Improving cancer immunotherapy in prostate cancer by modulating T cell function through targeting the galectin-1.

Our study aimed to elucidate the role of Galectin-1 (Gal-1) role in the immunosuppressive tumor microenvironment (TME) of prostate cancer (PCa). Our previous findings demonstrated a correlation between elevated Gal-1 expression and advanced PCa stages. In this study, we also observed that Gal-1 is expressed around the tumor stroma and its expression level is associated with PCa progression. We identified that Gal-1 could be secreted by PCa cells, and secreted Gal-1 has the potential to induce T cell apoptosis. Gal-1 knockdown or inhibition of Gal-1 function by LLS30 suppresses T cell apoptosis resulting in increased intratumoral T cell infiltration. Importantly, LLS30 treatment significantly improved the antitumor efficacy of anti-PD-1 in vivo. Mechanistically, LLS30 binds to the carbohydrate recognition domain (CRD) of Gal-1, disrupting its binding to CD45 leading to the suppression of T cell apoptosis. In addition, RNA-seq analysis revealed a novel mechanism of action for LLS30, linking its tumor-intrinsic oncogenic effects to anti-tumor immunity. These findings suggested that tumor-derived Gal-1 contributes to the immunosuppressive TME in PCa by inducing apoptosis in effector T cells. Targeting Gal-1 with LLS30 may offer a strategy to enhance anti-tumor immunity and improve immunotherapy.

Galectin-1 orchestrates an inflammatory tumor-stroma crosstalk in hepatoma by enhancing TNFR1 protein stability and signaling in carcinoma-associated fibroblasts.

Most cases of hepatocellular carcinoma (HCC) arise with the fibrotic microenvironment where hepatic stellate cells (HSCs) and carcinoma-associated fibroblasts (CAFs) are critical components in HCC progression. Therefore, CAF normalization could be a feasible therapy for HCC. Galectin-1 (Gal-1), a ß-galactoside-binding lectin, is critical for HSC activation and liver fibrosis. However, few studies has evaluated the pathological role of Gal-1 in HCC stroma and its role in hepatic CAF is unclear. Here we showed that Gal-1 mainly expressed in HCC stroma, but not cancer cells. High expression of Gal-1 is correlated with CAF markers and poor prognoses of HCC patients. In co-culture systems, targeting Gal-1 in CAFs or HSCs, using small hairpin (sh)RNAs or an therapeutic inhibitor (LLS30), downregulated plasminogen activator inhibitor-2 (PAI-2) production which suppressed cancer stem-like cell properties and invasion ability of HCC in a paracrine manner. The Gal-1-targeting effect was mediated by increased a disintegrin and metalloprotease 17 (ADAM17)-dependent TNF-receptor 1 (TNFR1) shedding/cleavage which inhibited the TNF-α → JNK → c-Jun/ATF2 signaling axis of pro-inflammatory gene transcription. Silencing Gal-1 in CAFs inhibited CAF-augmented HCC progression and reprogrammed the CAF-mediated inflammatory responses in a co-injection xenograft model. Taken together, the findings uncover a crucial role of Gal-1 in CAFs that orchestrates an inflammatory CSC niche supporting HCC progression and demonstrate that targeting Gal-1 could be a potential therapy for fibrosis-related HCC.

Immunosuppressive Roles of Galectin-1 in the Tumor Microenvironment.

Evasion of immune surveillance is an accepted hallmark of tumor progression. The production of immune suppressive mediators by tumor cells is one of the major mechanisms of tumor immune escape. Galectin-1 (Gal-1), a pivotal immunosuppressive molecule, is expressed by many types of cancer. Tumor-secreted Gal-1 can bind to glycosylated receptors on immune cells and trigger the suppression of immune cell function in the tumor microenvironment, contributing to the immune evasion of tumors. The aim of this review is to summarize the current literature on the expression and function of Gal-1 in the human tumor microenvironment, as well as therapeutics targeting Gal-1.

Cancer-associated fibroblasts-derived exosomal miR-3656 promotes the development and progression of esophageal squamous cell carcinoma via the ACAP2/PI3K-AKT signaling pathway.

Esophageal squamous cell carcinoma (ESCC) is one of the most common gastrointestinal tumors, accounting for almost half a million deaths per year. Cancer-associated fibroblasts (CAFs) are the major constituent of the tumor microenvironment (TME) and dramatically impact ESCC progression. Recent evidence suggests that exosomes derived from CAFs are able to transmit regulating signals and promote ESCC development. In this study, we compared different the component ratios of miRNAs in exosomes secreted by CAFs in tumors and with those from normal fibroblasts (NFs) in precancerous tissues. The mRNA level of hsa-miR-3656 was significantly upregulated in the former exosomes. Subsequently, by comparing tumor cell development in vitro and in vivo, we found that the proliferation, migration and invasion capabilities of ESCC cells were significantly improved when miR-3656 was present. Further target gene analysis confirmed ACAP2 was a target gene regulated by miR-3656 and exhibited a negative regulatory effect on tumor proliferation. Additionally, the downregulation of ACAP2 triggered by exosomal-derived miR-3656 further promotes the activation of the PI3K/AKT and ß-catenin signaling pathways and ultimately improves the growth of ESCC cells both in vitro and in xenograft models. These results may represent a potential therapeutic target for ESCC and provide a new basis for clinical treatment plans.

CAMK2N1 suppresses hepatoma growth through inhibiting E2F1-mediated cell-cycle signaling.

Human kinome/phosphatome screen identified CAMK2N1 genes suppressing the development of human hepatocellular carcinoma (HCC). CAMK2N1 downregulation was found in 47% HCCs and associated with poor prognosis. The downregulation was mainly attributed to its genome deletion (28.4%) and DNA hypermethylation of its promoter (12.5%). Silencing and ectopic expression of CAMK2N1 respectively enhanced and suppressed cell proliferation, colony formation, and xenograft tumor growth in nude mice. Comparative proteomics revealed that CAMK2N1 silencing transcriptionally deregulated the genes regulated by E2F1 (89 out of the 114 E2F-signaling targets, P = 8.8E-240). The promoter assays revealed that CAMK2N1 suppressed E2F1-mediated transcriptional activities. CAMK2N1 silencing induced cyclins D/E expression, whereas its ectopic expression induced P27/KIP1 expression and suppressed the cell cycle. CAMK2N1 was translocated from the nuclei to the cytoplasm when cell proliferation reached the stationary phase, where its functions as an endogenous inhibitor of CAMK2. In conclusion, CAMK2NA is a novel 1p36 tumor suppressor gene that inhibits E2F1 transcriptional activities and induces P27/KIP1 expression. CAMK2N1-CAMK2 signaling forms a mechanism that restricts the cell cycle progression. Its deregulation could lead to tumorigenesis and might serve as promising therapeutic targets.

A cyclic peptide retards the proliferation of DU145 prostate cancer cells in vitro and in vivo through inhibition of FGFR2.

In malignancies, fibroblast growth factor receptors (FGFRs) signaling is reinforced through overexpression of fibroblast growth factors (FGFs) or their receptors. FGFR2 has been proposed as a target for cancer therapy, because both the expression and activation of FGFR2 are boosted in various malignant carcinomas. Although several chemicals have been designed against FGFR2, they did not exhibit enough specificity and might bring potential accumulated toxicity. In this study, we developed an epitope peptide (P5) and its cyclic derivative (DcP5) based on the structure of FGF2 to limit the activation of FGFR2. The anticancer activities of P5 and DcP5 were examined in vitro and in vivo. Our results demonstrated that P5 significantly inhibited the cell proliferation in FGFR2-dependent manner in DU145 cells and retarded tumor growth in DU145 xenograft model with negligible toxicity toward normal organs. Further investigations found that the Gln4 and Glu6 residues of P5 bind to FGFR2 to abolish its activation. Moreover, we developed the P5 cyclic derivative, DcP5, which achieved reinforced stability and anticancer activity in vivo. Our findings suggest P5 and its cyclic derivative DcP5 as potential candidates for anticancer therapy.

Glypican-3: A molecular marker for the detection and treatment of hepatocellular carcinoma☆.

Hepatocellular carcinoma (HCC) is a malignant tumor with a fairly poor prognosis (5-year survival of less than 50%). Using sorafenib, the only food and drug administration (FDA)-approved drug, HCC cannot be effectively treated; it can only be controlled at most for a couple of months. There is a great need to develop efficacious treatment against this debilitating disease. Glypican-3 (GPC3), a member of the glypican family that attaches to the cell surface by a glycosylphosphatidylinositol anchor, is overexpressed in HCC cases and is elevated in the serum of a large proportion of patients with HCC. GPC3 expression contributes to HCC growth and metastasis. Furthermore, several different types of antibodies targeting GPC3 have been developed. The aim of this review is to summarize the current literatures on the GPC3 expression in human HCC, molecular mechanisms of GPC3 regulation and antibodies targeting GPC3.

Galectin-1 inhibition induces cell apoptosis through dual suppression of CXCR4 and Ras pathways in human malignant peripheral nerve sheath tumors.

BACKGROUND: The Ras signaling pathway is commonly dysregulated in human malignant peripheral nerve sheath tumors (MPNSTs). It is well known that galectin-1 (Gal-1) is essential to stabilize membrane Ras and thereby induce the activation of Ras. However, the role of Gal-1 in MPNST progression remains unknown. The aim of this study was to examine whether Gal-1 knockdown could have an effect on the Ras signaling pathway. METHODS: Cell viability, apoptosis assay, and colony formation were performed to examine the effects of inhibition of Gal-1 in MPNST cells. We used a human MPNST xenograft model to assess growth and metastasis inhibitory effects of Gal-1 inhibitor LLS2. RESULTS: Gal-1 was upregulated in MPNST patients and was highly expressed in MPNST cells. Knockdown of Gal-1 by small interfering (si)RNA in Gal-1 expressing MPNST cells significantly reduces cell proliferation through the suppression of C-X-C chemokine receptor type 4 (CXCR4) and the rat sarcoma viral oncogene homolog (RAS)/extracellular signal-regulated kinase (ERK) pathway, which are important oncogenic signaling in MPNST development. Moreover, Gal-1 knockdown induces apoptosis and inhibits colony formation. LLS2, a novel Gal-1 allosteric small molecule inhibitor, is cytotoxic against MPNST cells and was able to induce apoptosis and suppress colony formation in MPNST cells. LLS2 treatment and Gal-1 knockdown exhibited similar effects on the suppression of CXCR4 and RAS/ERK pathways. More importantly, inhibition of Gal-1 expression or function by treatment with either siRNA or LLS2 resulted in significant tumor responses in an MPNST xenograft model. CONCLUSION: Our results identified an oncogenic role of Gal-1 in MPNST and that its inhibitor, LLS2, is a potential therapeutic agent, applied topically or systemically, against MPNST.

Targeting Galectin-1 Impairs Castration-Resistant Prostate Cancer Progression and Invasion.

Purpose: The majority of patients with prostate cancer who are treated with androgen-deprivation therapy (ADT) will eventually develop fatal metastatic castration-resistant prostate cancer (mCRPC). Currently, there are no effective durable therapies for patients with mCRPC. High expression of galectin-1 (Gal-1) is associated with prostate cancer progression and poor clinical outcome. The role of Gal-1 in tumor progression is largely unknown. Here, we characterized Gal-1 functions and evaluated the therapeutic effects of a newly developed Gal-1 inhibitor, LLS30, in mCRPC.Experimental Design: Cell viability, colony formation, migration, and invasion assays were performed to examine the effects of inhibition of Gal-1 in CRPC cells. We used two human CRPC xenograft models to assess growth-inhibitory effects of LLS30. Genome-wide gene expression analysis was conducted to elucidate the effects of LLS30 on metastatic PC3 cells.Results: Gal-1 was highly expressed in CRPC cells, but not in androgen-sensitive cells. Gal-1 knockdown significantly inhibited CRPC cells' growth, anchorage-independent growth, migration, and invasion through the suppression of androgen receptor (AR) and Akt signaling. LLS30 targets Gal-1 as an allosteric inhibitor and decreases Gal-1-binding affinity to its binding partners. LLS30 showed in vivo efficacy in both AR-positive and AR-negative xenograft models. LLS30 not only can potentiate the antitumor effect of docetaxel to cause complete regression of tumors, but can also effectively inhibit the invasion and metastasis of prostate cancer cells in vivoConclusions: Our study provides evidence that Gal-1 is an important target for mCRPC therapy, and LLS30 is a promising small-molecule compound that can potentially overcome mCRPC. Clin Cancer Res; 24(17); 4319-31. ©2018 AACR.

A Novel Galectin-1 Inhibitor Discovered through One-Bead Two-Compound Library Potentiates the Antitumor Effects of Paclitaxel in vivo.

Through the one-bead two-compound (OB2C) ultra-high-throughput screening method, we discovered a new small-molecule compound LLS2 that can kill a variety of cancer cells. Pull-down assay and LC/MS-MS indicated that galectin-1 is the target protein of LLS2. Galectin-1 is known to be involved in the regulation of proliferation, apoptosis, cell cycle, and angiogenesis. Binding of LLS2 to galectin-1 decreased membrane-associated H-Ras and K-Ras and contributed to the suppression of pErk pathway. Importantly, combination of LLS2 with paclitaxel (a very important clinical chemotherapeutic agent) was found to exhibit synergistic activity against several human cancer cell lines (ovarian cancer, pancreatic cancer, and breast cancer cells) in vitro Furthermore, in vivo therapeutic study indicated that combination treatment with paclitaxel and LLS2 significantly inhibits the growth of ovarian cancer xenografts in athymic mice. Our results presented here indicate that the OB2C combinatorial technology is a highly efficient drug screening platform, and LLS2 discovered through this method can be further optimized for anticancer drug development. Mol Cancer Ther; 16(7); 1212-23. ©2017 AACR.

The miR-200b-ZEB1 circuit regulates diverse stemness of human hepatocellular carcinoma.

Accumulating evidence suggests that human hepatocellular carcinoma (HCC) can be derived from cancer stem cells (CSCs), which contribute to tumor initiation, metastasis, chemoresistance, and recurrence. A great variety of HCC CSCs resulting in diverse clinical manifestations have been reported. However, how CSC diversity is regulated and generated remains unclear. Here we report that the miR-200b-ZEB1 circuit is closely involved with the induction and maintenance of a diverse group of CSCs. We found that miR-200b downregulation occurred in early HCC and associated with poor prognosis. The downregulation was attributable to genome deletion and promoter methylation of the miR-200a/b/429 gene. Ectopic expression of miR-200b or silencing of ZEB1 led to a decrease in CD13+ and CD24+ HCC CSCs and an increase in EpCAM+ HCC CSCs. Mechanistically, miR-200b directly suppressed BMI1 and ZEB1 expressions. ZEB1 recognized promoters of CD13, CD24, and EpCAM genes resulting in CD13 and CD24 upregulation and EpCAM downregulation. Neither miR-200b nor ZEB1 had obvious effects on CD133 or CD90 expression. Silencing CD13 or CD24 expression suppressed tumorigenicity of HCC cells. Ectopic expression of CD24 reversed the suppression of tumorigenicity by ectopic expression of miR-200b. Clinically, miR-200b downregulation was coupled with ZEB1 upregulation in approximately two-thirds of HCC patients. ZEB1 expression was positively correlated with CD13 and CD24 expressions in HCCs, while miR-200b expression was positively correlated with EpCAM. Our findings suggest that the miR-200b-ZEB1 circuit is a master regulator of diverse stemness of HCC, which differentiates HCCs into those containing CD13+ /CD24+ CSCs from those containing EpCAM+ CSCs.

The integrin-binding defective FGF2 mutants potently suppress FGF2 signalling and angiogenesis.

We recently found that integrin αvß3 binds to fibroblast growth factor (FGF)-αvß31 (FGF1), and that the integrin-binding defective FGF1 mutant (Arg-50 to glutamic acid, R50E) is defective in signalling and antagonistic to FGF1 signalling. R50E suppressed angiogenesis and tumour growth, suggesting that R50E has potential as a therapeutic. However, FGF1 is unstable, and we had to express R50E in cancer cells for xenograft study, since injected R50E may rapidly disappear from circulation. We studied if we can develop antagonist of more stable FGF2. FGF2 is widely involved in important biological processes such as stem cell proliferation and angiogenesis. Previous studies found that FGF2 bound to αvß3 and antagonists to αvß3 suppressed FGF2-induced angiogenesis. However, it is unclear how FGF2 interacts with integrins. Here, we describe that substituting Lys-119/Arg-120 and Lys-125 residues in the predicted integrin-binding interface of FGF2 to glutamic acid (the K119E/R120E and K125E mutations) effectively reduced integrin binding to FGF2. These FGF2 mutants were defective in signalling functions (ERK1/2 activation and DNA synthesis) in NIH3T3 cells. Notably they suppressed, FGF2 signalling induced by WT FGF2 in endothelial cells, suggesting that the FGF2 mutants are antagonists. The FGF2 mutants effectively suppressed tube formation in vitro, sprouting in aorta ring assays ex vivo and angiogenesis in vivo The positions of amino acids critical for integrin binding are different between FGF1 and FGF2, suggesting that they do not interact with integrins in the same manner. The newly developed FGF2 mutants have potential as anti-angiogenic agents and useful tools for studying the role of integrins in FGF2 signalling.

Design, synthesis, and application of OB2C combinatorial peptide and peptidomimetic libraries.

The "one-bead two-compound" (OB2C) combinatorial library is constructed on topologically segregated trifunctional bilayer beads such that each bead has a fixed cell-capturing ligand and a random library compound co-displayed on its surface and a chemical coding tag (bar code) inside the bead. An OB2C library containing thousands to millions of compounds can be synthesized and screened concurrently within a short period of time. When live cells are incubated with such OB2C libraries, every bead will be coated with a monolayer of cells. The cell membranes of the captured cells facing the bead surface are exposed to the library compounds tethered to each bead. A specific biochemical or cellular response can be detected with an appropriate reporter system. The OB2C method enables investigators to rapidly discover synthetic molecules that not only interact with cell-surface receptors but can also stimulate or inhibit downstream cell signaling. To demonstrate this powerful method, one OB2C peptide library and two OB2C peptidomimetic libraries were synthesized and screened against Molt-4 lymphoma cells to discover "death ligands." Apoptosis of the bead-bound cells was detected with immunocytochemistry using horseradish peroxidase (HRP)-conjugated anti-cleaved caspase-3 antibody and 3,3'-diaminobenzidine as a substrate. Two novel synthetic "death ligands" against Molt-4 cells were discovered using this OB2C library approach.

MicroRNA-214 downregulation contributes to tumor angiogenesis by inducing secretion of the hepatoma-derived growth factor in human hepatoma.

BACKGROUND & AIMS: Unusual hypervascularity is a hallmark of human hepatocellular carcinoma (HCC). Although microRNA-214 (miR-214) is upregulated in other human cancers, it is downregulated in HCC. We elucidated the biological and clinical significance of miR-214 downregulation in HCC. METHODS: MicroRNAs deregulated in HCC were identified using array-based microRNA profiling. A luciferase reporter assay confirmed target association between miR-214 and the hepatoma-derived growth factor (HDGF). Tube formation and in vivo angiogenesis assays validated the roles of miR-214/HDGF in angiogenesis. RESULTS: miR-214 downregulation was associated with higher tumor recurrence and worse clinical outcomes. Ectopic expression of miR-214 suppressed xenograft tumor growth and microvascularity of the tumors and their surrounding tissues. The genes downregulated by ectopic expression of miR-214 were involved in the regulation of apoptosis, cell cycle, and angiogenesis. Integrated analysis disclosed HDGF as a downstream target of miR-214. Conditioned medium of HCC cells contained bioactivity to stimulate tube formation of human umbilical vein endothelial cells, which was abolished by pretreatment of the conditioned media with HDGF antibodies, suppression of HDGF expression or ectopic expression of miR-214 in the donor HCC cells. The angiogenic activity of the conditioned media, lost by ectopic expression of miR-214 in the donor cells, was restored by supplementation with recombinant HDGF. In vivo tumor angiogenesis assays showed significant suppression of tumor vascularity by ectopic expression of miR-214. CONCLUSIONS: A novel role of microRNA in tumorigenesis is identified. Downregulation of miR-214 contributes to the unusual hypervascularity of HCC via activation of the HDGF paracrine pathway for tumor angiogenesis.

Neuregulin/erythroblastic leukemia viral oncogene homolog 3 autocrine loop contributes to invasion and early recurrence of human hepatoma.

UNLABELLED: Intrahepatic metastasis is the primary cause of the high recurrence and poor prognosis of human hepatocellular carcinoma (HCC). However, neither its molecular mechanisms nor markers for its prediction before hepatectomy have been identified. We recently revealed up-regulation of erythroblastic leukemia viral oncogene homolog 3 (ERBB3) in human HCC. Here we examined the clinical and biological significance of ERBB3 in HCC. Up-regulation of ERBB3 in HCC was strongly associated with male gender (P < 0.001), chronic hepatitis B (P = 0.002), microscopic vascular invasion (P = 0.034), early recurrence (P = 0.003), and worse prognosis (P = 0.004). Phosphorylated ERBB3 and its ligands [neuregulins (NRGs)] were detected in both HCC tissues and cells. Phosphorylation of ERBB3 could be induced by conditioned media of HCC cells and abolished by the pretreatment of conditioned media with anti-NRG antibodies or by the silencing of the endogenous NRG expression of the donor HCC cells. Human epidermal growth factor receptor 2 was required for ERBB3 phosphorylation. The downstream phosphoinositide 3-kinase/v-akt murine thymoma viral oncogene homolog pathways were primarily elicited by NRG1/ERBB3 signaling, whereas the mitogen-activated protein kinase/extracellular signal-regulated kinase pathways were elicited by both epidermal growth factor/epidermal growth factor receptor and NRG1/ERBB3 signaling. The activation and silencing of ERBB3-dependent signaling had potent effects on both the migration and invasion of HCC cells, but neither had significant effects on the proliferation of HCC cells, tumor formation, or tumor growth in vitro and in vivo. CONCLUSION: The constitutive activation of ERBB3-dependent signaling via the NRG1/ERBB3 autocrine loop plays a crucial role in the regulation of cell motility and invasion, which contribute to intrahepatic metastasis and early recurrence of HCC. ERBB3 is a marker for the prediction of intrahepatic metastasis and early recurrence. ERBB3-dependent signaling is a candidate target for the treatment of microscopic vascular invasion and for the prevention of HCC recurrence.

Aberrant activation of nuclear factor of activated T cell 2 in lamina propria mononuclear cells in ulcerative colitis.

AIM: To investigate the role of nuclear factor of activated T cell 2 (NFAT2), the major NFAT protein in peripheral T cells, in sustained T cell activation and intractable inflammation in human ulcerative colitis (UC). METHODS: We used two-dimensional gel-electrophoresis, immunohistochemistry, double immunohistochemical staining, and confocal microscopy to inspect the expression of NFAT2 in 107, 15, 48 and 5 cases of UC, Crohn's disease (CD), non-specific colitis, and 5 healthy individuals, respectively. RESULTS: Up-regulation with profound nucleo-translocation/activation of NFAT2 of lamina propria mononuclear cells (LPMC) of colonic mucosa was found specifically in the affected colonic mucosa from patients with UC, as compared to CD or NC (P < 0.001, Kruskal-Wallis test). Nucleo-translocation/activation of NFAT2 primarily occurred in CD8+T, but was less prominent in CD4+ T cells or CD20+B cells. It was strongly associated with the disease activity, including endoscopic stage (tau = 0.2145, P = 0.0281) and histologic grade (tau = 0.4167, P < 0.001). CONCLUSION: We disclose for the first time the nucleo-translocation/activation of NFAT2 in lamina propria mononuclear cells in ulcerative colitis. Activation of NFAT2 was specific for ulcerative colitis and highly associated with disease activity. Since activation of NFAT2 is implicated in an auto-regulatory positive feedback loop of sustained T-cell activation and NFAT proteins play key roles in the calcium/calcineurin signaling pathways, our results not only provide new insights into the mechanism for sustained intractable inflammation, but also suggest the calcium-calcineurin/NFAT pathway as a new therapeutic target for ulcerative colitis.

Comparative proteomic studies on the pathogenesis of human ulcerative colitis.

Ulcerative colitis (UC) is a chronic inflammatory disorder primarily affecting the colon mucosa. Its etiology and pathogenesis remain unclear. We used 2-DE and MS to identify differentially expressed proteins among the UC active, UC inactive, nonspecific colitis, and normal colon mucosa. Thirteen down-regulated and six up-regulated proteins were identified. Of the down-expressed proteins, eight (heat-shock protein 90 (HSPA9B), heat-shock protein 60 (HSPD1), H+-transporting two-sector ATPase (ATP5B), prohibitin (PHB), mitochondrial malate dehydrogenase (MDH2), voltage-dependent anion-selective channel protein 1 (VDAC1), thioredoxin peroxidase (PRDX1), and thiol-specific antioxidant (PRDX2)) were mitochondrial proteins, three (ATP5B, MDH2, triosephosphate isomerase) were involved in energy generation, three (PRDX1, PRDX2, SELENBP1) were cellular antioxidants, and six (HSPD1, HSPA9B, PRDX1, PRDX2, PHB, VDAC1) were stress-response proteins. Transmission electron microscopy revealed pathological alterations of mitochondrial ultrastructures even before the global colonocyte changes in the UC colon mucosa. PHB, an essential mitochondrial component protein, was down-expressed in the disease active as well as inactive colon mucosa from the patients of UC, indicative of an early event of mitochondrial changes during UC development. In contrast, aberrant activation of NFAT and ectopic expression of potential immunogenic proteins (tumor rejection antigen 1 and poliovirus receptor related protein 1) were found in the UC-diseased colon mucosa. Our findings suggest the implications of colonocyte mitochondrial dysfunction and perturbed mucosa immune regulation in the pathogenesis of UC and provide potential targets for the development of a new therapy.

Complete genomic sequence of the temperate bacteriophage PhiAT3 isolated from Lactobacillus casei ATCC 393.

The complete genomic sequence of a temperate bacteriophage PhiAT3 isolated from Lactobacillus (Lb.) casei ATCC 393 is reported. The phage consists of a linear DNA genome of 39,166 bp, an isometric head of 53 nm in diameter, and a flexible, noncontractile tail of approximately 200 nm in length. The number of potential open reading frames on the phage genome is 53. There are 15 unpaired nucleotides at both 5' ends of the PhiAT3 genome, indicating that the phage uses a cos-site for DNA packaging. The PhiAT3 genome was grouped into five distinct functional clusters: DNA packaging, morphogenesis, lysis, lysogenic/lytic switch, and replication. The amino acid sequences at the NH2-termini of some major proteins were determined. An in vivo integration assay for the PhiAT3 integrase (Int) protein in several lactobacilli was conducted by constructing an integration vector including PhiAT3 int and the attP (int-attP) region. It was found that PhiAT3 integrated at the tRNAArg gene locus of Lactobacillus rhamnosus HN 001, similar to that observed in its native host, Lb. casei ATCC 393.

Dys-regulation of clusterin in human hepatoma is not associated with tumorigenesis but is secondary to cell response to external tresses.

It becomes feasible to perform genome-wide differential gene or protein expression in the post genome era. However, little has been addressed on the effects of external stresses and microenvironment alterations on the outcomes of gene and protein expression. To identify downregulated genes during hepatoma development, we combined the cDNA representational difference analysis (RDA) and reverse Northern blot analysis identifying eight genes. Of interest, the expression of the clusterin gene was either down or upregulated in 8 and 7 out of the 20 hepatoma tissues, respectively. Further analysis revealed that its expression was independent of patients' age, gender, causes of liver disease, tumor size, tumor histological stage, or clinical outcome, but was strongly associated with the methods of hepatectomy procedures. In vitro studies disclosed that the clusterin mRNA was increased twofold in early exponential phase of cell proliferation followed by downregulation in the subsequent quiescence phase, whereas it was rapidly increased up to twelvefold upon UV-induced apoptosis. These results suggest that dys-regulation of the clusterin gene in human hepatoma was most likely due to cellular responses to external stresses especially during the procedures for sample collection rather than any correlation to hepatoma development or progression. Our findings that external stresses or microenvironmental changes could greatly affect gene or protein expression offer a general caution to all the studies conducted via genomic and proteomic approaches.