Paolo Laccetti

Inhibitory effects of cannabinoid CB1 receptor stimulation on tumor growth and metastatic spreading: actions on signals involved in angiogenesis and metastasis

Stimulation of cannabinoid CB1 receptors by 2‐methyl‐arachidonyl‐2′‐fluoro‐ethylamide (Met‐F‐AEA) inhibits the growth of a rat thyroid cancer cell‐derived tumor in athymic mice by inhibiting the activity of the oncogene product p21ras. Here we report that Met‐F‐AEA also blocks the growth of tumors previously induced in nude mice by the s.c. injection of the same rat thyroid carcinoma cells. Met‐F‐AEA significantly inhibited, in tumors as well as transformed cells, the expression of the vascular endothelial growth factor, an angiogenetic factor known to be up‐regulated by p21ras, as well as of one of its receptors, flt‐1/VEGFR‐1. The levels of the cyclin‐dependent kinase inhibitor p27(kip1), which is down‐regulated by p21ras, were instead increased by Met‐F‐AEA. All these effects were antagonized by the selective CB1 receptor antagonist SR141716A. Met‐F‐AEA inhibited in vitro the growth of a metastasis‐derived thyroid cancer cell line more potently than a primary cancer cell line. Therefore, the hypothesis that CB1 receptor stimulation interferes not only with angiogenesis but also with metastatic processes was tested in a widely used model of metastatic infiltration in vivo, the Lewis lung carcinoma (3LL) in C57Bl/6 mice. Three weeks from the paw injection of 3LL cells, Met‐F‐AEA reduced significantly the number of metastatic nodes, in a way antagonized by SR141716A. Our findings indicate that CB1 receptor agonists might be used therapeutically to retard tumor growth in vivo by inhibiting at once tumor growth, angiogenesis, and metastasis.

Cellular and molecular crosstalk between leptin receptor and estrogen receptor-α in breast cancer: molecular basis for a novel therapeutic setting

Obesity is associated with an increased risk of breast cancer. A number of adipocytokines are increased in obesity causing low-level chronic inflammation associated with an increased risk of tumors. The adipocytokine leptin shows profound anti-obesity and pro-inflammatory activities. We have hypothesized that in common obesity, high circulating leptin levels might contribute to an increased risk of breast cancer by affecting mammary cell proliferation and survival. Leptin exerts its activity not only through leptin receptor (LepR), but also through crosstalk with other signaling systems implicated in tumorigenesis. In this study, we focused our attention on the relationship between the leptin/LepR axis and the estrogen receptor-alpha (ERalpha). To this aim, we utilized two human breast cancer cell lines, one ERalpha-positive cell line (MCF 7) and the other ERalpha-negative cell line (MDA-MB 231). We observed that the two cell lines had a different sensitivity to recombinant leptin (rleptin): on MCF 7 cells, rleptin induced a strong phosphorylation of the signal transducer and activator of transcription (STAT) 3 and of the extracellular related kinase 1/2 pathways with an increased cell viability and proliferation associated with an increased expression of ERalpha receptor. This response was not present in the MDA-MB 231 cells. The effects induced by leptin were lost when LepR was neutralized using either a monoclonal inhibitory antibody to LepR or LepR gene-silencing siRNA. These data suggest that there is a bidirectional communication between LepR and ERalpha, and that neutralization and/or inactivation of LepR inhibits proliferation and viability of human breast cancer cell lines. This evidence was confirmed by ex vivo studies, in which we analyzed 33 patients with breast cancer at different stages of disease, and observed that there was a statistically significant correlation between the expression of LepR and ERalpha. In conclusion, this study suggests a crosstalk between LepR and ERalpha, and could envisage novel therapeutic settings aimed at targeting the LepR in breast cancers.

A Fully Human Antitumor ImmunoRNase Selective for ErbB-2-Positive Carcinomas

We report the preparation and characterization of a novel, fully human antitumor immunoRNase (IR). The IR, a human RNase and fusion protein made up of a human single chain variable fragment (scFv), is directed to the ErbB-2 receptor and overexpressed in many carcinomas. The anti-ErbB-2 IR, named hERB-hRNase, retains the enzymatic activity of the wild-type enzyme (human pancreatic RNase) and specifically binds to ErbB-2-positive cells with the high affinity (Kd = 4.5 nm) of the parental scFv. hERB-hRNase behaves as an immunoprotoxin and on internalization by target cells becomes selectively cytotoxic in a dose-dependent manner at nanomolar concentrations. Administered in five doses of 1.5 mg/kg to mice bearing an ErbB-2-positive tumor, hERB-hRNase induced a dramatic reduction in tumor volume. hERB-hRNase is the first fully human antitumor IR produced thus far, with a high potential as a poorly immunogenic human drug devoid of nonspecific toxicity, directed against ErbB-2-positive malignancies.

A human, compact, fully functional anti-ErbB2 antibody as a novel antitumour agent.

A new human, compact antibody was engineered by fusion of a human, antitumour ErbB2-directed scFv with a human IgG1 Fc domain. Overexpression of the ErbB2 receptor is related to tumour aggressiveness and poor prognosis. This new immunoagent meets all criteria for a potential anticancer drug: it is human, hence poorly or not immunogenic; it binds selectively and with high affinity to target cells, on which it exerts an effective and selective antiproliferative action, including both antibody-dependent and complement-dependent cytotoxicity; it effectively inhibits tumour growth in vivo. Its compact molecular size should provide for an efficient tissue penetration, yet suitable to a prolonged serum half-life.

Preliminary tests of a prototype system for optical and radionuclide imaging in small animals

We have assembled a prototype system for multimodal (radionuclide and optical) in vivo planar imaging of small animals (mice) using single photon emission radiotracers (Tc-99m) and a fluorescent marker (hematoporphyrin). Preliminary tests of the separate (optical and radionuclide) prototype imaging systems are presented, aimed at assessing their features and at determining the experimental protocol for in vivo imaging. Tests were performed on anesthetized healthy or tumor bearing mice. The gamma radiation detector is a small area (11 /spl times/ 11 mm/sup 2/) hybrid pixel detector based on the Medipix1 ASIC readout technology (64 /spl times/ 64 square pixels of 170 /spl mu/m by side), bump-bonded to a 300 /spl mu/m thick silicon detector. High spatial resolution in radioimaging (in the order of 1 mm) is achieved in vivo with a pinhole tungsten collimator (0.35 mm diameter, 90/spl deg/ acceptance angle, field of view of over 20 mm at 10 mm source distance). A future setup will use the Medipix2 hybrid detector (256 /spl times/ 256 square pixels, 55 /spl mu/m by side) bump-bonded to a 1 mm thick CdTe pixel detector. The laser-induced in vivo fluorescence imaging system comprises a pulsed light source (Nd:YAG laser, /spl lambda/=532 nm, energy/pulse = 30 mJ, pulse width = 50 ps, repetition rate = 10 Hz) used to excite the fluorescence emission (600-760 nm) of injected hematoporphyrin compound, a low sensitivity CCD camera and a commercial image analysis system. Images of normal and tumor regions are acquired by using a cut-on filter (/spl lambda/>600 nm). Digital image subtraction then enhances the tumor contrast with respect to the background. The final experimental protocol, only partly implemented here, includes independent and then combined optical/radio imaging of control mice and of a solid tumoral area (human thyroid derived anaplastic carcinoma) after injection of the radiotracer and/or of the fluorophore. In this work, the accumulation of the radionuclide in selected organs and of the fluorophore in the tumor provides the signal contrast in the two imaging modalities. Fluorescence spectroscopy of excised tissue samples is also performed to help the interpretation of fluorescence images. Results of in vivo combined imaging on tumor in mice will be shown in a next paper.

Highly selective toxic and proapoptotic effects of two dimeric ribonucleases on thyroid cancer cells compared to the effects of doxorubicin.

The lack of selectivity of conventional antitumour drugs against cancer cells is responsible for their high toxicity. The development of new tumour-specific drugs is therefore highly needed. We tested the cytotoxic effects and the nature of cell death induced by a naturally dimeric bovine RNase and a newly engineered dimeric human RNase upon three genetically well-defined normal and malignant thyroid cell systems. RNases effects were compared with those of doxorubicin, a conventional antineoplastic drug. Our results show significant and selective proapoptotic effects exerted on tumour cells by both RNases, the strength of their cytotoxic and apoptotic activity being directly related to the degree of cell malignancy. No toxic effects were observed upon normal cells. Doxorubicin showed, instead, cytotoxic and apoptotic effects also against normal cells. The in vitro results were corroborated by the antitumour action of both dimeric RNases towards a malignant human thyroid tumour grown in nude mice. These results indicate a selective action of dimeric RNases against cancer cells and suggest the potential application of these molecules or their derivatives to the treatment of aggressive subtypes of thyroid cancer.

High-Resolution

We report on tests of a radionuclide imaging system for in vivo investigations in small animals with low-energy photons as from 125I (27-35 keV). Imaging optics features a high-resolution coded aperture mask and a fine pitch hybrid pixel detector (silicon 300-mum or 700-mum thick, or CdTe 1 mm thick) of the Medipix2 series (55 mum pitch, 256 x 256 pixels). The coded aperture had 480 70-mum holes in 100-mum-thick tungsten. Laboratory tests with a 109Cd 22 keV source and a microfocus X-ray tube (35 kVp, Mo anode) show a system resolution of about 110 mum at magnification m = 2.12 and a sensitivity improvement of 30:1 as compared to a 300-mum pinhole collimator. The field of view also depends on magnification: in the experiments presented, it varied from 6 mm (m = 2.12) to 21 mm (m = 0.66). 125I in vivo mouse thyroid imaging with the 70 mum coded aperture, a 300 mum pinhole and a 100 mum parallel hole collimator was also performed to obtain a qualitative comparison. This low energy, semiconductor-based, compact gamma-ray imaging system can be used as a gamma-ray sub-millimeter resolution imager for energies below about 35 keV and it is the basic imaging unit of a small animal Single Photon Emission Computed Tomography system (MediSPECT) built at University of Napoli Federico II and Istituto Nazionale Fisica Nucleare (INFN), Napoli.

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Background:Overexpression of ErbB2 receptor in breast cancer is associated with disease progression and poor prognosis. Trastuzumab, the only humanised anti-ErbB2 antibody currently used in breast cancer, has proven to be effective; however, a relevant problem for clinical practice is that a high fraction of breast cancer patients shows primary or acquired resistance to trastuzumab treatment.Methods:We tested on trastuzumab-resistant cells two novel human anti-tumour immunoconjugates engineered in our laboratory by fusion of a human anti-ErbB2 scFv, termed Erbicin, with either a human RNase or the Fc region of a human IgG1. Both Erbicin-derived immunoagents (EDIAs) are selectively cytotoxic for ErbB2-positive cancer cells in vitro and vivo, target an ErbB2 epitope different from that recognised by trastuzumab and do not show cardiotoxic effects.Results:We report that EDIAs are active also on trastuzumab-resistant tumour cells both in vitro and in vivo, most likely because of the different epitope recognised, as EDIAs, unlike trastuzumab, were found to be able to inhibit the signalling pathway downstream of ErbB2.Conclusion:These results suggest that EDIAs are immunoagents that could not only fulfil the therapeutic need of patients ineligible to trastuzumab treatment due to cardiac dysfunction but also prove to be useful for breast cancer patients unresponsive to trastuzumab treatment.

I Small Animal Imaging With a Coded Aperture and a Hybrid Pixel Detector

Histone methylation changes and formation of chromatin loops involving enhancers, promoters and 3′ end regions of genes have been variously associated with active transcription in eukaryotes. We have studied the effect of activation of the retinoic A receptor, at the RARE–promoter chromatin of CASP9 and CYP26A1 genes, 15 and 45 min following RA exposure, and we found that histone H3 lysines 4 and 9 are demethylated by the lysine-specific demethylase, LSD1 and by the JMJ-domain containing demethylase, D2A. The action of the oxidase (LSD1) and a dioxygenase (JMJD2A) in the presence of Fe++ elicits an oxidation wave that locally modifies the DNA and recruits the enzymes involved in base and nucleotide excision repair (BER and NER). These events are essential for the formation of chromatin loop(s) that juxtapose the RARE element with the 5′ transcription start site and the 3′ end of the genes. The RARE bound-receptor governs the 5′ and 3′ end selection and directs the productive transcription cycle of RNA polymerase. These data mechanistically link chromatin loops, histone methylation changes and localized DNA repair with transcription.

Two novel human anti-ErbB2 immunoagents are active on trastuzumab-resistant tumours.

Dual Oxidases (DUOX) 1 and 2 are efficiently expressed in thyroid, gut, lung and immune system. The function and the regulation of these enzymes in mammals are still largely unknown. We report here that DUOX 1 and 2 are expressed in human neuroblastoma SK-N-BE cells as well as in a human oligodendrocyte cell line (MO3-13) and in rat brain and they are induced by platelet derived growth factor (PDGF). The levels of DUOX 1 and 2 proteins and mRNAs are induced by reactive oxygen species (ROS) produced by the membrane NADPH oxidase. As to the mechanism, we find that PDGF stimulates membrane NADPH oxidase to produce ROS, which stabilize DUOX1 and 2 mRNAs and increases the levels of the proteins. Silencing of gp91phox (NOX2), or of the other membrane subunit of NADPH oxidase, p22phox, blocks PDGF induction of DUOX1 and 2. These data unravel a novel mechanism of regulation of DUOX enzymes by ROS and identify a circuitry linking NADPH oxidase activity to DUOX1 and 2 levels in neuroblastoma cells.