Dysprosium-Modified Tobacco Mosaic Virus Nanoparticles for Ultra-High-Field Magnetic Resonance and Near-Infrared Fluorescence Imaging of Prostate Cancer.

The increasing prevalence of ultra-high-field magnetic resonance imaging (UHFMRI) in biomedical research and clinical settings will improve the resolution and diagnostic accuracy of MRI scans. However, better contrast agents are needed to achieve a satisfactory signal-to-noise ratio. Here, we report the synthesis of a bimodal contrast agent prepared by loading the internal cavity of tobacco mosaic virus (TMV) nanoparticles with a dysprosium (Dy3+) complex and the near-infrared fluorescence (NIRF) dye Cy7.5. The external surface of TMV was conjugated with an Asp-Gly-Glu-Ala (DGEA) peptide via a polyethylene glycol linker to target integrin α2ß1. The resulting nanoparticle (Dy-Cy7.5-TMV-DGEA) was stable and achieved a high transverse relaxivity in ultra-high-strength magnetic fields (326 and 399 mM-1 s-1 at 7 and 9.4 T, respectively). The contrast agent was also biocompatible (low cytotoxicity) and targeted PC-3 prostate cancer cells and tumors in vitro and in vivo as confirmed by bimodal NIRF imaging and T2-mapping UHFMRI. Our results show that Dy-Cy7.5-TMV-DGEA is suitable for multiscale MRI scanning from the cellular level to the whole body, particularly in the context of UHFMRI applications.

Expression of core 3 synthase in human pancreatic cancer cells suppresses tumor growth and metastasis.

Core 3-derived glycans, a major type of O-glycan expressed by normal epithelial cells of the gastrointestinal tract, are downregulated during malignancy because of loss of expression of functional ß3-N-acetylglucosaminyltransferase-6 (core 3 synthase). We investigated the expression of core 3 synthase in normal pancreas and pancreatic cancer and evaluated the biological effects of re-expressing core 3 synthase in pancreatic cancer cells that had lost expression. We determined that pancreatic tumors and tumor cell lines have lost expression of core 3 synthase. Therefore, we re-expressed core 3 synthase in human pancreatic cancer cells (Capan-2 and FG) to investigate the contribution of core 3 glycans to malignant progression. Pancreatic cancer cells expressing core 3 synthase showed reduced in vitro cell proliferation, migration and invasion compared to vector control cells. Expression of core 3 O-glycans induced altered expression of ß1 integrin, decreased activation of focal adhesion kinase, led to the downregulation of expression of several genes including REG1α and FGFR3 and altered lamellipodia formation. The addition of a GlcNAc residue by core 3 synthase leads to the extension of the tumor-associated Tn structure on MUC1. Orthotopic injection of FG cells expressing core 3 synthase into the pancreas of nude mice produced significantly smaller tumors and decreased metastasis to the surrounding tissues compared to vector control FG cells. These findings indicate that expression of core 3-derived O-glycans in pancreatic cancer cells suppresses tumor growth and metastasis through modulation of glycosylation of mucins and other cell surface and extracellular matrix proteins.

Prostate tumor cells with cancer progenitor properties have high telomerase activity and are rapidly killed by telomerase interference.

BACKGROUND: Cancer progenitor cells (CPCs) have been postulated to promote treatment resistance and disease progression in prostate and other malignancies. We investigated whether the enzyme telomerase, which is active in cancer cells and in normal stem cells, plays an important role in CPC which can be exploited to neutralize these cells. METHODS: We used flow cytometry and assays of gene expression, clonogenicity, and invasiveness to isolate and characterize a putative CPC subpopulation from freshly resected human prostatectomy specimens. Telomerase activity was measured by qPCR-based Telomeric Repeat Amplification Protocol (TRAP). Telomerase interference was achieved by ectopic expression of a mutated telomerase RNA construct which reprograms telomerase to generate "toxic" uncapped telomeres. Treated cells were assayed for apoptosis, proliferation in culture, and xenograft tumor formation. RESULTS: CPC in prostate tumors expressed elevated levels of genes associated with a progenitor phenotype and were highly clonogenic and invasive. Significantly, CPC telomerase activity was 20- to 200-fold higher than in non-CPC from the same tumors, and CPC were exquisitely sensitive to telomerase interference which induced rapid apoptosis and growth inhibition. Similarly, induction of telomerase interference in highly tumorigenic CPC isolated from a prostate cancer cell line abrogated their ability to form tumor xenografts. CONCLUSIONS: Human prostate tumors contain a CPC subpopulation with markedly elevated telomerase activity which renders them acutely susceptible to telomerase interference. These findings offer the first tumor-derived and in vivo evidence that telomerase may constitute a CPC "Achilles heel" which may ultimately form the basis for more effective new CPC-targeting therapies.

Regional administration of oncolytic Echovirus 1 as a novel therapy for the peritoneal dissemination of gastric cancer.

The dissemination of malignant gastric cells to the peritoneum occurs frequently, usually as an early event in disease, and results in poor patient prognosis. Surgery and chemotherapy offer limited therapeutic success. The low-pathogenic human enterovirus, Echovirus 1 (EV1), is an oncolytic virus that selectively targets and destroys malignant prostate and ovarian cancer xenografts in vivo. Lytic EV1 infection requires the cell surface expression of alpha(2)beta(1), an integrin involved in the dissemination of gastric cancer cells to the peritoneum. Herein, we evaluated the capacity of EV1 for anti-neoplastic cell action in gastric peritoneal carcinomatosis. Flow cytometric analysis demonstrated that alpha(2)beta(1) was abundantly surface expressed on a panel of gastric cancer cell lines, rendering the majority of lines highly susceptible to in vitro lytic EV1 infection and supportive of efficient viral progeny production. A bioluminescent MKN-45-Luc SCID mouse model of peritoneal dissemination was developed to allow real-time non-invasive monitoring of peritoneal tumor burden. Employing this mouse model, we demonstrated a therapeutic dose-response for escalating oncolytic EV1 doses. Taken together, these results emphasize the exciting potential for EV1 as a single or adjunct therapy for the control of the peritoneal dissemination of gastric cancer.

Reduced growth and integrin expression of prostate cells cultured with lycopene, vitamin E and fish oil in vitro.

Integrins are transmembrane proteins that facilitate the interaction of cells with the extracellular environment. They have also been implicated in cancer progression. The effects of nutrients thought to be involved in the prevention of prostate cancer on integrin expression have not been determined. Prostate cancer cell lines representing a range of malignancy from normal (RWPE-1) to highly invasive phenotypes (22Rv1 < LNCaP < PC-3) were cultured with or without lycopene (10 nM), vitamin E (5 microm) or fish oil (100 microm) for 48 h. Growth and integrin (alpha2beta1, alphavbeta3 and alphavbeta5) expression were assessed using Trypan Blue exclusion and monoclonal antibodies combined with flow cytometry. Vitamin E enhanced (P < 0.001) whereas fish oil reduced the growth of all the cell lines tested (P < 0.001). Lycopene had no effect on growth. All the malignant cell lines exhibited lower expression of alpha2beta1 with the addition of lycopene to culture media. Supplemental fish oil reduced alpha2beta1 in most invasive cell lines (LNCaP and PC-3). Each nutrient at physiological levels reduced integrins alphavbeta3 and alphavbeta5 in most invasive cell lines (PC-3). The results suggest that integrins may represent an additional target of bioactive nutrients and that the effects of nutrients may be dependent on the type of cell line used.

Osteoblast differentiation is enhanced in rotary cell culture simulated microgravity environments.

PURPOSE: As the aging population increases, more people will become reliant on regenerative dental medicine for implant therapy. The objective of this study was to test the hypothesis that 3D rotary cell culture (RCC) environments created by simulated microgravity would enhance osteogenic gene expression using integrin mediated pathways. MATERIALS AND METHODS: Human embryonic palatal mesenchymal (HEPM, ATCC 1486) pre-osteoblasts were cultured in either RCC to create 3D environments or in 2D monolayers for 72 hours. Gross phenotypic analysis was performed using Alizarin Red S staining for calcium and microscopy. Real-time PCR analysis was used to detect differences in osteoblast gene expression. Aggregates developed in 3D RCC environments were treated with or without antibody to the collagen-I integrin receptor alpha2beta1 to determine whether this molecular pathway might contribute to the development of a mineralized matrix. RESULTS: Microscopic analysis demonstrated that RCC environments promoted 3D aggregate formation by 72 hours without any scaffold. The mass appeared osseous-like with a white, shiny, translucent surface. The center was amorphous with areas of vacuolization, tubule-like structures, and fibrous-like extensions. Real-time PCR data showed that 3D environments enhanced osteogenic gene expression as compared with 2D monolayer culturing conditions. At 72 hours, changes in levels of osteogenic gene expression were noted. Cbfa1, a necessary transcription factor for osteoblast differentiation, was expressed 33% higher (p= 0.26); Collagen 1, 69% higher (p= 0.05); Osterix, 49% higher (p= 0.001); and BSPII, 54% higher (p= 0.001) than osteoblasts cultured for 72 hours in standard 2D monolayer conditions. When cultured in the presence of collagen alpha2beta1 integrin receptor antibody, 3D aggregates had decreased levels of mineralization as compared with non-treated aggregates. CONCLUSION: RCC enhances osteoblast differentiation using integrin mediated pathways.

The maturity of intestinal neomucosa: integrin expression and ultrastructural aspects.

BACKGROUND/PURPOSE: The maturity of neomucosa growing on a serosal surface for the treatment of short bowel syndrome still is questionable. The aim of this study was to evaluate the intestinal neomucosa to assess its histologic maturity. METHODS: A 6-cm-long isolated ileal segment (IS) was prepared in 8 Wistar albino-type rats. The IS was divided from the antimesenteric side, and 2 intestinal tubes were established, which shared a common wall and a common pedicle. After ileal biopsy sampling for the control group (CG), the IS was fashioned into a mucous fistula. Eight weeks later, all the rats were killed, and the ISs were investigated for neomucosal growth. Sections were prepared with periodic acid shift (PAS) and H & E staining for light microscopy. They also were evaluated by transmission electron microscopy. The microscopic morphology of the 2 groups was evaluated. Immunohistochemical staining was performed to show the expression of the tissue beta1, alpha3 and alpha2beta1 integrin subunits of both the neomucosa (NS) and control group (CG) segments. RESULTS: Sections of the NS showed a well-arranged columnar epithelial cell layer with goblet cells that were generally located superficially and with a complete basement membrane. Under the electron microscope, the sections from the NS group showed an epithelial cell layer with proper microvilli of the same height, although they were shorter than those of the CG, and tight intercellular junctions between the epithelial cells. Significant differences between the NS and CG groups were found in the measurements of villus width at base, microvillus surface, and microvillus height. The lamina propria consisted of rich collagen fibers and active fibroblasts in the NS group. In the immunohistochemical staining, although beta1 integrine showed a dense distribution (+++) in the lamina propria, particularly localizing at the depth of the tunica mucosa layer, alpha3 integrin was observed to have a less dense immunoreactivity (++) in both groups. The expression of alpha2beta1 integrin showed slight and dispersed (+) staining. CONCLUSIONS: The NS showed histologic maturity and ultimate structural similarity with the native small bowel mucosa, which provides strong indirect evidence for the proper functioning of the neomucosa.

Matrix metalloproteinase 1 interacts with neuronal integrins and stimulates dephosphorylation of Akt.

Several studies have demonstrated that matrix metalloproteinases (MMPs) are cytotoxic. The responsible mechanisms, however, are not well understood. MMPs may promote cytotoxicity through their ability to disrupt or degrade matrix proteins that support cell survival, and MMPs may also cleave substrates to generate molecules that stimulate cell death. In addition, MMPs may themselves act on cell surface receptors that affect cell survival. Among such receptors is the alpha(2)beta(1) integrin, a complex that has previously been linked to leukocyte death. In the present study we show that human neurons express alpha(2)beta(1) and that pro-MMP-1 interacts with this integrin complex. We also show that stimulation of neuronal cultures with MMP-1 is associated with a rapid reduction in the phosphorylation of Akt, a kinase that can influence caspase activity and cell survival. Moreover, MMP-1-associated dephosphorylation of Akt is inhibited by a blocking antibody to the alpha(2) integrin, but not by batimastat, an inhibitor of MMP-1 enzymatic activity. Such dephosphorylation is also stimulated by a catalytic mutant of pro-MMP-1. Additional studies show that MMP-1 causes neuronal death, which is significantly diminished by both a general caspase inhibitor and anti-alpha(2) but not by batimastat. Together, these results suggest that MMP-1 can stimulate dephosphorylation of Akt and neuronal death through a non-proteolytic mechanism that involves changes in integrin signaling.

Modulation in vitro of keratinocyte integrins by interferon-alpha and interferon-gamma.

BACKGROUND: Interferon-alpha and -gamma are glycoproteins with antiviral and immunoregulatory properties. In vitro studies have shown a role for these cytokines in the regulation of epidermal keratinocyte growth and differentiation. In the same way, integrins are adhesion molecules which regulate keratinocyte proliferation and differentiation. AIM: To determine whether the regulatory activity of interferons on keratinocyte proliferation and differentiation is related to a modulation of keratinocyte integrins. METHODS: Two different methods were used: monolayers and reconstituted skin, incubated either with 1,200 U/mL interferon-alpha or 500 U/mL interferon-gamma or control medium for 48 h. The integrin expression was assessed by flow cytometry and immunohistochemistry. RESULTS: In monolayers, only the alpha3 subunit was significantly inhibited by interferon-gamma. In reconstituted skin, where keratinocytes are differentiated, both interferons had an inductive effect on beta1 expression and interferon-alpha had an inhibitory effect on alpha6 expression. CONCLUSION: Interferon-alpha and -gamma induce a modulatory effect on alpha3, alpha6 and beta1 which appears to be related to the state of differentiation. Moreover, the decreased expression of alpha6 and alpha3 could be one of the mechanisms involved in the formation of bullous lesions during long-term interferon therapy.

VLA-2 and VLA-5 cell adhesion molecules expression in CD34+ cells from umbilical cord blood and from bone marrow.

BACKGROUND/AIMS: Umbilical cord blood contains a large number of early hematopoietic cells with high proliferating capacity, that has been used as an alternative to bone marrow transplantation. The aim of this study is to investigate the number of two cell adhesion molecules in cord blood and in bone marrow. METHODS: We investigated two integrins, named VLA-2 and VLA-5 (Very Late Appearing Antigen), expressed in the surface of CD34+ cells. The CD34+ cells, isolated with MACS CD34+ isolation kit, were labelled with the appropriate monoclonal antibodies. RESULTS: Cell adhesion molecules showed highly expressed in both cord blood and bone marrow CD34+ cells. CONCLUSION: There are no significant differences between the two sources of CD34+ populations.