Identification of a small-molecule compound that inhibits homodimerization of oncogenic NAC1 protein and sensitizes cancer cells to anticancer agents.

Nucleus accumbens-associated protein-1 (NAC1) is a transcriptional repressor encoded by the NACC1 gene, which is amplified and overexpressed in various human cancers and plays critical roles in tumor development, progression, and drug resistance. NAC1 has therefore been explored as a potential therapeutic target for managing malignant tumors. However, effective approaches for effective targeting of this nuclear protein remain elusive. In this study, we identified a core unit consisting of Met7 and Leu90 in NAC1's N-terminal domain (amino acids 1-130), which is critical for its homodimerization and stability. Furthermore, using a combination of computational analysis of the NAC1 dimerization interface and high-throughput screening (HTS) for small molecules that inhibit NAC1 homodimerization, we identified a compound (NIC3) that selectively binds to the conserved Leu-90 of NAC1 and prevents its homodimerization, leading to proteasomal NAC1 degradation. Moreover, we demonstrate that NIC3-mediated down-regulation of NAC1 protein sensitizes drug-resistant tumor cells to conventional chemotherapy and enhances the antimetastatic effect of the antiangiogenic agent bevacizumab both in vitro and in vivo These results suggest that small-molecule inhibitors of NAC1 homodimerization may effectively sensitize cancer cells to some anticancer agents and that NAC1 homodimerization could be further explored as a potential therapeutic target in the development of antineoplastic agents.

Soluble PD-1 is associated with aberrant regulation of T cells activation in aplastic anemia.

Engagement of the membrane program death-1 (PD-1) receptor by its ligands suppresses T cell proliferation and cytokine production. Aberrant over-expression of costimulatory molecules, including PD-1, has been associated with persistent activation of self-reactive T cells in autoimmune diseases. However, the mechanism underlying the dysfunction of PD-1 in the regulation of T-cell activation in such diseases remains unclear. Here, we report the overexpression of CD4(+) and CD8(+) T cell PD-1 and elevated serum levels of soluble PD-1 in aplastic anemia (AA) patients. Detailed characterization of soluble PD-1 revealed that it corresponded to an alternative splice variant PD-1Deltaex3, which lacks the transmembrane domain but has a soluble extracellular domain of the PD-1 molecule. In a further study, PD-1 fusion protein displayed the ability of increasing the proliferation of T cells in vitro, which suggested that soluble PD-1 might serve as an autoimmune antibody to block the function of membrane-bound PD-1 on T cells and lead to aberrant T cell proliferation. Our study revealed a novel pathogenic pathway in which the function of overexpressed PD-1 to restrict over-self-reaction is counteracted by the excessive production of soluble PD-1.

eEF-2 Kinase-targeted miR-449b confers radiation sensitivity to cancer cells.

The roles of microRNA in regulation of various biological processes and in modulation of therapeutic effects have been widely appreciated. In this study, we found a positive correlation between miR-449 b expression and radiation sensitivity in cancer cells and in tumor specimens from patients. We showed that eEF-2 kinase, a negative regulator of global protein synthesis, is a target of miR-449 b. Introducing a miR-449 b mimic into cancer cells led to suppression of eEF-2 kinase expression, leading to increases of protein synthesis and depletion of cellular ATP. Further, we demonstrated that the miR-449 b mimic rendered the cancer cells more sensitive to ionizing radiation both in vitro (cell culture) and in vivo (animal xenograft model). Moreover, the radiation sensitivity conferred by miR-449 b could be blunted by cycloheximide, an inhibitor of protein synthesis, or by direct delivery of ATP liposome, supporting eEF-2 kinase as a mediator of the radio-sensitizing effects of miR-449 b. These results indicate that miR-449 b, which is frequently down-regulated in radio-resistant cancers, may represent a new critical determinant of radio-sensitivity.

Silencing of NAC1 Expression Induces Cancer Cells Oxidative Stress in Hypoxia and Potentiates the Therapeutic Activity of Elesclomol.

In order to survive under conditions of low oxygen, cancer cells can undergo a metabolic switch to glycolysis and suppress mitochondrial respiration in order to reduce oxygen consumption and prevent excessive amounts of reactive oxygen species (ROS) production. Nucleus accumbens-1 (NAC1), a nuclear protein of the BTB/POZ gene family, has pivotal roles in cancer development. Here, we identified that NAC1-PDK3 axis as necessary for suppression of mitochondrial function, oxygen consumption, and more harmful ROS generation and protects cancer cells from apoptosis in hypoxia. We show that NAC1 mediates suppression of mitochondrial function in hypoxia through inducing expression of pyruvate dehydrogenase kinase 3 (PDK3) by HIF-1α at the transcriptional level, thereby inactivating pyruvate dehydrogenase and attenuating mitochondrial respiration. Re-expression of PDK3 in NAC1 absent cells rescued cells from hypoxia-induced metabolic stress and restored the activity of glycolysis in a xenograft mouse model, and demonstrated that silencing of NAC1 expression can enhance the antitumor efficacy of elesclomol, a pro-oxidative agent. Our findings reveal a novel mechanism by which NAC1 facilitates oxidative stress resistance during cancer progression, and chemo-resistance in cancer therapy.

CD8+ T lymphocytes required for enhanced survival of Trypanosoma cruzi-infected mice at elevated environmental temperature.

C3H mice, which are highly susceptible to infection with the Brazil strain of Trypanosoma cruzi, not only survive infection when maintained at elevated environmental temperature, but also experience reduced parasitemias, reduced tissue pathology, and enhanced immune responsiveness. The contribution of CD8+ T cells to this phenomenon was investigated using the in vivo cell-depletion technique. The depletion of CD8+ T lymphocytes with purified monoclonal antibody resulted in an abrogation of the protective effects of elevated environmental temperature during T. cruzi infection, as evidenced by high parasitemia levels and 100% mortality in monoclonal antibody-treated mice.

[Effect of total glucosides of Centella asiatica on antagonizing liver fibrosis induced by dimethylnitrosamine in rats].

OBJECTIVE: To investigate the anti-liver fibrosis effect of total glucosides of Centella asiatica (GCA) in experimental rats. METHODS: Rat liver fibrosis model was induced by injecting dimethylnitrosamine (DMN) intraperitoneally for 6 weeks. Rats were randomly divided into 6 groups, the normal group, the model group, the positive control group treated by colchicine, and the three GCA groups treated by high, moderate and low dosage of GCA through gastrogavage started simultaneously with the modeling. At the end of the experiment, levels of serum total protein (TP), albumin (ALB), alanine transaminase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), laminin (LN) were measured, and the pathology of liver tissue was observed. RESULTS: The liver function in the GCA groups was improved, the levels of serum ALT, AST, HA were significantly lower than those in the model group (P < 0.05). Histopathological observation showed that GCA has significant anti-liver fibrosis effect. CONCLUSION: GCA has significant preventive and therapeutic effect on DMN induced liver fibrosis in rats.

Association between polymorphisms in PDCD1 gene and aplastic anemia in Chinese Han population.

Single nucleotide polymorphism (SNP) of programmed cell death 1 (PD-1, encoded by PDCD1) has been reported to be associated with several autoimmune diseases including rheumatoid arthritis (RA), Graves' disease and multiple sclerosis (MS). In order to study the correlation between PD-1 gene polymorphism and aplastic anemia in a Chinese Han population, two SNPs, PD-1.1 G/A (rs36084323) and PD-1.6 G/A (rs10204525), were genotyped in 166 patients with aplastic anemia and 144 healthy controls by direct sequencing. All genotype distributions in both patients and controls were in Hardy-Weinberg equilibrium. Associations of genotypes and alleles with aplastic anemia were analyzed. The results suggested that the G allele of PD-1.1 was associated with an increased risk for aplastic anemia, while SNP of PD-1.6 was not associated with aplastic anemia in a Chinese Han population.

[Preparation of two mouse anti-human PD-1 monoclonal antibodies and identification of their biological characteristics].

AIM: To prepare mouse anti-human PD-1 monoclonal antibodies (mAbs) and identify their biological characteristics. METHODS: The BALB/c mice were immunized with the transfected cell line PD-1/L929. The cells were fused with Sp2/0 using monoclonal antibody techniques and the positive clones were screened by FCS with PD-1/L929. The secreted anti-PD-1 mAbs were identified through rapid isotyping analysis, karyotype analysis, Western blot, competitive inhibition test, indirect immunofluorescence assay, and tumor cell lines detection. RESULTS: Two mouse anti-human PD-1 hybridomas were obtained and their secreted mAbs were named (1F2 and 5F10). Their biological characteristics suggested that they could recognize a protein with approximate molecular weight 55 000 on PD-1/L929 cell lines and different epitopes. 1F2 could recognize PD-1 molecules expressed on SKHep-1 and 7721 while 5F10 could recognize Raji cells. CONCLUSION: Two mouse anti-human PD-1 hybridoma cell lines and their secreted monoclonal antibodies have been successfully obtained and identified.