Antitumoral effect of maintained neutrophilia induced by rhG-CSF in a murine model of pancreatic cancer.

Although the protumoral functions of polymorphonuclear neutrophils are well known, some now-forgotten studies report antitumoral roles for these cells. The present work examines the antitumoral effect of maintained neutrophilia induced via the injection of recombinant human granulocyte colony stimulating factor (rhG-CSF, 100 µg/kg/day) in a Panc-1 subcutaneous xenograft murine model of pancreatic cancer. This treatment was compared with gemcitabine administration (120 mg/kg every two days) and a saline control (n = 6-7 mice per group). Compared to the controls, both the rhG-CSF- and gemcitabine-treated mice showed significantly suppressed tumor growth by day 4 (p < 0.001 and p = 0.013 respectively). From a mean starting volume of 106.9 ± 3.1 mm3 for all treatment groups, the final mean tumor volumes reached were 282.0 ± 30.7 mm3 for the rhG-CSF-treated mice, 202.6 ± 18.1 mm3 for the gemcitabine-treated mice and 519.4 ± 62.9 mm3 for the control mice (p < 0.004 and p < 0.01, respectively, vs. control). The rhG-CSF-treated tumors showed higher percentage necrosis than those treated with gemcitabine (37.4 ± 4.6 vs. 7.5 ± 3.0; p < 0.001). This is the first report of a clear anti-tumoral effect of rhG-CSF when used in monotherapy against pancreatic cancer. Since rhG-CSF administration is known to be associated with very few adverse events, it may offer an attractive alternative in the clinical treatment of pancreatic cancer.

Gene expression signatures and molecular markers associated with clinical outcome in locally advanced head and neck carcinoma.

The purpose of this study was to identify molecular markers associated with tumor recurrence and survival in patients with locally advanced head and neck squamous cell carcinoma (HNSCC). We studied the expression profile of 63 pre-treatment tumor biopsies obtained from locally advanced HNSCCs treated with standard treatments. Cluster analysis identified three tumor subtypes associated with significant differences in local recurrence-free survival (LRFS) (P<0.001), progression free-survival (PFS) (P<0.009) and overall survival (OS) (P<0.004). Tumor subtype 1, associated with short LRFS, PFS and OS, showed features of epithelial-mesenchymal transition and undifferentiation. It also overexpressed genes involved in cell adhesion, NF-κB and integrin signalling. Tumor subtype 3, associated with longer LRFS, PFS and OS, showed a high degree of differentiation and overexpressed genes located in chromosomal regions 19q13 and 1q21. Tumor subtype 2, which had an intermediate clinical outcome between subtype 1 and subtype 3, overexpressed genes involved in branching morphogenesis. Finally, we validated the association between gene cluster classification and patient survival using Gene Set Enrichment Analysis and two HNSCC data sets obtained from two independent patient cohorts. In conclusion, we generated a gene prognostic signature associated with survival in locally advanced patients using the expression profile of the pre-treatment tumor biopsy. Independent prospective studies would be necessary to assess if the proposed survival signature could help to guide clinical management of HNSCC.

Mouse models in oncogenesis and cancer therapy.

Animal models have been critical in the study of the molecular mechanisms of cancer and in the development of new antitumor agents; nevertheless, there is still much room for improvement. The relevance of each particular model depends on how close it replicates the histology, physiological effects, biochemical pathways and metastatic pattern observed in the same human tumor type. Metastases are especially important because they are the main determinants of the clinical course of the disease and patient survival, and are the target of systemic therapy. The generation of clinically relevant models using the mouse requires their humanization, since differences exist in transformation and oncogenesis between human and mouse. Although genetically modified (GM) mice have been instrumental in understanding the molecular mechanisms involved in tumor initiation, they have been less successful in replicating advanced cancer. Moreover, a particular genetic alteration frequently leads to different tumor types in human and mouse and to lower metastastatic rates in GM mice than in humans. These findings question the capacity of current GM mouse carcinoma models to predict clinical response to therapy. On the other hand, orthotopic (ORT) xenografts of human tumors, or tumor cell lines, in nude mice reproduce the histology and metastatic pattern of most human tumors at advanced stage. Using ex vivo genetic manipulation of human tumor cells, ORT models can be used to molecularly dissect the metastatic process and to evaluate in vivo tumor response to therapy, using non-invasive procedures. Nevertheless, this approach is not useful in the study of the initial stages of tumorigenesis or the contribution of the immune system in this process. Despite ORT models are more promising than the most commonly used subcutaneous xenografts in preclinical drug development, their capacity to predict clinical response to antitumor agents remains to be studied. Humanizing mouse models of cancer will most likely require the combined use of currently available methodologies.

Day/night variations of dopamine ocular content during Xenopus laevis ontogeny.

Concentration of dopamine (DA) and its metabolite, 3,4-dihydroxyphenylacetic acid is quantified by high-pressure liquid chromatography with a coulometric detection system in the eye of Xenopus laevis through ontogeny and in adults at two times during photocycle (midday and midnight). Ocular dopaminergic activity remains low during pre- and prometamorphosis and significantly rises in postmetamorphic froglets. This increase is more pronounced at midnight than at midday. The dualism of DA content versus DA release in Xenopus ocular tissue is studied in an eyecup culture system. On a 24-h cycle of DA release from adult Xenopus eyecups the highest DA release by eyecups is produced during daytime, and significantly decreases in darkness. From these results it can be concluded that in spite of the early development of the retinal dopaminergic system in the ontogeny of Xenopus, the final maturation must occur during the metamorphic climax. Endogenous DA release is significantly inhibited by light offset, which explains the higher ocular DA content found at midnight as compared to midday in postmetamorphic froglets and adults.

Peripheral nerve soluble esterases are spontaneously reactivated after inhibition by paraoxon: implications for a new definition of neuropathy target esterase.

Soluble extracts of chicken peripheral nerve contain detectable amounts of phenyl valerate esterase (PVase) activity (about 2000 nmol/min per g of fresh tissue). More than 95% of this activity is inhibited in assays where substrate has been added to a preincubated mixture of tissue with the non-neuropathic organophosphorus compound (OP) paraoxon (O,O'-diethyl p-nitrophenyl phosphate): residual activity includes soluble neuropathy target esterase (S-NTE) which, by definition, is considered resistant to long-term progressive (covalent) inhibition by paraoxon. However we have previously shown that paraoxon strongly interacts with S-NTE so interfering with its sensitivity to other inhibitors. We now show that, surprisingly, removal of paraoxon by ultrafiltration ('P' tissue) in order to avoid such an interference results in the reappearance of about 65% of total original soluble PVase activity which is inhibited in the presence of this OP. Although a purely reversible non-progressive inhibition might be suspected, kinetic analysis data show a time-progressive inhibition which suggests that such PVase(s) covalently bind paraoxon. Also a time-dependent recovery due to spontaneous reactivation of the PVase activity was observed after dilution of the inhibitor. Gel filtration chromatography of 'P' tissue in Sephacryl S-300 shows that the reactivated activity is associated with proteins of about 100-kDa mass which include S-NTE and an, as yet, unknown number of other PVases. The implications of these findings in the definition of NTE in a target tissue for the so-called organophosphorus-induced delayed polyneuropathy (OPIDP) are discussed.

Discrimination of carboxylesterases of chicken neural tissue by inhibition with a neuropathic, non-neuropathic organophosphorus compounds and neuropathy promoter.

Carboxylesterases are enzymes present in neural and other tissues that are sensitive to organophosphorus compounds. The esterase activity in particulate forms, resistant to paraoxon and sensitive to mipafox have been implicated in the initiation of organophosphorus-induced delayed polyneuropathy (OPIDP) and is called neuropathy target esterase (P-NTE). Certain esterases inhibitors such as phenylmethylsulfonyl fluoride (PMSF), can also irreversibly inhibit P-NTE and by this mechanism PMSF 'protects' from further effect of neuropathic OPs. However, if PMSF is dosed after a low non-neuropathic dose of a neuropathic OP, its neurotoxicity is 'promoted', causing severe neuropathy. The molecular target of promotion has not yet been identified and it has been shown that it is unlikely to be the P-NTE. In order to discriminate the different esterases, we used non-neuropathic (paraoxon), and neuropathic organophosphorus compounds (mipafox, DFP) and a neuropathy promoter (PMSF). They were used alone or in concurrent inhibition to study particulate and soluble fractions of brain, spinal cord and sciatic nerve of chicken. From the experimental data, a matrix was constructed and equations deduced to estimate the proportions of the different potential activity fractions that can be discriminated by their sensitivity to the tested inhibitors. It was deduced that only combinations of up to three inhibitors can be used for the analysis with consistent results. In all tissues, inside the paraoxon sensitive activity, most of the activity was sensitive either to mipafox, to PMSF or both. In all fractions, except brain soluble fractions, within the paraoxon resistant activity, a mipafox sensitive component was detected that is operationally considered NTE (P-NTE and S-NTE in particulate and soluble fractions, respectively). Most of this activity was also sensitive to PMSF, and this should be considered the target of organophosphorus inducing neuropathy and of PMSF protective effect. Either in brain and spinal cord, a significant amount of the activity resistant to 40 microM paraoxon and 250 microM mipafox (usually called 'C' activity) is sensitive to PMSF. It could be a good candidate to contain the target of the promotion effect of PMSF as well as the S-NTE activity that is also PMSF sensitive.

Chromatographic discrimination of soluble neuropathy target esterase isoenzymes and related phenyl valerate esterases from chicken brain, spinal cord, and sciatic nerve.

Neuropathy target esterase (NTE) activity is operatively defined in this work as the phenyl valerate esterase (PVase) activity resistant to 40 microM paraoxon but sensitive to 250 microM mipafox. Gel filtration chromatography with Sephacryl S-300 of the soluble fraction from spinal cord showed two PVase peaks containing NTE activity (S-NTE1 and S-NTE2). The titration curve corresponding to inhibition by mipafox was studied over the 1-250 microM range, in the presence of 40 microM paraoxon. The data revealed that S-NTE1 and S-NTE2 have different sensitivities to mipafox with I50 (30 min) values of 1.7 and 19 microM, respectively. This was similar to the pattern observed in the soluble fraction from sciatic nerve with two components (Vo peak, or S-NTE1; and 100-K peak, or S-NTE2) with different sensitivity to mipafox. However, in the brain soluble fraction, only the high-molecular-mass (>700-kDa) peak or S-NTE1 was obtained. It showed an I50 of 5.2 microM in the mipafox inhibition curve. The chromatographic profile was different on changing the pH in the subcellular fractionation. When the homogenized tissue was centrifuged at pH 6.8, the Vo peak activity decreased in the soluble fraction from these nerve tissues. This suggests that the Vo peak could be related to materials partly solubilized from membranes at higher pH. The chromatographic pattern and mipafox sensitivity suggest that the different tissues have a different NTE isoform composition. S-NTE2 should be a different entity than S-NTE1 and particulate NTE. The potential role of soluble forms in the mechanism of initiation or promotion of neuropathy due to organophosphorus remain unknown.

Mu-opioid receptor is involved in beta-endorphin-induced feeding in goldfish.

The present study evaluated the central effects of selective opioid receptor subtype agonists and antagonists on food intake in satiated goldfish. Significant increases in feeding behavior occurred in goldfish injected with beta-endorphin, the kappa agonist, U-50488, the delta agonist, [D-Pen2,D-Pen5]enkephalin (DPEN), and the mu agonist, [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO). On the other hand, the different receptor antagonists used: nor-binaltorphamine (nor-BNI) for kappa, 7-benzidilidenenaltrexone (BNTX) for delta 1, naltriben for delta 2, beta-funaltrexamine (beta-FNA) for mu, and naloxonazine for mu 1, by themselves, did not modify ingestion or slightly reduced it. The feeding stimulation by beta-endorphin was antagonized by beta-FNA and naloxonazine, but not by nor-BNI, BNTX, or naltriben. These data indicate that the mu-opioid receptor is involved in the modulation of the feeding behavior in goldfish.

The galanin-induced feeding stimulation is mediated via alpha 2-adrenergic receptors in goldfish.

In the present study we examined the effects of intracerebroventricular (ICV) or intraperitoneal (IP) galanin administration on food intake in satiated goldfish, at different time intervals after injections, 0-2, 2-8 and 0-8 h. We found that food intake was increased by ICV administered galanin (1 and 3.33 micrograms) at 2 and 8 h postinjection, while no modifications on feeding were observed after intraperitoneal injection in any of the studied time intervals. The galanin receptor antagonist, galantide, blocked the galanin-induced feeding increase. Pretreatment with yohimbine (alpha 2-adrenergic receptor antagonist), but not with prazosin (alpha 1-adrenergic receptor antagonist) antagonized the stimulatory effect of galanin on ingestive behavior. These results suggest that galanin is involved in the central regulation of feeding in goldfish, being the food intake stimulatory effect mediated by the alpha 2-adrenergic system.