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D Belotti

Publications and source records attributed to D Belotti.

At least 19 recordsLinked to original sources
PubMed2003

Expression levels of vascular endothelial growth factor, matrix metalloproteinases 2 and 9 and tissue inhibitor of metalloproteinases 1 and 2 in the plasma of patients with ovarian carcinoma.

We measured the levels of the vascular endothelial growth factor (VEGF), matrix metalloproteinases type 2 and type 9 (MMP-2 and MMP-9) and tissue inhibitors of matrix metalloproteinase 1 and 2 (TIMP-1 and TIMP-2) in the plasma of patients with ovarian carcinoma (n=40), in other gynaecological pathologies (n=30) and in the plasma of healthy volunteers (n=26). MMP-2 and MMP-9 (pro and active forms) gelatinolytic activity was measured by zymography. Enzyme-linked immunosorbent assays (ELISA) were used to assay soluble VEGF and TIMPs. Preoperative plasma VEGF levels were significantly higher in patients with ovarian cancer than in healthy volunteers (P<0.0001) or patients with a benign gynaecological pathology (P<0.0001). The expression of pro-MMP-9 was higher in the plasma of ovarian cancer patients than in the plasma of women with non-malignant disease (P=0.01) or healthy women (P<0.0002). Pro-MMP-2 was detected in the plasma of ovarian cancer patients, but levels did not differ from those in non-malignant disease or healthy donor samples. Plasma TIMP-1 and TIMP-2 levels were significantly higher in patients with ovarian carcinomas than in healthy volunteers (P<0.0001 and P=0.006, respectively) or in the patients with a non-malignant pathology (P<0.0001 and P=0.002, respectively). Sub-group analysis showed that VEGF and pro-MMP-9 were higher in the plasma of patients with serous carcinomas than other histological types. Furthermore, plasma VEGF and pro-MMP-9 levels were higher in the plasma of cancer patients with thrombocytosis. Throughout the study, and in the univariate analysis, no correlation was found between the VEGF, MMP and TIMP levels. Only TIMP-1 was associated with a poor survival and mortality risk.

Adult↗
PubMed2003

Low efficiency of a newly introduced high-density microparticles method for B cell depletion in multiple myeloma patients undergoing autologous hematopoietic stem cell transplantation.

Autologous peripheral blood stem cell (PBSC) transplantation proved to increase complete remission (CR) and DFS in multiple myeloma (MM) patients. CD34(+) cell selection has been used to reduce possible myeloma cell contamination in the graft, but it has not been showed to offer substantial advantages when compared to unpurged grafts; on the contrary, an increase of infectious complications was observed. We investigated the feasibility of a new negative-selection method in this setting. B cell negative selection was performed by using Eligix B cell HDM method. B cell contamination in the yield and in the final product was investigated by flow cytometry. Three patients with newly diagnosed MM entered the study. CD34(+) cell recovery in the three procedures was 73, 97, and 106%, and CD3(+) cell recovery was 88, 86, and 102%, respectively. CD20(+) cell depletion was 100% in all procedures, while CD19(+) cell depletion was 0.37, 1.21, and 0.07 log, respectively. We found an unexpected unreliability and a low efficiency in this B cell depletion method and suggest the need for further extensive testing before its introduction in the preclinical and clinical settings, at least in MM patients. In fact, reasons of such unsatisfactory results are still controversial: platelet contamination/activation in the preselection product, plasma protein interference, reduced CD19 antigen expression on immature B cells, lack of specificity of anti-CD19 monoclonal antibodies, instable binding between anti-CD19-coated high-density microparticles (HDM) and CD19 antigen may, alone or in combination, be involved in the system's low performance.

Antigens, CD19↗
PubMed1999

HOXC5 and HOXC8 expression are selectively turned on in human cervical cancer cells compared to normal keratinocytes.

A growing number of data have sustained the involvement of homeobox genes expression deregulation in cancer. In this study, we have performed an exhaustive survey of the expression of the 39 class I HOX genes expressed in normal and malignant human cervix keratinocytes. Using RT-PCR, we observed that the vast majority (34/39) of HOX genes are expressed in normal keratinocytes. Only HOXA2, HOXA7, HOXC5, HOXC8 and HOXD12 were found to be silent. Interestingly, this pattern is conserved in the transformed keratinocytes (SiHa cells) except for the appearance of HOXC5 and HOXC8 mRNA. The HOXC5 and HOXC8 expression was also observed in two other transformed keratinocytes cell lines of independent origins, Eil-8 and 18-11S3, and confirmed by in situ hybridization. Our data add weight to the body of evidence attributing to a specific adult tissue a particular combination of expressed HOX genes and suggest that HOXC5 and/or HOXC8 could be involved in the process leading to the transformation of cervical keratinocytes.

Cell Line, Transformed↗
PubMed1998

Expression and modulation of homeobox genes from cluster B in endothelial cells.

Angiogenesis is a complex phenomenon likely to be under the strict control of a group of transcription factor(s). Homeobox (HOX)-containing proteins have been identified as regulators controlling the coordinated expression of genes involved in organ development and tissue differentiation. In this study, we have demonstrated that human umbilical vein endothelial cells (HUVEC) express 8 of the 10 HOX genes contained in cluster B. Treatment of HUVEC with tissue plasminogen activator (TPA), an agent known to induce morphologic changes in endothelial cells, or vascular endothelium growth factor (VEGF), a proliferative and angiogenesis inducer, results in a specific time-dependent modulation of the eight HOX genes identified. Interestingly, neither basic fibroblast growth factor, an endothelial proliferative agent, nor TNP-470, a fumagillin derivative with potent antiendothelial cell proliferation properties, affected expression of these HOX genes. Specific modulation of HOX genes by differentiating agents but not by proliferative or antiproliferative molecules suggests that they could be involved in the control of the genetic program that coordinates the construction of new blood vessels.

Base Sequence↗
PubMed1997

Inhibition of the ABL kinase activity blocks the proliferation of BCR/ABL+ leukemic cells and induces apoptosis.

The BCR/ABL fusion protein transforms myeloid stem cells. Both chronic myelogenous leukemias (CML) and a subset of acute lymphoblastic leukemias (ALL) are associated with the expression of BCR/ABL proteins. This knowledge has not yet been translated into any specific tool to control ABL driven neoplastic cells growth. CGP57148B is an ATP-competitive inhibitor of the ABL protein kinase; it has been shown to inhibit the kinase activity of ABL both in vitro and in vivo and to inhibit the growth of v-abl and bcr/abl transfectants, as well as the in vitro formation of bone marrow (BM)-derived colonies in the presence of growth factors in some CML patients. These studies were performed to investigate the activity of CGP57148B on the spontaneous proliferation of both fresh and cultured, leukemic and normal, BCR/ABL positive and negative cells, and to study its mechanism of action. Six cell lines derived from BCR/ABL+ leukemias (K562, BV173, KCL22, KU812, MC3, LAMA84), thirteen BCR/ABL negative lines, both neoplastic (KG1, SU-DHL-1, U937, Daudi, NB4, NB4.306) and derived from normal cells (PHA blasts, LAK, fibroblasts, LCL, renal epithelial cells, endothelial cells, CD34(+) cells), and 14 fresh leukemic samples were tested using a tritiated thymidine uptake assay. The in vivo phosphorylation of the BCR/ABL protein was evaluated by western blot, while apoptosis was detected by the annexin V/propidium binding test. The induction of differentiation was assayed by immunofluorescence using multiple antibodies. All six BCR/ABL+ lines showed a dose dependent inhibition of their spontaneous proliferative rate, which was not accompanied by differentiation. The treatment caused, within minutes, dephosphorylation of the BCR/ABL protein, followed in 16-24 hours by a decrease in cycling cells and induction of apoptosis. No significant inhibition of DNA synthesis was observed in any BCR/ABL negative normal or neoplastic line at concentrations </=3 microM, with the exception of fibroblasts and CD34 cells. Proliferation inhibition was observed also when using fresh samples obtained from two Ph+ ALL and 12 consecutive CML patients. Induction of apoptosis was observed in these samples too. The activity of CGP57148B can be monitored in ex vivo isolated or cultured cells using a simple and reproducible assay, without the need for exogenously added growth factors. This molecule possibly exerts its effects through the inhibition of the kinase activity of BCR/ABL and the subsequent initiation of apoptosis, without inducing cell differentiation. Some normal cells are also affected. These data support the use of CGP57148B in initial clinical studies; possible toxic effects on BM and fibroblast-derived cells will have to be closely monitored. The in vivo monitoring of patients will have to be focused on the induction of apoptosis in leukemic cells.

Apoptosis↗
PubMed1997

The 140-kilodalton antiangiogenic fragment of thrombospondin-1 binds to basic fibroblast growth factor.

Thrombospondin-1 (TSP) inhibits the angiogenic activity of basic fibroblast growth factor (bFGF). Here we address the hypothesis of a direct interaction between TSP and bFGF. Gel permeation chromatography and cross-linking experiments demonstrated that bFGF binds to TSP in solution. bFGF also bound to immobilized TSP in a solid-phase assay. Binding was dose-dependent, with a Kd in the nanomolar range, and was inhibited by anti-TSP antibodies. The 140-kDa carboxyl-terminal fragment of TSP, but not the 25-kDa heparin-binding fragment, fully retained the bFGF binding capacity. Accordingly, binding was inhibited by monoclonal antibodies directed against this fragment. Heparin completely blocked bFGF binding to TSP and to the 140-kDa fragment. TSP and its 140-kDa fragment inhibited the binding of bFGF to endothelial cells at concentrations (> or = 100 nM) that inhibited endothelial cell proliferation but not motility. Low-affinity binding was inhibited more than high-affinity binding (up to 76 and 41% inhibition, respectively), and the inhibition was reversed by anti-TSP antibodies. Vitronectin and transforming growth factor beta, potentially associated with TSP, did not affect bFGF binding to endothelial cells. Although TSP did not affect the activation of the high-affinity receptors, it reduced the long-term internalization of bFGF. We conclude that TSP binds to bFGF through a domain within its 140-kDa fragment, a mechanism that might affect bFGF interaction with endothelial cells, activity, and association with the extracellular matrix.

Animals↗
PubMed1996

Isolation from a multigene family of the active human gene of the metastasis-associated multifunctional protein 37LRP/p40 at chromosome 3p21.3.

The 37 kD precursor of the 67 kD laminin receptor (37LRP) is a polypeptide whose expression is consistently upregulated in aggressive carcinoma. Interestingly, the 37LRP appears to be a multifunctional protein involved in the translational machinery and has also been identified as p40 ribosome-associated protein. Although highly conserved cDNAs corresponding to this polypeptide have been isolated from several species including vertebrates, invertebrates, plants and prokaryotes, characterization of any of the corresponding active genes has never been reported. In this study, we have cloned an intron-containing fragment which permitted us to isolate the active 37LRP/p40 human gene. This gene contains seven exons and six introns. Ribonuclease protection experiments suggest multiple transcription start sites. The promoter area does not bear a TATA box but contains four Sp1 sites. The first intron is also GC rich containing five Sp1 sites. Intron 4 contains the full sequence of the small nuclear RNA E2 and two Alu sequences are found in intron 3. Fluorescent in situ hybridization localized the 37LRP/p40 active gene on chromosome 3 in the locus 3p21.3 which, interestingly, is a hot spot for genetic alterations in several cancers and particularly in small cell lung carcinoma.

Amino Acid Sequence↗
PubMed1996

Shedding of the 67-kD laminin receptor by human cancer cells.

The 67-kD laminin receptor (67LR) is a cell membrane-associated molecule exhibiting high affinity for the basement membrane glycoprotein, laminin. While export of the 67LR toward the extracellular matrix has been recently suggested by electron microscopy studies, there is to date no evidence of shedding of the 67LR from cells. Using two monoclonal antibodies directed against the 67LR, we developed a double-determinant radioimmunoassay that demonstrates that the 67LR is released from cancer cells into the culture medium. The shed molecule exhibited the same apparent molecular weight as that of the membrane-associated 67LR, suggesting that no proteolytic cleavage is involved in the process. Furthermore, we demonstrate that the 67LR is not anchored to the membrane through a glycolsyl-phosphatidylinositol bridge. However, the observation that lactose increased the release of 67LR suggests that a lectin-type interaction is involved in the cell membrane association of this laminin binding protein and the cell surface. Interestingly, the released 67LR recovered after HPLC gel filtration was found free as well as associated to high molecular weight complexes. The free 67LR retained its ability to bind to the cell surface. Our study is the first demonstration that the 67LR is effectively shed by cancer cells. The released free 67LR could play an important role in modulating interactions between cancer cells and laminin during tumor invasion and metastasis.

Antibodies, Monoclonal↗
PubMed1996

Anthracycline drugs and MDR expression in human leukemia.

We investigated the expression of P-glycoprotein (P-gp) in 50 adults with de novo acute myeloid leukemia (AML) at the initial diagnosis in order to further define the relationship between the presence of P-gp on leukemic cells and the efficacy of two different anthracycline drugs, Daunorubicin (DNR) and Idarubicin (IRR), in terms of remission, induction and survival. We found that 30 (60%) of the 50 patients were negative for P-gp expression (group 1) and 20 patients (40%) were positive (group 2) for P-gp expression by MRK16MoAb using a cut of 10% positive cells. Among the 50 patients, 35 (70%) obtained complete remission (CR); depending on P-gp expression the CR rate was 80% for group 1 and 45% for group 2 (p < 0.005). The median duration of overall survival (OS) was 20 months for patients in group 1, compared to 10 months for patients in group 2 (p < 0.005). Regarding the anthracycline used, no difference in CR has been observed in patients of group 1 (75% CTR with DNR versus 90% CR with IDR); on the contrary in group 2 we observed 40% CR with DNR versus 70% CR with IDR (p < 0.005). No significant difference has been achieved in group 1 terms of median duration of overall survival between DNR and IDR regimen; on the contrary the median duration of OS in patients of group 2 treated with IDR regimen was significantly longer than DNR regimen (p < 0.005). These results confirm the prognostic value of P-gp expression in AML at diagnosis and we suggest that Idarubicin could be a valid anthracycline drug for reversing multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Expression of the 67 kD laminin receptor in human ovarian carcinomas as defined by a monoclonal antibody, MLuC5.

Previous immunohistochemical data from our laboratory have demonstrated that expression of the 67 kD laminin receptor (67LR), a cancer-associated, high-affinity laminin-binding protein, is upregulated in ovarian carcinoma cells compared with normal serosal cells, and that this increased expression in cancer cells could be related to patient outcome. The aim of this study was to validate MLuC5, a monoclonal antibody that recognises the 67LR, as a tool to perform future immunohistochemical studies on larger populations of ovarian carcinoma patients. Expression of the 67LR was determined in 51 primary human ovarian carcinoma samples using immunohistochemistry and MLuC5. The 67LR was detected in ovarian carcinoma cell clusters of variable extent. Analysis of the data determined that 67LR expression was significantly increased in the samples from patients with disease progression, compared with those with no evidence of disease after completion of primary therapy, and in pooled grade 2 and 3 tumours compared to borderline and grade 1 tumours (P < 0.05, chi-squared test). No other significant correlation between 67LR expression and other clinicopathological parameters could be established. These data suggest that the 67LR is correlated to ovarian tumour progression. Detection of the 67LR using this monoclonal antibody could constitute an interesting parameter in prognosis determination of ovarian cancer.

Adenocarcinoma↗
PubMed1996

Paclitaxel (Taxol(R)) inhibits motility of paclitaxel-resistant human ovarian carcinoma cells.

The effect of paclitaxel on the adhesive and motility properties of human ovarian carcinoma cell lines was investigated. Paclitaxel significantly inhibited the motility of OVCAR 5, SK-OV-3, and HOC-1OTC ovarian carcinoma cell lines (IC50 = 2.1 x 10(-8), 2 x 10(-9), and 1.9 x 10(-8) m, respectively) but did not affect the adhesion of these cells to the subendothelial matrix. The association between inhibition of motility and cytotoxic activity was investigated using an A2780 subclone (1A9) and three paclitaxel-resistant variants (designated 1A9/PTX22, 1A9/PTX10, and 1A9/PTX18). Although paclitaxel did not significantly affect the adhesion to subendothelial matrix of the sublines, it completely inhibited their migration. Inhibition of migration was similar in 1A9 cells and the resistant sublines, with an IC50 of 1 x 10(-8) for 1A9 cells and 5.4 x 10(-9), 1.1 x 10(-8), and 5.2 x 10(-9) m for 1A9/PTX22, 1A9/PTX10, and 1A9/PTX18, respectively. Paclitaxel inhibited motility induced by soluble attractant (chemotaxis) and immobilized attractant (haptotaxis). Inhibition of cell motility occurred in the absence of an antiproliferative effect, because higher concentrations of paclitaxel were required to inhibit tumor cell proliferation (IC50 = 1.9 x 10(-7) and 4.6 x 10(-6), 1 x 10(-5), and 3.1 x 10(-6) m for 1A9 and 1A9/PTX22, 1A9/PTX10, and 1A9/PTX18, respectively). These data show that paclitaxel is a potent inhibitor of ovarian carcinoma cell motility and that this activity is independent of its cytotoxic activity.

Antineoplastic Agents, Phytogenic↗
PubMed1996

The microtubule-affecting drug paclitaxel has antiangiogenic activity.

Endothelial cell migration is a critical event during angiogenesis, and inhibitors of cell motility can affect the angiogenic process. Paclitaxel (Taxol(R)), a microtubule-stabilizing antineoplastic cytotoxic drug, inhibits motility and invasiveness of several cell types. The aim of this study was to investigate the effect of paclitaxel on endothelial cell functions and on angiogenesis. In vivo, paclitaxel (20-28 mg/kg i.v.) significantly inhibited the angiogenic response induced by tumor cell supernatant embedded in a pellet of reconstituted basement membrane (Matrigel) injected s.c. into C57BL/6N mice. In vitro, paclitaxel inhibited endothelial cell proliferation, motility, invasiveness, and cord formation on Matrigel in a dose-dependent manner. The antiangiogenic activity of paclitaxel was not linked to its cytotoxicity, since inhibition of endothelial cell chemotaxis and invasiveness occurred at drug concentrations which did not affect endothelial cell proliferation. Another cytotoxic drug, cisplatin, that inhibited endothelial cell proliferation in vitro, did not affect angiogenesis in vivo. These data indicate that paclitaxel has a strong antiangiogenic activity, a property that might contribute to its antineoplastic activity in vivo.

Animals↗
PubMed1995

Loss of blast cell procoagulant activity and improvement of hemostatic variables in patients with acute promyelocytic leukemia administered all-trans-retinoic acid.

All-trans-retinoic acid (ATRA) induces complete remission (CR) in up to 90% of acute promyelocytic leukemia (APL) patients with rapid amelioration of the bleeding syndrome. Previous studies indicate that ATRA treatment in vitro of the APL NB4 cell line can affect their procoagulant activity (PCA). To assess whether ATRA has this effect also in vivo, we prospectively studied the PCA of bone marrow blasts from APL patients on therapy with ATRA alone or associated with chemotherapy. Samples were obtained before, during, and after ATRA. To characterize the coagulopathy, we measured a series of plasma hemostatic variables before and during the first two weeks of therapy, as follows: (1) markers of hypercoagulability; (2) natural anticoagulants; (3) fibrinolysis proteins; and (4) elastase. The results by enzymatic and immunologic methods show that both total (tissue factor-like) and factor VII-independent (cancer procoagulant-like) blast cell PCAs, present before therapy, were reduced during (69% and 65% decrement, respectively) and virtually undetectable after ATRA. The plasma hemostatic assessment of patients before treatment was elevated hypercoagulability markers, low mean protein C, normal fibrinolysis proteins, and increased elastase. After starting ATRA, hypercoagulability markers were reduced within 4 to 8 days, protein C augmented, the overall fibrinolytic balance was unmodified, and elastase remained elevated. These results were not different either with or without chemotherapy and are consistent with the clinical findings of rapid improvement of the coagulopathy.

Adolescent↗
PubMed1995

Inhibition of angiogenesis and murine hemangioma growth by batimastat, a synthetic inhibitor of matrix metalloproteinases.

BACKGROUND: The importance of matrix metalloproteinases in angiogenesis, tumor growth, and metastasis is well known. However, little is known about the role of matrix metalloproteinases in the formation of hemangiomas and about the possible therapeutic use of matrix metalloproteinase inhibitors in aggressive vascular tumors. PURPOSE: To study the role of matrix metalloproteinase in vascular tumors, we tested the antineoplastic activity of a synthetic inhibitor of matrix metalloproteinases, batimastat, on an experimental model of hemangioma, formed by murine endothelioma cells transformed by polyoma middle-T oncogene (eEnd.1). METHODS: The effect of batimastat was studied in vivo on the formation of hemorrhaging, cavernous hemangiomas by eEnd.1 endothelioma cells injected subcutaneously in nude mice and on the angiogenic response induced by an endothelioma cell supernatant embedded in a pellet of reconstituted basement membrane (Matrigel). The effect of batimastat was investigated in vitro on endothelial cell proliferation, motility, and invasion of a layer of Matrigel. RESULTS: Daily treatment with batimastat (30, 3, and 0.3 mg/kg at the site of eEnd.1 cell injection) inhibited tumor growth, with increased doubling time. The carboxamide derivative of batimastat, BB-374, a poor inhibitor of matrix metalloproteinase activity, was less active in reducing hemangioma growth. Histologic analysis of treated tumors indicated a reduction in the size of blood-filled spaces and in hemorrhage. Batimastat also inhibited the angiogenic response induced by cultured eEnd.1 endothelioma cell supernatant embedded in a pellet of Matrigel. Batimastat significantly inhibited endothelial cell invasion in vitro through a layer of Matrigel, but it showed no direct cytotoxic activity. CONCLUSIONS: Batimastat reduces in vivo growth of experimental hemangiomas, most probably by blocking endothelial cell recruitment by the transformed cells or by interfering with cell organization in vascular structures. IMPLICATIONS: These results confirm the importance of matrix metalloproteinase in endothelial cell recruitment that occurs in angiogenesis and in the formation of vascular tumors and suggest a therapeutic potential for synthetic matrix metalloproteinase inhibitors.

Animals↗
PubMed1995

Prognostic significance of laminin production in relation with its receptor expression in human breast carcinomas.

Laminin is a basement membrane glycoprotein whose expression has been widely related to cancer progression. Laminin production by primary breast carcinomas was investigated using immunohistochemistry on archival specimens from a retrospective series with long term follow-up. Laminin production was found to be independent of the clinical and pathological variables analyzed, whereas a statistically significant direct association with the expression of the laminin receptor and a negative association with the differentiation-related antigen Ca-MBr8 were observed. Survival analysis indicated that laminin positivity by itself has no prognostic significance. However, when analyzed together with the laminin receptor expression, laminin was associated with a good prognosis in receptor-negative tumors and with the worst prognosis in receptor-positive tumors.

Breast Neoplasms↗
PubMed1995

Design, synthesis and evaluation of D,D-peptidase and beta-lactamase inhibitors: azapeptides, oxapeptides and related heterocycles.

Reactive molecules susceptible to form stable acyl enzyme intermediates with D,D-peptidases and beta-lactamases were designed as potential irreversible inhibitors of Penicillin Sensitive Enzymes (PSEs). The structures examined were a series of azapeptides and oxapeptides, both analogs of the D-Ala-D-Ala substrate, and some heterocycles, such as imidazolidinones and oxazolidinones, both analogs of the beta-lactam antibiotics. The various strategies investigated for their synthesis are described and discussed. Some biological results are reported.

Alanine↗