A Novel Time-Dependent CENP-E Inhibitor with Potent Antitumor Activity

Centromere-associated protein E (CENP-E) regulates both chromosome congression and the spindle assembly checkpoint (SAC) during mitosis. The loss of CENP-E function causes chromosome misalignment, leading to SAC activation and apoptosis during prolonged mitotic arrest. Here, we describe the biological and antiproliferative activities of a novel small-molecule inhibitor of CENP-E, Compound-A (Cmpd-A). Cmpd-A inhibits the ATPase activity of the CENP-E motor domain, acting as a time-dependent inhibitor with an ATP-competitive-like behavior. Cmpd-A causes chromosome misalignment on the metaphase plate, leading to prolonged mitotic arrest. Treatment with Cmpd-A induces antiproliferation in multiple cancer cell lines. Furthermore, Cmpd-A exhibits antitumor activity in a nude mouse xenograft model, and this antitumor activity is accompanied by the elevation of phosphohistone H3 levels in tumors. These findings demonstrate the potency of the CENP-E inhibitor Cmpd-A and its potential as an anticancer therapeutic agent.     

B7-H1 and a Mathematical Model for Cytotoxic T Cell and Tumor Cell Interaction

The surface protein B7-H1, also called PD-L1 and CD274, is found on carcinomas of the lung, ovary, colon, and melanomas but not on most normal tissues. B7-H1 has been experimentally determined to be an antiapoptotic receptor on cancer cells, where B7-H1-positive cancer cells have been shown to be immune resistant, and in vitro experiments and mouse models have shown that B7-H1-negative tumor cells are significantly more susceptible to being repressed by the immune system. We derive a new mathematical model for studying the interaction between cytotoxic T cells and tumor cells as affected by B7-H1. By integrating experimental data into the model, we isolate the parameters that control the dynamics and obtain insights on the mechanisms that control apoptosis.     

A20 Controls Macrophage to Elicit Potent Cytotoxic CD4+ T Cell Response

Emerging evidence indicates that CD4⁺ T cells possess cytotoxic potential for tumor eradication and perforin/granzyme-mediated cytotoxicity functions as one of the important mechanisms for CD4⁺ T cell-triggered cell killing. However, the critical issue is how the cytotoxic CD4⁺ T cells are developed. During the course of our work that aims at promoting immunostimulation of APCs by inhibition of negative regulators, we found that A20-silenced Mф drastically induced granzyme B expression in CD4⁺ T cells. As a consequence, the granzyme-highly expressing CD4⁺ T cells exhibited a strong cytotoxic activity that restricted tumor development. We found that A20-silenced Mф activated cytotoxic CD4⁺ T cells by MHC class-II restricted mechanism and the activation was largely dependent on enhanced production of IFN-γ.     

Human Antigen-Specific Regulatory T Cells Generated by T Cell Receptor Gene Transfer

Background

Therapies directed at augmenting regulatory T cell (Treg) activities in vivo as a systemic treatment for autoimmune disorders and transplantation may be associated with significant off-target effects, including a generalized immunosuppression that may compromise beneficial immune responses to infections and cancer cells. Adoptive cellular therapies using purified expanded Tregs represents an attractive alternative to systemic treatments, with results from animal studies noting increased therapeutic potency of antigen-specific Tregs over polyclonal populations. However, current methodologies are limited in terms of the capacity to isolate and expand a sufficient quantity of endogenous antigen-specific Tregs for therapeutic intervention. Moreover, FOXP3+ Tregs fall largely within the CD4+ T cell subset and are thus routinely MHC class II-specific, whereas class I-specific Tregs may function optimally in vivo by facilitating direct tissue recognition.

Methodology/Principal Findings

To overcome these limitations, we have developed a novel means for generating large numbers of antigen-specific Tregs involving lentiviral T cell receptor (TCR) gene transfer into in vitro expanded polyclonal natural Treg populations. Tregs redirected with a high-avidity class I-specific TCR were capable of recognizing the melanoma antigen tyrosinase in the context of HLA-A*0201 and could be further enriched during the expansion process by antigen-specific reactivation with peptide loaded artificial antigen presenting cells. These in vitro expanded Tregs continued to express FOXP3 and functional TCRs, and maintained the capacity to suppress conventional T cell responses directed against tyrosinase, as well as bystander T cell responses. Using this methodology in a model tumor system, murine Tregs designed to express the tyrosinase TCR effectively blocked antigen-specific effector T cell (Teff) activity as determined by tumor cell growth and luciferase reporter-based imaging.

Conclusions/Significance

These results support the feasibility of class I-restricted TCR transfer as a promising strategy to redirect the functional properties of Tregs and provide for a more efficacious adoptive cell therapy.     

Novel Gallate Triphenylphosphonium Derivatives with Potent Antichagasic Activity

Chagas disease is one of the most neglected tropical diseases in the world, affecting nearly 15 million people, primarily in Latin America. Only two drugs are used for the treatment of this disease, nifurtimox and benznidazole. These drugs have limited efficacy and frequently induce adverse effects, limiting their usefulness. Consequently, new drugs must be found. In this study, we demonstrated the in vitro trypanocidal effects of a series of four gallic acid derivatives characterized by a gallate group linked to a triphenylphosphonium (TPP⁺) moiety (a delocalized cation) via a hydrocarbon chain of 8, 10, 11, or 12 atoms (TPP⁺-C₈, TPP⁺-C₁₀, TPP⁺-C₁₁, and TPP⁺-C₁₂, respectively). We analyzed parasite viability in isolated parasites (by MTT reduction and flow cytometry) and infected mammalian cells using T. cruzi Y strain trypomastigotes. Among the four derivatives, TPP⁺-C₁₀ and TPP⁺-C₁₂ were the most potent in both models, with EC₅₀ values (in isolated parasites) of 1.0 ± 0.6 and 1.0 ± 0.7 μM, respectively, and were significantly more potent than nifurtimox (EC₅₀ = 4.1 ± 0.6 μM). At 1 μM, TPP⁺-C₁₀ and TPP⁺-C₁₂ induced markers of cell death, such as phosphatidylserine exposure and propidium iodide permeabilization. In addition, at 1 μM, TPP⁺-C₁₀ and TPP⁺-C₁₂ significantly decreased the number of intracellular amastigotes (TPP⁺-C₁₀: 24.3%, TPP⁺-C₁₂: 19.0% of control measurements, as measured by DAPI staining) and the parasite’s DNA load (C₁₀: 10%, C₁₂: 13% of control measurements, as measured by qPCR). Based on the previous mode of action described for these compounds in cancer cells, we explored their mitochondrial effects in isolated trypomastigotes. TPP⁺-C₁₀ and TPP⁺-C₁₂ were the most potent compounds, significantly altering mitochondrial membrane potential at 1 μM (measured by JC-1 fluorescence) and inducing mitochondrial transition pore opening at 5 μM. Taken together, these results indicate that the TPP⁺-C₁₀ and TPP⁺-C₁₂ derivatives of gallic acid are promising trypanocidal agents with mitochondrial activity.     

L1 Cell Adhesion Molecule-Specific Chimeric Antigen Receptor-Redirected Human T Cells Exhibit Specific and Efficient Antitumor Activity against Human Ovarian Cancer in Mice

New therapeutic modalities are needed for ovarian cancer, the most lethal gynecologic malignancy. Recent clinical trials have demonstrated the impressive therapeutic potential of adoptive therapy using chimeric antigen receptor (CAR)-redirected T cells to target hematological cancers, and emerging studies suggest a similar impact may be achieved for solid cancers. We sought determine whether genetically-modified T cells targeting the CE7-epitope of L1-CAM, a cell adhesion molecule aberrantly expressed in several cancers, have promise as an immunotherapy for ovarian cancer, first demonstrating that L1-CAM was highly over-expressed on a panel of ovarian cancer cell lines, primary ovarian tumor tissue specimens, and ascites-derived primary cancer cells. Human central memory derived T cells (T_CM) were then genetically modified to express an anti-L1-CAM CAR (CE7R), which directed effector function upon tumor antigen stimulation as assessed by in vitro cytokine secretion and cytotoxicity assays. We also found that CE7R⁺ T cells were able to target primary ovarian cancer cells. Intraperitoneal (i.p.) administration of CE7R⁺ T_CM induced a significant regression of i.p. established SK-OV-3 xenograft tumors in mice, inhibited ascites formation, and conferred a significant survival advantage compared with control-treated animals. Taken together, these studies indicate that adoptive transfer of L1-CAM-specific CE7R⁺ T cells may offer a novel and effective immunotherapy strategy for advanced ovarian cancer.     

Modification of Cytotoxic Lymphocytes with T Cell Receptor Specific for Minor Histocompatibility Antigen ACC-1Y

Molecular Biology - Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative therapy for hematopoietic malignancies. The graft-derived donor lymphocytes are capable of...     

Combined TLR2/4-Activated Dendritic/Tumor Cell Fusions Induce Augmented Cytotoxic T Lymphocytes

Induction of antitumor immunity by dendritic cell (DC)-tumor fusion cells (DC/tumor) can be modulated by their activation status. In this study, to address optimal status of DC/tumor to induce efficient antigen-specific cytotoxic T lymphocytes (CTLs), we have created various types of DC/tumor: 1) un-activated DC/tumor; 2) penicillin-killed Streptococcus pyogenes (OK-432; TLR4 agonist)-activated DC/tumor; 3) protein-bound polysaccharides isolated from Coriolus versicolor (PSK; TLR2 agonist)-activated DC/tumor; and 4) Combined OK-432- and PSK-activated DC/tumor. Moreover, we assessed the effects of TGF-β1 derived from DC/tumor on the induction of MUC1-specific CTLs. Combined TLR2- and TLR4-activated DC/tumor overcame immune-suppressive effect of TGF-β1 in comparison to those single activated or un-activated DC/tumor as demonstrated by: 1) up-regulation of MHC class II and CD86 expression on DC/tumor; 2) increased fusion efficiency; 3) increased production of fusions derived IL-12p70; 4) activation of CD4⁺ and CD8⁺ T cells that produce high levels of IFN-γ; 5) augmented induction of CTL activity specific for MUC1; and 6) superior efficacy in inhibiting CD4⁺CD25⁺Foxp3⁺ T cell generation. However, DC/tumor-derived TGF-β1 reduced the efficacy of DC/tumor vaccine in vitro. Incorporating combined TLRs-activation and TGF-β1-blockade of DC/tumor may enhance the effectiveness of DC/tumor-based cancer vaccines and have the potential applicability to the field of adoptive immunotherapy.     

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the radionuclide Chromium 51 (⁵¹Cr) to label target cells. These targets are then exposed to effector cells and the release of ⁵¹Cr from target cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of ⁵¹Cr. As effector cells, we used an activated autoreactive clonal population of CD8⁺ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with ⁵¹Cr labeled target NIT cells for 16 hours, release of ⁵¹Cr was recorded to calculate specific lysis Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37°C, with 5% CO₂, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.     

Biosynthesis of Sibiromycin,a Potent Antitumor Antibiotic

Pyrrolobenzodiazepines, a class of natural products produced by actinomycetes, are sequence selective DNA alkylating compounds with significant antitumor properties. Among the pyrrolo[1,4]benzodiazepines (PBDs) sibiromycin, one of two identified glycosylated PBDs, displays the highest affinity for DNA and the most potent antitumor properties. Despite the promising antitumor properties clinical trials of sibiromycin were precluded by the cardiotoxicity effect in animals attributed to the presence of the C-9 hydroxyl group. As a first step toward the development of sibiromycin analogs, we have cloned and localized the sibiromycin gene cluster to a 32.7-kb contiguous DNA region. Cluster boundaries tentatively assigned by comparative genomics were verified by gene replacement experiments. The sibiromycin gene cluster consisting of 26 open reading frames reveals a “modular” strategy in which the synthesis of the anthranilic and dihydropyrrole moieties is completed before assembly by the nonribosomal peptide synthetase enzymes. In addition, the gene cluster identified includes open reading frames encoding enzymes involved in sibirosamine biosynthesis, as well as regulatory and resistance proteins. Gene replacement and chemical complementation studies are reported to support the proposed biosynthetic pathway.Pyrrolo[1,4]benzodiazepines (PBDs) are a class of natural products found in actinomycetes (Fig. ​(Fig.1)1) and defined by a common pyrrolo[1,4]benzodiazepine ring system (41). They are sequence-selective DNA alkylating agents with significant antitumor properties (21). Once in the minor groove of DNA an aminal bond is formed between the electrophilic C-11 of a PBD and the exocyclic N-2 of a guanine base in a double-stranded DNA (20). Formation of the PBD-DNA complex causes very little distortion of the double-helical structure of DNA (20), and as such this complex is less readily repaired by DNA repair proteins compared to DNA adducts with other alkylating agents (4), significantly contributing to the potency of PBDs. Successful syntheses of PBD analogs have been reported, but synthetic procedures for the more chemically diverse PBDs are laborious and have modest yields (1, 44). In addition, a chemical synthesis for glycosylated PBDs has not yet been accomplished. Structure-activity relationship studies on the synthetically and naturally produced PBDs showed that the C-9 hydroxylation present in anthramycin is the source of the cardiotoxic properties of this compound (Fig. ​(Fig.1)1) (3, 17, 26, 38). These studies also showed that O glycosylation at C7 significantly enhanced DNA-binding affinity (Fig. ​(Fig.1)1) (17). The only known glycosylated PBDs are sibiromycin and sibanomicin produced by Streptosporangium sibiricum and Micromonospora sp., respectively, both containing a sibirosamine moiety (16, 35). Only the producer of sibiromycin is commercially available. A loose correlation between DNA binding affinity and cytotoxicity has been shown with naturally and synthetically produced PBDs (42). Sibiromycin has the highest DNA binding affinity and cytotoxicity with 50% inhibitory concentrations varying from 4 to 1.7 pM in leukemia, plasmacytoma, and ovarian cancer cell lines (42). Despite its potency, further testing of sibiromycin is precluded due to the presence of C-9 hydroxyl group responsible for the cardiotoxic properties. In order to generate analogs of glycosylated PBDs by combinatorial biosynthesis and to exploit their potency, we chose to characterize the sibiromycin gene cluster.Open in a separate windowFIG. 1.(A) Pyrrolobenzodiazepine common ring system. (B) Metabolic precursors and chemical structures of sibiromycin, anthramycin, tomaymycin, and lincomycin A.The metabolic precursors of the pyrrolobenzodiazepine ring of three PBDs (anthramycin, sibiromycin, and tomaymycin) were identified by feeding experiments to be l-tryptophan via the kynurenine pathway for the anthranilate moiety and l-tyrosine for the hydropyrrole moiety (11), suggesting a common biosynthetic pathway for these moieties in PBDs. The tyrosine-to-hydropyrrole transformation has been also identified by feeding studies in the biosynthesis of lincomycin, a lincosamide antibiotic (2) (Fig. ​(Fig.1B).1B). Despite the sequencing of the biosynthetic gene clusters of anthramycin (10) and lincomycin (37), limited functional assignment of open reading frames (ORFs) and elucidation of the biosynthetic pathways were reported partly due to the presence of several gene products with no significant similarities to functionally characterized enzymes. We reasoned that we could take advantage of the identification of the sibiromycin gene cluster not only to try to lay the groundwork for the production of analogs of sibiromycin by combinatorial biosynthesis but also to establish the biosynthetic pathways of the anthranilate and the hydropyrrole moieties by a comparative analysis of the PBDs and lincomycin gene clusters. To help in this analysis, we have also utilized the gene cluster of another PBD, tomaymycin, whose characterization is reported in the accompanying study (24a). The comparative analysis takes advantage of the presence of similarity and differences at the anthranilate and hydropyrrole moieties among these natural products (Fig. ​(Fig.1).1). For example, both anthramycin and sibiromycin contain C-8 methyl and C-9 hydroxyl substituents not present in tomaymycin. However, tomaymycin shares with sibiromycin a C-7 hydroxyl substituent. Therefore, homologous proteins involved in C-9 hydroxylation are expected to be present in the anthramycin and sibiromycin gene cluster but absent in the tomaymycin gene cluster. We applied a similar approach for the biosynthesis of the hydropyrrole moiety using also the lincomycin gene cluster.In the present study, we describe the cloning and sequencing of the sibiromycin gene cluster, the first biosynthetic gene cluster for a glycosylated PBD. Gene replacement experiments were used to confirm that the identified gene cluster was involved in sibiromycin biosynthesis, to define the boundaries of the sibiromycin gene cluster, and to elucidate the biosynthesis of the anthranilate moiety. Using the comparative approach, we were able not only to elucidate the sibiromycin biosynthetic pathway with a certain degree of confidence but also to assign ORFs in the anthramycin gene cluster contributing to the determination of the anthramycin biosynthetic pathway. The proposed biosynthetic pathway for the anthranilic moiety was supported by gene replacement and chemical complementation studies. The data reported here provide the basis for future studies on the enzymes involved in the biochemistry present in these pathways and for combinatorial biosynthetic experiments for the production of glycosylated PBDs.     

Potent Anti-Inflammatory Activity of Novel Microtubule-Modulating Brominated Noscapine Analogs

Noscapine, a plant-derived, non-toxic, over-the-counter antitussive alkaloid has tubulin-binding properties. Based upon the structural resemblance of noscapine to colchicine, a tubulin-binding anti-inflammatory drug, noscapine and its semi-synthetic brominated analogs were examined for in vitro anti-inflammatory activity. Brominated noscapine analogs were found to inhibit cytokine and chemokine release from macrophage cell lines but did not affect cell viability. Brominated noscapine analogs demonstrated anti-inflammatory properties in both TLR- and non-TLR induced in vitro innate immune pathway inflammation models, mimicking septic and sterile infection respectively. In addition, electron microscopy and immunoblotting data indicated that these analogs induced robust autophagy in human macrophages. This study is the first report to identify brominated noscapines as innate immune pathway anti-inflammatory molecules.     

shRNA Expression Plasmids Generated by a Novel Method Efficiently Induce Gene-Specific Knockdown in a Silkworm Cell Line

RNAi knockdown by using shRNA expression plasmids is widely used to determine the function of individual genes in mammals. Here we developed a simple method to create an IR DNA in a U6 small nuclear RNA promoter-based parent vector using a single-stranded IR DNA with short hairpin structure and Bst DNA polymerase. Furthermore, we demonstrated that the shRNA expression plasmids constructed by our method effectively induced target-specific RNAi in the silkworm cell line. We also found that sequence preference in the silkworm cell line was much lower than in mammalian cells and shRNA-induced RNAi was influenced by the length of the stem region.     

一种新型肿瘤血管抗体Fab的基因克隆与表达

鼠单克隆抗体AA98是我室研制的一株新型抗肿瘤血管抗体,体内实验证明它可以抑制血管生成,抑制肿瘤生长。从分泌抗体AA98的杂交瘤细胞中提取总RNA,采用逆转录PCR(RT-PCR)分别扩增重链Fd及轻链κ,并进行序列测定。将Fd和κ链依次与噬菌粒pComb3H连接,构建pComb3H-AA98Fab表达载体。在大肠杆菌中表达了AA98Fab。免疫印迹表明该Fab片段识别分子量为100kD的蛋白,具有原抗体AA98的抗原特异性。AA98Fab是研究抗体AA98作用机理的工具,也为制备重组免疫毒素,尝试肿瘤血管靶向治疗实验打下了基础。     

海南文昌清澜港红树林真菌抗B16肿瘤细胞活性菌株的筛选

从海南文昌清澜港红树林采集78份植物组织和土壤样品。对样品进行真菌分离, 共分离得到真菌608株, 采用目测法对其体外抗肿瘤细胞活性进行检测, 发现81株红树林真菌对小鼠黑色素瘤B16细胞有不同程度的抑制作用, 占总分离菌株的13.32%。结果表明, 从红树植物杯萼海桑中分离得到真菌菌株的数量最多, 而从红树植物银叶树分离得到的抗肿瘤细胞活性菌株数量最多。     

海南文昌清澜港红树林真菌抗B16肿瘤细胞活性菌株的筛选

从海南文昌清澜港红树林采集78份植物组织和土壤样品。对样品进行真菌分离, 共分离得到真菌608株, 采用目测法对其体外抗肿瘤细胞活性进行检测, 发现81株红树林真菌对小鼠黑色素瘤B16细胞有不同程度的抑制作用, 占总分离菌株的13.32%。结果表明, 从红树植物杯萼海桑中分离得到真菌菌株的数量最多, 而从红树植物银叶树分离得到的抗肿瘤细胞活性菌株数量最多。     

Antitumor Activity of IMC-038525, a Novel Oral Tubulin Polymerization Inhibitor

Microtubules are a well-validated target for anticancer therapy. Molecules that bind tubulin affect dynamic instability of microtubules causing mitotic arrest of proliferating cells, leading to cell death and tumor growth inhibition. Natural antitubulin agents such as taxanes and Vinca alkaloids have been successful in the treatment of cancer; however, several limitations have encouraged the development of synthetic small molecule inhibitors of tubulin function. We have previously reported the discovery of two novel chemical series of tubulin polymerization inhibitors, triazoles (Ouyang et al. Synthesis and structure-activity relationships of 1,2,4-triazoles as a novel class of potent tubulin polymerization inhibitors. Bioorg Med Chem Lett. 2005; 15:5154-5159) and oxadiazole derivatives (Ouyang et al. Oxadiazole derivatives as a novel class of antimitotic agents: synthesis, inhibition of tubulin polymerization, and activity in tumor cell lines. Bioorg Med Chem Lett. 2006; 16:1191-1196). Here, we report on the anticancer effects of a lead oxadiazole derivative in vitro and in vivo. In vitro, IMC-038525 caused mitotic arrest at nanomolar concentrations in epidermoid carcinoma and breast tumor cells, including multidrug-resistant cells. In vivo, IMC-038525 had a desirable pharmacokinetic profile with sustained plasma levels after oral dosing. IMC-038525 reduced subcutaneous xenograft tumor growth with significantly greater efficacy than the taxane paclitaxel. At efficacious doses, IMC-038525 did not cause substantial myelosuppression or peripheral neurotoxicity, as evaluated by neutrophil counts and changes in myelination of the sciatic nerve, respectively. These data indicate that IMC-038525 is a promising candidate for further development as a chemotherapeutic agent.