Search

Background. Chronic lymphocytic leukaemia (CLL), the most common form of leukaemia in adults in Western countries, is characterized by the clonal expansion of B cells. Despite major advances in CLL therapy/diagnosis, the medical approach to CLL can be further improved. Here we explore the potential diagnostic/therapeutic role of the elongation factor 1 A (eEF1A) in CLL. Two major isoforms of eEF1A proteins exist: the ubiquitous eEF1A1 and the tissue-specialized eEF1A2. Beside their role in the elongation step of translation, both isoforms are involved in different cellular processes such as cell proliferation and apoptosis. Whereas both eEF1A isoforms play a role in solid and hematologic human tumors, nothing is known in CLL. Methods. eEF1A1/eEF1A2 amounts were quantitated by quantitative real time PCR and western blotting in the lymphocytes of 46 CLL patients vs 26 normal control. eEF1A1 functional role in CLL was investigated in a cellular (MEC-1) and in a subcutaneous xenograft animal model of CLL via its targeting by an aptamer (GT75) or a siRNA (siA1), we previously developed. As control molecules an inactive aptamer (CT75) or siRNA (siGL2) were used. Results. At the mRNA level, eEF1A1 but not eEF1A2 was significantly (p=0,0081) more elevated in CLL lymphocytes compared to control. At the protein level, both eEF1A1 and eEF1A2 were more elevated (p=0,028) in CLL lymphocytes compared to control. Moreover, eEF1A1 but not eEF1A2 protein levels were higher (p=0,0042) in patient which died during the study compared to those surviving. Finally, eEF1A1 targeting by either GT75 or siA1 resulted in MEC-1 viability down regulation (p=0,04) mostly due to autophagy stimulation. In vivo, GT75 or siA1 resulted in tumor growth down-regulation (p=0.014) and extension of animal survival (p=0.014), demonstrating the functional role of eEF1A1 in CLL. Conclusions. The increase of eEF1A1/eEF1A2 protein in lymphocytes of CLL patient cells suggests a role as possible novel CLL markers. The increase of eEF1A1 protein in dead vs surviving patients may confer to eEF1A1 also the role of a novel prognostic marker. This, together with the involvement of eEF1A1 in MEC-1 survival in vitro and in vivo, opens the possibility to consider eEF1A1 also as a novel therapeutic target in CLL.

EFFECTS OF CHLORAMBUCIL/HYDROXYCHLOROQUINE-LOADED ANTI-CD20 NANOPARTICLES ON A HUMAN/MOUSE MODEL OF BURKITT LYMPHOMA Mezzaroba N,1 Mansilla E,2 Zorzet S,1 Larsen G,3 Tripodo C,4 Núñez L,5 Marín GH,2 Nabergoj M,6 Tedesco F,1 Pozzato G,6 Macor P1 1 Department of Life Science, University of Trieste, Italy; 2 CUCAIBA, Ministry of Health, La Plata, Buenos Aires, Argentina; 3 LNK Chemsolutions, Lincoln Nebraska, USA and Bio-Target, Chicago, USA; 4 Department of Human Pathology, University of Palermo, Italy; 5 University of Chicago, Chicago, USA and BioTarget, Chicago, USA; 6 Department of Medical and Surgical Sciences, University of Trieste, Italy Introduction. In Burkitt lymphoma (BL) chemotherapy shows variable responses since a fraction of younger patients are resistant or relapse and the patients over 40 have difficulties to follow the therapy. In nonresponders/relapsers BL cells often express low level of CD20 and/or mutant/deleted p53. To overcome these problems, we developed a new therapeutic approach in which the efficacy of chemotherapy is associated to the specificity and low side-effects of antibody-based therapy. This approach lies in biodegradable nanoparticles (NP) coated with an antibody to target the neoplastic cells and filled with Hydroxychloroquine (HCQ) (a p53-independent toxic substance) and chlorambucil (CLB) as modern “Trojan Horses” to destroy the cancer cells. Methods. NP were prepared by the copolymerization of polylactic acid and polycaprolactone (diameter:250nm). The NP with HCQ and CLB were prepared by encapsulation at concentrations of 165 µg per mg of polymer inside their core, and then modified by anti-CD20 adsorption. In vitro study: To investigate the ability of NP to kill Burkitt lymphoma cell lines, 5 x 105 BJAB cells were incubated for 48 hours and the number of residual viable cells was determined with MTT assay. Apoptosis was evaluated by analyzing PARP-1 cleavage and Annexin V detection. In vivo study: A xenograft model was developed in SCID mice inoculated i.p. with 2 x 106 labeled or unlabeled BJAB cells; a fraction of mice was treated with NP and all animals examined twice weekly up to 120 days. A small-animal time-domain eXplore Optix pre-clinical imager was used for the invivo and ex-vivo evaluation of labeled BJAB cell dissemination and Ritcy5.5 distribution. At 120 days the animals were sacrificed and peritoneal mass and organs, lymph nodes and bones were collected for immunohistochemical analysis. Results. In vitro study: The binding NP on cell surface and their internalization were documented with confocal microscopy. In BJAB cells, HCQ and CLM as free substances induced cell death in 82% and 38% respectively, while NP containing only 5,4 g of HCQ and CLB killed 100% of cells. This effect is due to the synergic proapoptotic effect as shown by Annexin V detection and by PARP activation. The effects of NP was tested on CD20-negative cell lines CHO, HUVEC, MEL-28, LoVo: No cell cytotoxicity was detectable using NP, whereas free HCQ+CLB induce cell killing between 58 and 71%. In vivo study: The intra-peritoneal injection of BJAB cells induced a fast (within 4 days) tumor cells spread in different organs and, without therapy, all animals died in 50-70 days as well as those receiving free HCQ and CLM. Conversely, survival at 120 days of the mice receiving 4 or 8 i.p. injections of NP was 50 and 90% respectively. Conclusions. These results indicate that NP are able to cure even the highly proliferating BL cell line BJAB in both in vitro and in vivo models. This suggests the introduction of this promising technology into human clinical trial.

DEVELOPMENT OF CHLORAMBUCIL/HYDROXYCHLOROQUINE-LOADED ANTI-CD20 NANOPARTICLES FOR THE TREATMENT OF CHRONIC LYMPHOCYTIC LEUKEMIA : IN VITRO MODEL Mezzaroba N,1 Mansilla E,2 Zorzet S,1 Calvaruso M,3 Tripodo C,3 Núñez L,4 Tedesco F,1 Nabergoj M,6 Sblattero D,5 Granzotto M,7 Pozzato G,6 Macor P1 1 Department of Life Science, University of Trieste, Italy; 2 CUCAIBA, Ministry of Health, La Plata, Buenos Aires, Argentina; 3 Department of Human Pathology, University of Palermo, Italy; 4 University of Chicago, Chicago, USA and BioTarget, Chicago, USA; 5 Department of Medical Sciences, University of Eastern Piedmont, Novara, Italy; 6 Department of Medical and Surgical Sciences, University of Trieste; 7 Department of Molecular and Laboratory Medicine, IRCCSS Burlo Garofolo, Trieste, Italy Introduction. The actual challenge of tumor therapy is to design new therapeutic agents able to maximize the efficacy minimizing the adverse effects. Nanoparticles (NP) have emerged as important tools to modify the release profile for a large number of drugs. The aim of this study was to determine whether anti-CD20-coated biocompatible FDA-approved NP loaded with Chlorambucil (CLB) and Hydroxychloroquine (HCQ) could induce apoptosis of the B-Chronic Lymphocytic Leukemia (CLL) cell line MEC-1, as well as of circulating neoplastic B-cells isolated from patients with B-CLL. Methods. Nanoparticles were prepared by the copolymerization of polylactic acid and polycaprolactone and had an diameter of 250 nm. The NP with HCQ and CLB were prepared by encapsulation of the two drugs at concentrations of 165 µg per mg of polymer inside their core, and then modified by anti-CD20 adsorption. The NP were resuspended in PBS with 10% Bovine Serum Albumin (BSA) and maintained at 4°C before use. To investigate the ability of NP to affect cell viability and induce cellular apoptosis, MEC-1 cells (2 x 105) were incubated with anti-CD20 coated and CLB-HCQ loaded NP for 48 hours and the residual viable cells was determined with MTT assay. Cell death program activation was also evaluated by analyzing PARP-1 cleavage and Annexin V detection. Monoclonal B-cells were obtained from 44 patients affected by CLL. In each case the IGHV gene mutational status was assessed using standard methods and CD38 and ZAP-70 expressions were determined by flow cytometry. Interphase FISH was performed on nuclei preparations of PBMC and each case was investigated for 13q deletion, 11q deletion, 17p deletion or presence of trisomy 12. Results. Anti-CD20 NP with HCQ and CLB were able to induce a strong reduction (95%) in cell viability of MEC-1 cells. The cytotoxicity was present in a dose-dependent manner and was apoptosis-dependent as confirmed by the PARP- 1 activation and by the binding of Annexin V on cell surface. The same high cytotoxicity rate (>90%) was found also in patient-derived B-cells. The killing rate was independent from IGVH mutational status or from ZAP70 and CD38 expression or from the presence of 17p deletion. An efficient cytotoxicity was present even in CLL B-lymphocytes expressing low CD20 antigen levels on cell surface. The effects of NP were also tested on CD20-negative human and animal cells (CHO, HUVEC, MEL- 28, LoVo cell lines) and no specific cytotoxicity was obtained. On the contrary, incubation of CD20-negative cells with free HCQ + CLB induced cell killing between 58 and 71%. Conclusions. These results indicate that anti-CD20 coated and CLB-HCQ loaded NP show a high toxicity rate in CLL cell line as well as in B-cell obtained from CLL patients. The toxic effect is present also in 17p deleted cases, usually resistant to chemo-immuno-therapy. These results should be confirmed in animal models in order to introduce this promising technology into human clinical trials

Photodynamic therapy (PDT) is a non-invasive therapeutic modality used in a various number of diseases and cancer. It involves the systemic or topic administration of a photosensitizer, followed by irradiation with light. The activated photosensitizer converts oxygen to singlet oxygen and/or reactive oxygen species (ROS) which lead to cell death and tissue necrosis. One aim of PDT research is the discovery of new photosensitizers possessing minimal dark cytotoxicity, high photodynamic properties, improved pharmacokinetics, preferential retention in diseased instead of healthy tissues, chemical stability and a good cellular uptake .We recently focused our efforts on pheophorbide a (Pba), a chlorophyll derivative. Pba is characterized by a stronger absorption between 650-700 nm, in the tissuepenetrating wavelength range. For in vivo applications the capacity of the photosensitiser to reach in the diseased tissues becomes critical, in particular when a large peritoneal area is interested as occurring in carcinomatosis and sarcomatosis. To improve the pharmacokinetic and the activity of the photosensitizer we conjugated Pba to polyethylene glycol (PEG). In vivo the pharmacokinetic analysis performed on living female C57/BL6 mice bearing a subcutaneous melanoma mass, showed that injected mPEG-Pba distributes all over the body, with an higher uptake in the tumor respect to free Pba. Moreover, preliminary data suggest that PEG-Pba in mice bearing B78-H1 amelanotic melanoma reduces the tumor growth after light activation in comparison with Pba.

The present invention describes conjugates of hyaluronic acid (HA) with an antitumor agent (AT) for use in the treatment and/or prevention of brain metastatising tumors. In these conjugates, the conjugation linkage is preferably of covalent type, and may involve any free positions on the hyaluronic acid molecule, in particular the Carbon 6 of the N-acetyl-glucosamine units, the carboxyl group of the glucuronic acid units, the amino group of the deacetylated-N-acetyl glucosamine units, any free hydroxyl groups, etc. The antitumor agent may be chosen among drugs know for antitumor activity, like methotrexate, camptothecin, etc. Pharmaceutical compositions and kits for performing said treatments are also disclosed.

Inibizione delle metastasi di un melanoma murino da complessi di rutenio e down-regulation dell'invasione cellulare.

Intratumor (i.t.) injection of 35 mg/kg/day NAMI-A for six consecutive days to CBA mice bearing i.m. implants of MCa mammary carcinoma reduces primary tumor growth and particularly lung metastasis formation, causing 60% of animals to be free of macroscopically detectable metastases. The i.t. treatment allows study of the effects of NAMI-A on in vivo tumor cells exposed to millimolar concentrations for a relatively prolonged time. Under these conditions, NAMI-A reduces the number of CD44 tumor cells and changes tumor cell phenotype to a lower aggressive behavior, as shown by scanning electron microscopy analysis. On primary tumor site, NAMI-A causes unbalance between 2n and aneuploid cells in favor of lymphocytes. Furthermore, in tumor tissue, nitric oxide production is increased and active matrix metalloproteinase 9 is decreased, and these effects are accompanied by a reduced hemoglobin concentration. These data are in agreement with the reduction of tumor invasion and metastasis and suggest the therapeutic usefulness of NAMI-A in neoadjuvant or tumor reduction treatments for preventing metastasis formation. These data further stress the usefulness of intratumor treatments as experimental preclinical model for studying in vivo the mechanism of tumor cell interactions after prolonged exposure to ruthenium-based compounds to be developed for metastasis inhibition.

In mice bearing Lewis lung carcinoma, rotational and restraint stress specifically increases the formation of lung metastasis, and restraint stress markedly attenuates the antitumor effects of cyclophosphamide. The aim of this investigation was therefore to examine the effects of restraint stress on tumor metastasis in mice bearing MCa mammary carcinoma, and on the effectiveness of CCNU and DTIC. Restraint stress increases MCa mammary carcinoma metastasis, causes a marked reduction in cyclophosphamide activity, and a minor attenuation of the effects of CCNU and DTIC. The possible occurrence of seasonal factors, observed for the increase by rotational stress of Lewis lung carcinoma metastasis, was also determined for cyclophosphamide effectiveness. The survival time of control mice is longer in February than in June, and is not appreciably modified by rotational stress. The effects of cyclophosphamide are similar in both seasonal periods, and are similarly attenuated by rotational stress. The seasonal effects of rotational stress, and the reduction of the effects of cyclophosphamide caused by rotational stress, are accompanied by corresponding variations in the number of CD3+ and CD4+ splenic T-lymphocyte subsets and in the CD4+/CD8+ ratio, respectively. The reported effects of stress on tumor progression and on the effectiveness of cyclophosphamide thus appear to occur via modulation of immune responses of the host directed against the tumor. These data appear of interest for their experimental implications, and suggest the opportunity to consider the role that the stress during treatment may play in determining the effectiveness of clinical antitumor chemotherapy.

CLINICAL EXPERIMENTAL METASTASIS

The therapeutic activity of the combined treatment with surgery and ICRF-159, measured in terms of increase of the survival time, has been tested in mice bearing two Lewis lung carcinoma lines having different potential to spontaneously metastasize (M1087 high; BM21548 low). As expected from the characteristics of this drug, a significant prolongation of the survival time of the treated hosts can be achieved only after presurgical treatment; the overall effect is greater using the tumor line with low metastatic ability. The response of the two tumor lines used to postoperative treatment with Cyclophosphamide also depends upon the tumor line used. The preoperative treatment of the animals with ICRF-159 markedly increases the response to Cyclophosphamide of the low responding M1087 line.

The tissue levels of two proteolytic enzymes, plasminogen activator and cathepsin B - like cysteine proteinase, which were found to be increased in malignant tumors and to be proportional to tumor metastatic potential in some instances, have been determined in a panel of solid metastasizing tumors in mice. The examination of B16 melanoma, MCa mammary carcinoma and of two lines of Lewis lung carcinoma with widely different potential to spontaneously metastasize, showed no correlation between metastatic potential and the tissue content of the proteinases considered. The treatment of the animals with cytotoxic antitumor drugs (CCNU, GANU, cisplatin, and cyclophosphamide) or with antimetastatic drugs acting with a mechanism unrelated with cytotoxicity (ICRF 159 and DM-COOK) caused only marginal inhibition in some instances, whereas no meaningful pattern of inhibition either based in terms of metastatic potential of the tumor or on drug mechanism of action was recognizable. A direct involvement of the two proteinases examined in the process of metastasis in the tumor panel used is thus not apparent, although a more complex interaction with other latent proteinases and inhibitors might be operative.

The aim of this work was to determine the possible participation of adrenergic responses to the increase in tumor dissemination induced by rotational stress using drugs affecting monoaminergic function. The growth of the primary tumor and the formation of lung metastasis were determined in mice implanted with Lewis lung carcinoma, subjected to rotational stress and treated with the adrenergic neuron blocker reserpine, the alpha-receptor blocker phenoxybenzamine, and the beta-blocker propranolol. Treatment with reserpine markedly reduced the formation of spontaneous lung metastasis and completely abolished the increase in metastases caused by rotational stress without direct effect on tumor cells or blood vessels. Phenoxybenzamine and propranolol caused opposite effects on tumor progression. Previous immunosuppression by cyclophosphamide in mice with tumors reduced the antimetastatic effects of reserpine. These results suggesting the importance of the adrenergic modulation of immune resistance factors in controlling metastasis development and indicate a possible role in monitoring the use and effects of monoaminergic drugs in cancer patients.

The antitumor effects of therapy with peptidoglycan monomer, combined with surgery were investigated. Timing od administration plays an important role on the overall effectiveness of such therapy.

The present invention is related to Ruthenium derived antitumoral compounds with high antimetastatic activity and with low toxicity for the host. It is described the use of inorganic Ruthenium complexes in the therapy of solid tumors, in particular of those tumors characterized by high metastatizing ability. Pharmaceutical compositions containing Ruthenium complexes in formulations suitable for the administration and a kit for the preparation of such complexes at the moment of use or immediately before use, are moreover described.