Effects of different pedalling techniques on muscle fatigue and mechanical efficiency during prolonged cycling.

The present study aimed to test the influence of the pedalling technique on the occurrence of muscular fatigue and on the energetic demand during prolonged constant-load cycling exercise. Subjects performed two prolonged (45 min) cycling sessions at constant intensity (75% of maximal aerobic power). In a random order, participants cycled either with their preferred technique (PT) during one session or were helped by a visual force-feedback to modify their pedalling pattern during the other one (FB). Index of pedalling effectiveness was significantly (P<0.05) improved during FB (41.4 ± 5.5%); compared with PT (36.6 ± 4.1%). Prolonged cycling induced a significant reduction of maximal power output, which was greater after PT (-15 ± 9%) than after FB (-7 ± 12%). During steady-state FB, vastus lateralis muscle activity was significantly (P<0.05) reduced, whereas biceps femoris muscles activities increased compared with PT. Gross efficiency (GE) did not significantly differ between the two sessions, except during the first 15 min of exercise (FB: 19.0 ± 1.9% vs PT: 20.2 ± 1.9%). Although changes in muscular coordination pattern with feedback did not seem to influence GE, it could be mainly responsible for the reduction of muscle fatigue after prolonged cycling.

New pharmacological strategies against metastatic spread.

Although metastatic spread is the most frequent cause of death in cancer patients, there are very few drugs specifically targeting this process. Bases for a new antimetastatic drug discovery strategy are weak because a great number of unknowns characterize the complete understanding of the metastatic cascade mechanisms. Moreover, the current experimental models are too simplistic and do not account for the complexity of the phenomenon. Some targets have been identified but too few are validated. Among them, the metastasis suppressor genes seem to be the most promising. In spite of this, during recent years, a dozen of molecules, which fulfil the definition of a specific metastatic drug that inhibits the metastases without altering the growth of the primary tumour (which can be eradicated by surgery), have been identified and assessed for the proof of the concept. The continuation of this effort would benefit in terms of efficiency, if the objectives were defined more precisely. It is particularly important to distinguish molecules that prevent spread of the metastatic cells of the early-stage primary tumour from the ones which induce a regression of the established metastases or to inhibit the transition from disseminated occult tumour cells to dormant micrometastasis. This second goal is a priori more relevant in the current clinical setting where the detection of early metastatic spread is very difficult, and therefore would call for greater effort on the part of the scientific community.

Polyphenols purified from the Brazilian aroeira plant (Schinus terebinthifolius, Raddi) induce apoptotic and autophagic cell death of DU145 cells.

Polyphenols extracted from many plants have shown antiproliferative and antitumor activities in a wide range of carcinogenesis models. The antiproliferative effects of polyphenols purified from the Brazilian aroeira plant (Schinus terebinthifolius, Raddi) were investigated on the androgen-insensitive DU145 human prostatic carcinoma cell line. A F3 fraction purified from leaf extract inhibited the DU145 cell proliferation more than 30-fold compared to the crude extract. By flow cytometric analysis, the polyphenol fraction was demonstrated to induce G0/G1 cell growth arrest and cell apoptosis. This apoptosis was evidenced by caspase 3 stimulation in F3-treated cells as compared to crude extract treated cells. The acid phosphatase activity of lysosomes was strongly activated in the lysosomal fraction of the F3-treated DU145 cells. This lysosomal activation, together with the appearance of autophagic vacuoles, suggests that "type 2 physiological cell death" was also involved in this antiproliferative effect. HPLC analysis of this F3 fraction showed 18 different subfractions. Among these subfractions, F3-3, F3-7 and F3-13 strongly inhibited DU145 cell proliferation in a dose-dependent manner. However, the nature of these polyphenols remains unknown since only one (Isoquercitrin) of the tested pure polyphenols co-migrated with F3-13. Since lysosomotropic drugs are considered as possible regulators of lysosome activity, aroeira polyphenols could target lysosomes of prostatic cancer cells to induce autophagic cell death.

Alternagin-C, a disintegrin-like protein from the venom of Bothrops alternatus, modulates alpha2ß1 integrin-mediated cell adhesion, migration and proliferation

The alpha2ß1 integrin is a major collagen receptor that plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Alternagin-C (ALT-C), a disintegrin-like protein purified from the venom of the Brazilian snake Bothrops alternatus, competitively interacts with the alpha2ß1 integrin, thereby inhibiting collagen binding. When immobilized in plate wells, ALT-C supports the adhesion of fibroblasts as well as of human vein endothelial cells (HUVEC) and does not detach cells previously bound to collagen I. ALT-C is a strong inducer of HUVEC proliferation in vitro. Gene expression analysis was done using an Affimetrix HU-95A probe array with probe sets of 10,000 human genes. In human fibroblasts growing on collagen-coated plates, ALT-C up-regulates the expression of several growth factors including vascular endothelial growth factor, as well as some cell cycle control genes. Up-regulation of the vascular endothelial growth factor gene and other growth factors could explain the positive effect on HUVEC proliferation. ALT-C also strongly activates protein kinase B phosphorylation, a signaling event involved in endothelial cell survival and angiogenesis. In human neutrophils, ALT-C has a potent chemotactic effect modulated by the intracellular signaling cascade characteristic of integrin-activated pathways. Thus, ALT-C acts as a survival factor, promoting adhesion, migration and endothelial cell proliferation after binding to alpha2ß1 integrin on the cell surface. The biological activities of ALT-C may be helpful as a therapeutic strategy in tissue regeneration as well as in the design of new therapeutic agents targeting alpha2ß1 integrin.

Alternagin-C, a disintegrin-like protein from the venom of Bothrops alternatus, modulates alpha2beta1 integrin-mediated cell adhesion, migration and proliferation.

The alpha2beta1 integrin is a major collagen receptor that plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Alternagin-C (ALT-C), a disintegrin-like protein purified from the venom of the Brazilian snake Bothrops alternatus, competitively interacts with the alpha2beta1 integrin, thereby inhibiting collagen binding. When immobilized in plate wells, ALT-C supports the adhesion of fibroblasts as well as of human vein endothelial cells (HUVEC) and does not detach cells previously bound to collagen I. ALT-C is a strong inducer of HUVEC proliferation in vitro. Gene expression analysis was done using an Affimetrix HU-95A probe array with probe sets of approximately 10,000 human genes. In human fibroblasts growing on collagen-coated plates, ALT-C up-regulates the expression of several growth factors including vascular endothelial growth factor, as well as some cell cycle control genes. Up-regulation of the vascular endothelial growth factor gene and other growth factors could explain the positive effect on HUVEC proliferation. ALT-C also strongly activates protein kinase B phosphorylation, a signaling event involved in endothelial cell survival and angiogenesis. In human neutrophils, ALT-C has a potent chemotactic effect modulated by the intracellular signaling cascade characteristic of integrin-activated pathways. Thus, ALT-C acts as a survival factor, promoting adhesion, migration and endothelial cell proliferation after binding to alpha2beta1 integrin on the cell surface. The biological activities of ALT-C may be helpful as a therapeutic strategy in tissue regeneration as well as in the design of new therapeutic agents targeting alpha2beta1 integrin.

Integrative analysis of gene expression patterns predicts specific modulations of defined cell functions by estrogen and tamoxifen in MCF7 breast cancer cells.

To explore the mechanisms whereby estrogen and antiestrogen (tamoxifen (TAM)) can regulate breast cancer cell growth, we investigated gene expression changes in MCF7 cells treated with 17beta-estradiol (E2) and/or with 4-OH-TAM. The patterns of differential expression were determined by the ValiGen Gene IDentification (VGID) process, a subtractive hybridization approach combined with microarray validation screening. Their possible biologic consequences were evaluated by integrative data analysis. Over 1000 cDNA inserts were isolated and subsequently cloned, sequenced and analyzed against nucleotide and protein databases (NT/NR/EST) with BLAST software. We revealed that E2 induced differential expression of 279 known and 28 unknown sequences, whereas TAM affected the expression of 286 known and 14 unknown sequences. Integrative data analysis singled out a set of 32 differentially expressed genes apparently involved in broad cellular mechanisms. The presence of E2 modulated the expression patterns of 23 genes involved in anchors and junction remodeling; extracellular matrix (ECM) degradation; cell cycle progression, including G1/S check point and S-phase regulation; and synthesis of genotoxic metabolites. In tumor cells, these four mechanisms are associated with the acquisition of a motile and invasive phenotype. TAM partly reversed the E2-induced differential expression patterns and consequently restored most of the biologic functions deregulated by E2, except the mechanisms associated with cell cycle progression. Furthermore, we found that TAM affects the expression of nine additional genes associated with cytoskeletal remodeling, DNA repair, active estrogen receptor formation and growth factor synthesis, and mitogenic pathways. These modulatory effects of E2 and TAM upon the gene expression patterns identified here could explain some of the mechanisms associated with the acquisition of a more aggressive phenotype by breast cancer cells, such as E2-independent growth and TAM resistance.

Transendothelial migration of two metastatic breast carcinoma cells depend on the SDF-lalpha-CXCR4 complexes.

The role of the SDF-1alpha chemokine-CXCR4 receptor system on tumor cell transendothelial migration was studied by culturing metastatic breast tumor cell lines, MDA-MB-231 and MDA-MB-435, either alone or on a HUVEC monolayer pre-established on a "Transwell" filter. After a 24-hour culture in the presence or absence of SDF-1alpha, tumor cell transmigration rates were compared. We showed that: CXCR4 is present on both cell lines; MDA-MB-231 but not MDA-MB-435 cell migration is stimulated by increasing concentrations of SDF-1alpha; neutralizing anti-CXCR4 antibody inhibits the SDF-1alpha chemoattractant effect; CXCR4 expression, measured by cytofluorometry, was enhanced after treatment with SDF-1alpha on MDA-MB-231 cells but remained unchanged on MDA-MB-435 cells; Scatchard analysis evidences 8.10(5) and 2.10(5) high affinity sites (KD range: 20 to 30 nM) on, respectively, MDA-MB-231 and MDA-MB-435 cells. These significant differences could explain the distinctive transendothelial migration responses of these two cell lines in the presence of SDF-1alpha.

Carboxymethyl benzylamide dextran inhibits angiogenesis and growth of VEGF-overexpressing human epidermoid carcinoma xenograft in nude mice.

Vascular endothelial growth factor (VEGF) expression is elevated in a wide variety of solid tumours. Inhibition of VEGF activities is able to reduce angiogenesis and tumour growth. We have recently shown in vitro that carboxymethyl dextran benzylamide (CMDB7) prevents the binding of VEGF(165) to its cell surface receptors and thus inhibits VEGF activities on endothelial cells. In the present study, we explored the effects of CMDB7 on highly aggressive human epidermoid carcinoma A431 cells known to overexpress epidermal growth factor receptors (EGFRs) and produce a high amount of VEGF and a minor quantity of bFGF. In vitro, CMDB7 blocked the mitogenic activity of A431-conditioned medium on endothelial cells. Concerning A431 cells, CMDB7 inhibited their proliferation and the VEGF(165) binding to them. In vivo, administration of CMDB7 (10 mg kg(-1)) three times per week for 2 weeks inhibited the growth of A431 xenografts in nude mice by 73% as compared to the control group. Immunostaining of endothelial cells with mouse-specific GSL-1 lectin in tumour sections revealed that CMDB7 also inhibited the density of intratumour endothelial cells by 66%. These findings demonstrate that CMDB7 has an efficient antiangiogenic and antitumour action in vivo even when tumour cells produce a high level of VEGF and EGFRs.

Inhibition of epidermoid carcinoma A431 cell growth and angiogenesis in nude mice by early and late treatment with a novel dextran derivative.

We investigated the effect of a new dextran derivative, phenylacetate carboxymethyl benzylamide dextran (NaPaC), on epidermoid carcinoma A431 cells secreting a large quantity of angiogenic factor, vascular endothelial growth factor (VEGF). In vitro, NaPaC inhibited the proliferation of A431 cells (IC(50)=5 micro M). Also, NaPaC decreased the binding of radiolabelled VEGF(165) to endothelial cells (IC(50)=0.2 micro M). In vivo, we explored the effects of NaPaC (15 mg kg(-1)) on A431 xenograft growth starting the drug administration at the time of tumour cell inoculation (early treatment) and 1 week later, when tumours were well established (late treatment). Early treatment was more efficient on tumour inhibition (70% vs control) than late treatment (50% vs control). Early and late NaPaC-treatment increased the aponecrosis in tumour by 70 and 30%, respectively. Whatever treatment, NaPaC inhibited the intratumour endothelial cell density in the same manner. In contrast, vessel area was decreased only when NaPaC was injected early (35%). These results show that NaPaC has a potent inhibitory effect, dependent on treatment outset, on epidermoid carcinoma growth associated with an intratumour microvascular network diminution and an aponecrosis increase. As this drug is nontoxic at efficient dose, it offers interesting perspectives for the therapy of malignant lesions.

Growth inhibition of human melanoma tumor cells by the combination of sodium phenylacetate (NaPA) and substituted dextrans and one NaPA-dextran conjugate.

We have studied the cytostatic effects of sodium phenylacetate (NaPA) in association with several substituted dextrans on human tumor melanoma 1205LU cells. We show that NaPA alone inhibits the growth of these cells (IC50 = 3.9 mM) while a weak inhibitory effect appears at a concentration of 37 microM (10 microg/ml) for a dextran methyl carboxylate benzylamide (LS17-DMCB). The precursors of LS17-DMCB [T40 Dextran and carboxymethyl dextran (LS17-DMC)] did not affect the growth of 1205LU cells. To potentiate the inhibitory activity of NaPA at low concentrations (below 5.6 mM), we have tested NaPA and LS17-DMCB in physical mixture (association) or linked together covalently (this conjugate is termed 'LS17-NaPaC'). We have observed an increase of the 1205LU cell growth inhibition effect with NaPA in association (IC50 1.8 mM). For a concentration of 5 mM of NaPA (free in the case of association or linked in the case of conjugate), the association with dextran derivative exhibits a 4.6-fold higher efficacy than with NaPA alone (9 versus 41% surviving fraction), while the conjugate is 1.3-fold smaller (52% growth inhibition). By performing isobologram analysis of the IC50 data, we have shown a synergistic effect for a particular molar ratio of NaPA and LS17-DMCB (NaPA:LS17-DMCB = 0.35).

Sodium phenylacetate enhances the inhibitory effect of dextran derivative on breast cancer cell growth in vitro and in nude mice.

Sodium phenylacetate (NaPa) and carboxymethyl benzylamide dextran derivative (CMDB(LS4)) are able to inhibit growth of breast tumour cells. In this study, we explored whether the combination of NaPa and CMDB(LS4)may enhance their respective inhibitory effects on the MCF-7ras cell growth in vitro and in vivo. NaPa inhibited MCF-7ras cell proliferation by reducing the DNA replication concomitantly with a recruitment of cells in G0/G1 phase and by inducing apoptosis in a dose- and time-dependent manner. The addition of CMDB(LS4)potentiated the NaPa antiproliferative effect in the manner dependent on the ratio of CMDB(LS4)and NaPa concentrations. In nude mice, CMDB(LS4)(150 mg kg(-1)) or NaPa (40 mg kg(-1)) administrated twice a week, for 7 weeks inhibited MCF-7ras xenograft growth by 40% and 60%, respectively. The treatment by both, CMDB(LS4)and NaPa, decreased tumour growth by 83% without any toxicity. To better understand the mechanism of NaPa and CMDB(LS4)action we assessed their effect on mitogenic activity of MCF-7ras conditioned medium (CM) on BALBC/3T3 fibroblasts. CMDB(LS4)added to the CM, inhibited its mitogenic activity whereas NaPa had an anti-mitogenic effect when CM was prepared from MCF-7ras cells pretreated with NaPa. Thus, the antiproliferative effects of NaPa and CMDB(LS4)involve 2 different mechanisms explaining, at least in part, the possible synergism between them. Overall, this study points to the potential use of a combination of dextran derivatives with NaPa to inhibit the breast tumour growth.

Vascular endothelial growth factor 165 (VEGF(165)) activities are inhibited by carboxymethyl benzylamide dextran that competes for heparin binding to VEGF(165) and VEGF(165).KDR Complexes.

We have previously shown that carboxymethyl dextran benzylamide (CMDB7), a heparin-like molecule, inhibits the growth of tumors xenografted in nude mice, angiogenesis, and metastasis by altering the binding of angiogenic growth factors, including platelet-derived growth factor, transforming growth factor beta, and fibroblast growth factor 2, to their specific receptors. In this study, we explore the effect of CMDB7 on the most specific angiogenic growth factor, vascular endothelial growth factor 165 (VEGF(165)). We demonstrate here that CMDB7 inhibits the mitogenic effect of VEGF(165) on human umbilical vein endothelial cells (HUV-ECs) by preventing the VEGF(165)-induced VEGF receptor-2 (KDR) autophosphorylation and consequently a specific intracellular signaling. In competition experiments, the binding of (125)I-VEGF(165) to HUV-ECs is inhibited by CMDB7 with an IC(50) of 2 microm. Accordingly, CMDB7 inhibits the cross-linking of (125)I-VEGF(165) to the surface of HUV-ECs, causing the disappearance of both labeled complexes, 170-180 and 240-250 kDa. We show that CMDB7 increases the electrophoretic mobility of VEGF(165), thus evidencing formation of a stable complex with this factor. Moreover, CMDB7 reduces the (125)I-VEGF(165) binding to coated heparin-albumin and prevents a heparin-induced increase in iodinated VEGF(165) binding to soluble (125)I-KDR-Fc chimera. Concerning KDR, CMDB7 has no effect on (125)I-KDR-Fc electrophoretic migration and does not affect labeled KDR-Fc binding to coated heparin-albumin. In the presence of VEGF(165), (125)I-KDR-Fc binding to heparin is enhanced, and under these conditions, CMDB7 interferes with KDR binding. These data indicate that CMDB7 effectively inhibits the VEGF(165) activities by interfering with heparin binding to VEGF(165) and VEGF(165).KDR complexes but not by direct interactions with KDR.

Could 99mTc-MIBI be used to visualize the apoptotic MCF7 human breast cancer cells?

Defects in key components of apoptotic pathways provide a survival advantage to cells and have been implicated as important factors in tumorogenesis. As therapeutic drug-induced apoptosis is a key component in treatment of most cancers, alterations in apoptotic pathways may be critical to drug resistance. The question is: would it be possible to distinguish apoptotic cells and resistant cells with a same radiotracer? In this study, we investigated the ability of sodium phenylacetate (NaPa), a natural cytostatic proapoptotic metabolite, to induce apoptosis in MCF7 human breast cancer cells. Then, we tested the 99mTc-MIBI accumulation in these apoptotic cells. Annexin V-FITC was used to identify apoptotic cells by flow cytometry. Ours results demonstrated that a 72 hr treatment of MCF7 cells with 40 mM NaPa induced apoptosis in 60% of cells. In a parallel way, 99mTc-MIBI accumulation in NaPa treated cells decreased for concentrations higher than 20 mM NaPa. Thus, 99mTc-MIBI accumulation decreased correlatively with the increasing percentage of apoptotic cells obtained by treatment of MCF7 cells with NaPa. These data demonstrate that NaPa induced apoptosis in MCF7 cells and that 99mTc-MIBI is a negative tracer of apoptosis: the more MCF7 cells were engaged in the apoptotic pathway, the more 99mTc-MIBI accumulation decreased in these MCF7 apoptotic cells.

Carboxymethyl benzylamide dextran and tamoxifen combination inhibits tumor growth and angiogenesis.

We showed previously that a carboxymethyl dextran benzylamide (CMDB7) blocks angiogenesis of MDA-MB-435 carcinoma and its lung metastases in nude mice. In this study, we examined the combination effects of CMDB7 and tamoxifen (TAM) on cell proliferation, tumor growth, and angiogenesis on the MCF-7RAS cells. We showed that CMDB7 and TAM acted in a synergistic manner to inhibit the growth of MCF-7RAS cells, blocking them in G(0)/G(1) phase of the cell cycle. For 7 weeks, the CMDB7- (300 mg/kg/week) and TAM- (20 mg/kg/week) treated groups showed tumor growth inhibition of about 66% and 76%, respectively. Combined treatments with CMDB7 and TAM block the tumor development by 94% and induce a complete regression of 4 of 8 mice. Histological analysis showed markedly less neovascularization (88%) in the tumors treated with a combination of CMDB7 and TAM. This antiangiogenic activity was further demonstrated by direct inhibition of endothelial cell proliferation. Overall, this study points to the potential use of a combination of CMDB7 and TAM to inhibit tumor angiogenesis that can prevent tumor progression.

Breast carcinoma cell uptake and biodistribution of technetium-99m-carboxymethyl benzylamide dextran.

Carboxymethyl Benzylamide Dextran (CMDB7) displayed an in vitro growth inhibitory activity on breast tumor cells. CMDB7 is able to disrupt the interaction of angiogenic growth factors (FGF2, TGF beta and PDGF) with their membrane receptors. This compound blocks the angiogenesis of MDA-MB435 carcinoma xenografted in mammary fat pad and their lung metastases in nude mice. In this work, we studied the uptake of CMDB7 labeled with 99mTc in cultured human breast cancer MCF-7 cell line and the highly tumorigenic MCF-7ras cell line (Ha-ras-transfected MCF-7 cells) and the in vivo distribution in MCF-7ras tumor-bearing mice. The 99mTc-CMDB7 are stable and the intracellular concentration is time-dependent and reaches a plateau at 180 minutes. 99mTc CMDB7 uptake is much higher in MCF-7ras cells than MCF-7 cells. Since CMDB7 is internalized and could also inhibit cell proliferation by acting at nuclear sites, we investigated the MCF-7ras nuclear localization after cell fractionation. Cell fractionation revealed a cytoplasmic and nuclear internalization of CMDB7. The tumor uptakes of 99mTc-CMDB7 were 0.34%, 0.72% and 0.62% of the administrated doses per gram of tumor tissue at 1 hour, 3 hour and 5 hours respectively after their injection. The blood clearance of 99mTc CMDB7 was very rapid and the liver, spleen and kidney uptakes were very weak. These results confirm the absence of toxicity of CMDB7 and the usefulness of CMDB7 in cancer therapy by targeting breast tumors.

Decrease of breast cancer cell invasiveness by sodium phenylacetate (NaPa) is associated with an increased expression of adhesive molecules.

Sodium phenylacetate (NaPa), a non-toxic phenylalanine metabolite, has been shown to induce in vivo and in vitro cytostatic and antiproliferative effects on various cell types. In this work, we analysed the effect of NaPa on the invasiveness of breast cancer cell (MDA-MB-231, MCF-7 and MCF-7 ras). Using the highly invasive breast cancer cell line MDA-MB-231, we demonstrated that an 18-hour incubation with NaPa strongly inhibits the cell invasiveness through Matrigel (86% inhibition at 20 mM of NaPa). As cell invasiveness is greatly influenced by the expression of urokinase (u-PA) and its cell surface receptor (u-PAR) as well as the secretion of matrix metalloproteinases (MMP), we tested the effect of NaPa on these parameters. An 18-hour incubation with NaPa did not modify u-PA expression, either on MDA-MB-231 or on MCF-7 and MCF-7 ras cell lines, and induced a small u-PA decrease after 3 days of treatment of MDA-MB-321 with NaPa. In contrast, an 18 h incubation of MDA-MB-231 increased the expression of u-PAR and the secretion of MMP-9. As u-PAR is a ligand for vitronectin, a composant of the extracellular matrix, these data could explain the increased adhesion of MDA-MB-231 to vitronectin, while cell adhesivity of MCF-7 and MCF-7 ras was unmodified by NaPa treatment. NaPa induced also an increased expression of both Lymphocyte Function-Associated-1 (LFA-1) and Intercellular Adhesion Molecule-1 (ICAM-1), which was obvious from 18 hour incubation with NaPa for the MDA-MB-231 cells, but was delayed (3 days) for MCF-7 and MCF-7 ras. Only neutralizing antibodies against LFA-1 reversed the decreased invasiveness of NaPa-treated cells. Therefore we can conclude that the strong inhibition of MDA-MB-231 invasiveness is not due to a decrease in proteases involved in cell migration (u-PA and MMP) but could be related both to the modification of cell structure and an increased expression of adhesion molecules such as u-PAR and LFA-1.

Proteomic detection of changes in protein synthesis induced by fibroblast growth factor-2 in MCF-7 human breast cancer cells.

Fibroblast growth factor-2 (FGF-2) is a potent regulator of breast cancer cell growth through stimulation of tyrosine kinase receptors and activation of the mitogen-activated protein kinase cascade. In the present study, we have investigated changes in protein synthesis induced by FGF-2 stimulation of the prototypic human breast cancer cell line MCF-7. Using high-resolution two-dimensional electrophoresis of (35)S amino acid metabolically labeled proteins and computerized analysis of 2D autoradiograms, we found that four proteins were up-regulated within the first 12 h of FGF-2 stimulation. Mass spectrometry analysis (MALDI-TOF and MS-MS) of tryptic fragments and database searches allowed the identification of these FGF-2-regulated proteins as the heat shock proteins HSP90 and HSP70, the proliferating cell nuclear antigen (PCNA), and the transcriptionaly controlled tumor protein (TCTP). We then analyzed the distribution of these proteins in various cancerous and normal breast epithelial cells. Interestingly, the four FGF-2-regulated proteins were found to be constitutively up-regulated in ras-transfected MCF-7 cells, indicating their relevance to the up-regulation of cellular proliferation. Moreover, HSP90 and PCNA were found at higher levels in cancerous cells than in normal cells. The role of HSP90 was further investigated using the specific inhibitor geldanamycin. We showed that the functionality of HSP90 is strictly required in order to obtain FGF-2 mitogenic stimulation in MCF-7 cells, indicating the crucial role played by this molecular chaperone in the control of breast cancer cell growth. Finally, these results show that proteomic analysis is a valuable method for identifying potential markers or therapeutic targets related to cancer growth.

Functional Rac-1 and Nck signaling networks are required for FGF-2-induced DNA synthesis in MCF-7 cells.

The effects of Fibroblast Growth Factor-2 (FGF-2) on breast cancer cell DNA synthesis are controversial. To elucidate the mechanisms by which FGF-2 stimulates or inhibits DNA synthesis, we analysed FGF-2 signaling pathways in breast cancer MCF-7 and MCF-7 cells overexpressing Ha-Ras (MCF-7ras). We found that FGF-2-induction of DNA synthesis correlates with Ras transient activation, FRS-2 tyrosine phosphorylation and low level of expression of p66Shc. In addition, Nck-associated proteins are highly tyrosine phosphorylated and JNK reaches a higher level of activation when FGF-2 triggers DNA synthesis. Interestingly upon FGF-2 treatment, JNK activation and DNA synthesis are dependent on Rac-1 activity. These results confirm that in MCF-7 cells, induction of DNA synthesis by FGF-2 requires a transient activation of the Ras/MAPK cascade and demonstrates for the first time that intact Rac-1 and Nck signaling networks are required.

Sodium phenylacetate (NaPa) induces modifications of the proliferation, the adhesion and the cell cycle of tumoral epithelial breast cells.

Sodium phenylacetate (NaPa), a physiological product of phenylalanine metabolism, present in micromolar concentrations in human plasma, has been shown to induce in vivo and in vitro cytostatic antiproliferative effects at millimolar concentrations. Cadherin molecules are powerful invasion suppressor molecules and the reduction of E-cadherin expression plays an important role in the invasion and metastasis of human breast cancer. In this study, we demonstrated, on one hand, that NaPa stimulated aggregation by increasing the expression of E-cadherin at the surface of breast cancer MCF-7ras cells transformed by Ha-ras oncogene and inhibited its expression in MCF-7 cells. We demonstrated that NaPa increased the formation of MCF-7ras cell aggregates and did not alter the formation of MCF-7 cell aggregates. By Northern blot, we demonstrated that the E-cadherin expression was not regulated at the transcriptional level. On the other hand, we analyzed the cell cycle of these 2 cell lines after NaPa treatment and showed that NaPa induced arrest at the G1/S phase in both MCF-7 and MCF-7ras cells. bFGF increased the growth of MCF-7 cells, but inhibited MCF-7ras cell proliferation. NaPa treatment suppressed the stimulation of MCF-7 cell proliferation and increased MCF-7ras cell growth inhibition. We have demonstrated a new target of NaPa action in blocking the cell cycle of tumor cells in G0/G1. We suggest that the anti-proliferative effect of NaPa associated to the restoration of the cadherin function in human mammary carcinoma cells indicates that NaPa could be a novel therapeutic agent in breast cancer.

Effects of the binding of a dextran derivative on fibroblast growth factor 2: secondary structure and receptor-binding studies.

CMDB (carboxymethyldextran-benzylamide) are dextrans statistically substituted with carboxymethyl and benzylamide groups which can mimick some of the biological properties of heparin. It has previously been shown that CMDB inhibit autocrine growth of breast tumor cells (Bagheri-Yarmand et al., Biochem. Biophys. Res. Commun. 239: 424-428, 1997) and selectively displace fibroblast growth factor 2 (FGF-2) from its receptor. Here, we used circular dichroism and fluorescence anisotropy measurements to show that the conformation of FGF-2 was significantly altered upon its binding to CMDB and to short CMDB fragments prepared within this study. CMDB and fragments formed a stable 1:1 complex with FGF-2, with affinities being estimated as 20+/-10 nM from fluorescence anisotropy analysis. No such a complex was formed with insulin-like growth factor (IGF-1) or epidermal growth factor (EGF). CMDB competed with the FGF-2 receptor for binding to FGF-2 but did not disturb the binding of IGF-1 and EGF to their receptors. Thus, our results highlight the selectivity of CMDB and their fragments towards FGF-2. Heparin, however, competes with CMDB and their fragments for binding to FGF-2. The carboxymethyl and benzylamide groups of these molecules likely interact directly with a heparin-binding region of FGF-2. The resulting change in conformation disturbs the binding of FGF-2 to its receptor and consecutively its mitogenic activity.