Nucleic acid aptamer-guided cancer therapeutics and diagnostics: the next generation of cancer medicine

Conventional anticancer therapies, such as chemo- and/or radio-therapy are often unable to completely eradicate cancers due to abnormal tumor microenvironment, as well as increased drug/radiation resistance. More effective therapeutic strategies for overcoming these obstacles are urgently in demand. Aptamers, as chemical antibodies that bind to targets with high affinity and specificity, are a promising new and novel agent for both cancer diagnostic and therapeutic applications. Aptamer-based cancer cell targeting facilitates the development of active targeting in which aptamer-mediated drug delivery could provide promising anticancer outcomes. This review is to update the current progress of aptamer-based cancer diagnosis and aptamer-mediated active targeting for cancer therapy in vivo, exploring the potential of this novel form of targeted cancer therapy.

Evaluation of glycated glucagon-like peptide-1(7-36)amide in intestinal tissue of normal and diabetic animal models

Glucagon-like peptide-1(7-36)amide (tGLP-1) is an important insulin-releasing hormone of the enteroinsular axis which is secreted by endocrine L-cells of the small intestine following nutrient ingestion. The present study has evaluated tGLP-1 in the intestines of normal and diabetic animal models and estimated the proportion present in glycated form. Total immunoreactive tGLP-1 levels in the intestines of hyperglycaemic hydrocortisone-treated rats, streptozotocin-treated mice and ob/ob mice were similar to age-matched controls. Affinity chromatographic separation of glycated and non-glycated proteins in intestinal extracts followed by radioimmunoassay using a fully crossreacting anti-serum demonstrated the presence of glycated tGLP-1 within the intestinal extracts of all control animals (approximately 19%., of total tGLP-1 content). Chemically induced and spontaneous animal models of diabetes were found to possess significantly greater levels of glycated tGLP-1 than controls, corresponding to between 24-71% of the total content. These observations suggest that glycated tGLP-1 may be of physiological significance given that such N-terminal modification confers resistance to DPP IV inactivation and degradation, extending the very short half-life (

Animal models for genetic neuromuscular diseases

The neuromuscular disorders are a heterogeneous group of genetic diseases, caused by mutations in genes coding sarcolemmal, sarcomeric, and citosolic muscle proteins. Deficiencies or loss of function of these proteins leads to variable degree of progressive loss of motor ability. Several animal models, manifesting phenotypes observed in neuromuscular diseases, have been identified in nature or generated in laboratory. These models generally present physiological alterations observed in human patients and can be used as important tools for genetic, clinic, and histopathological studies. The mdx mouse is the most widely used animal model for Duchenne muscular dystrophy (DMD). Although it is a good genetic and biochemical model, presenting total deficiency of the protein dystrophin in the muscle, this mouse is not useful for clinical trials because of its very mild phenotype. The canine golden retriever MD model represents a more clinically similar model of DMD due to its larger size and significant muscle weakness. Autosomal recessive limb-girdle MD forms models include the SJL/J mice, which develop a spontaneous myopathy resulting from a mutation in the Dysferlin gene, being a model for LGMD2B. For the human sarcoglycanopahties (SG), the BIO14.6 hamster is the spontaneous animal model for delta-SG deficiency, whereas some canine models with deficiency of SG proteins have also been identified. More recently, using the homologous recombination technique in embryonic stem cell, several mouse models have been developed with null mutations in each one of the four SG genes. All sarcoglycan-null animals display a progressive muscular dystrophy of variable severity and share the property of a significant secondary reduction in the expression of the other members of the sarcoglycan subcomplex and other components of the Dystrophin-glycoprotein complex. Mouse models for congenital MD include the dy/dy (dystrophia-muscularis) mouse and the allelic mutant dy(2J)/dy(2J) mouse, both presenting significant reduction of alpha 2-laminin in the muscle and a severe phenotype. The myodystrophy mouse (Large(myd)) harbors a mutation in the glycosyltransferase Large, which leads to altered glycosylation of alpha-DG, and also a severe phenotype. Other informative models for muscle proteins include the knockout mouse for myostatin, which demonstrated that this protein is a negative regulator of muscle growth. Additionally, the stress syndrome in pigs, caused by mutations in the porcine RYR1 gene, helped to localize the gene causing malignant hypertermia and Central Core myopathy in humans. The study of animal models for genetic diseases, in spite of the existence of differences in some phenotypes, can provide important clues to the understanding of the pathogenesis of these disorders and are also very valuable for testing strategies for therapeutic approaches.

Immunocytochemical study of Ki-67 as a prognostic marker in canine mammary neoplasia

Background: Canine mammary tumors are challenging for clinicians and pathologists because of complex histologic classification, low specificity of cytologic diagnosis, and unpredictable biological behavior. In histologic specimens, expression of tumor proliferation marker Ki-67, a nuclear nonhistone protein, has been shown to have prognostic value for canine mammary tumors and to correlate with malignancy and low survival rates. Objective: The objective of this study was to measure the proliferation index of canine mammary tumors by immunochemical detection of Ki-67 in cytologic specimens and to determine its relationship to clinical and pathologic variables and patient outcome. Methods: Spontaneous mammary tumors from 31 female dogs were surgically excised. Imprint specimens for cytologic evaluation were wet-fixed in ethanol; histologic specimens were prepared routinely. Immunostaining was performed with the PH 177 monoclonal antibody against Ki-67; proliferation index was graded from negative to +++. Dogs were followed for 18 months. Multivariate logistic regression analysis was used to determine correlations between immunocytochemical results, tumor and clinical variables, and patient outcome. Results: Ki-67 proliferation indices in cytologic specimens were significantly lower for nonmalignant tumors than for malignant tumors. High index values of Ki-67 were positively correlated with metastasis, death from neoplasia, low disease-free survival rates, and low overall survival rate. With the exception of 4 specimens for which cellularity was insufficient, positive expression of Ki-67 in cytologic specimens correlated with that of histologic specimens. Conclusions: The prognostic value of the Ki-67 index in canine mammary tumors by using wet-fixed cytology imprint specimens was similar to that observed previously for histologic specimens. Immunocytochemical detection of Ki-67 could improve the accuracy and value of cytology by providing safe and rapid information about malignancy and patient outcome. © 2004 American Society for Veterinary Clinical Pathology.

Chalcone JAI-51 improves efficacy of synchrotron microbeam radiation therapy of brain tumors

Microbeam radiation therapy (MRT), a preclinical form of radiosurgery, uses spatially fractionated micrometre-wide synchrotron-generated X-ray beams. As MRT alone is predominantly palliative for animal tumors, the effects of the combination of MRT and a newly synthesized chemotherapeutic agent JAI-51 on 9L gliosarcomas have been evaluated. Fourteen days (D14) after implantation (D0), intracerebral 9LGS-bearing rats received either MRT, JAI-51 or both treatments. JAI-51, alone or immediately after MRT, was administered three times per week. Animals were kept up to ∼20 weeks after irradiation or sacrificed at D16 or D28 after treatment for cell cycle analysis. MRT plus JAI-51 increased significantly the lifespan compared with MRT alone (p = 0.0367). JAI-51 treatment alone had no effect on rat survival. MRT alone or associated with JAI-51 induced a cell cycle blockade in G2/M (p < 0.01) while the combined treatment also reduced the proportion of G0/G1 cells. At D28 after irradiation, MRT and MRT/JAI-51 had a smaller cell blockade effect in the G2/M phase owing to a significant increase in tumor cell death rate (<2c) and a proportional increase of endoreplicative cells (>8c). The combination of MRT and JAI-51 increases the survival of 9LGS-bearing rats by inducing endoreduplication of DNA and tumor cell death; further, it slowed the onset of tumor growth resumption two weeks after treatment.

Antitumor effect of Bothrops jararaca venom

MANY experimental studies have been carried out using snake venoms for the treatment of animal tumors, with controversial results. While some authors have reported an antitumor effect of treatment with specific snake venom fractions, others have reported no effects after this treatment. The aim of this study was to evaluate the effect of Bothrops jararaca venom (BjV) on Ehrlich ascites tumor (EAT) cells in vivo and in vitro. In the in vivo study, Swiss mice were inoculated with EAT cells by the intraperitoneal (i.p.) route and treated with BjV venom (0.4 mg/kg, i.p.), on the 1st, 4th, 7th, 10th, and 13th days. Mice were evaluated for total and differential cells number on the 2nd, 5th, 8th, 11th and 14th days. The survival time was also evaluated after 60 days of tumor growth. In the in vitro study, EAT and normal peritoneal cells were cultivated in the presence of different BjV concentrations (2.5, 5.0, 10.0, 20.0, 40.0, and 80 mug) and viability was verified after 3, 6, 12 and 24 h of cultivation. Results were analyzed statistically by the Kruskal-Wallis and Tukey tests at the 5% level of significance. It was observed that in vivo treatment with BjV induced tumor growth inhibition, increased animal survival time, decreased mortality, increased the influx of polymorphonuclear leukocytes on the early stages of tumor growth, and did not affect the mononuclear cells number. In vitro treatment with BjV produced a dose-dependent toxic effect on EAT and peritoneal cells, with higher effects against peritoneal cells. Taken together, our results demonstrate that BjV has an important antitumor effect. This is the first report showing this in vivo effect for this venom.

Characterization of canine dendritic cells in healthy, atopic, and non-allergic inflamed skin

Atopic dermatitis in humans and dogs is a chronic relapsing allergic skin disease. Dogs show a spontaneous disease similar to the human counterpart and represent a model to improve our understanding of the immunological mechanisms, the pathogenesis of the disease, and new therapy development. The aim of the study was to determine the frequency and phenotype of dendritic cells (DC) in the epidermis and dermis of healthy, canine atopic dermatitis lesional, and non-allergic inflammatory skin to further validate the model and to obtain insights into the contribution of DC to the pathogenesis of skin diseases in dogs. We first characterized canine skin DC using flow-cytometric analysis of isolated skin DC combined with an immunohistochemical approach. A major population of canine skin dendritic cells was identified as CD1c(+)CD11c(+)CD14(-)CD80(+)MHCII(+)MAC387(-) cells, with dermal DC but not Langerhans cells expressing CD11b. In the epidermis of lesional canine atopic dermatitis and non-allergic inflammatory skin, we found significantly more dendritic cells compared with nonlesional and control skin. Only in canine atopic dermatitis skin did we find a subset of dendritic cells positive for IgE, in the epidermis and the dermis. Under all inflammatory conditions, dermal dendritic cells expressed more CD14 and CD206. MAC387(+) putative macrophages were absent in healthy but present in inflamed skin, in particular during non-allergic diseases. This study permits a phenotypic identification and differentiation of canine skin dendritic cells and has identified markers and changes in dendritic cells and macrophage populations related to allergic and non-allergic inflammatory conditions. Our data suggest the participation of dendritic cells in the pathogenesis of canine atopic dermatitis similar to human atopic dermatitis and further validate the only non-murine spontaneous animal model for this disease.

THE ROLE OF TYROSINE PHOSPHORYLATION IN THE FUNCTIONS OF THE TUMOR SUPPRESSOR GPRC5A

The retinoic acid inducible G protein coupled receptor family C group 5 type A (GPRC5A) is expressed preferentially in normal lung tissue but its expression is suppressed in the majority of human non-small cell lung cancer cell lines and tissues. This differential expression has led to the idea that GPRC5A is a potential tumor suppressor. This notion was supported by the finding that mice with a deletion of the Gprc5a gene develop spontaneous lung tumors. However, there are various tumor cell lines and tissue samples, including lung, that exhibit higher GPRC5A expression than normal tissues and some reports by other groups that GPRC5A transfection increased cell growth and colony formation. Obviously, GPRC5A has failed to suppress the development of the tumors and the growth of the cell lines where its expression is not suppressed. Since no mutations were detected in the coding sequence of GPRC5A in 20 NSCLC cell lines, it’s possible that GPRC5A acts as a tumor suppressor in the context of some cells but not in others. Alternatively, we raised the hypothesis that the GPRC5A protein may be inactivated by posttranslational modification(s) such as phosphorylation. It is well established that Serine/Threonine phosphorylation of G protein coupled receptors leads to their desensitization and in a few cases Tyrosine phosphorylation of GPCRs has been linked to internalization. Others reported that GPRC5A can undergo tyrosine phosphorylation in the cytoplasmic domain after treatment of normal human mammary epithelial cells (HMECs) with epidermal growth factor (EGF) or Heregulin. This suggested that GPRC5A is a substrate of EGFR. Therefore, we hypothesized that tyrosine phosphorylation of GPRC5A by activation of EGFR signaling may lead to its inactivation. To test this hypothesis, we transfected human embryo kidney (HEK) 293 cells with GPRC5A and EGFR expression vectors and confirmed that GPRC5A can be tyrosine phosphorylated after activation of EGFR by EGF. Further, we found that EGFR and GPRC5A can interact either directly or through other proteins and that inhibition of the EGFR kinase activity decreased the phosphorylation of GPRA5A and the interaction between GPRC5A and EGFR. In c-terminal of GPRC5A, There are four tyrosine residues Y317, Y320, Y347, Y350. We prepared GPRC5A mutants in which all four tyrosine residues had been replaced by phenylalanine (mutant 4F) or each individual Tyr residue was replaced by Phe and found that Y317 is the major site for EGFR mediated phosphorylation in the HEK293T cell line. We also found that EGF can induce GPRC5A internalization both in H1792 transient and stable cell lines. EGF also partially inactivates the suppressive function of GPRC5A on cell invasion activity and anchorage-independent growth ability of H1792 stable cell lines. These finding support our hypothesis that GPRC5A may be inactivated by posttranslational modification- tyrosine phosphorylation.

Longitudinal extension of myelomalacia by intramedullary and subdural hemorrhage in a canine model of spinal cord injury

BACKGROUND CONTEXT In canine intervertebral disc (IVD) extrusion, a spontaneous animal model of spinal cord injury, hemorrhage is a consistent finding. In rodent models, hemorrhage might be involved in secondary tissue destruction by biochemical mechanisms. PURPOSE This study aimed to investigate a causal association between the extents of intramedullary, subdural and epidural hemorrhage and the severity of spinal cord damage following IVD extrusion in dogs. STUDY DESIGN/SETTING A retrospective study using histologic spinal cord sections from 83 dogs euthanized following IVD extrusion was carried out. METHODS The degree of hemorrhage (intramedullary, subdural, epidural), the degree of spinal cord damage in the epicenter (white and gray matter), and the longitudinal extent of myelomalacia were graded. Associations between the extent of hemorrhage and the degree of spinal cord damage were evaluated statistically. RESULTS Intramedullary and subdural hemorrhages were significantly associated with the degree of white (p<.001/ p=.004) and gray (both p<.001) matter damage, and with the longitudinal extension of myelomalacia (p<.001/p=.005). Intriguingly, accumulation of hemorrhagic cord debris inside or dorsal to a distended and ruptured central canal in segments distant to the epicenter of the lesion was observed exhibiting a wave-like pattern on longitudinal assessment. The occurrence of this debris accumulation was associated with high degrees of tissue destruction (all p<.001). CONCLUSIONS Tissue liquefaction and increased intramedullary pressure associated with hemorrhage are involved in the progression of spinal cord destruction in a canine model of spinal cord injury and ascending or descending myelomalacia. Functional and dynamic studies are needed to investigate this concept further.

Fibulin-2 stabilizes tumor extracellular matrix and drives malignant progression of lung adenocarcinoma

The ECM of epithelial carcinomas undergoes structural remodeling during periods of uncontrolled growth, creating regional heterogeneity and torsional stress. How tumors maintain ECM integrity in the face of dynamic biophysical forces is still largely unclear. This study addresses these deficiencies using mouse models of human lung adenocarcinoma. Spontaneous lung tumors were marked by disorganized basement membranes, dense collagen networks, and increased tissue stiffness. Metastasis-prone lung adenocarcinoma cells secreted fibulin-2 (Fbln2), a matrix glycoprotein involved in ECM supra-molecular assembly. Fibulin-2 depletion in tumor cells decreased the intra-tumoral abundance of matrix metalloproteinases and reduced collagen cross-linking and tumor compressive properties resulting in inhibited tumor growth and metastasis. Fbln2 deposition within intra-tumoral fibrotic bands was a predictor of poor clinical outcome in patients. Collectively, these findings support a feed-forward model in which tumor cells secrete matrix-stabilizing factors required for the assembly of ECM that preferentially favors malignant progression. To our knowledge, this is the first evidence that tumor cells directly regulate the integrity of their surrounding matrix through the secretion of matrix-stabilizing factors such as fibulin-2. These findings open a new avenue of research into matrix assembly molecules as potential therapeutic targets in cancer patients.

A tumor-selective somatostatin analog (TT-232) with strong in vitro and in vivo antitumor activity.

We report a series of new in vitro and in vivo data proving the selective antitumor activity of our somatostatin structural derivative, TT-232. In vitro, it inhibited the proliferation of 20 different human tumor cell lines in the range of 50-95% and induced a very strong apoptosis. In vivo TT-232 was effective on transplanted animal tumors (Colon 26, B16 melanoma, and S180 sarcoma) and on human tumor xenografts. Treatment of MDA-MB-231 human breast cancer xenografted in mice with low submaximal doses of TT-232 [0.25 and 0.5 mg/kg of body weight (b.w.)] caused an average 80% decrease in the tumor volume resulting in 30% tumor-free animals surviving for longer than 200 days. Treatment of prostate tumor (PC-3) xenografted animals with 20 mg/kg of b.w. of TT-232 for 3 weeks resulted in 60% decrease in tumor volume and 100% survival even after 60 days, while 80% of nontreated animals perished. We have demonstrated that TT-232 did not bind to the membrane preparation of rat pituitary and cortex and had no antisecretory activity. TT-232 was not toxic at a dose of 120 mg/kg of b.w. in mice. Long-term incubation (24 h) of tumor cells with TT-232 caused significant inhibition of tyrosine kinases in good correlation with the apoptosis-inducing effect. The level of p53 or KU86 did not change following TT-232 treatment, suggesting a p53-independent apoptotic effect. Preincubation of human breast cancer cells (MDA-MB-453) with TT-232 for 2 h decreased the growth factor receptor autophosphorylation. All of these data suggest that TT-232 is a promising and selective antitumor agent.

Malignant conversion of chemically transformed normal human cells.

Two structurally unrelated chemicals, aflatoxin B1 and propane sultone, transformed human foreskin cells to a stage of anchorage-independent growth. Isolation from agar and repopulation in monolayer culture of these transformed cells was followed by transfection with a cDNA library, which resulted in cells that exhibited an altered epithelioid morphology. Chemically transformed/nontransfected cells and transfected normal cells did not undergo a significant morphological change. These epithelioid-appearing, transfected cells, when inoculated into nude mice, form progressively growing tumors. The tumors are histopathologically interpreted as carcinomas. All of the first generation tumors in the surrogate hosts exhibited characteristic rates of growth similar to those of transplants of spontaneous human tumors. In the second generation of tumor xenografts, the progressively growing tumors derived from the transfected cells exhibited a more rapid rate of growth. Southern analysis and reverse transcription PCR confirmed that a 1.3-kb genetic element was integrated into the genome and was actively being transcribed. Examination of the metaphase chromosomes in normal human cells revealed that the genetic element responsible for this conversion was located at site 31-32 of the q arm of chromosome 7. The DNA sequence of this 1.3-kb genetic element contains a coding region for 79 amino acids and a long 3'-untranslated region and appears to be identical to CATR1.3 isolated from tumors produced by methyl methanesulfonate-converted, nontransplantable human tumor cells.

Spontaneous canine transmissible venereal tumor: cell morphology and influence on P-glycoprotein expression

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Three-dimensional magnetic resonance reconstruction images before and after surgical therapy of spontaneous canine brain tumors

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)