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1.
Infez Med ; 20 Suppl 2: 35-42, 2012.
Article in English | MEDLINE | ID: mdl-23042004

ABSTRACT

Clonal B-cell populations in non-lymphomatous processes have been sporadically reported in enlarged reactive lymph nodes and mucosa-associated lymphoid cell populations. These generally small clones are considered non-malignant proliferations of B-lymphocytes determined by an abnormal response to bacterial or viral antigen stimulation. In cases reported in literature, clonality was detected by light chain assessment and or by polymerase chain reaction (PCR) analysis of immunoglobulin heavy chain (IgH) gene in histologically and clinically proven non lymphomatous processes. In this study the clinical, cytological, phenotypical and pathological features of three HIV patients in which non-lymphomatous clonal B-cell populations detected in enlarged lymph nodes are reported. All the patients complained for later cervical lymph nodes enlargement, positive at the FDG-positron emission tomography scan. Fine needle cytology, coupled with flow cytometry showed atypical lymphoid cell proliferations and kappa (2 cases) or lambda (1 case) light chain restriction. Reactive, non lymphomatous nature of these processes were then proven by histological control in two cases and by clinical follow-up in the last one; corresponding clinical and pathological aspects are discussed. Clonal B-cell populations in non-lymphomatous processes can sporadically occur in enlarged reactive lymph nodes in immunodeficiency as well as in autoimmune processes. Awareness of the phenomenon and attention should be paid in the evaluation of corresponding pathological features and in the clinical management of corresponding patients.


Subject(s)
Acquired Immunodeficiency Syndrome/immunology , Acquired Immunodeficiency Syndrome/pathology , B-Lymphocytes , Lymph Nodes/pathology , Adult , Female , Humans , Male , Retrospective Studies
2.
Infez Med ; 20 Suppl 2: 26-34, 2012.
Article in English | MEDLINE | ID: mdl-23042003

ABSTRACT

The efficacy and safety of low dose oral valgancyclovir (VGCV) as cytomegalovirus (CMV) reactivation prophylaxis was retrospectively evaluated in 32 consecutive patients which underwent allogeneic HLA-matched related and unrelated hematopoietic stem cell transplantation (HSCT). Thirty HSCT recipients showed pretransplant CMV seropositivity. Fifteen received a myeloablative conditioning regimen, while seventeen patients received a reduced-intensity conditioning regimen. Twenty-one patients received graft-versus-host disease (GVHD) prophylaxis with cyclosporin A (CsA) and methotrexate (MTX), and the others CsA with MTX and anti-thymocyte globulin. CMV infection was monitored weekly using polymerase chain reaction (PCR). VGCV was administered orally at a dose of 450 mg daily for six months. Six patients developed a positive CMV-PCR on average 56 days after HSCT successfully treated with VGCV at 1800 mg/day, except one who developed fatal gastrointestinal CMV disease. At the time of CMV reactivation, four patients had been affected by grade II-IV acute GVHD and two by an extensive chronic GVHD. No significant specific VGCV-related toxicity was encountered. Seven patients presented hematological toxicity which did not require drug discontinuation. Our data suggest that low dose VGCV is safe and effective as CMV reactivation prophylaxis in allogeneic HSCT recipients. These results require further validation in prospective randomized studies.


Subject(s)
Antiviral Agents/administration & dosage , Cytomegalovirus Infections/etiology , Cytomegalovirus Infections/prevention & control , Cytomegalovirus/drug effects , Cytomegalovirus/physiology , Ganciclovir/analogs & derivatives , Hematopoietic Stem Cell Transplantation/adverse effects , Virus Activation/drug effects , Adolescent , Adult , Antiviral Agents/pharmacology , Female , Ganciclovir/administration & dosage , Ganciclovir/pharmacology , Humans , Male , Middle Aged , Prospective Studies , Valganciclovir , Young Adult
3.
Ann Oncol ; 20(5): 906-12, 2009 May.
Article in English | MEDLINE | ID: mdl-19155242

ABSTRACT

BACKGROUND: Preoperative chemoradiation is now standard treatment for stages II-III rectal cancer. Capecitabine (CAP) and oxaliplatin (OX) are synergistic with radiotherapy (RT) and active in colorectal neoplasms. PATIENTS AND METHODS: Two cycles of CAP 825 mg/m(2) b.i.d. (days 1-14) and OX 50 mg/m(2) (days 1 and 8) every 3 weeks were given concomitantly with pelvic conformal RT (45 Gy). Patients with a > or =T3 and/or node-positive rectal tumour were eligible. The pathologic tumour response was defined according to the tumour regression grade (TRG) scale. RESULTS: Forty-six patients were enrolled. Gastrointestinal adverse events were mostly G1-G2; only two patients experienced G3 vomiting and diarrhoea and six patients had G1 peripheral neuropathy. Haematological toxicity was rare. G2 proctitis and anal pain occurred in two patients. Pathological complete response (TRG1) was observed in nine patients (20.9%; 95% CI 8.7%-33.1%); TRG2 in 19 patients (44.2%); TRG3 in 12 patients (27.9%); and TRG4 in three patients (7%). Overall, nine patients recurred: five with distant metastases, one with local recurrence, and three with both local recurrence and distant metastases. CONCLUSIONS: CAP-OX-RT as preoperative treatment for rectal cancer induces a remarkable rate of complete or near-complete pathologically documented response and is well tolerated.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Digestive System Surgical Procedures , Radiotherapy, Conformal , Rectal Neoplasms/therapy , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Capecitabine , Chemotherapy, Adjuvant , Deoxycytidine/administration & dosage , Deoxycytidine/analogs & derivatives , Disease-Free Survival , Female , Fluorouracil/administration & dosage , Fluorouracil/analogs & derivatives , Humans , Male , Middle Aged , Neoadjuvant Therapy , Neoplasm Metastasis , Neoplasm Recurrence, Local , Organoplatinum Compounds/administration & dosage , Oxaliplatin , Radiotherapy, Conformal/adverse effects , Rectal Neoplasms/drug therapy , Rectal Neoplasms/mortality , Rectal Neoplasms/pathology , Rectal Neoplasms/radiotherapy , Rectal Neoplasms/surgery , Time Factors , Treatment Outcome
4.
Ann Oncol ; 20(5): 874-8, 2009 May.
Article in English | MEDLINE | ID: mdl-19179552

ABSTRACT

BACKGROUND: Although most BRCA sequence variants are clearly deleterious and unequivocally pathogenetic, several are still classified as variants of unknown significance. PATIENTS AND METHODS: We followed families undergoing oncogenetic counseling from risk identification to risk definition by genetic testing and risk management. RESULTS: We identified two germline mutations in the BRCA2 gene in a woman with breast and ovarian cancer. One sequence alteration was 859/G>A in exon 7 (V211I). The other second sequence alteration (IVS13-2A>T) affected the splicing site in intron 13. The latter alteration is not yet listed in the Breast Cancer Information Core database. RT-PCR resulted in transcription of a sequence lacking exon 7 and a subsequent anomalous stop codon in exon 9 thereby confirming altered messenger RNA (mRNA) maturation. Amplification of the mutation in intron 13 resulted in transcription of a sequence lacking exon 14 and an anomalous stop codon in exon 15 thereby confirming altered mRNA maturation. Both mutations led to a truncated BRCA2 protein in its carboxy-terminal region. CONCLUSION: The two BRCA2 mutations identified affect mRNA splicing fidelity and play a pathogenetic role in breast and ovarian cancer.


Subject(s)
BRCA2 Protein/genetics , Breast Neoplasms/genetics , Exons , Genetic Counseling , Genetic Testing , Germ-Line Mutation , Ovarian Neoplasms/genetics , RNA Splice Sites , Apoptosis Regulatory Proteins , BRCA1 Protein/genetics , Breast Neoplasms/pathology , Breast Neoplasms/therapy , DNA Mutational Analysis , Female , Gene Expression Regulation, Neoplastic , Genetic Predisposition to Disease , Genetic Testing/methods , Humans , Middle Aged , Ovarian Neoplasms/pathology , Ovarian Neoplasms/therapy , Pedigree , Phenotype , Prognosis , Risk Assessment
6.
Br J Cancer ; 99(3): 473-80, 2008 Aug 05.
Article in English | MEDLINE | ID: mdl-18665191

ABSTRACT

We investigated the antitumour effect and ability to overcome the resistance to anti-EGFR drugs of enzastaurin, an inhibitor of VEGFR-dependent PKCbeta signalling. Enzastaurin was evaluated alone and in combination with the EGFR inhibitor gefitinib, on growth and signalling protein expression in human cancer cells sensitive and resistant to anti-EGFR drugs, both in vitro and in nude mice. We demonstrated the marked inhibitory activity of enzastaurin against GEO colon and PC3 prostate cancer cells and their gefitinib-resistant counterparts GEO-GR and PC3-GR, accompanied by inhibition of pAkt and its effector pp70S6K, pGSK3beta and VEGF expression and secretion. Moreover, enzastaurin showed a cooperative effect with gefitinib in parental and in gefitinib-resistant cells. Remarkably, these results were confirmed in vivo, where enzastaurin showed antitumour activity and cooperativity with gefitinib in mice grafted with GEO and GEO-GR tumours, incrementing their median survival and inhibiting the aforesaid protein expression and secretion in tumour specimens. In conclusion, enzastaurin by interfering with signalling proteins implicated in EGFR drug resistance markedly cooperates with gefitinib in sensitive and gefitinib-resistant tumours, thus overcoming and reverting such resistance and providing a rational basis for its development in patients resistant to anti-EGFR drugs.


Subject(s)
Antineoplastic Agents/pharmacology , ErbB Receptors/antagonists & inhibitors , Indoles/pharmacology , Animals , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Line, Tumor , Culture Media, Conditioned , Drug Screening Assays, Antitumor , Enzyme-Linked Immunosorbent Assay , Gefitinib , Humans , Mice , Mice, Nude , Phosphatidylinositol 3-Kinases/metabolism , Quinazolines/pharmacology , Vascular Endothelial Growth Factor A/metabolism
7.
J Mol Cell Cardiol ; 44(2): 270-80, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18191142

ABSTRACT

Impaired glucose uptake is associated with both cardiac hypertrophy and contractile dysfunction, but whether there are common underlying mechanisms linking these conditions is yet to be determined. Using a 'gene dose' Cre-Lox GLUT4-deficient murine model, we examined the effect of suppressed glucose availability on global myocardial gene expression and glycolysis substrate bypass on the function of isolated perfused hearts. Performance of hearts from 22- to 60-week-old male GLUT4 knockout (KO, >95% reduction in GLUT4), GLUT4 knockdown (KD, 85% reduction in cardiac GLUT4) and C57Bl/6 wild-type (WT) controls was measured ex vivo in Langendorff mode perfusion. DNA microarray was used to profile mRNA expression differences between GLUT4-KO and GLUT4-KD hearts. At 22 weeks, GLUT4-KO hearts exhibited cardiac hypertrophy and impaired contractile function ex vivo, characterized by a 40% decrease in developed pressure. At 60 weeks, dysfunction was accentuated in GLUT4-KO hearts and evident in GLUT4-KD hearts. Exogenous pyruvate (5 mM) restored systolic pressure to a level equivalent to WT (GLUT4-KO, 176.8+/-13.2 mmHg vs. WT, 146.4+/-9.56 mmHg) in 22-week-old GLUT4-KO hearts but not in 60-week-old GLUT4-KO hearts. In GLUT4-KO, DNA microarray analysis detected downregulation of a number of genes centrally involved in mitochondrial oxidation and upregulation of other genes indicative of a shift to cytosolic beta-oxidation of long chain fatty acids. A direct link between cardiomyocyte GLUT4 deficiency, hypertrophy and contractile dysfunction is demonstrated. These data provide mechanistic insight into the myocardial metabolic adaptations associated with short and long-term insulin resistance and indicate a window of opportunity for substrate intervention and functional 'rescue'.


Subject(s)
Glucose Transporter Type 4/deficiency , Glucose/metabolism , Myocardium/metabolism , Aging/drug effects , Animals , Blood Pressure/drug effects , Body Weight/drug effects , Down-Regulation/drug effects , Energy Metabolism/drug effects , Energy Metabolism/genetics , Heart Rate/drug effects , In Vitro Techniques , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Myocardial Contraction/drug effects , Myocardium/pathology , Organ Size/drug effects , Pyruvic Acid/pharmacology , Substrate Specificity/drug effects , Time Factors , Up-Regulation/drug effects
8.
Hum Gene Ther ; 18(9): 821-36, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17845130

ABSTRACT

Successful gene therapy approaches for metachromatic leukodystrophy (MLD), based either on hematopoietic stem/progenitor cells (HSPCs) or direct central nervous system (CNS) gene transfer, highlighted a requirement for high levels of arylsulfatase A (ARSA) expression to achieve correction of disease manifestations in the mouse model. Full assessment of the safety of ARSA expression above physiological levels thus represents a prerequisite for clinical translation of these approaches. Here, using lentiviral vectors (LVs), we generated two relevant models for the stringent evaluation of the consequences of ARSA overexpression in transduced cells. We first demonstrated that ARSA overexpression in human HSPCs does not affect their clonogenic and multilineage differentiation capacities in clonogenic assays and in a neonatal hematochimeric mouse model. Further, we studied ARSA overexpression in all body tissues by generating transgenic mice overexpressing the ARSA enzyme by LV up to 15-fold above the normal range and carrying multiple copies of LV in their genome. Characterization of these mice demonstrated the safety of ARSA overexpression in two main gene therapy targets, HSPCs and neurons, with maintenance of the complex functions of the hematopoietic and nervous system in the presence of supraphysiological enzyme levels. The activity of other sulfatases dependent on the same common activator, sulfatase-modifying factor-1 (SUMF1), was tested in ARSA-overexpressing HSPCs and in transgenic mice, excluding the occurrence of saturation phenomena. Overall, these data indicate that from the perspective of clinical translation, therapeutic levels of ARSA overexpression can be safely achieved. Further, they demonstrate an experimental platform for the preclinical assessment of the safety of new gene therapy approaches.


Subject(s)
Cerebroside-Sulfatase/metabolism , Genetic Therapy , Leukodystrophy, Metachromatic/genetics , Leukodystrophy, Metachromatic/therapy , Animals , Animals, Newborn , Antigens, CD34/immunology , Antigens, CD34/metabolism , Blotting, Southern , Cell Differentiation , Cell Lineage , Cell Proliferation , Cerebroside-Sulfatase/adverse effects , Cerebroside-Sulfatase/analysis , Colony-Forming Units Assay , Feasibility Studies , Genetic Vectors , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Humans , Lentivirus/genetics , Leukodystrophy, Metachromatic/metabolism , Leukodystrophy, Metachromatic/pathology , Mice , Mice, Transgenic , Models, Animal , Neurons/cytology , Neurons/metabolism , Polymerase Chain Reaction , Spleen/cytology , Transduction, Genetic
9.
Mol Cell Biochem ; 295(1-2): 217-28, 2007 Jan.
Article in English | MEDLINE | ID: mdl-16944307

ABSTRACT

Although the causal relationship between insulin resistance (IR) and hypertension is not fully resolved, the importance of IR in cardiovascular dysfunction is recognized. As IR may follow excess sucrose or fructose diet, the aim of this study was to test whether dietary starch substitution with sucrose results in myocardial dysfunction in energy substrate utilization and contractility during normoxic and post-ischemic conditions. Forty-eight male Wistar rats were randomly allocated to three diets, differing only in their starch to sucrose (S) ratio (13, 2 and 0 for the Low S, Middle S and High S groups, respectively), for 3 weeks. Developed pressure and rate x pressure product (RPP) were determined in Langendorff mode-perfused hearts. After 30 min stabilization, hearts were subjected to 25 min of total normothermic global ischemia, followed by 45-min reperfusion. Oxygen consumption, beta-oxidation rate (using 1-13C hexanoate and Isotopic Ratio Mass Spectrometry of CO2 produced in the coronary effluent) and flux of non-oxidative glycolysis were also evaluated. Although fasting plasma glucose levels were not affected by increased dietary sucrose, high sucrose intake resulted in increased plasma insulin levels, without significant rise in plasma triglyceride and free fatty acid concentrations. Sucrose-rich diet reduced pre-ischemic baseline measures of heart rate, RPP and non-oxidative glycolysis. During reperfusion, post-ischemic recovery of RPP was impaired in the Middle S and High S groups, as compared to Low S, mainly due to delayed recovery of developed pressure, which by 45 min of reperfusion eventually resumed levels matching Low S. At the start of reperfusion, delayed post-ischemic recovery of contractile function was accompanied by: (i) reduced lactate production; (ii) decreased lactate to pyruvate ratio; (iii) increased beta-oxidation; and (iv) depressed metabolic efficiency. In conclusion, sucrose rich-diet increased plasma insulin levels, in intact rat, and increased cardiac beta-oxidation and coronary flow-rate, but reduced glycolytic flux and contractility during normoxic baseline function of isolated perfused hearts. Sucrose rich-diet impaired early post-ischemic recovery of isolated heart cardiac mechanical function and further augmented cardiac beta-oxidation but reduced glycolytic and lactate flux.


Subject(s)
Dietary Sucrose/administration & dosage , Glycolysis , Hyperinsulinism/pathology , Myocardial Contraction/physiology , Myocardial Ischemia/pathology , Myocardium/metabolism , Animals , Blood Glucose/metabolism , Body Weight/drug effects , Dietary Sucrose/pharmacology , Glycolysis/drug effects , In Vitro Techniques , Insulin/blood , Lactic Acid/metabolism , Lipids/blood , Male , Myocardial Contraction/drug effects , Myocardial Ischemia/chemically induced , Myocardial Reperfusion Injury , Organ Size/drug effects , Oxidation-Reduction/drug effects , Pyruvic Acid/metabolism , Rats , Rats, Wistar , Triglycerides/blood
10.
Ann Oncol ; 16 Suppl 4: iv61-68, 2005 May.
Article in English | MEDLINE | ID: mdl-15923432

ABSTRACT

BACKGROUND: Epidermal growth factor receptor (EGFR) inhibitors are in clinical development in cancer treatment. Preclinical studies have shown potential antitumor efficacy of these agents in combination with chemotherapy or with radiotherapy. However, controversial results have been obtained in different clinical trials. MATERIALS AND METHODS: The effects on proliferation, cell cycle distribution and induction of apoptosis of three different anti-EGFR agents (gefitinib, ZD6474, cetuximab) were evaluated in different sequences of combination with either a platinum derivative (cisplatin, carboplatin, oxaliplatin) or a taxane (docetaxel, paclitaxel) in KYSE30 cells, a model of a human cancer cell line with a functional EGFR autocrine pathway. RESULTS: The combination of a cytotoxic drug with an EGFR inhibitor caused different antiproliferative effects on KYSE30 cancer cells depending on the treatment schedule. An antagonistic effect was observed when treatment with each EGFR inhibitor was done before chemotherapy. In contrast, a synergistic antiproliferative activity was obtained when chemotherapy was followed by treatment with EGFR antagonists. This effect was accompanied by potentiation of apoptosis and arrest of the surviving cancer cells in the G(2)/M phases of the cell cycle. CONCLUSIONS: This study provides a rationale for the evaluation of a potentially synergistic sequence of cytotoxic drugs and EGFR inhibitors in a clinical setting.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents/therapeutic use , Carcinoma, Squamous Cell/drug therapy , ErbB Receptors/antagonists & inhibitors , Esophageal Neoplasms/drug therapy , Piperidines/therapeutic use , Quinazolines/therapeutic use , Antibodies, Monoclonal, Humanized , Carboplatin/therapeutic use , Cell Cycle/drug effects , Cell Line, Tumor , Cetuximab , Cisplatin/therapeutic use , Docetaxel , Dose-Response Relationship, Drug , Gefitinib , Humans , Organoplatinum Compounds/therapeutic use , Oxaliplatin , Paclitaxel/therapeutic use , Taxoids/therapeutic use
11.
Br J Cancer ; 92(9): 1644-9, 2005 May 09.
Article in English | MEDLINE | ID: mdl-15856038

ABSTRACT

The aim of the study was to assess the toxicity and the clinical activity of biweekly oxaliplatin in combination with infusional 5-fluorouracil (5-FU) and folinic acid (FA) administered every 2 weeks (FOLFOX-4 regimen) in patients with advanced gastric cancer (AGC). A total of 61 previously untreated AGC patients were treated with oxaliplatin 85 mg m(-2) on day 1, FA 200 mg m(-2) as a 2 h infusion followed by bolus 5-FU 400 mg m(-2) and a 22 h infusion of 5-FU 600 mg m(-2), repeated for 2 consecutive days every 2 weeks. All patients were assessable for toxicity and response to treatment. Four (7%) complete responses and 19 partial responses were observed (overall response rate, 38%). Stable disease was observed in 22 (36%) patients, with progressive disease in the other six (10%) patients. Median time to progression (TTP) and median overall survival (OS) were 7.1 and 11.2 months, respectively. National Cancer Institute Common Toxicity Criteria grade 3 and 4 haematologic toxicities were neutropenia, anaemia and thrombocytopenia in 36, 10 and 5% of the patients, respectively. Grade 3 peripheral neuropathy was recorded in three (5%) patients. FOLFOX-4 is an active and well-tolerated chemotherapy. Response rate (RR), TTP and OS were comparable with those of other oxaliplatin-based regimens, suggesting a role for this combination in gastric cancer.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Fluorouracil/therapeutic use , Leucovorin/therapeutic use , Organoplatinum Compounds/therapeutic use , Stomach Neoplasms/drug therapy , Adenocarcinoma/drug therapy , Aged , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Carcinoma, Signet Ring Cell/drug therapy , Drug Administration Schedule , Female , Fluorouracil/adverse effects , Humans , Leucovorin/adverse effects , Male , Middle Aged , Neoplasm Metastasis , Neutropenia/chemically induced , Organoplatinum Compounds/adverse effects , Stomach Neoplasms/pathology , Survival Rate
12.
Eur J Cancer ; 39(14): 2103-9, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12957467

ABSTRACT

In the present paper, we investigated the relationship between the growth inhibitory effects of recombinant interferon-alpha2b (rIFN-alpha2b) and poly (ADPR) polymerase-1 (PARP-1) activity in the human squamous KB cancer cell line. Growth inhibition of the KB cells mediated by 1000 IU/ml of rIFN-alpha2b was accompanied by a transient rise in PARP-1 specific activity 24 h after rIFN-alpha2b treatment, confirmed by both the increase of intracellular poly (ADP-ribose) content and the PARP-1 auto-modification level. At longer times of incubation, the onset of apoptosis accompanied KB cell growth inhibition, as demonstrated by both flow cytometry and western-blotting analysis showing an 89 kDa apoptotic fragment of PARP-1. Moreover, pretreatment of the cells with the PARP-1 inhibitor, 3-aminobenzamide (3-ABA), at non-cytotoxic concentrations (1 mM), reduced the cell-growth inhibition, cell-cycle perturbation and apoptosis caused by rIFN-alpha2b. Taken together, these results strongly suggest that PARP-1 may be directly involved in the effects of rIFN-alpha2b in the KB cancer cell line.


Subject(s)
Antineoplastic Agents/therapeutic use , Carcinoma, Squamous Cell/drug therapy , Interferon-alpha/therapeutic use , Poly(ADP-ribose) Polymerases/metabolism , Blotting, Western , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Cell Cycle , Cell Division , Enzyme Activation , Flow Cytometry , Fluorescent Antibody Technique , Humans , Interferon alpha-2 , KB Cells , Recombinant Proteins , Tumor Cells, Cultured
13.
Cell Death Differ ; 10(2): 218-29, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12700650

ABSTRACT

The mechanisms of tumor cell resistance to interferon-alpha (IFNalpha) are at present mostly unsolved. We have previously demonstrated that IFNalpha induces apoptosis on epidermoid cancer cells and EGF antagonizes this effect. We have also found that IFNalpha-induced apoptosis depends upon activation of the NH(2)-terminal Jun kinase-1 (Jnk-1) and p(38) mitogen-activated protein kinase, and that these effects are also antagonized by EGF. At the same time, IFNalpha increases the expression and function of the epidermal growth factor receptor (EGF-R). Here we report that the apoptosis induced by IFNalpha occurs together with activation of caspases 3, 6 and 8 and that EGF also antagonizes this effect. On the basis of these results, we have hypothesized that the increased EGF-R expression and function could represent an inducible survival response that might protect tumor cells from apoptosis caused by IFNalpha via extracellular signal regulated kinase 1 and 2 (Erk-1/2) cascades. We have found an increased activity of Ras and Raf-1 in IFNalpha-treated cells. Moreover, IFNalpha induces a 50% increase of the phosphorylated isoforms and enzymatic activity of Erk-1/2. We have also demonstrated that the inhibition of Ras activity induced by the transfection of the dominant negative Ras plasmid RASN17 and the inhibition of Mek-1 with PD098059 strongly potentiates the apoptosis induced by IFNalpha. Moreover, the selective inhibition of this pathway abrogates the counteracting effect of EGF on the IFNalpha-induced apoptosis. All these findings suggest that epidermoid tumor cells counteract the IFNalpha-induced apoptosis through a survival pathway that involves the hyperactivation of the EGF-dependent Ras->Erk signalling. The selective targeting of this pathway appears to be a promising approach in order to enhance the antitumor activity of IFNalpha.


Subject(s)
Apoptosis/drug effects , Epidermal Growth Factor/metabolism , Interferon-alpha/pharmacology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases/metabolism , ras Proteins/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Caspases/metabolism , Cell Survival , Enzyme Activation , Enzyme Inhibitors/pharmacology , Epidermal Growth Factor/pharmacology , ErbB Receptors/drug effects , ErbB Receptors/metabolism , Flavonoids/pharmacology , Humans , Interferon-alpha/metabolism , KB Cells , Mitogen-Activated Protein Kinase 1/drug effects , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/drug effects , Proto-Oncogene Proteins c-raf/analysis , Proto-Oncogene Proteins c-raf/drug effects
14.
Ann Oncol ; 13(3): 392-8, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11996469

ABSTRACT

BACKGROUND: DNA damage caused by platinum agents is frequently followed by induction of topoisomerase I, providing a rationale for use of platinum-based compounds with topoisomerase I inhibitors. MATERIALS AND METHODS: We studied the effect of a sequential schedule of oxaliplatin on day I and topotecan on days 2-5, in human colon and ovarian cancer cells in vitro, in nude mice bearing human cancer xenografts and finally in cancer patients in a phase I trial. RESULTS: We demonstrated a supra-additive effect of this combination on inhibition of colony formation and induction of apoptosis in vitro. We then demonstrated that the two agents in combination markedly inhibit tumor growth in nude mice. We translated these results into a clinical setting, conducting a phase I study in cancer patients with oxaliplatin 85 mg/m2 on day 1 and topotecan at doses escalating from 0.5 to 1.5 mg/m2 on days 2-5. Sixty cycles of treatment were administered to 18 patients affected prevalently by ovarian and colorectal cancer. Combination with topotecan 1.5 mg/m2 caused a dose-limiting toxicity. Therefore the maximum tolerated dose of topotecan was 1.25 mg/m2, at which six patients experienced a mild hematological and gastrointestinal toxicity. We also obtained evidence of clinical activity, particularly in ovarian cancer. CONCLUSIONS: Our results provide a solid biological and clinical rationale for a phase II trial at the recommended doses of oxaliplatin 85 mg/m2 and topotecan 1.25 mg/m2, possibly in ovarian cancer patients.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Colorectal Neoplasms/drug therapy , Ovarian Neoplasms/drug therapy , Tumor Cells, Cultured/drug effects , Adult , Aged , Animals , Apoptosis/drug effects , Cell Cycle/drug effects , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Female , Humans , Male , Maximum Tolerated Dose , Mice , Mice, Inbred BALB C , Middle Aged , Organoplatinum Compounds/administration & dosage , Oxaliplatin , Topotecan/administration & dosage
15.
Asia Pac J Clin Nutr ; 10(3): 212-5, 2001.
Article in English | MEDLINE | ID: mdl-11708311

ABSTRACT

Studies of the therapeutic efficacy of coenzyme Q10 (CoQ10) have been confounded by the variable bioavailability of numerous CoQ10 preparations. The aims of the present study were to determine the early serum levels attained by two different preparations of CoQ10, a soybean oil-based preparation and a complex micelle emulsion and to assess whether these preparations of oral CoQ10 influence plasma lipid profiles. Twelve healthy individuals received 300 mg CoQ10 daily of either preparation for 7 days in a double-blind cross-over design with a 21-day washout period. Blood samples to determine serum levels of CoQ10 and lipids were taken at baseline, after 24 h and 7 days. Both preparations induced significant increases in serum CoQ10 levels at 24 h and 7 days. These were for soy oil: baseline 0.27 +/- 0.03 mol/L, 24 h 0.50 +/- 0.04 mol/L (180%) and 7 days 0.80 +/- 0.05 mol/L (291%), mean +/- SEM: for emulsion: baseline 0.29 +/- 0.03 mol/L, 24 h 0.45 +/- 0.03 mol/L (150%) and 7 days 0.79 +/- 0.06 mol/L (270%). There were no significant differences between CoQ10 levels for the two preparations at either time point. There was no change in any of the serum lipids following the 7 days treatment. We conclude that administration of either a soy oil suspension or a complex emulsion of CoQ10 increases serum levels to the therapeutic range within 1 week.


Subject(s)
Antioxidants/pharmacology , Lipids/blood , Ubiquinone/analogs & derivatives , Ubiquinone/pharmacology , Adult , Antioxidants/analysis , Antioxidants/pharmacokinetics , Biological Availability , Coenzymes , Cross-Over Studies , Double-Blind Method , Emulsions , Female , Humans , Male , Middle Aged , Soybean Oil , Treatment Outcome , Ubiquinone/blood , Ubiquinone/pharmacokinetics
16.
Nat Cell Biol ; 3(10): 867-73, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11584267

ABSTRACT

Stretching of cardiac muscle modulates contraction through the enhancement of the Ca2+ transient, but how this occurs is still not known. We found that stretching of myocytes modulates the elementary Ca2+ release process from ryanodine-receptor Ca2+-release channels (RyRCs), Ca2+ sparks and the electrically stimulated Ca2+ transient. Stretching induces PtdIns-3-OH kinase (PI(3)K)-dependent phosphorylation of both Akt and the endothelial isoform of nitric oxide synthase (NOS), nitric oxide (NO) production, and a proportionate increase in Ca2+-spark frequency that is abolished by inhibiting NOS and PI(3)K. Exogenously generated NO reversibly increases Ca2+-spark frequency without cell stretching. We propose that myocyte NO produced by activation of the PI(3)K-Akt-endothelial NOS axis acts as a second messenger of stretch by enhancing RyRC activity, contributing to myocardial contractile activation.


Subject(s)
Calcium Signaling/physiology , Calcium/metabolism , Myocardial Contraction/physiology , Myocardium/metabolism , Nitric Oxide/metabolism , Protein Serine-Threonine Kinases , Animals , Electric Stimulation , Enzyme Inhibitors/pharmacology , Fluorescent Dyes/metabolism , Male , Myocardial Contraction/drug effects , Myocardium/cytology , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type III , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Rats , Rats, Sprague-Dawley , S-Nitroso-N-Acetylpenicillamine/pharmacology , Spectrometry, Fluorescence
17.
Novartis Found Symp ; 235: 172-96; discussion 196-201, 217-20, 2001.
Article in English | MEDLINE | ID: mdl-11280025

ABSTRACT

Excitation of cardiac cells is accompanied by Ca2+ influx which triggers a transient increase in cytosolic [Ca2+], (Cai), and contraction. While the amplitudes of the Cai transient and contraction increase with the extent of cell Ca2+ loading, excess Ca2+ loading leads to dysregulation of Ca2+ homeostasis, impaired contraction, arrhythmia and cell death. The cell Ca2+ load is determined by membrane structure and permeability characteristics, the intensity of stimuli that modulate Ca2+ influx or efflux via regulatory function of proteins within membranes, and reactive oxygen species (ROS), which affect both membrane structure and function. Cardiocytes of senescent hearts exhibit a reduced threshold for pathologic manifestations of excess Ca2+ loading during stimulation (physiologic or pharmacologic) that increases Ca2+ influx, e.g. in response to neurotransmitters, post-ischaemic reperfusion, or oxidative stress. Cell 'remodelling' is one cause of the relative Ca2+ intolerance of cardiocytes in the senescent heart; cells increase in size and changes occur in the amounts of proteins that regulate Ca2+ handling due, in part, to altered gene expression; another cause is a change in the composition of membranes in which Ca2+ regulatory proteins reside, e.g. an increase in membrane omega 6:omega 3 polyunsaturated fatty acids (PUFA); a third cause is an enhanced likelihood for intracellular generation of ROS. Each class of these determinants changes with ageing and reduces the threshold for Ca2+ overload to occur with the older heart. The risk of excess Ca2+ loading within the senescent heart can potentially be reduced by gene therapy to restore Ca2+ regulatory proteins, by diet to reverse the membrane omega 6:omega 3 PUFA imbalance, or by antioxidants.


Subject(s)
Aging , Heart/physiopathology , Myocardium/metabolism , Animals , Calcium/metabolism , Fatty Acids, Unsaturated , Humans , Myocardial Ischemia/metabolism , Myocardial Ischemia/physiopathology
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20.
Cell Growth Differ ; 11(10): 517-26, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11063125

ABSTRACT

Retinoic acid (RA) treatment of embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) induces growth arrest and terminal differentiation along the neuronal pathway. In the present study, we provide a functional link between RA and p27 function in the control of neuronal differentiation in NT2/D1 cells. We report that RA enhances p27 expression, which results in increased association with cyclin E/cyclin-dependent kinase 2 complexes and suppression of their activity; however, antisense clones, which have greatly reduced RA-dependent p27 inducibility (NT2-p27AS), continue to synthesize DNA and are unable to differentiate properly in response to RA as determined by lack of neurite outgrowth and by the failure to modify surface antigens. As to the mechanism involved in RA-dependent p27 upregulation, our data support the concept that RA reduces p27 protein degradation through the ubiquitin/proteasome-dependent pathway. Taken together, these findings demonstrate that in embryonal carcinoma cells, p27 expression is required for growth arrest and proper neuronal differentiation.


Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Embryonal/drug therapy , Carcinoma, Embryonal/pathology , Cell Cycle Proteins , Neurons/pathology , Tretinoin/pharmacology , Tumor Suppressor Proteins , Antineoplastic Agents/therapeutic use , Carcinoma, Embryonal/genetics , Carcinoma, Embryonal/metabolism , Cell Differentiation/drug effects , Cyclin-Dependent Kinase Inhibitor p27 , Cyclin-Dependent Kinases/antagonists & inhibitors , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Humans , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Neurons/metabolism , Proteasome Endopeptidase Complex , Tretinoin/therapeutic use , Tumor Cells, Cultured
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