Zhongyun Dong

Cutting edge : role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins

The Toll-like receptor (TLR) family acts as pattern recognition receptors for pathogen-specific molecular patterns (PAMPs). TLR2 is essential for the signaling of a variety of PAMPs, including bacterial lipoprotein/lipopeptides, peptidoglycan, and GPI anchors. TLR6 associates with TLR2 and recognizes diacylated mycoplasmal lipopeptide along with TLR2. We report here that TLR1 associates with TLR2 and recognizes the native mycobacterial 19-kDa lipoprotein along with TLR2. Macrophages from TLR1-deficient (TLR1−/−) mice showed impaired proinflammatory cytokine production in response to the 19-kDa lipoprotein and a synthetic triacylated lipopeptide. In contrast, TLR1−/− cells responded normally to diacylated lipopeptide. TLR1 interacts with TLR2 and coexpression of TLR1 and TLR2 enhanced the NF-κB activation in response to a synthetic lipopeptide. Furthermore, lipoprotein analogs whose acylation was modified were preferentially recognized by TLR1. Taken together, TLR1 interacts with TLR2 to recognize the lipid configuration of the native mycobacterial lipoprotein as well as several triacylated lipopeptides.

MACROPHAGE-DERIVED METALLOELASTASE IS RESPONSIBLE FOR THE GENERATION OF ANGIOSTATIN IN LEWIS LUNG CARCINOMA

To determine the mechanism responsible for the in vivo production of angiostatin that inhibits growth and metastasis in Lewis lung carcinoma (3LL), we implanted 3LL variant cells into the subcutis of syngeneic C57BL/6 mice. The tumors were infiltrated by macrophages and expressed high levels of steady-state mRNA for metalloelastase (MME). Successive passages (more than three) of cultures established from the tumors resulted in complete depletion of macrophages; steady-state MME mRNA, elastinolytic activity, and production of angiostatin (in the presence of plasminogen) were correspondingly reduced. Coculture of macrophages with either 3LL cells or their conditioned media containing granulocyte-macrophage colony-stimulating factor resulted in secretion of MME and production of angiostatin by the macrophages, suggesting that angiostatin is produced by tumor-infiltrating macrophages whose MME expression is stimulated by tumor cell-derived granulocyte-macrophage colony-stimulating factor.

Lipopolysaccharide-induced metastatic growth is associated with increased angiogenesis, vascular permeability and tumor cell invasion.

Endotoxin/lipopolysaccharide (LPS), a cell wall component of Gram‐negative bacteria, is a potent inflammatory stimulus. We previously reported that LPS increased the growth of experimental metastases in a murine tumor model. Here, we examined the effect of LPS exposure on key determinants of metastasis—angiogenesis, tumor cell invasion, vascular permeability, nitric oxide synthase (NOS) and matrix metalloproteinase 2 (MMP2) expression. BALB/c mice bearing 4T1 lung metastases were given an intraperitoneal (i.p.) injection of 10 μg LPS or saline. LPS exposure resulted in increased lung weight and incidence of pleural lesions. LPS increased angiogenesis both in vivo and in vitro. Vascular permeability in lung tissue was increased 18 hr after LPS injection. LPS increased inducible nitric oxide synthase (iNOS) and MMP2 expression in lung tumor nodules. 4T1 cells transfected with green fluorescent protein (4T1‐GFP) were injected via lateral tail vein. LPS exposure resulted in increased numbers of 4T1‐GFP cells in mouse lung tissue compared to saline controls, an effect blocked by the competitive NOS inhibitor, NG methyl‐L‐arginine (NMA). LPS‐induced growth and metastasis of 4T1 experimental lung metastases is associated with increased angiogenesis, vascular permeability and tumor cell invasion/migration with iNOS expression implicated in LPS‐induced metastasis.

Fluorescent, Superparamagnetic Nanospheres for Drug Storage, Targeting, and Imaging: A Multifunctional Nanocarrier System for Cancer Diagnosis and Treatment

For early cancer diagnosis and treatment, a nanocarrier system is designed and developed with key components uniquely structured at nanoscale according to medical requirements. For imaging, quantum dots with emissions in the near-infrared range (∼800 nm) are conjugated onto the surface of a nanocomposite consisting of a spherical polystyrene matrix (∼150 nm) and the internally embedded, high fraction of superparamagnetic Fe(3)O(4) nanoparticles (∼10 nm). For drug storage, the chemotherapeutic agent paclitaxel (PTX) is loaded onto the surfaces of these composite multifunctional nanocarriers by using a layer of biodegradable poly(lactic-co-glycolic acid) (PLGA). A cell-based cytotoxicity assay is employed to verify successful loading of pharmacologically active drug. Cell viability of human, metastatic PC3mm2 prostate cancer cells is assessed in the presence and absence of various multifunctional nanocarrier populations using the MTT assay. PTX-loaded composite nanocarriers are synthesized by conjugating anti-prostate specific membrane antigen (anti-PSMA) for targeting. Specific detection studies of anti-PSMA-conjugated nanocarrier binding activity in LNCaP prostate cancer cells are carried out. LNCaP cells are targeted successfully in vitro by the conjugation of anti-PSMA on the nanocarrier surfaces. To further explore targeting, the nanocarriers conjugated with anti-PSMA are intravenously injected into tumor-bearing nude mice. Substantial differences in fluorescent signals are observed ex vivo between tumor regions treated with the targeted nanocarrier system and the nontargeted nanocarrier system, indicating considerable targeting effects due to anti-PSMA functionalization of the nanocarriers.

Modulation of tumor cell response to chemotherapy by the organ environment

The outcome of cancer metastasis depends on the interaction of metastatic cells with various host factors. The implantation of human cancer cells into anatomically correct (orthotopic) sites in nude mice can be used to ascertain their metastatic potential. While it is clear that vascularity and local immunity can retard or facilitate tumor growth, we have found that the organ environment also influences tumor cell functions such as production of degradative enzymes. The organ microenvironment can also influence the response of metastases to chemotherapy. It is not uncommon to observe the regression of cancer metastases in one organ and their continued growth in other sites after systemic chemotherapy. We demonstrated this effect in a series of experiments using a murine fibrosarcoma, a murine colon carcinoma, and a human colon carcinoma. The tumor cells were implanted subcutaneously or into different visceral organs. Subcutaneous tumors were sensitive to doxorubicin (DXR), whereas lung or liver metastases were not. In contrast, sensitivity to 5-FU did not differ between these sites of growth. The differences in response to DXR between s.c. tumors (sensitive) and lung or liver tumors (resistant) were not due to variations in DXR potency or DXR distribution. The expression of the multidrug resistance-associated P-glycoprotein as determined by flow cytometric analysis of tumor cells harvested from lesions in different organs correlated inversely with their sensitivity to DXR: increased P-glycoprotein was associated with overexpression ofmdr1 mRNA. However, the organ-specific mechanism for upregulatingmdr1 and P-glycoprotein has yet to be elucidated.

ACTIVATION OF NITRIC OXIDE SYNTHASE GENE FOR INHIBITION OF CANCER METASTASIS

The process of cancer metastasis consists of multiple sequential and highly selective steps. The vast majority of tumor cells that enter the circulation die rapidly and only a few survive and proliferate to form distant metastases. This survival is not random. Metastases are clonal in origin and are produced by specialized subpopulations of cells that preexist in a heterogeneous primary tumor. Metastatic cells of the murine K‐1735 melanoma survive in the circulation to produce experimental lung metasteses, whereas nonmetastatic cells do not. After incubation with different cytokines or LPS, nonmetastatic cells exhibit a high level of inducible nitric oxide synthase (iNOS) activity and nitric oxide (NO) production, whereas metastatic cells do not. To provide direct evidence for the inverse correlation between the production of endogenous NO and the ability of K‐1735 cells to produce metastasis in syngeneic mice, highly metastatic clone 4 cells (C4.P), which express low levels of iNOS, were transfected with a functional iNOS (C4.L8), inactive mutated iNOS (C4.S2), or neomycin resistance (C4.Neo) genes in medium containing 3 mM NMA. C4.P, C4.Neo3, and C4.S2.3 cells were highly metastatic, whereas C4.L8.5 cells were not. Moreover, C4.L8.5 cells produced slow‐growing subcutaneous tumors in nude mice, whereas the other three cell lines produced fast‐growing tumors. In vitro studies indicated that the expression of iNOS in C4.L8.5 cells was associated with apoptosis. Multiple intravenous injections of liposomes containing a synthetic lipopeptide up‐regulated iNOS expression in murine M5076 reticulum sarcoma cells growing as hepatic metastases. The induction of iNOS was associated with the complete regression of the lesions. Collectively, these data demonstrate that the expression of iNOS in tumor cells is associated with apoptosis, suppression of tumorigenicity, abrogation of metastasis, and regression of established hepatic metastases.

Revolutionizing biodegradable metals

Development of biodegradable metal implants is a complex problem because it combines engineering and medical requirements for a material. This article discusses the development of sensing and corrosion control techniques that can help in the design of biodegradable metallic implants. Biodegradable metallic implants dissolve as new tissue is formed. One of the most important factors in the design of biodegradable implants is to study the active interface, which should be monitored and controlled to address the medical concern of biocompatibility. Thus miniaturized and nanotechnology-based sensors that measure the activities of the degradation process and the formation of tissue are discussed for use with in vitro and in vivo experiments. These sensors can monitor chemical components and also cell activity and can provide new knowledge about biodegradable interfaces and how to actively control the interface to provide the best bioactivity to regenerate new tissue in a short time. Development of new alloys, nano-materials, miniature sensors, corrosion control coatings, and auxiliary applications such as biodegradable drug delivery capsules is expected to open up a new era in the engineering of materials for medicine.

Nanotube electrodes and biosensors

This article reviews the state of the art in carbon nanotube electrode and biosensor research. Carbon nanotubes have unique mechanical, electrical, and geometrical properties that are ideal for developing different types of nanoscale electrodes and biosensors. Carbon nanotube synthesis and subsequent functionalization strategies to immobilize special biomolecules are discussed first. Then different types of carbon nanotube biosensors and electroanalytical methods are reviewed particularly considering their capabilities for low detection limits, point-of-care applications, and label-free use. Detection strategies for proteins and nucleic acids, as well as mammalian and bacterial cells are also outlined. We conclude with some speculations and predictions on future exciting and challenging directions for nanotube biosensor research and applications.

Activation of tumoricidal properties in macrophages by lipopolysaccharide requires protein-tyrosine kinase activity

The purpose of these studies was to determine whether triggering murine peritoneal macrophages to a tumoricidal state by lipopolysaccharide (LPS) requires protein‐tyrosine phosphorylation. The LPS‐triggered activation of mouse macrophages to lyse syngeneic B16 melanoma cells was significantly inhibited in a dose‐ dependent manner by the protein‐tyrosine kinase (PTK) inhibitors genistein, herbimycin A, and tyrphostin. Genistein was effective only when added to macrophages prior to or simultaneously with LPS. Genistein potently inhibited the productive interaction of macrophages with LPS but had only a minor effect on the action of interferon‐ γ. The effects of genistein on LPS‐triggered macrophage activation were not due to nonspecific changes in macrophage metabolism or toxicity because genistein did not prevent lysis of tumor cells by activated macrophages, nor did it reduce the capacity of macrophages to phagocytose antibody‐opsonized sheep erythrocytes. Western blot analysis with antiphosphotyrosine monoclonal antibody revealed that incubation of macrophages with LPS produced a rapid increase in tyrosine phosphorylation of several proteins and that the induced phosphorylation could be inhibited by effective concentrations of genistein, herbimycin A, or tyrphostin. Taken together, these data indicate that protein‐tyrosine phosphorylation plays an important role in LPS‐induced tumoricidal activation of macrophages.

Adenovirus-mediated interferon-β gene therapy suppresses growth and metastasis of human prostate cancer in nude mice

The purpose of this study was to determine the effects of interferon-β (IFN-β) gene transfer on the growth of PC3MM2 human prostate cancer cells in nude mice. Intralesional delivery of an adenoviral vector encoding murine IFN-β (AdIFN-β), but not a vector encoding bacterial β-galactosidase (AdLacZ), suppressed PC3MM2 tumors in a dose-dependent manner. At the highest dose (2×10 9 plaque-forming units, PFU), a single injection of AdIFN-β (but not AdLacZ) suppressed orthotopic PC3MM2 tumors and development of metastasis by 80%, and eradicated the tumors in 20% of mice. Immunohistochemical staining showed that AdIFN-β–treated tumors contained fewer microvessels, fewer proliferating cells, and more apoptotic cells than did the control tumors. Compared with controls, tumors injected with AdIFN-β expressed higher levels of IFN-β and inducible nitric oxide synthase (iNOS) and lower levels of basic fibroblast growth factor (bFGF) and transforming growth factor β1 (TGF-β1). In vitro analysis indicated that expression of bFGF and TGF-β1 in PC3MM2 cells could be suppressed by the nitric oxide donor sodium nitroprusside. These data suggest that intratumoral delivery of the IFN-β gene with adenoviral vectors could be an effective therapy for prostate cancer and that tumor suppression by AdIFN-β correlated with up-regulation of iNOS and down-regulation of angiogenesis. Cancer Gene Therapy (2001) 8, 497–505