To prepare and evaluate the liver-targeted drug delivery system of Bufalin with higher liver uptake and stronger antitumor activity against hepatocellular carcinoma.

Multiple carcinomas including breast, ovarian, colon, lung and stomach cancer, overexpress the hyaluronic acid (HA) receptor, CD44. Overexpression of CD44 contributes to key cancer processes including tumor invasion, metastasis, recurrence, and chemoresistance. Herein, we devised novel targeted nanoparticles (NPs) for delivery of anticancer chemotherapeutics, comprised of self-assembling Maillard reaction-based conjugates of HA and bovine serum albumin (BSA). HA served as the hydrophilic block, and as the ligand for actively targeting cancer cells overexpressing CD44. We demonstrate that Maillard reaction-based covalent conjugates of BSA-HA self-assemble into NPs, which efficiently entrap hydrophobic cytotoxic drugs including paclitaxel and imidazoacridinones. Furthermore, BSA-HA conjugates stabilized paclitaxel and prevented its aggregation and crystallization. The diameter of the NPs was < 15 nm, thus enabling CD44 receptor-mediated endocytosis. These NPs were selectively internalized by ovarian cancer cells overexpressing CD44, but not by cognate cells lacking this HA receptor. Moreover, free HA abolished the endocytosis of drug-loaded BSA-HA conjugates. Consistently, drug-loaded NPs were markedly more cytotoxic to cancer cells overexpressing CD44 than to cells lacking CD44, due to selective internalization, which could be competitively inhibited by excess free HA. Finally, a CD44-targeted antibody which blocks receptor activity, abolished internalization of drug-loaded NPs. In conclusion, a novel cytotoxic drug-loaded nanomedicine platform has been developed, which is based on natural biocompatible biopolymers, capabale of targeting cancer cells with functional surface expression of CD44.

Sufficient knowledge regarding cellular and molecular basis of lung cancer progression and metastasis would help in the development of novel and effective strategies for the treatment of lung cancer. 4HPR is a synthetic retinoid with potential anti-tumor activity but is still limited because of its poor bioavailability. The use of albumin as a complexing agent for a hydrophobic drug is expected to improve the water solubility and consequently their bioavailability.This study investigated the antitumor activity of a novel complex between albumin and 4-HPR in a mouse model of human lung cancer and focuses on role and mechanism of Cav-1 mainly involved in regulating cancer and ACSVL3 mainly connected with tumor growth. Their expressions were assayed by immunohistochemistry and qRT-PCR, to demonstrate the reduction of the tumor growth following the drug treatment. Our results showed a high antitumor activity of 4HPR-HSA by reduction of the volume of tumor mass and the presence of a high level of apoptotic cell by TUNEL assay. The downregulation of Cav-1 and ACSVL3 suggested a reduction of tumor growth. In conclusion, we demonstrated the great potential of 4HPR-HSA in the treatment of lung cancer. More data about the mechanism of drug delivery the 4HPR-HSA are necessary.

Tumor cells rely on high concentrations of amino acids to support their growth and proliferation. Although increased macropinocytic uptake and lysosomal degradation of the most abundant serum protein, albumin, in Ras-transformed cells can meet these demands, it is not understood how the majority of tumor cells that express wild type Ras achieve this. In the current study we reveal that the neonatal Fc receptor, FcRn, regulates tumor cell proliferation through the ability to recycle its ligand, albumin. By contrast with normal epithelial cells, we show that human FcRn is present at very low or undetectable levels in the majority of tumor cell lines analyzed. Remarkably, shRNA-mediated ablation of FcRn expression in an FcRn-positive tumor cell line results in a substantial growth increase of tumor xenografts, whereas enforced expression of this receptor by lentiviral transduction has the reverse effect. Moreover, intracellular albumin and glutamate levels are increased by the loss of FcRn-mediated recycling of albumin, combined with hypoalbuminemia in tumor-bearing mice. These studies identify a novel role for FcRn as a suppressor of tumor growth and have implications for the use of this receptor as a prognostic indicator and therapeutic target.

Phosphorylation of p62/SQSTM1 (p62) on Serine 349 (P-Ser349 p62) as well as proteasome dysfunction have been shown to activate the cell protective Keap1/Nrf2 pathway. We showed previously that BAY 11-7085-induced human synovial fibroblast cell death includes autophagy and p62 downregulation. In this work, we have studied expression of P-Ser349 p62 in human synovial fibroblasts. Results showed that P-Ser349 p62 was not detected in synovial cell extracts unless cells were cultured in the presence of proteasome inhibitor (MG132). MG132 revealed P-Ser349 p62 turnover, that was further increased by concomitant autophagy inhibition and markedly enhanced in serum starved cells. Starvation sensitized synovial fibroblasts to BAY 11-7085 while MG132 protected both non-starved and starved cells from BAY 11-7085-induced cell death. Lentivirus mediated overexpression of phosphorylation-mimetic p62 mutant S349E markedly protected synovial fibroblasts from BAY 11-7085. Inhibitor of Keap1-P-S349 p62 interaction, K67, had synergistic effect with MG132. Starvation increased p62 molecular weight, that was reversed by serum and bovine serum albumin re-feeding. Furthermore, starvation markedly induced RAD23B. Increased endo-β-N-acetylglucosaminidase (ENGase) turnover was detected in starved synovial fibroblasts. PNGase F treatment produced faster migration p62 form in human synovial tissue extracts but starvation-like p62 form of higher molecular weight in synovial cell extracts. Co-transfection of NGLY1, with p62 or p62 mutants S349A and S349E markedly stabilized p62 expressions in HEK293 cells. Tunicamycin upregulated p62 and protected synovial fibroblasts from BAY 11-7085-induced cell death. These results showed that P-Ser349 p62 has pro-survival role in human synovial fibroblasts and that de-glycosylation events are involved in p62 turnover.

CD19 is overexpressed in most human B cell malignancies and considered an important tumor marker for diagnosis and treatment. Aptamers are oligonucleotides that may potentially serve as tumor-homing ligand for targeted cancer therapy with excellent affinity and specificity. In this study, we selected a novel CD19 aptamer (LC1) that was a 59-nucleotide single strand DNA. The aptamer could bind to recombinant CD19 protein with a Kd of 85.4 nM, and had minimal cross reactivity to bovine serum albumin (BSA) or ovalbumin (OVA). Moreover, the aptamer was found capable of binding with the CD19-positive lymphoma cells (Ramos and Raji), but not the CD19-negative cell lines (Jurkat and NB4). An aptamer-doxorubicin complex (Apt-Dox) was also formulated, and selectively delivered doxorubicin to CD19-positive lymphoma cells in vitro. The results indicate that aptamer LC1 can recognize CD19-positive tumor cells and may potentially function as a CD19-targeting ligand.

The study aims to explore the effects of TLR-2/NF-κB signaling pathway on the occurrence of degenerative knee osteoarthritis (OA). Degenerative knee OA and normal cartilage samples were collected from patients with degenerative knee OA receiving total knee arthroplasty and amputation. Expressions of TLR-2, NF-κB and MMP-13 were determined by qRT-PCR and immunochemistry. The chondrocytes were divided into control, IL-1β, IL-1β + anti-TLR-2 and IL-1β + PDTC groups. MTT assay and flow cytometry were performed to determine proliferation and apoptosis of the chondrocytes. Expressions of TLR-2, NF-κB and MMP-13 were measured by Western blotting. ELISA was conducted to detect the expressions of related inflammatory factors. The positive expressions of TLR, NF-κB and MMP13 were associated with body mass index (BMI), family history, exercise, and WOMAC scores of OA patients. Logistic regression analysis showed that OA influencing factors were TLR, NF-κB, MMP13, BMI, family history and exercise. Compared with normal chondrocytes, the expressions of TLR-2, NF-κB, MMP-13 and related inflammatory factors increased in degenerative knee OA. The chondrocytes in the IL-1β + anti-TLR-2 and IL-1β + PDTC groups showed lower apoptosis rates than those in the IL-1β group. Compared with the control group, increased expressions of TLR-2, NF-κB, phosphorylated-NF-κB (p-NF-κB), MMP-13, IL-1, IL-6 and TNF-α were found in the IL-1β group. In the IL-1β + anti-TLR-2 and IL-1β + PDTC groups, decreased expressions of NF-κB, p-NF-κB, MMP-13, IL-1, IL-6 and TNF-α were found compared with those in the IL-1β group. TLR-2/NF-κB signaling pathway contributes to the occurrence of degenerative knee OA.

To study the structural relationship between annexin A4 and the Lewis y antigen and compare their expression and significance in ovarian clear cell carcinoma, and to explore how annexin A4 fucose glycosylation effects the interaction between annexin A4 and NF-kB p50, and how it promotes tumour progression of ovarian clear cell carcinoma.

Heparanase (HPSE), a heparan sulfate-specific endo-β-D-glucuronidase, plays an important role in tumor cell metastasis through the degradation of extracellular matrix heparan sulfate proteoglycans. Suramin, a polysulfonated naphthylurea, is an inhibitor of HPSE with suramin analogues. Our objective was to analyze the HPSE involvement in gastric signet ring cell adenocarcinoma (SRCA) invasion. High expression of HPSE mRNA and protein was found in the tumor and in ascites of SRCA as well as in KATO-III cell line. Beside of collagen-I, growth factors (TGF-β1 and VEGF-A, except FGF-2) and epithelial mesenchymal transition (EMT) markers (Snail, Slug, Vimentin, α-SMA and Fibronectin, except E-cadherin) were found higher in main nodules of SRCA as compared to peritumoral sites. Among MDR proteins, MDR-1 and LRP (lung resistance protein) were highly expressed in tumor cells. The formation of 3D cell spheroids was found to be correlated with their origin (adherent or non-adherent KATO-III). After treatment of KATO-III cells with a HPSE inhibitor (suramin), cell proliferation and EMT-related markers, besides collagen-1 expression, were down regulated. In conclusion, in SRCA, HPSE via an autocrine secretion is involved in acquisition of mesenchymal phenotype and tumor cell malignancy. Therefore, HPSE could be an interesting pharmacological target for the treatment of SRCA.

ALOS4, a unique synthetic cyclic peptide without resemblance to known integrin ligand sequences, was discovered through repeated biopanning with pIII phage expressing a disulfide-constrained nonapeptide library. Binding assays using a FITC-labeled analogue demonstrated selective binding to immobilized αvβ3 and a lack of significant binding to other common proteins, such as bovine serum albumin and collagen. In B16F10 cell cultures, ALOS4 treatment at 72 h inhibited cell migration (30%) and adhesion (up to 67%). Immunofluorescent imaging an ALOS4-FITC analogue with B16F10 cells demonstrated rapid cell surface binding, and uptake and localization in the cytoplasm. Daily injections of ALOS4 (0.1, 0.3 or 0.5 mg/kg i.p.) to mice inoculated with B16F10 mouse melanoma cells in two different cancer models, metastatic and subcutaneous tumor, resulted in reduction of lung tumor count (metastatic) and tumor mass (subcutaneous) and increased survival of animals monitored to 45 and 60 days, respectively. Examination of cellular activity indicated that ALOS4 produces inhibition of cell migration and adhesion in a concentration-dependent manner. Collectively, these results suggest that ALOS4 is a structurally-unique selective αvβ3 integrin ligand with potential anti-metastatic activity.

We hypothesized that cancer tissue immunogens - antigens capable of inducing specific antibody production in patients - are promising targets for development of precision diagnostics and humoral immunotherapies. We developed an innovative immuno-proteomic strategy and identified new immunogenic markers of colon cancer. Proteins from cancers and matched normal tissues were separated by 2D gel electrophoresis and blotted with serum antibodies from the same patients. Antibody-reactive proteins were sequenced by mass spectrometry and validated by Western blotting and immunohistochemistry. 170 serum antibody-reactive proteins were identified only in cancerous but not matched normal. Among these, proteasome subunit alpha type 1 (PSA1), leucine aminopeptidase 3 (LAP3), annexin A3 (ANXA3), and maspin (serpin B5) were reproducibly found in tissues from three patients. Differential expression patterns were confirmed in samples from eight patients with various stages of colon adenocarcinoma and liver metastases. These tumor-resident proteins and/or their associated serum antibodies may be promising markers for colon cancer screening and early diagnosis. Furthermore, tumor tissue-specific antibodies could potentially be exploited as immunotherapeutic targets against cancer. More generally, proteomic profiling of antibody-inducing cancer-associated immunogens represents a powerful generic method for uncovering the tumor antigen-ome, i.e., the totality of immunogenic tumor-associated proteins.

Aberrant Ras-MAPK signaling from receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor-2 (HER2), is a hallmark of triple negative breast cancer (TNBC); thus providing rationale for targeting the Ras-MAPK pathway. Components of this EGFR/HER2-Ras-Raf-Mek-Erk pathway were co-targeted in the MDA-MB-231 and MDA-MB-468 human TNBC cell lines, and in vitro effects on signaling and cytotoxicity, as well as in vivo effects on xenograft tumor growth and metastasis were assessed. The dual EGFR/HER2 inhibitor lapatinib (LPN) displayed greater cytotoxic potency and MAPK signaling inhibition than the EGFR inhibitor erlotinib, suggesting both EGFR and HER2 contribute to MAPK signaling in this TNBC model. The Raf inhibitor sorafenib (SFN) or the Mek inhibitor U0126 suppressed MAPK signaling to a greater extent than LPN; which correlated with greater cytotoxic potency of SFN, but not U0126. However, U0126 potentiated the cytotoxic efficacy of LPN and SFN in an additive and synergistic manner, respectively. This in-series Raf-Mek co-targeting synergy was recapitulated in orthotopic mouse xenografts, where SFN and the Mek inhibitor selumitinib (AZD6244) inhibited primary tumor growth and pulmonary metastasis. Raf and Mek co-inhibition exhibits synergy in TNBC models and represent a promising combination therapy for this aggressive breast cancer type.

The deposition of the activating H3K4me3 and repressive H3K27me3 histone modifications within the same promoter, forming a so-called bivalent domain, maintains gene expression in a repressed but transcription-ready state. We recently reported a significantly increased incidence of bivalency following an epithelial-mesenchymal transition (EMT), a process associated with the initiation of the metastatic cascade. The reverse process, known as the mesenchymal-epithelial transition (MET), is necessary for efficient colonization. Here, we identify numerous genes associated with differentiation, proliferation and intercellular adhesion that are repressed through the acquisition of bivalency during EMT, and re-expressed following MET. The majority of EMT-associated bivalent domains arise through H3K27me3 deposition at H3K4me3-marked promoters. Accordingly, we show that the expression of the H3K27me3-demethylase KDM6A is reduced in cells that have undergone EMT, stem-like subpopulations of mammary cell lines and stem cell-enriched triple-negative breast cancers. Importantly, KDM6A levels are restored following MET, concomitant with CDH1/E-cadherin reactivation through H3K27me3 removal. Moreover, inhibition of KDM6A, using the H3K27me3-demethylase inhibitor GSK-J4, prevents the re-expression of bivalent genes during MET. Our findings implicate KDM6A in the resolution of bivalency accompanying MET, and suggest KDM6A inhibition as a viable strategy to suppress metastasis formation in breast cancer.

Galectin-1 (Gal-1) has been reported to be an independent prognostic indicator of poor survival in gastric cancer and overexpression of Gal-1 enhances the invasiveness of gastric cancer cells. However, the downstream mechanisms by which Gal-1 promotes invasion remains unclear. Moreover, the function of Gal-1 in the epithelial-mesenchymal transition (EMT) in gastric cancer has not yet been elucidated. In this study, we observed Gal-1 expression was upregulated and positively associated with metastasis and EMT markers in 162 human gastric cancer tissue specimens. In vitro studies showed Gal-1 induced invasion, the EMT phenotype and activated the non-canonical hedgehog (Hh) pathway in gastric cancer cell lines. Furthermore, our data revealed that Gal-1 modulated the non-canonical Hh pathway by increasing the transcription of glioma-associated oncogene-1 (Gli-1) via a Smoothened (SMO)-independent manner, and that upregulation of Gal-1 was strongly associated with gastric cancer metastasis. We conclude that Gal-1 promotes invasion and the EMT in gastric cancer cells via activation of the non-canonical Hh pathway, suggesting Gal-1 could represent a promising therapeutic target for the prevention and treatment of gastric cancer metastasis.

Pulmonary arterial hypertension (PAH) is a lethal disease that often affects the young. Although Bone Morphogenetic Protein Receptor Type 2 gene (BMPR2) mutations are related with idiopathic and heritable PAH, the low penetrance and variable expressivity in PAH suggest the existence of other genetic and/or environmental factors. In this study, we aimed to identify novel genetic factors associated with PAH, irrespective of BMPR2 mutation. We performed genome-wide association study (GWAS) in a Japanese population comprising 44 individuals with idiopathic and heritable PAH, and 2,993 controls. Seven loci identified in the genome-wide study were submitted to the validation study, and a novel susceptibility locus, PDE1A|DNAJC10, was identified that maps to 2q32.1 (rs71427857, P = 7.9 × 10-9, odds ratio in the validation study = 5.18; 95% CI 1.86 - 14.42). We also found the augmentation of PDE1A protein in distal remodeled pulmonary artery walls in idiopathic PAH patients. Given that phosphodiesterase 5 inhibitors are effective for the treatment of idiopathic and heritable PAH, our findings suggest that PDE1A could be a novel therapeutic target of PAH.

Vaccination is a potential approach for prevention and control of disease in fish. The use of genetically engineered vaccines is an effective method and a green intervention to control bacterial infection in aquaculture. However, efforts to develop these vaccines are limited by the lack of conserved protective antigens. In this study, three candidate immunogens (Srr, NeuA, and Hsp) of the pathogenic Streptococcus iniae strain DGX07 isolated from diseased channel catfish were identified and analyzed. Molecular cloning, expression, and purification of candidate antigen genes were carried out to obtain the candidate immunogens in the form of recombinant subunit vaccines. Western blotting was performed to evaluate immunogenicity in vitro and channel catfish were vaccinated by intraperitoneal injection and the specific antibody titers and relative percent of survival were determined to evaluate immune protection in vivo. The results showed that these three candidate immunogens were expressed correctly as recombinant proteins fused with His tags, with molecular weights of 70 kDa for Srr, 86 kDa for NeuA, and 51 kDa for Hsp, respectively. Moreover, each immunogen was predicted to be located either extracellularly or on the surface of S. iniae, and were able to offer protection against S. iniae infection in the form of recombinant subunit vaccines with adjuvant ISA763, especially Srr, with a relative percent of survival of 70% for Srr, 55% for NeuA, and 50% for Hsp, respectively.

The socioeconomic burden of chronic back pain related to intervertebral disc (IVD) disease is high and current treatments are only symptomatic. Minimally invasive strategies that promote biological IVD repair should address this unmet need. Notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) during IVD maturation and degeneration. The regenerative potential of NC-secreted substances on CLCs and mesenchymal stromal cells (MSCs) has already been demonstrated. However, identification of these substances remains elusive. Innovatively, this study exploits the regenerative NC potential by using healthy porcine NC-derived matrix (NCM) and employs the dog as a clinically relevant translational model. NCM increased the glycosaminoglycan and DNA content of human and canine CLC aggregates and facilitated chondrogenic differentiation of canine MSCs in vitro. Based on these results, NCM, MSCs and NCM+MSCs were injected in mildly (spontaneously) and moderately (induced) degenerated canine IVDs in vivo and, after six months of treatment, were analyzed. NCM injected in moderately (induced) degenerated canine IVDs exerted beneficial effects at the macroscopic and MRI level, induced collagen type II-rich extracellular matrix production, improved the disc height, and ameliorated local inflammation. MSCs exerted no (additive) effects. In conclusion, NCM induced in vivo regenerative effects on degenerated canine IVDs. NCM may, comparable to demineralized bone matrix in bone regeneration, serve as 'instructive matrix', by locally releasing growth factors and facilitating tissue repair. Therefore, intradiscal NCM injection could be a promising regenerative treatment for IVD disease, circumventing the cumbersome identification of bioactive NC-secreted substances.

Barrett's esophagus (BE) is metaplasia of the squamous epithelium to a specialized columnar epithelium. BE progresses through low- and high-grade dysplasia before developing into esophageal adenocarcinoma. The BE microenvironment is not well defined. We compare 12 human clinical BE and adjacent normal squamous epithelium biopsies using single cell immunophenotyping by flow cytometry. A cassette of 19 epithelial and immune cell markers was used to detect differences between cellular compartments in normal and BE tissues. We found that the BE microenvironment has an immunological landscape distinct from adjacent normal epithelium. BE has an increased percentage of epithelial cells with a concomitant decrease in the percentage of immune cells, accompanied by a shift in the immune landscape from a predominantly T cell rich microenvironment in normal tissue to a B cell rich landscape in BE tissue. Hierarchical clustering separates BE and normal samples into two discrete groups based upon our 19-marker panel, but also reveals unexpected, shared phenotypes for three patients. Our results suggest that flow based single cell analysis may have the potential for revealing clinically relevant differences between BE and normal adjacent tissue, and that surface immunophenotypes could identify specific subpopulations from dysplastic tissue for further investigation.

Esophageal squamous cell carcinoma (ESCC) has a high morbidity in China, accounting for 90% of all esophageal carcinoma cases. Hence, identifying drug targets for prevention and treatment of ESCC is essential. Due to its critical role in the regulation of cell apoptosis, Mcl-1 holds great potential as a target for treatment against ESCC. In current study, we used a 4-nitroquinoline-1-oxide (4-NQO)-induced ESCC mouse model of test whether A-1210477, a Mcl-1 small molecular inhibitor, could repress ESCC development. We showed that A-1210477 treatment decreased ESCC formation and animal weight loss in a dose dependent manner. We detected decreased cellular proliferation in A-1210477-treated ESCC tissue by Ki67 expression. Moreover, A-1210477 treatment increased the number of apoptotic cells in ESCC tissues. Our study clearly demonstrates the contribution of Mcl-1 to ESCC development through promoting cell proliferation and inhibition of apoptosis, and provides a strong evidence for further evaluation of A-1210477 for treating ESCC.

The association between metabolic diseases and the risk of developing cancer is emerging. However, the impact of long pentraxin-3 (PTX3) on dyslipidemia-associated tumor metastasis remains unknown. In this study, we found that oleate induced PTX3 expression and secretion through the activation of Akt/NF-κB pathway in head and neck squamous cell carcinomas (HNSCCs). The activation of NF-κB was essential for the oleate-induced stabilization of PTX3 mRNA. In addition, both the depletion of PTX3 and the inhibition of NF-κB significantly inhibited oleate-induced tumor cell migration and invasion. The enhancement of binding between tumor and endothelial cells was observed in oleate-treated cells but not in the depletion and neutralization of PTX3 with siPTX3 and anti-PTX3 antibodies, respectively. The levels of oleate-induced epithelial-mesenchymal transition (EMT) markers, such as vimentin and MMP-3, were significantly reduced in PTX3-depleted cells. Knocking down vimentin also repressed oleate-induced HNSCC invasion. Furthermore, the depletion of PTX3 blocked the oleate-primed metastatic seeding of tumor cells in the lungs. These results demonstrate that oleate enhances HNSCC metastasis through the PTX3/vimentin signaling axes. The inhibition of PTX3 could be a potential strategy for the treatment of dyslipidemia-mediated HNSCC metastasis.