxyvitamin D3 signaling, H3 and H12 play an important role in the course of conformational change resulting in the provision of interfaces for dimerization, coactivator (CoA), corepressor (CoR), and hTAFII 28. In this paper we provide a detailed description of the amino acid residues stabilizing the structure and taking part in conformational change of VDR LBD according to functional domains.

Relationship of structure and function of DNA-binding domain in vitamin D receptor.

While the structure of the DNA-binding domain (DBD) of the vitamin D receptor (VDR) has been determined in great detail, the roles of its domains and how to bind the motif of its target genes are still under debate. The VDR DBD consists of two zinc finger modules and a C-terminal extension (CTE), at the end of the C-terminal of each structure presenting α-helix. For the first zinc finger structure, N37 and S-box take part in forming a dimer with 9-cis retinoid X receptor (RXR), while V26, R50, P-box and S-box participate in binding with VDR response elements (VDRE). For the second zinc finger structure, P61, F62 and H75 are essential in the structure of the VDR homodimer with the residues N37, E92 and F93 of the downstream of partner VDR, which form the inter-DBD interface. T-box of the CTE, especially the F93 and I94, plays a critical role in heterodimerization and heterodimers-VDRE binding. Six essential residues (R102, K103, M106, I107, K109, and R110) of the CTE α-helix of VDR construct one interaction face, which packs against the DBD core of the adjacent symmetry mate. In 1,25(OH)2D3-activated signaling, the VDR-RXR heterodimer may bind to DR3-type VDRE and ER9-type VDREs of its target gene directly resulting in transactivation and also bind to DR3-liked nVDRE of its target gene directly resulting in transrepression. Except for this, 1α,25(OH)2D3 ligand VDR-RXR may bind to 1αnVDRE indirectly through VDIR, resulting in transrepression of the target gene. Upon binding of 1α,25(OH)2D3, VDR can transactivate and transrepress its target genes depending on the DNA motif that DBD binds.

Enhanced Peptide delivery into cells by using the synergistic effects of a cell-penetrating Peptide and a chemical drug to alter cell permeability.

Cell-penetrating peptides (CPPs) are short, often hydrophilic peptides that can deliver many kinds of molecules into cells and that are likely to serve as a useful tool of future biotherapeutics. However, CPPs application is limited because of insufficient transduction efficiency and unpredictable cellular localization. Here, we investigated the enhancement of 1,2-benzisothiazolin-3-one (BIT) on the uptake of a synthetic fluorescent TAT and a TAT-conjugated green fluorescent protein (GFP) or pro-apoptotic peptide KLA and evaluated its toxicity in various cell lines. Our results showed that BIT pretreatment can enhance the penetration efficiency of TAT and its fusion peptide. In addition, the fluorescence of the peptide conjugate at effective doses was well-distributed in the intracellular of different cell lines without membrane perforation or detectable cytotoxicity. The internalization of the peptides was serum-dependent and temperature-independent. These findings imply that BIT may serve as a newly found delivery enhancer that is suitable for improving the penetration of CPPs.

Hes1, an important gene for activation of hepatic stellate cells, is regulated by Notch1 and TGF-ß/BMP signaling.

AIM: To determine the role of Notch1 and Hes1 in regulating the activation of hepatic stellate cells (HSCs) and whether Hes1 is regulated by transforming growth factor (TGF)/bone morphogenetic protein (BMP) signaling. METHODS: Immunofluorescence staining was used to detect the expression of desmin, glial fibrillary acidic protein and the myofibroblastic marker α-smooth muscle actin (α-SMA) after freshly isolated, normal rat HSCs had been activated in culture for different numbers of days (0, 1, 3, 7 and 10 d). The expression of α-SMA, collagen1α2 (COL1α2), Notch receptors (Notch1-4), and the Notch target genes Hes1 and Hey1 were analyzed by reverse transcriptase-polymerase chain reaction. Luciferase reporter assays and Western blot were used to study the regulation of α-SMA, COL1α1, COL1α2 and Hes1 by NICD1, Hes1, CA-ALK3, and CA-ALK5 in HSC-T6 cells. Moreover, the effects of inhibiting Hes1 function in HSC-T6 cells using a Hes1 decoy were also investigated. RESULTS: The expression of Notch1 and Hes1 mRNAs was significantly down-regulated during the culture of freshly isolated HSCs. In HSC-T6 cells, Notch1 inhibited the promoter activities of α-SMA, COL1α1 and COL1α2. On the other hand, Hes1 enhanced the promoter activities of α-SMA and COL1α2, and this effect could be blocked by inhibiting Hes1 function with a Hes1 decoy. Furthermore, co-transfection of pcDNA3-CA-ALK3 (BMP signaling activin receptor-like kinase 3) and pcDNA3.1-NICD1 further increased the expression of Hes1 compared with transfection of either vector alone in HSC-T6 cells, while pcDNA3-CA-ALK5 (TGF-ß signaling activin receptor-like kinase 5) reduced the effect of NICD1 on Hes1 expression. CONCLUSION: Selective interruption of Hes1 or maintenance of Hes1 at a reasonable level decreases the promoter activities of α-SMA and COL1α2, and these conditions may provide an anti-fibrotic strategy against hepatic fibrosis.

Non-viral methods for generating integration-free, induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells were created from mouse fibroblasts by induced expression of Yamanaka factors, Oct3/4, Sox2, Klf4, and c-Myc. This technique has quickly resulted in an exponential increase in the amount of pluripotency studies, and has provided a valuable tool in regenerative medicine. At the same time, many methodologies to generate iPS cells have been reported, and are comprised mainly of viral methods and non-viral methods. Although viral methods may not be applicable for clinical applications, various nonviral methods have been reported in recent years, including DNA vector-based approaches, transfection of mRNA, transduction of reprogramming proteins, and use of small molecule compounds. This review summarizes and evaluates these non-viral methods.

Cell-penetrating Peptide-mediated therapeutic molecule delivery into the central nervous system.

The blood-brain barrier (BBB), a dynamic and complex barrier formed by endothelial cells, can impede the entry of unwanted substances - pathogens and therapeutic molecules alike - into the central nervous system (CNS) from the blood circulation. Taking into account the fact that CNS-related diseases are the largest and fastest growing unmet medical concern, many potential protein- and nucleic acid-based medicines have been developed for therapeutic purposes. However, due to their poor ability to cross the BBB and the plasma membrane, the above-mentioned bio-macromolecules have limited use in treating neurological diseases. Finding effective, safe, and convenient ways to deliver therapeutic molecules into the CNS is thus urgently required. In recent decades, much effort has been expended in the development of drug delivery technologies, of which cell-penetrating peptides (CPPs) have the most promising potential. The present review covers the latest advances in CPP delivery technology, and provides an update on their use in CNS-targeted drug delivery.

Generation of transgene-free induced pluripotent stem cells with non-viral methods.

Induced pluripotent stem (iPS) cells were originally generated from mouse fibroblasts by enforced expression of Yamanaka factors (Oct3/4, Sox2, Klf4, and c-Myc). The technique was quickly reproduced with human fibroblasts or mesenchymal stem cells. Although having been showed therapeutic potential in animal models of sickle cell anemia and Parkinson's disease, iPS cells generated by viral methods do not suit all the clinical applications. Various non-viral methods have appeared in recent years for application of iPS cells in cell transplantation therapy. These methods mainly include DNA vector-based approaches, transfection of mRNA, and transduction of reprogramming proteins. This review summarized these non-viral methods and compare the advantages, disadvantages, efficiency, and safety of these methods.

Enhancement of TAT cell membrane penetration efficiency by dimethyl sulphoxide.

Cell penetrating peptides (CPPs) are promising tools for transducing presynthesized therapeutic molecules which possess low membrane permeability. The poor efficiency of cellular uptake and unexpected cellular localization are still the main obstacles to the development of drug delivery by using CPPs. In this study, we investigated the effect of a penetration enhancer, dimethylsulfoxide (DMSO), on the penetrating efficiency of a synthetic TAT peptide or the TAT fusion protein. FITC-labeled TAT and TAT-GFP were added to 10% DMSO or 100 microM chloroquine pretreated cells, fluorescence uptake into culturing cells was observed using fluorescence microscopy, FACS or quantitatively analyzed by a fluorescence spectrum. 10% DMSO treatment markedly increased internalization of TAT into cells and appeared in a well-distributed pattern throughout the cytosol and nucleus without membrane perforating or detectable cytotoxicity, the enhancement effect by 10% DMSO was reduced by endocytosis inhibitors including ammonium chloride and sodium azide. 10% DMSO also enhanced TAT-Apoptin induced apoptosis of carcinoma cells. These findings implicated that DMSO can be a novel delivery enhancer appropriate for CPP penetration.