γ-Tocopherol inhibits human prostate cancer cell proliferation by up-regulation of transglutaminase 2 and down-regulation of cyclins.

To establish a system to study differentiation therapy drugs, we used the androgen-independent human prostate PC-3 tumor cell line as a target and α- and γ-tocopherol as inducers. Effects of α- and γ-tocopherol on the cell cycle, proliferation and differentiation, were examined. A more significant growth inhibition activity for γ- than for α-tocopherol was observed. Flow cytometry analysis of α- and γ-tocopherol-treated prostate carcinoma PC3 cells showed decreased progression into the S-phase. This effect, particularly evident for γ-tocopherol, was associated with an up-regulation and increased activity of transglutaminase 2 (TG2), a reduced DNA synthesis and a remarkable decreased levels of cyclin D1 and cyclin E. Activation of TG2 suggests that γ-tocopherol has an evident differentiative capacity on PC3 cells, leading to an increased expression of TG2, and reduced cyclin D1 and cyclin E levels, affecting cell cycle progression. It is feasible that up-regulation and activation of TG2, associated with a reduced proliferation, are parts of a large-scale reprogramming that can attenuate the malignant phenotype of PC3 cells in vitro. These data suggest further investigation on the potential use of this γ-form of vitamin E as a differentiative agent, in combination with the common cytotoxic treatments for prostate cancer therapy.

eIF5A isoforms and cancer: two brothers for two functions?

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the unusual amino acid hypusine [N(ε)-(4-amino-2-hydroxybutyl)lysine]. The role of hypusine formation in the eIF5A protein in the regulation of cell proliferation and apoptosis is addressed in the present review. Moreover, vertebrates carry two genes that encode two eIF5A isoforms, eIF5A-1 and eIF5A-2, which, in humans, are 84% identical. However, the biological functions of these two isoforms may be significantly different. In fact, eIF5A-1 is demonstrable in most cells of different histogenesis, whereas eIF5A-2 protein is detectable only in certain human cancer cells or tissues, suggesting its role as a potential oncogene. In this review we focus our attention on the involvement of eIF5A-1 in the triggering of an apoptotic program and in the regulation of cell proliferation. In addition, the potential oncogenic role and prognostic significance of eIF5A-2 in the prediction of the survival of cancer patients is described. eIF5A-1 and/or the eIF5A-2 isoform may serve as a new molecular diagnostic or prognostic marker or as a molecular target for anti-cancer therapy.

Everolimus is an active agent in medullary thyroid cancer: a clinical and in vitro study.

Everolimus, an mTOR inhibitor, which has been demonstrated to induce anti-tumour effects in different types of neuroendocrine tumours, has never been evaluated in patients with medullary thyroid cancer (MTC). The aim of this study was to evaluate the in vitro and in vivo effects of everolimus in combination with octreotide in MTC. Two patients with progressive metastatic MTC and high calcitonin levels were treated with everolimus 5-10 mg/day. Both patients were under treatment with octreotide LAR at the study entry. An in vitro study was also performed to assess everolimus effects on MTC cell lines (TT and MZ-CRC-1 cells). A tumour response was observed in both patients. Serum calcitonin decreased by 86% in patient 1 and by 42% in patient 2. In TT and MZ-CRC-1 cells, everolimus induced a significant dose-dependent inhibition in cell proliferation. This effect seems to be related to a cell cycle arrest in G(0) /G(1) phase in both cell lines and to the induction of cellular senescence in TT cells. Everolimus in combination with octreotide may be active as anti-tumour therapy in patients with progressive metastatic MTC, suggesting to further evaluate this agent in MTC patients in a large prospective study.

Cetuximab ± chemotherapy enhances dendritic cell-mediated phagocytosis of colon cancer cells and ignites a highly efficient colon cancer antigen-specific cytotoxic T-cell response in vitro.

Cetuximab is a human/mouse chimeric IgG1 monoclonal antibody (mAb) to epidermal growth factor receptor, approved for colorectal carcinoma treatment in combination with chemotherapy. The immune-mediated effects elicited by its human fraction of crystallization moiety might critically contribute to the overall anti-tumor effectiveness of the antibody. We therefore investigated cetuximab ability to promote colon cancer cell opsonization and phagocytosis by human dendritic cells (DCs) that are subsequently engaged in antigen-cross presentation to cytotoxic T-lymphocyte (CTL) precursors. Human colon cancer cell lines were evaluated for susceptibility to DC-mediated phagocytosis before and after treatment with chemotherapy ± cetuximab in vitro. Human DCs loaded with control or drug-treated cetuximab-coated colon cancer cells were used to in vitro generate cytotoxic T cell clones from peripheral blood mononuclear cells of human leucocyte antigen-A(*)02.01(+) donors. T-cell cultures were characterized for immune-phenotype and tumor-antigen specific CTL activity. The results confirmed that treatment of tumor cells with irinotecan + L-folinate + 5-flurouracil (ILF) or with gemcitabine + ILF increased tumor antigen expression. Moreover, malignant cells exposed to chemotherapy and cetuximab were highly susceptible to phagocytosis by human DCs and were able to promote their activation. The consequent DC-mediated cross-priming of antigens derived from mAb-covered/drug-treated cancer cells elicited a robust CTL anti-tumor response. On the basis of our data, we suggest a possible involvement of CTL-dependent immunity in cetuximab anti-cancer effects.

High disease relapse after bDMARD spacing in psoriatic arthritis compared to rheumatoid arthritis and axial spondyloarthritis patients: real-life data from BIOPURE registry.

The objective is to evaluate the effectiveness of a spacing strategy of bDMARDs in a cohort of selected patients in disease remission or low-disease activity (LDA) without glucocorticoids affected with rheumatoid arthritis (RA), psoriatic arthritis (PsA) and axial spondyloarthritis (axSpA). This was a single-centre study carried out on patients prospectively enrolled in the biologic Apulian registry. Patients whose disease was in remission or LDA without taking glucocorticoids during the previous 6 months and who had agreed to increase the time interval between bDMARD doses were included in this study. Demographic and clinical characteristics were recorded at baseline and at 3, 6 and 12 months of follow-up. Endpoint of the study was the survival of spacing doses in the time lag of the study. Failure of spacing was defined as the first flare of disease. Thirty-seven RA, 28 PsA and 20 axSpA patients underwent bDMARD spacing according to a local strategy. During the follow-up, 5 RA, 6 PsA and 4 axSpA patients had a joint flare, but further 5 PsA patients manifested a skin relapse. Global persistence was 86.5% for RA (MST = 41 (95% CI: 37-45) months) and 80% for axSpA patients (MST = 36 (95% CI: 31-42) months). PsA patients showed a lower persistence, being of 60.7% (MST = 30 (95% CI: 23-36) months) (log-rank test, p = 0.03). Dose reduction by spacing bDMARD doses may be a feasible approach in patients with persistent remission/LDA activity. However, PsA patients might have greater odds of spacing failure because of skin psoriasis relapse. Key Points • Spacing of bDMARDs may be a feasible strategy for some patients with rheumatoid arthritis, psoriatic arthritis and axial spondyloarthritis who achieve the target and withdrawn glucocorticoids. • Psoriatic arthritis patients showed lower persistence because of both articular and skin relapses.

Bovine serum amine oxidase and spm potentiate docetaxel and interferon-alpha effects in inducing apoptosis on human cancer cells through the generation of oxidative stress.

It was previously demonstrated that bovine serum amine-oxidase (BSAO) and SPM (SPM) addition to cancer cells induces cell growth inhibition and over-run the multi-drug resistance (MDR) phenotype through the oxidative stress caused by polyamine metabolites. In this study, it is reported that BSAO/SPM enzymatic system antagonizes the survival pathway induced by either docetaxel (DTX) or interferon alpha (IFNalpha) in human epidermoid cancer KB cells. The combination of BSAO/SPM with either DTX or IFNalpha had a synergistic effect on cell growth inhibition through apoptosis in both human epidermoid KB and breast cancer MCF-7 cell lines. The effects of the BSAO/SPM-DTX combination on apoptosis were caspase 3 and 9-dependent and were paralleled by the enhancement of intracellular O(2-), nitric oxide levels and of lipo-oxidation. The scavenger moiety N-acetyl-cysteine antagonized the effects on apoptosis and cell growth inhibition induced by the combination suggesting a role of the oxidative products of SPM. These effects occurred together with a decrease of the physiological scavenger MnSOD and an increase of both p38 kinase activity and DNA damage. The results suggest that DTX and IFNalpha could sensitize tumour cells to the oxidative stress and apoptosis induced by BSAO/SPM through the induction of a survival ras-dependent pathway and the consequent elevation of the intracellular polyamine pool. These data allow the design of new therapeutic strategy based on the use of this combination in human neoplasms.

Possible antioxidant role of SPA therapy with chlorine-sulphur-bicarbonate mineral water.

The aim of our research was to analyze the antioxidant role and efficacy of thermal or salus per aquam (spa) therapy with chlorine-sulphur-bicarbonate mineral water. The study has been performed on 30 rats. The animals were randomized in three groups, each of them composed by ten animals, denominated A, B and C. The A group was the control group and was not subjected to any specific treatment (placebo); the B group has been treated with a standard cycle of hydropinics treatment with mineral water of Therme of Stabia in Castellammare (Naples, Italy) denominated STABIA; the C group was treated with a standard cycle of hydropinic treatment with mineral water of Therme of Stabia in Castellammare (Naples, Italy) denominated SULFUREA. After two weeks of treatment all the rats were sacrificed and blood was collected for the plasmatic determination of reactive oxygen species (ROS). The results demonstrated a significant (P < 0.05) reduction of ROS in B (374 Carr. U. +/-73) and C group (399 carr. U. +/-62) treated with mineral waters if compared with control group (571 + 69 Carr. U.). In conclusion this study suggests a possible antioxidant effect of chlorine-sulphur-bicarbonate spa hydropinic treatment with a consequent suitable intestinal physiology, with reduction of the functional and organic modifications that can lead to pathological disorders of the gastroenteric diseases in whose pathogenesis the oxidative stress can develop an important role.

C-Raf antagonizes apoptosis induced by IFN-alpha in human lung cancer cells by phosphorylation and increase of the intracellular content of elongation factor 1A.

Interferon alpha (IFNalpha) induces both apoptosis and a counteracting epidermal growth factor Erk-dependent survival response in cancer cells. In this report, IFNalpha increased eukaryotic elongation factor 1A (eEF-1A) protein expression by inhibition of eEF-1A degradation via a proteasome-dependent pathway. The reduction of the expression level of eEF-1A by RNA interference enhanced the apoptosis induced by IFNalpha on the same cells. Moreover, IFNalpha induced the phosphorylation of both serine and threonine in eEF-1A. These effects were paralleled by an increased co-immunoprecipitation and colocalization of eEF-1A with C-Raf. The suppression of C-Raf kinase activity with the inhibitor BAY 43-9006 completely antagonized the increase of both eEF-1A phosphorylation and expression and of C-Raf/eEF-1A colocalization induced by IFNalpha and enhanced apoptosis and eEF-1A ubiquitination. Cell transfection with the mutated K48R ubiquitin increased EF-1A expression and desensitized tumor cells to the modulating effects of IFNalpha. The dynamic simulation of 3Dstructure of eEF-1A identified putative serine and threonine phosphorylation sites. In conclusion, the interaction between eEF-1A and C-Raf increases eEF-1A stability and induces a survival activity.

Impairment of the metastatic activity of melanoma cells by transglutaminase-catalyzed incorporation of polyamines into laminin and Matrigel.

Previously published evidences highlighted the effect of transglutaminase (TG, EC 2.3.2.13) activation on the reduction of the in vitro adhesive and invasive behaviour of murine B16-F10 melanoma cells, as well as in vivo. Here, we investigated the influence of spermidine (SPD) incorporation by TG into basement membrane components i.e. laminin (LN) or Matrigel (MG), on the adhesion and invasion of B16-F10 melanoma cells by these TG/SPD-modified substrates. The adhesion assays showed that cell binding to the TG/SPD-modified LN was reduced by 30%, when compared to untreated LN, whereas the reduction obtained using TG/SPD-modified MG was 35%. Similarly, tumor cell invasion by the Boyden chamber system through TG/SPD modified LN or MG was respectively reduced by 45%, and by 69%. Evaluation of matrix metalloproteinase (gelatinases MMP-2 and MMP-9) activities by gel-zymography showed that MMP-2 activity was unaffected, while MMP-9 activity was reduced by about 32% using TG/SPD-modified substrate. These results strongly suggest that the observed antiinvasive effect of TG activation in the host may be ascribed to the covalent incorporation of polyamines, which led to the post-translational modification of some components of the cell basement membrane. This modification may interfere with the metastatic property of melanoma cells, affecting the proteolytic activity necessary for their migration and invasion activities.

Experimental study on vasoactive intestinal peptide (VIP) and its diaminopropane bound (VIP-DAP) analog in solution.

Bioactive peptides represent an exciting area of research in the fields of biochemistry and medicine and in particular the VIP/PACAP network appears to be of interest. Vasoactive intestinal peptide (VIP) is a pleiotropic factor that exerts a physiological regulatory influence and is involved in the pathogenesis of several human disorders. In this paper we have reported structural characterization of VIP by experimental and computational methods as well as a comparative analysis of the peptide with its transglutaminase catalyzed analog VIP-Diaminopropane (VIP-DAP).

Adenylate cyclase/cAMP pathway downmodulation counteracts apoptosis induced by IFN-alpha in human epidermoid cancer cells.

We have reported previously that interferon-alpha (IFN-alpha) induces apoptosis that is counteracted by an epidermal growth factor (EGF) --> Ras --> extracellular signal-regulated kinase (ERK)-dependent survival response in human epidermoid cancer KB cells. We have studied the effects of the cytokine on the cAMP-dependent pathway in these cells. A decrease in the intracellular cAMP levels was recorded in KB cells treated with IFN-alpha, whereas forskolin induced an increase in the production of cAMP that was reduced in the presence of IFN-alpha, suggesting a reduction in the activity of adenylate cyclase (AC) induced by IFN-alpha. These effects were paralleled by significant change in the expression of some AC catalytic subunit(s) and by reduction in the activity of protein kinase A (PKA). 8-Br-cAMP completely antagonized the reduction of PKA activity induced by IFN-alpha, whereas PKA inhibitor KT5720 enhanced the reduction of the enzyme activity induced by IFN-alpha. We have found that IFN-alpha induced a decrease in cAMP response element binding protein (CREB) phosphorylation without changes in its total expression. The concomitant treatment with IFN-alpha and 8-Br-cAMP potentiated and KT5720 counteracted apoptosis induced by IFN-alpha alone. In conclusion, these data suggest that the decrease in AC/cAMP pathway activity is a survival response to the apoptosis induced by IFN-alpha. Therefore, this pathway could represent a target to enhance the antitumor activity of IFN-alpha.

Anticancer drugs and hyperthermia enhance cytotoxicity induced by polyamine enzymatic oxidation products.

A correlation between regulation of cell proliferation and polyamine metabolism is described. The latter can enter protein synthesis through the modification of eukaryotic initiation factor 5A (eIF5A) and the formation of the peculiar amino acid hypusine. Specific inhibitors of hypusine formation induce apoptosis that can be potentiated by the combination with cytokines such as interferonalpha (IFNalpha) that itself decreases hypusine synthesis. We have also demonstrated that the concomitant treatment of cancer cells with IFNalpha and the protein synthesis inhibitor fusion protein TGFalpha/Pseudomonas Aeruginosa toxin synergize in inducing cancer cell growth inhibition. Another way used by polyamines to induce apoptosis is the generation of intracellular oxidative stress through the interaction with bovine serum amine oxidase (BSAO). This enzyme used simultaneously to spermine induces apoptosis, necrosis, inhibition of cell proliferation and inhibition of DNA and protein synthesis in several cell types. The enzymatic oxidation products of polyamine, H2O2 and aldehyde(s) cause these effects. We have recently found that the cytotoxicity of anti-cancer agents, either etoposide or docetaxel, in cancer cells is potentiated in the presence of BSAO/Spermine. In conclusion, polyamine metabolites could be useful in the design of new therapeutic strategies.

Unusual exophytic subependymoma in the bulbo-cerebellar angle. Case report.

Subependymoma was first described by Scheinker in 1945; it frequently occurs in the ventricles and rarely in the spinal canal representing 0.7% of all central nervous system tumours. Most of these intraventricular tumours are subclinical entities, remaining of small size and discovered at autopsy with 0.4%incidence. We report a case of subependymoma with a completely exophytic growth from the foramen of Luscka: only a similar one has been described in the literature but with a lesser cysternal involvement. Neuroradiological and anatomopathological features of subependymoma are discussed.

Inhibition of deoxyhypusine hydroxylase by polyamines and by a deoxyhypusine peptide.

The inhibition of deoxyhypusine hydroxylase was studied in vitro. Of the polyamines tested, spermine and its homologue thermine exhibited the strongest inhibition against the enzyme from rat testis. Kinetic analysis revealed that the inhibition by spermine was competitive (Ki, 0.25 +/- 0.02 mM) with respect to the deoxyhypusine protein substrate. Spermidine and its homologue caldine were also inhibitors, but less potent ones than spermine. The spermidine analogues with one or both primary amino groups replaced by the cyano group did not inhibit. A number of diamines, including putrescine, were found to display little or no inhibition. The observed effects of naturally occurring polyamines on deoxyhypusine hydroxylase activity is consistent with a suggestion of regulation of this enzymic activity by cellular levels of polyamines. A synthetic peptide Lys-Thr-Gly-deoxyhypusine-His-Gly-His-Ala-Lys, the amino acid sequence of which corresponds to that surrounding hypusine in eukaryotic initiation factor 4D, was found to display competitive-type inhibition (Ki, 0.44 +/- 0.02 mM) against deoxyhypusine hydroxylase from Chinese hamster ovary cells. Free hypusine and deoxyhypusine, on the other hand, possessed no inhibitory properties. A peptide analogous to the deoxyhypusine nonapeptide with lysine in place of deoxyhypusine had little effect on enzyme activity. The preparation of a derivative of deoxyhypusine, suitably protected for use in the solid-phase synthesis of deoxyhypusine peptides, is described.

The active site of deoxyhypusyl hydroxylase: use of catecholpeptides and their component chelator and peptide moieties as molecular probes.

The final step of hypusine formation in the eukaryotic translation initiation factor 4D (eIF-4D) is mediated by the enzyme deoxyhypusyl hydroxylase. In an effort to find specific inhibitors for this enzyme, we have studied the effects of two catecholpeptides, N alpha-acetyl-N delta-(3,4-dihydroxybenzoyl)-L-Orn-L-Pro-Gly (compound I) and N alpha-acetyl-N delta-(2,3-dihydroxybenzoyl)-L-Orn-L-Pro-Gly (compound II). Their structures were designed for anchorage to the enzyme s active site, utilizing the catechol-mediated chelation of a putative, enzyme-bound metal ion. Both compounds were found to strongly inhibit hypusine formation in vitro. Compound I was about seven times more potent than compound II, whereas the component peptide itself showed no intrinsic inhibitory activity even at concentrations as high as 1 mM. When used in conjugation with a chelating catechol moiety, however, it gave a 17- and an 8-fold enhancement of the half-maximal inhibition mediated by the chelating moieties per se, i.e. the 3,4- and the 2,3-dihydroxybenzoyl esters, respectively. The mode of inhibition by compound I was competitive with respect to the unhydroxylated precursor of eIF-4D and showed a Ki value of 32 microM +/- 3.4 microM. These catecholpeptides are the most efficient peptide antagonists of deoxyhypusyl hydroxylase known at present. They allow an assessment of the enzyme's active site organization and provide the first experimental evidence that a metal ion constitutes an integral part of its catalytic center.

Interferon-alpha induces apoptosis in human KB cells through a stress-dependent mitogen activated protein kinase pathway that is antagonized by epidermal growth factor.

We have demonstrated that interferon-alpha2-recombinant (IFNalpha) at growth inhibitory concentrations enhances the expression and signalling activity of the epidermal growth factor receptor (EGF-R) in human epidermoid carcinoma KB cells. Here we report that KB cells exposed to IFNalpha underwent apoptotic cell death and this effect was antagonized by EGF. We have also found that IFNalpha enhanced the expression of heat shock proteins (HSP) HSP-70, HSP-90 and HSP-27 and activated the NH2-terminal Jun kinase-1 (JNK-1) and p38 mitogen activated protein kinase, the target enzymes of a stress-dependent intracellular transduction pathway. Moreover, the overexpression of the wild-type JNK-1, obtained through plasmid transfection of KB cells, induced apoptosis which was potentiated by the exposure of wild-type JNK-1 (JNK-1wt)-transfected cells to IFNalpha. All these effects were neutralized by the addition of EGF to parental and JNK-1wt-transfected KB cells exposed to IFNalpha. In conclusion, EGF has a protective effect on KB cells from apoptosis while antagonizing a stress response elicited by IFNalpha and targeted on the stress pathway terminal kinases.

EGF activates an inducible survival response via the RAS-> Erk-1/2 pathway to counteract interferon-alpha-mediated apoptosis in epidermoid cancer cells.

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.

Docetaxel induces p53-dependent apoptosis and synergizes with farnesyl transferase inhibitor r115777 in human epithelial cancer cells.

Docetaxel (Taxotere, DTX) is a promoter of apoptosis in cancer cells. Since cytotoxic mechanisms of DTX are not yet fully understood, we have investigated the effects of DTX on apoptosis and ras-->Erk-mediated signal transduction in human epidermoid KB, colon HT-29 and breast HCC1937 cancer cells. We have found that the exposure to 0.78 or 1.56 or 2.5 ng/ml DTX for 48 h induced apoptosis and growth inhibition in about 50 % of KB, HCC1937 and HT-29 cell population, respectively. In these experimental conditions, PARP and caspase 3 cleavage was also showed in all cell lines. KB and HCC1937 cells express a wild type p53 while HT-29 display a mutated form. Interestingly, we have found that DTX reduces the expression of mutated p53 in HT-29 and increases the expression of wild type in KB and HCC1937 cells. Moreover, DTX reduces ubiquitination of the wild type p53 in KB and HCC1937 cells and increases the ubiquitin-conjugated form of mutated p53 in HT-29 cells. Furthermore, exposure of cancer cells to DTX for 48 h increases the expression and activity of Ras and up-regulates Raf-1 and the phosphorylated isoforms of Erk-1/2. On the bases of these data, we have hypothesized that the increased activity of the ras-->erk-dependent pathway induced by DTX could be a protective signalling from the apoptosis caused by the drug. Therefore, we have used R115777, a farnesyl transferase inhibitor that inactivates ras, in combination with DTX. The combined treatment with DTX and R115777 resulted in a strong synergism in growth inhibition in the three cell lines. These data suggest the use of the combination in these therapeutic settings even if further experiments are required for the clinical translation.

Isoprenylation of intracellular proteins as a new target for the therapy of human neoplasms: preclinical and clinical implications.

Cell proliferation, differentiation, and survival are regulated by a number of extracellular hormones, growth factors, and cytokines in complex organisms. The transduction of the signals by these factors from the outside to the nucleus often requires the presence of small intracellular proteins (i.e. ras and other small G proteins) that are linked to the plasma membrane through a isoprenyl residue that functions as hydrophobic anchor. Isoprenylation is a complex process regulated by different enzymatic steps that could represent potential molecular targets for anti-cancer strategies. In the present paper the different transduction pathways regulated by some isoprenylated proteins such as ras and other small G proteins are described. Moreover, the molecular mechanisms of the isoprenylation process and the mode of action of the different isoprenylation inhibitors are discussed with attention to statins, farnesyltransferase inhibitors (FTI) and aminobisphosphonates. The role of different candidate targets in the determination of anti-tumour effects by FTIs is also described in order to define potential molecular markers predictor of clinical response. On the basis of several preclinical data, new strategies based on multi-step enzyme inhibition or on target prioritization are proposed in order to enhance the anti-tumour activity of agents inhibiting isoprenylation. Finally, a summary of the principal data on clinical trials based on the use of FTIs and statins is given. In conclusion, the inhibition of isoprenylation is an attractive, but still not completely investigated therapeutic alternative that requires optimization for the translation in the current treatment of neoplasms.

Differential expression and cytoplasm/membrane distribution of endoglin (CD105) in human tumour cell lines: Implications in the modulation of cell proliferation.

Endoglin (CD105, an accessory component of the TGF-beta receptor complex) expression and distribution on different human tumour cells and its role in cellular proliferation were evaluated. We examined: 1) sixteen human carcinoma cell lines, 2) eight human sarcoma cell lines, 3) five miscellaneous tumour cell lines. HECV (endothelial cells) were employed as a positive control for endoglin expression. Normal Human Dermal Fibroblasts (NHDF) and 293 cells (epithelial kidney cells) were used as normal controls for connective and epithelial tissues, respectively. The results showed that CD105 was poorly expressed in the majority of human carcinoma cells (10/16), whereas it was highly expressed in most human sarcoma cells (7/8), and differently expressed by miscellaneous tumour cell lines. These data reflect endoglin expression by the normal counterparts of tumour cell lines, i.e. NHDF and 293 cells. However, CD105 levels in sarcoma cell lines, even though consistently lower than in NHDF, were significantly higher than those observed in carcinoma cells. Interestingly, CD105 presented a strong expression in the cytoplasm of MDA-MB-453 (breast carcinoma), NPA (papillary thyroid carcinoma), COLO-853 (melanoma) and SaOS-2 (osteosarcoma), but was weakly expressed on their cell membrane. This differential expression in the cytoplasm and on the membrane of some tumour cells, suggests a complex mechanism of translocation for this protein. The analysis of clonal growth in soft agar of some cell lines, characterized by high CD105 expression, showed an increased colony formation potential that was antagonized by the addition of anti-CD105 blocking mAb. The results indicated that endoglin is differentially expressed in human carcinoma and sarcoma cells and its overexpression modulates the proliferative rate of human solid tumour cells. Moreover, these data suggest that CD105 is involved in the regulation of TGF-beta effects in human solid malignancies, and therefore it could play an important role in tumour diagnosis and treatment.