Galactosylated hydroxyl-polyamidoamine dendrimer targets hepatocytes and improves therapeutic outcomes in a severe model of acetaminophen poisoning-induced liver failure.

Toxicity to hepatocytes caused by various insults including drugs is a common cause of chronic liver failure requiring transplantation. Targeting therapeutics specifically to hepatocytes is often a challenge since they are relatively nonendocytosing unlike the highly phagocytic Kupffer cells in the liver. Approaches that enable targeted intracellular delivery of therapeutics to hepatocytes have significant promise in addressing liver disorders. We synthesized a galactose-conjugated hydroxyl polyamidoamine dendrimer (D4-Gal) that targets hepatocytes efficiently through the asialoglycoprotein receptors in healthy mice and in a mouse model of acetaminophen (APAP)-induced liver failure. D4-Gal localized specifically in hepatocytes and showed significantly better targeting when compared with the non-Gal functionalized hydroxyl dendrimer. The therapeutic potential of D4-Gal conjugated to N-acetyl cysteine (NAC) was tested in a mouse model of APAP-induced liver failure. A single intravenous dose of a conjugate of D4-Gal and NAC (Gal-d-NAC) improved survival in APAP mice, decreased cellular oxidative injury and areas of necrosis in the liver, even when administered at the delayed time point of 8 h after APAP exposure. Overdose of APAP is the most common cause of acute hepatic injury and liver transplant need in the United States, and is treated with large doses of NAC administered rapidly within 8 h of overdose leading to systemic side effects and poor tolerance. NAC is not effective when treatment is delayed. Our results suggest that D4-Gal is effective in targeting and delivering therapies to hepatocytes and Gal-D-NAC has the potential to salvage and treat liver injury with a broader therapeutic window.

Rationally Designed Galactose Dendrimer for Hepatocyte-Specific Targeting and Intracellular Drug Delivery for the Treatment of Liver Disorders.

Over two million people die of liver disorders every year globally. Hepatocytes are the key cells affected in several acute and chronic liver diseases. The current clinical outcomes of liver-targeted nanoparticles are limited, necessitating the need to develop smart hepatocyte-targeted drug delivery systems. Here, we present the rational design and development of a hepatocyte-targeting glycodendrimer (GAL-24) built from biocompatible building blocks, using expedite and facile chemical methodology. GAL-24 is designed to inherently target asialoglycoprotein receptor 1 (ASGP-R) on hepatocytes and shows significant accumulation in the liver (20% of injected dose), just 1 h after systemic administration. This is highly specific to hepatocytes, with over 80% of hepatocytes showing GAL-24-Cy5 signal at 24 h. GAL-24-Cy5 maintains hepatocyte-targeting capabilities in both a mouse model of severe acetaminophen poisoning-induced hepatic necrosis and a rat model of nonalcoholic steatohepatitis (NASH). This GAL-24 nanoplatform holds great promise for improved drug delivery to hepatocytes to combat many liver disorders.

α-Actinin-4 Promotes the Progression of Prostate Cancer Through the Akt/GSK-3ß/ß-Catenin Signaling Pathway.

The first-line treatment for prostate cancer (PCa) is androgen ablation therapy. However, prostate tumors generally recur and progress to androgen-independent PCa (AIPC) within 2-3 years. α-Actinin-4 (ACTN4) is an actin-binding protein that belongs to the spectrin gene superfamily and acts as an oncogene in various cancer types. Although ACTN4 is involved in tumorigenesis and the epithelial-mesenchymal transition of cervical cancer, the role of ACTN4 in PCa remains unknown. We found that the ACTN4 expression level increased during the transition from androgen-dependent PCa to AIPC. ACTN4 overexpression resulted in enhanced proliferation and motility of PCa cells. Increased ß-catenin due to ACTN4 promoted the transcription of genes involved in proliferation and metastasis such as CCND1 and ZEB1. ACTN4-overexpressing androgen-sensitive PCa cells were able to grow in charcoal-stripped media. In contrast, ACTN4 knockdown using si-ACTN4 and ACTN4 nanobody suppressed the proliferation, migration, and invasion of AIPC cells. Results of the xenograft experiment revealed that the mice injected with LNCaPACTN4 cells exhibited an increase in tumor mass compared with those injected with LNCaPMock cells. These results indicate that ACTN4 is involved in AIPC transition and promotes the progression of PCa.

Tyrosine kinase inhibitor neratinib attenuates liver fibrosis by targeting activated hepatic stellate cells.

Liver fibrosis, a common outcome of chronic liver disease characterized by excessive accumulation of extracellular matrix (ECM), is a leading cause of mortality worldwide. The tyrosine kinase inhibitor neratinib is a human epidermal growth factor receptor 2 (HER2) inhibitor approved by the FDA for HER2-positive breast cancer treatment; however, it has not yet been evaluated for liver fibrosis treatment. We elucidated the anti-fibrotic effects of neratinib in hepatic stellate cells (HSCs) and in vivo models of CCl4-induced liver fibrosis. HSC activation is a key step in liver fibrogenesis and has a crucial role in collagen deposition, as it is primarily responsible for excessive ECM production. The effect of neratinib on HSC was evaluated in transforming growth factor (TGF-ß)-incubated LX-2 cells and culture-activated primary human HSCs. In vivo study results indicated that neratinib inhibited the inflammatory response, HSC differentiation, and collagen accumulation induced by CCl4. Moreover, the anti-fibrotic effects of neratinib were not associated with the HER2 signaling pathways. Neratinib inhibited FGF2 expression in activated HSCs and serum FGF2 level in the model, suggesting that neratinib possessed therapeutic potency against liver fibrosis and the potential for application against other fibrotic diseases.

α-Actinin-4 regulates cancer stem cell properties and chemoresistance in cervical cancer.

Cancer stem cells (CSCs) initiate tumors and possess the properties of self-renewal and differentiation. Since they are responsible for chemoresistance, CSCs are known to be a key factor in cancer recurrence. α-Actinin-4 (ACTN4) is an actin-binding protein that is involved in muscle differentiation and cancer metastasis. It promotes epithelial to mesenchymal transition and cell cycle progression via ß-catenin stabilization in cervical cancer. In the present study, we investigated the role of ACTN4 in regulating cancer cell stemness and chemoresistance in cervical cancer. Results from the gene expression database analysis showed that ACTN4 mRNA expression was elevated in cancerous cervices when compared with normal cervices. Furthermore, ACTN4 knockdown suppressed sphere formation and CSC proliferation. It also decreased CSC size and CD44high/CD24low cell population. ACTN4-knockdown CSCs were sensitive to anticancer drugs, which was observed by down-regulation of the ATP-binding cassette family G2 involved in drug resistance. Finally, ACTN4-knockdown CSCs formed reduced tumors in vivo when compared with control CSCs. Overall, these findings suggest that ACTN4 regulates CSC properties and contributes to chemoresistance in cervical cancer.

Targeting of dermal myofibroblasts through death receptor 5 arrests fibrosis in mouse models of scleroderma.

Scleroderma is an autoimmune rheumatic disorder accompanied by severe fibrosis in skin and other internal organs. During scleroderma progression, resident fibroblasts undergo activation and convert to α-smooth muscle actin (α-SMA) expressing myofibroblasts (MFBs) with increased capacity to synthesize collagens and fibrogenic components. Accordingly, MFBs are a major therapeutic target for fibrosis in scleroderma and treatment with blocking MFBs could produce anti-fibrotic effects. TLY012 is an engineered human TNF-related apoptosis-inducing ligand (TRAIL) which induces selective apoptosis in transformed cells expressing its cognate death receptors (DRs). Here we report that TLY012 selectively blocks activation of dermal fibroblasts and induces DR-mediated apoptosis in α-SMA+ MFBs through upregulated DR5 during its activation. In vivo, TLY012 reverses established skin fibrosis to near-normal skin architecture in mouse models of scleroderma. Thus, the TRAIL pathway plays a critical role in tissue remodeling and targeting upregulated DR5 in α-SMA+ MFBs is a viable therapy for fibrosis in scleroderma.

Roles of 14-3-3ß and γ in regulation of the glucocorticoid receptor transcriptional activation and hepatic gluconeogenesis.

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor that mediates the effects of glucocorticoids, and plays a crucial role in cell growth, development, inflammation, and gluconeogenesis. The 14-3-3 proteins bind to target proteins via phosphorylation, and influence many cellular events by altering their subcellular localization or by acting as chaperones. However, the mechanisms by which 14-3-3 proteins regulate GR transactivation and their involvement in gluconeogenesis remain uncharacterized. We found that 14-3-3ß and γ increased GR transcriptional activity and the promoter activities of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase in the presence of glucocorticoids. Inhibition of the endogenous 14-3-3ß and γ decreased dexamethasone- and cAMP-stimulated PEPCK expression. Further, both 14-3-3ß and γ increased glucose production in response to glucocorticoids. Our findings suggest that 14-3-3ß and γ function as positive regulators of GR transactivation and glucocorticoid-mediated hepatic gluconeogenesis.

Ataxin-1 is involved in tumorigenesis of cervical cancer cells via the EGFR-RAS-MAPK signaling pathway.

Ataxin-1 (ATXN1) is a coregulator protein within which expansion of the polyglutamine tract causes spinocerebellar ataxia type 1, an autosomal dominant neurodegenerative disorder. Previously, we reported that ATXN1 regulates the epithelial-mesenchymal transition of cervical cancer cells. In the present study, we demonstrate that ATXN1 is involved in cervical cancer tumorigenesis by promoting the proliferation of human cervical cancer cells. Chromatin immunoprecipitation assays showed that ATXN1 bound to the promoter region within cyclin D1 and activated cyclin D1 transcription, resulting in cell proliferation. ATXN1 promoted cyclin D1 expression through the EGFR-RAS-MAPK signaling pathway. Mouse xenograft tumorigenicity assays showed that ATXN1 downregulation inhibited tumorigenesis in cervical cancer cell lines in nude mice. Human cervical cancer tissue microarrays and immunohistochemical techniques showed that ATXN1 was significantly upregulated in many such tissues. Our results suggest that ATXN1 plays an important role in cervical cancer tumorigenesis and is a prognostic marker for cervical cancer.

Small leucine zipper protein functions as a negative regulator of estrogen receptor α in breast cancer.

The nuclear transcription factor estrogen receptor α (ERα) plays a critical role in breast cancer progression. ERα acts as an important growth stimulatory protein in breast cancer and the expression level of ERα is tightly related to the prognosis and treatment of patients. Small leucine zipper protein (sLZIP) functions as a transcriptional cofactor by binding to various nuclear receptors, including glucocorticoid receptor, androgen receptor, and peroxisome proliferator-activated receptor γ. However, the role of sLZIP in the regulation of ERα and its involvement in breast cancer progression is unknown. We found that sLZIP binds to ERα and represses the transcriptional activity of ERα in ERα-positive breast cancer cells. sLZIP also suppressed the expression of ERα target genes. sLZIP disrupted the binding of ERα to the estrogen response element of the target gene promoter, resulting in suppression of cell proliferation. sLZIP is a novel co-repressor of ERα, and plays a negative role in ERα-mediated cell proliferation in breast cancer.

Caveolin-1 deficiency induces premature senescence with mitochondrial dysfunction.

Paradoxical observations have been made regarding the role of caveolin-1 (Cav-1) during cellular senescence. For example, caveolin-1 deficiency prevents reactive oxygen species-induced cellular senescence despite mitochondrial dysfunction, which leads to senescence. To resolve this paradox, we re-addressed the role of caveolin-1 in cellular senescence in human diploid fibroblasts, A549, HCT116, and Cav-1-/- mouse embryonic fibroblasts. Cav-1 deficiency (knockout or knockdown) induced cellular senescence via a p53-p21-dependent pathway, downregulating the expression level of the cardiolipin biosynthesis enzymes and then reducing the content of cardiolipin, a critical lipid for mitochondrial respiration. Our results showed that Cav-1 deficiency decreased mitochondrial respiration, reduced the activity of oxidative phosphorylation complex I (CI), inactivated SIRT1, and decreased the NAD+ /NADH ratio. From these results, we concluded that Cav-1 deficiency induces premature senescence via mitochondrial dysfunction and silent information regulator 2 homologue 1 (SIRT1) inactivation.

Human leucine zipper protein promotes hepatic steatosis via induction of apolipoprotein A-IV.

The molecular mechanism of stress-induced hepatic steatosis is not well known. Human leucine zipper protein (LZIP) regulates the expression of genes involved in inflammation, cell migration, and stress response. The aim of this study was to determine the regulatory role of LZIP in stress-induced hepatic steatosis. We used a microarray analysis to identify LZIP-induced genes involved in hepatic lipid metabolism. LZIP increased the expression of apolipoprotein A-IV (APOA4) mRNA. In the presence of stress inducer, APOA4 promoter analysis was performed, and LZIP-induced lipid accumulation was monitored in mouse primary cells and human tissues. Under Golgi stress conditions, LZIP underwent proteolytic cleavage and was phosphorylated by AKT to protect against proteasome degradation. The stabilized N-terminal LZIP was translocated to the nucleus, where it directly bound to the APOA4 promoter, leading to APOA4 induction. LZIP-induced APOA4 expression resulted in increased absorption of surrounding free fatty acids. LZIP also promoted hepatic steatosis in mouse liver. Both LZIP and APOA4 were highly expressed in human steatosis samples. Our findings indicate that LZIP is a novel modulator of APOA4 expression and hepatic lipid metabolism. LZIP might be a therapeutic target for developing treatment strategies for hepatic steatosis and related metabolic diseases.-Kang, M., Kim, J., An, H.-T., Ko, J. Human leucine zipper protein promotes hepatic steatosis via induction of apolipoprotein A-IV.

Ataxin-1 regulates epithelial-mesenchymal transition of cervical cancer cells.

The mutant form of the protein ataxin-1 (ATXN1) causes the neurodegenerative disease spinocerebellar ataxia type-1. Recently, ATXN1 was reported to enhance E-cadherin expression in the breast cancer cell line MCF-7, suggesting a potential association between ATXN1 and cancer development. In the present study, we discovered a novel mechanism through which ATXN1 regulates the epithelial-mesenchymal transition (EMT) of cancer cells. Hypoxia-induced upregulation of the Notch intracellular domain expression decreased ATXN1 expression via MDM2-associated ubiquitination and degradation. In cervical cancer cells, ATXN1 knockdown induced EMT by directly regulating Snail expression, leading to matrix metalloproteinase activation and the promotion of cell migration and invasion. These findings provide insights into a novel mechanism of tumorigenesis and will facilitate the development of new and more effective therapies for cancer.

Identification and molecular characterization of cellular factors required for glucocorticoid receptor-mediated mRNA decay.

Glucocorticoid (GC) receptor (GR) has been shown recently to bind a subset of mRNAs and elicit rapid mRNA degradation. However, the molecular details of GR-mediated mRNA decay (GMD) remain unclear. Here, we demonstrate that GMD triggers rapid degradation of target mRNAs in a translation-independent and exon junction complex-independent manner, confirming that GMD is mechanistically distinct from nonsense-mediated mRNA decay (NMD). Efficient GMD requires PNRC2 (proline-rich nuclear receptor coregulatory protein 2) binding, helicase ability, and ATM-mediated phosphorylation of UPF1 (upstream frameshift 1). We also identify two GMD-specific factors: an RNA-binding protein, YBX1 (Y-box-binding protein 1), and an endoribonuclease, HRSP12 (heat-responsive protein 12). In particular, using HRSP12 variants, which are known to disrupt trimerization of HRSP12, we show that HRSP12 plays an essential role in the formation of a functionally active GMD complex. Moreover, we determine the hierarchical recruitment of GMD factors to target mRNAs. Finally, our genome-wide analysis shows that GMD targets a variety of transcripts, implicating roles in a wide range of cellular processes, including immune responses.

Antibody neutralization of cell-surface gC1qR/HABP1/SF2-p32 prevents lamellipodia formation and tumorigenesis.

We previously demonstrated that cell-surface gC1qR is a key regulator of lamellipodia formation and cancer metastasis. Here, we screened a monoclonal mouse antibody against gC1qR to prevent cell migration by neutralizing cell-surface gC1qR. The anti-gC1qR antibody prevented growth factor-stimulated lamellipodia formation, cell migration and focal adhesion kinase activation by inactivating receptor tyrosine kinases (RTKs) in various cancer cells such as A549, MDA-MB-231, MCF7 and HeLa cells. The antibody neutralization of cell-surface gC1qR also inhibited angiogenesis because the anti-gC1qR antibody prevented growth factor-stimulated RTK activation, lamellipodia formation, cell migration and tube formation in HUVEC. In addition, we found that A549 tumorigenesis was reduced in a xenograft mouse model by following the administration of the anti-gC1qR antibody. With these data, we can conclude that the antibody neutralization of cell-surface gC1qR could be a good therapeutic strategy for cancer treatment.

14-3-3ß and γ differentially regulate peroxisome proliferator activated receptor γ2 transactivation and hepatic lipid metabolism.

Peroxisome proliferator activated receptor (PPAR) γ2 plays important roles in glucose and lipid metabolism in hepatocytes. PPARγ2 is involved in metabolic disorders, including obesity, diabetes, and fatty liver disease. Although the 14-3-3 proteins participate in a variety of cell signal pathways, the roles of the 14-3-3 proteins in regulating PPARγ2 transactivation and hepatic lipid metabolism are unknown. We identified 14-3-3ß and γ as PPARγ2 transcriptional regulators. We found that 14-3-3ß and γ competitively interacted with the phosphorylated Ser273 of PPARγ2, which is important for regulating glucose and lipid metabolism. 14-3-3ß increased the transcriptional activity of PPARγ2 and enhanced the expression levels of PPARγ2 target genes involved in lipogenesis and lipid transport. In contrast, 14-3-3γ decreased PPARγ2 transactivation and reduced the expression levels of PPARγ2 target genes. A high concentration of free fatty acids increased PPARγ2 expression and lipid accumulation. 14-3-3ß enhanced hepatic lipogenesis, which is a major symptom of non-alcoholic fatty liver disease. However, 14-3-3γ suppressed hepatic lipid accumulation in the presence of high free fatty acids. These findings indicate that 14-3-3ß and γ are novel PPARγ2 regulators and are involved in hepatic lipid metabolism. 14-3-3ß and γ can be therapeutic target molecules to treat non-alcoholic fatty liver disease.

Small leucine zipper protein (sLZIP) negatively regulates skeletal muscle differentiation via interaction with α-actinin-4.

The small leucine zipper protein (sLZIP) plays a role in transcriptional regulation in various types of cells. However, the role of sLZIP in myogenesis is unknown. We identified α-actinin-4 (ACTN4) as a sLZIP-binding protein. ACTN4 functions as a transcriptional regulator of myocyte enhancer factor (MEF)2, which plays a critical role in expression of muscle-specific genes during skeletal muscle differentiation. We found that ACTN4 translocates to the nucleus, induces myogenic gene expression, and promotes myotube formation during myogenesis. The myogenic process is controlled by an association between myogenic factors and MEF2 transcription factors. ACTN4 increased expression of muscle-specific proteins via interaction with MEF2. However, sLZIP decreased myogenic gene expression and myotube formation during myogenesis via disruption of the association between ACTN4 and MEF2. ACTN4 increased the promoter activities of myogenic genes, whereas sLZIP abrogated the effect of ACTN4 on transcriptional activation of myogenic genes in myoblasts. The C terminus of sLZIP is required for interaction with the C terminus of ACTN4, based on deletion mutant analysis, and sLZIP plays a role in regulation of MEF2 transactivation via interaction with ACTN4. Our results indicate that sLZIP negatively regulates skeletal muscle differentiation via interaction with ACTN4 and that sLZIP can be used as a therapeutic target molecule for treatment of muscle hypertrophy and associated diseases.

Effects of protein kinase Cdelta and phospholipase C-gamma1 on monocyte chemoattractant protein-1 expression in taxol-induced breast cancer cell death.

Monocyte chemoattractant protein-1 (MCP-1) is a CC chemokine that plays an important role in immune cell migration. It has been reported that chemokines, including MCP-1, are involved in angiogenesis and metastasis. However, the exact role of chemokines in cancer development is still obscure. We investigated the involvement of MCP-1 in taxol-induced breast cancer cell death. The anti-cancer drug taxol induced MCF-7 breast cancer cell death. Treatment with taxol increased the mRNA expression level of MCP-1 in a dose- and time-dependent manner. Up-regulation of MCP-1 by taxol was augmented in cells treated with rottlerin, a specific inhibitor of protein kinase Cdelta (PKCdelta). In addition, taxol-induced MCP-1 expression was reduced by the ectopic expression of PKCdelta in a dose-dependent manner, indicating that PKCdelta plays a negative role in taxol-induced MCP-1 expression in MCF-7 cells. On the other hand, taxol-induced up-regulation of MCP-1 was reduced in cells treated with U73122, an inhibitor of phospholipase C (PLC), and ectopic expression of PLC-gamma1 increased the expression of MCP-1 in taxol-treated MCF-7 cells, indicating that PLC-gamma1 functions as a positive regulator in taxol-induced MCP-1 expression. These results indicate that MCP-1 is involved in taxol-induced breast cancer cell death and we propose that taxol induces up-regulation of MCP-1 by affecting both positive and negative regulatory signaling pathways.