Helicobacter pylori infection associates with a mucosal downregulation of ghrelin, negative regulator of Th1-cell responses.

BACKGROUND: Helicobacter pylori (Hp)-related gastritis is characterized by a predominant T helper (Th)1/Th17 cell immunity. Ghrelin (GR) has immunoregulatory properties and inhibits experimental Th cell-dependent pathology. AIMS: To evaluate whether Hp infection associates with changes in GR expression and whether GR negatively regulates Th1/Th17 cytokines during Hp infection. METHODS: GR expression was evaluated by real-time PCR in gastric biopsies taken from Hp-infected and Hp-uninfected patients and in gastric biopsies of Hp-negative subjects cultured with or without H. pylori culture supernatant. To examine whether GR regulates Hp-induced cytokine production, H. pylori-infected gastric biopsies were stimulated with GR, and interleukin (IL)-12, interferon (IFN)-γ and IL-4 transcripts were evaluated by real-time PCR. IL-12 and IFN-γ were also analyzed in lamina propria mononuclear cells (LPMCs) extracted from Hp-infected gastric biopsies and cultured with GR. RESULTS: GR RNA transcripts were reduced in biopsies from Hp-infected patients. Treatment of Hp-negative gastric biopsies with Hp culture supernatant reduced GR RNA expression. GR dose-dependently inhibited RNA expression of IL-12 and IFN-γ but not IL-4 in ex vivo cultures of mucosal explants and in cultures of gastric LPMCs from Hp-positive patients. CONCLUSIONS: GR is downregulated in the gastric mucosa of H. pylori-infected patients. Such a defect could contribute to sustain the ongoing Th1-cell response.

Carcinoembryonic antigen (CEA)-based cancer vaccines: recent patents and antitumor effects from experimental models to clinical trials.

Carcinoembryonic antigen (CEA), a glycosylated protein of MW 180 kDa, is overexpressed in a wide range of human carcinomas, including colorectal, gastric, pancreatic, non-small cell lung and breast carcinomas. Accordingly, CEA is one of several oncofetal antigens that may serve as a target for active anti-cancer specific immunotherapy. Experimental results obtained by employing animal models have supported the design of clinical trials using a CEA-based vaccine for the treatment of different types of human cancers. This review reports findings from experimental models and clinical evidence on the use of a CEA-based vaccine for the treatment of cancer patients. Among the diverse CEA-based cancer vaccines, DCs- and recombinant viruses-based vaccines seem the most valid. However, although vaccination was shown to induce a strong immune response to CEA, resulting in a delay in tumor progression and prolonged survival in some cancer patients, it failed to eradicate the tumor in most cases, owing partly to the negative effect exerted by the tumor microenvironment on immune response. Thus, in order to develop more efficient and effective cancer vaccines, it is necessary to design new clinical trials combining cancer vaccines with chemotherapy, radiotherapy and drugs which target those factors responsible for immunosuppression of immune cells. This review also discusses relevant patents relating to the use of CEA as a cancer vaccine.

Spontaneous immunogenicity of ribosomal P0 protein in patients with benign and malignant breast lesions and delay of mammary tumor growth in P0-vaccinated mice.

A common carboxyl-terminal epitope (C-22 P0) of the ribosomal P proteins (P0, P1 and P2) was shown to elicit autoantibodies in systemic lupus erythematosus (SLE) and in head and neck cancer patients. In this report we provide evidence for the in vivo immunogenicity of the P0 protein in breast cancer patients. Using recombinant P proteins, we demonstrated that sera from breast carcinoma patients (8/75) displayed significant reactivity to P0 protein when compared with healthy donor sera (0/45). Four out of the eight sera showed simultaneous reactivity to all P proteins. Breast benign tumor (3/17) and mammary hyperplasia (3/17) patient sera also showed significant reactivity to P proteins, thus suggesting that the occurrence of P protein autoantibodies might reveal mammary cell cycle dysregulation. Patient sera reacting with all P proteins recognized C-22 P0. Anti-P0 autoantibodies did not correlate with prognostic parameters of breast carcinomas. High level expression of C-22 P0 was found in mammary carcinomas compared with normal adjacent epithelium and benign lesions. To determine the antitumor activity of P0 as an immunogen, BALB-neuT transgenic mice displaying age-related breast cancer progression were vaccinated using xenogeneic P0 at the stage of mammary atypical hyperplasia. P0 vaccination significantly delayed the onset of mouse mammary tumors that overexpressed C-22 P0. Sera from P0 vaccinated mice recognized C-22 P0. Evidence for immunity to the P0 protein, its overexpression in carcinomas and its peculiar surface localization on cancer cells, along with its antitumor activity as an immunogen might be relevant for the use of P0 protein in monitoring cancer progression and in planning immunotherapeutic strategies.

The use of temozolomide for the treatment of malignant tumors: clinical evidence and molecular mechanisms of action.

Temozolomide (TMZ) is a monofunctional methylating agent which is spontaneously activated in aqueous solution into the dacarbazine metabolite 5-(3-methyl-1-triazeno)imidazole-4-carboxamide. This drug has been approved for the treatment of metastatic melanoma and glioblastoma multiforme, the latter in combination with radiotherapy. Furthermore, clinical trials have been performed to assess the activity of TMZ, alone or in combination, on brain metastatic solid tumors and leukaemias. This review will report clinical evidence on the use of TMZ for the treatment of different types of cancer; it also considers current knowledge on TMZ's molecular mechanisms of action of and discusses relevant patents relating to the same drug.

Local delivery of recombinant vaccinia virus encoding for neu counteracts growth of mammary tumors more efficiently than systemic delivery in neu transgenic mice.

Recombinant vaccinia virus has been widely employed as a cancer vaccine in several clinical trials. In this study we explored, employing BALB/c mice transgenic for the rat neu oncogene, the ability of the recombinant vaccinia virus neu (rV-neuT) vaccine to inhibit growth of neu+ mammary carcinomas and whether the efficacy of vaccination was dependent on: (a) carcinogenesis stage at which the vaccination was initiated; (b) number of vaccinations and (c) route of delivery (systemic vs. local). BALB-neuT mice were vaccinated one, two and three times by subcutaneous (s.c.) and intramammary gland (im.g.) injection with rV-neuT or V-wt (wild-type vaccinia virus) starting at the stage in which mouse mammary gland displays atypical hyperplasia, carcinoma in situ or invasive carcinoma. We demonstrated that vaccination using rV-neuT was more effective when started at an earlier stage of mammary carcinogenesis and after three vaccinations. The im.g. vaccination was more effective than the s.c. vaccination in inhibiting mammary carcinogenesis, eliciting anti-Neu antibodies, increasing anti-Neu IgG2a/G3 isotypes and inducing antibodies able to trigger mammary tumor cells apoptosis and antibody-dependent cellular cytotoxicity. The better protective ability of rV-neuT im.g. vaccination was associated with its capacity to induce a superior degree of in vivo mammary cancer cells apoptosis. Our research suggests that intratumoral vaccination using recombinant vaccinia virus could be employed to increase the activity of a genetic cancer vaccine. This study may have important implications for the design of cancer vaccine protocols for the treatment of breast cancer and of accessible tumors using recombinant vaccinia virus.

Topical use of sucralfate in epithelial wound healing: clinical evidences and molecular mechanisms of action.

Sucralfate is a basic aluminium salt of sucrose octasulphate which was orally employed for prevention and treatment of several gastrointestinal diseases including gastroesophageal reflux, gastric and duodenal ulcer. Recent studies have employed sucralfate as a topical drug for the healing of several types of epithelial wounds such as ulcers, inflammatory dermatitis, mucositis and burn wounds. Epithelial wound healing is a well orchestrated process involving hemostasis, inflammatory reaction, cell proliferation and tissue remodelling which leads to granulation tissue development and filling of the wound space. This report will review clinical evidences on the use of topical sucralfate for the management of epithelial lesions and deal with the current knowledge on the molecular mechanisms of action of this compound towards the epithelial wound healing process and will also discuss relevant patents.

Combined effects of 5-fluorouracil, folinic acid and oxaliplatin on the expression of carcinoembryonic antigen in human colon cancer cells: pharmacological basis to develop an active antitumor immunochemotherapy.

BACKGROUND: Five-fluorouracil (FU), mainly associated with leucovorin (L), plays an essential role in chemotherapy of colorectal carcinoma. Moreover, FU +/- L has been found to increase the expression of tumor-associated carcinoembryonic antigen (CEA), that may be an important target in therapeutic protocols of active specific immunotherapy. FU + L (FUL) are frequently combined with oxaliplatin (OXA) in advanced colon cancer patients. Thus, we investigated whether FUL in combination with OXA according to 2 different schedules may influence CEA expression in human colon cancer cells in vitro. METHODS: CEA protein expression was evaluated by cytofluorimetric and western blot analysis. Relative quantification of CEA mRNA was assessed by real time RT-PCR analysis. RESULTS: Levels of CEA protein and transcript were found to be higher in FUL-treated cells than in controls. However, when target cells were exposed to OXA before but not after FUL treatment, the up-regulation of CEA was partially inhibited. CONCLUSION: These results suggest that target cells must be exposed to OXA after but not before treatment with the fluoropyrimidine in order to exploit drug-induced up-regulation of CEA. This finding appears to provide useful information to design chemo-immunotherapy protocols based on FUL + OXA, combined with host's immunity against CEA directed cancer vaccines.

Endothelial function and arterial stiffness in normotensive normoglycemic first-degree relatives of diabetic patients are independent of the metabolic syndrome.

BACKGROUND AND AIM: The aim of the present study was to investigate endothelial function and arterial stiffness in normotensive normoglycemic first-degree relatives (offspring) of diabetic subjects and to explore the relationship with the metabolic syndrome and its components. METHODS AND RESULTS: Forty-five healthy normotensive normoglycemic subjects (aged 18-42 years), 29 first-degree relatives of diabetic subjects (FDR) and 16 with no parental history of type 2 diabetes mellitus were studied. Endothelial function was measured as flow-mediated dilation of the brachial artery (FMD) and arterial stiffness as carotid-femoral pulse wave velocity (PWV). Insulin resistance was calculated by homeostasis model assessment (HOMA). Plasma levels of inflammation markers (hsCRP, TNF-alpha, IL-1beta, CD40L, VCAM, and ICAM) were evaluated. Normotensive normoglycemic FDR presented a 33% lower flow-mediated dilation than the control group (9.8+/-5.2 vs. 16.2+/-7.6%, p<0.01). FMD was reduced in FDR, with or without insulin resistance, whereas arterial stiffness was significantly increased only in FDR with insulin resistance. To investigate the role of FDR status independently of altered components of the metabolic syndrome, subjects with no altered components of the metabolic syndrome were compared according to their FDR status: FDR subjects with no altered components of the metabolic syndrome presented a blunted endothelial function (lower FMD: 11.2+/-1.6 vs. 16.8+/-2.0%, p<0.05) and stiffer large arteries (higher PWV: 9.6+/-0.3 vs. 8.8+/-0.3m/s, p<0.05) than controls. CONCLUSION: Normoglycemic first-degree relatives of diabetic subjects have blunted endothelial function and increased stiffness of the large arteries. These alterations are already present at a very young age, before any alteration in glycemic control or blood pressure values can be detected, and are independent of the presence of the metabolic syndrome and its altered components.

Triazene compounds: mechanism of action and related DNA repair systems.

Triazene compounds of clinical interest (i.e. dacarbazine and temozolomide) are a group of alkylating agents with similar chemical, physical, antitumour and mutagenic properties. Their mechanism of action is mainly related to methylation of O(6)-guanine, mediated by methyldiazonium ion, a highly reactive derivative of the two compounds. The cytotoxic/mutagenic effects of these drugs are based on the presence of DNA O(6)-methylguanine adducts that generate base/base mismatches with cytosine and with thymine. These adducts lead to cell death, or if the cell survives, provoke somatic point mutations represented by C:G-->T:A transition in DNA helix. Triazene compounds have excellent pharmacokinetic properties and limited toxicity. Dacarbazine requires hepatic activation whereas temozolomide is spontaneously converted into active metabolite in aqueous solution at physiological pH. Moreover, temozolomide is fully active when administrated orally (100% bioavailability). The biological effects of triazene compounds and cell resistance to them depend on at least three DNA repair systems, (a) O(6)-alkylguanine-DNA-alkyltransferase, called also methyl-guanine methyl-transferase (MGMT); (b) mismatch repair (MMR), and (c) base excision repair (BER). MGMT is a small enzyme-like protein that removes small alkyl adducts from the O(6) position of DNA guanine through a stoichiometric and auto-inactivating reaction. This reaction consists in a covalent transfer of the alkyl group from the alkylated site in DNA to an internal cysteine residue of MGMT protein. High levels of MGMT are responsible for normal and tumour cell resistance to triazenes. Therefore, pre-treatment with MGMT inhibitors - i.e. O(6)-benzylguanine or O(6)-(4-bromotenyl)guanine (Lomeguatrib) - is followed by a great increase in the activity of triazenes against target cells expressing high MGMT levels. MMR is represented by a protein complex dedicated to the repair of biosynthetic errors generated during DNA replication. The MMR system recognizes base mismatches and insertion-deletion loops, cuts the nucleotide sequence containing the lesion, and restores the correct base sequence. Therefore, not only MGMT but also MMR is involved in target cell susceptibility to triazenes. However, the system does not suppress, but instead promotes the cytotoxic effects of triazenes. In fact, MMR is not able to repair the incorrect base pairing determined by treatment with triazenes and, according to a predominant hypothesis, it causes reiterated "futile" attempts of damage repair leading to the activation of cell cycle arrest and apoptosis. BER removes lesions due to cellular metabolism, or to physical or chemical agents. BER is able to repair N(7)-methylguanine and N(3)-methyladenine determined by treatment with triazenes. Therefore, triazene compounds can also kill tumour cells by a N(3)-methyladenine-mediated mechanism if BER activity is inhibited by chemical agents (i.e. PARP inhibitors). In conclusion, in selected cases, triazenes can represent a therapeutic alternative to treatment of neoplastic diseases including haematological malignancies. Moreover, the susceptibility of neoplastic cells to these compounds can be substantially increased through pharmacological modulation of the expression level and functional activity of DNA repair enzymes.

Novel role of triazenes in haematological malignancies: pilot study of Temozolomide, Lomeguatrib and IL-2 in the chemo-immunotherapy of acute leukaemia.

Previous studies indicated that dacarbazine and Temozolomide could be highly effective against refractory acute leukaemia. Their activity relies mainly on the generation of methyl adducts at the O(6)-position of guanine in DNA. High levels of O(6)-methylguanine-DNA methyltransferase (MGMT) or a defective mismatch repair (MMR) system, are associated with cellular resistance to triazenes. The MGMT inhibitor, O(6)-(4-bromothenyl)guanine (Lomeguatrib), can restore in vitro sensitivity to Temozolomide in MMR-proficient blasts. In the early 1970s we discovered that, in vivo, triazene compounds induce the appearance of novel transplantation antigens in murine leukaemia ("Chemical Xenogenization", CX). Non-self peptides presented by class I MHC molecules are generated by triazene-induced somatic mutations, affecting retroviral sequences that are detectable in the mouse genome. Moreover, preliminary experiments suggested that human cancer cells can also undergo CX. Therefore, we designed a chemo-immunotherapy strategy in leukaemic patients as follows: (a) cytoreduction and a hypothetical CX phase, i.e. treatment with Lomeguatrib (to suppress MGMT activity) and Temozolomide (to kill sensitive blasts and to presumably induce CX in resistant leukaemic cells); (b) immune response recovery phase using interleukin-2 (to possibly restore an immune response and take advantage of the hypothetical, triazene-induced CX). Here we present the results of pilot study which is in progress in patients with refractory/relapsed acute leukaemia. In all tested cases, Lomeguatrib suppressed MGMT activity in vivo. Six out of eight patients showed partial or complete disappearance of blast cells in peripheral blood or in bone marrow. We observed severe and long-lasting myelosuppression, accompanied by limited non-haematological toxicity. Up to now, two patients are alive (after 9 and 10 months, respectively), four died of opportunistic infections and two of progressive disease. This investigation confirms the potential role of triazenes in leukaemia and highlights the contribution of Lomeguatrib in overcoming drug resistance. Further studies are required to establish whether Temozolomide can induce CX in human leukaemia, and thus offer a new approach to control minimal residual disease.

Vascular, metabolic, and inflammatory abnormalities in normoglycemic offspring of patients with type 2 diabetes mellitus.

Endothelial dysfunction, insulin resistance, and elevated levels of circulating proinflammatory markers are among the earliest detectable abnormalities in people at risk for atherosclerosis. Accelerated atherosclerosis is a leading contributor to morbidity and mortality in type 2 diabetes mellitus, a complex genetic disorder. Therefore, we hypothesized that normoglycemic offspring of patients with type 2 diabetes mellitus (NOPD) may have impaired vascular and metabolic function related to an enhanced proinflammatory state. We compared NOPD (n = 51) with matched healthy control subjects without family history of diabetes (n = 35). Flow- and nitroglycerin-mediated brachial artery vasodilation were assessed by ultrasound to evaluate endothelium-dependent and -independent vascular function. Each subject also underwent an oral glucose tolerance test to evaluate metabolic function. Fasting levels of plasma adiponectin and circulating markers of inflammation (high-sensitivity C-reactive protein, CD40 ligand, interleukin 1beta, tumor necrosis factor alpha, vascular cell adhesion molecule 1, and intracellular adhesion molecule) were measured. Both NOPD and the control group had fasting glucose and insulin levels well within the reference range. However, results from oral glucose tolerance test and quantitative insulin sensitivity check index revealed that NOPD were insulin resistant with significantly impaired flow- and nitroglycerin-mediated dilation compared with the control group. Adiponectin levels were lower, whereas many circulating markers of inflammation were higher, in NOPD compared with the control group. Normoglycemic offspring of patients with type 2 diabetes mellitus have impaired vascular and metabolic function accompanied by an enhanced proinflammatory state that may contribute to their increased risk of diabetes and its vascular complications.

Combined effect of temozolomide and hyperthermia on human melanoma cell growth and O6-methylguanine-DNA methyltransferase activity.

Hyperthermic isolated limb perfusion (HILP) with L-phenylalanine mustard (L-PAM) represents an effective treatment for locally advanced melanoma of the limbs. However, regional chemotherapy of melanoma still needs to be improved. Temozolomide (TMZ) is a methylating agent that spontaneously decomposes into the active metabolite of dacarbazine, the most effective agent for the systemic treatment of melanoma. Tumor cells with high levels of O6-methylguanine-DNA methyltransferase (MGMT) and/or with a defective DNA mismatch repair (MMR) are resistant to TMZ. Inhibition of MGMT activity increases TMZ sensitivity of MMR-proficient, but not of MMR-deficient cells, while inhibition of base excision repair (BER) potentiates TMZ cytotoxicity in both cell types. Recent studies, performed in an animal model, have shown that TMZ is more effective than L-PAM when applied regionally and that hyperthermia can increase the antitumor activity of TMZ. In this study, three thermoresistant human melanoma cell lines, endowed with different MGMT activity and functional status of the MMR system, were treated with TMZ at 37 degrees C or 41.5 degrees C for 90 min, and then analyzed for cell growth and MGMT activity. Hyperthermia significantly enhanced TMZ cytotoxicity in MMR-proficient cells, either endowed or not with MGMT activity, and in MMR-deficient cells. Endogenous MGMT activity was not affected by hyperthermia that, however, enhanced the enzyme depletion induced by TMZ treatment. Moreover, MGMT recovery after drug removal was delayed in cells that had been treated at 41.5 degrees C. Taken together, these findings confirm the therapeutic potential of a combined treatment of hyperthermia and TMZ. They also suggest that inhibition of BER and/or increased DNA methylation may be involved in the thermal enhancement of TMZ cytotoxicity. Additional studies are necessary to better clarify the mechanisms underlying hyperthermia-induced potentiation of TMZ activity. However, the present investigation provides further support to the development of clinical trials of HILP with TMZ.

Concomitant impairment in endothelial function and neural cardiovascular regulation in offspring of type 2 diabetic subjects.

Endothelial function is impaired in first-degree relatives (FDRs) of patients with type 2 diabetes. Many states characterized by endothelial dysfunction are associated with increased cardiovascular sympathetic outflow. In this study, we investigated endothelial and autonomic nervous system (ANS) functioning in FDRs and tested the hypothesis that in basal condition, impaired endothelial function is associated with impaired cardiovascular ANS regulation. Flow-mediated endothelium-dependent and -independent vasodilation of the brachial artery was measured with high-resolution ultrasound in 27 otherwise healthy FDRs (14 men and 13 women; mean age 32 years) with normal oral glucose tolerance and in 15 age- and gender-matched control subjects. Cardiovascular ANS regulation was investigated by means of spectral analysis of heart rate and systolic blood pressure (SBP) variability. Baroreflex sensitivity was assessed by the spontaneous baroreflex sequences technique. Flow-mediated endothelium-dependent vasodilation was 9.4+/-1.0% in FDRs and 17.0+/-2.3% in control subjects (P=0.001). Low-frequency oscillations in SBP variability were 8.6+/-2.8 and 2.8+/-0.6 mm Hg in FDRs and controls, respectively (P=0.04). Baroreflex sensitivity was significantly less in FDRs than controls (22.8+/-2.7 versus 37.0+/-5.8, respectively; P=0.01). Change in vessel diameter was inversely correlated with the low-frequency component of SBP variability (r=-0.40; P=0.014). In healthy FDRs of diabetic patients there is a concomitant, possibly related, impairment in endothelial and ANS functioning, which manifests, indirectly, with increase in vascular sympathetic outflow and a depressed baroreflex, vagal, control of heart rate.

Resveratrol down-regulates the growth and telomerase activity of breast cancer cells in vitro.

A number of previous studies investigated the in vitro effects of resveratrol on malignant human breast epithelial cell replication. The aim of the present study was to evaluate the activity of resveratrol on human metastatic breast cancer cells. The study was performed on the MCF-7 tumor cell line. Cell growth, cell cycle perturbation and apoptosis were evaluated by trypan blue dye exclusion assay, flow cytometric analysis and confocal fluorescence microscopy. TRAP assay and Western blot analysis respectively detected levels of telomerase activity and levels of hTERT in intracellular compartments of MCF-7 cells treated with resveratrol. Resveratrol has a direct inhibitory effect on cell proliferation. The results demonstrate that the drug induces apoptosis in MCF-7 cells, in a time- and concentration-related manner. Our results also show that the growth-inhibitory effect of resveratrol on malignant cells is mainly due to its ability to induce S-phase arrest and apoptosis in association with reduced levels of telomerase activity. In particular, TRAP assay and Western blot analysis respectively showed that resveratrol treatment down-regulates the telomerase activity of target cells and the nuclear levels of hTERT, the reverse transcriptase subunit of the telomerase complex. In our experimental model of breast cancer, resveratrol shows direct antiproliferative and pro-apoptotic effects. Studies on telomerase function and intracellular hTERT distribution point out that this agent is endowed with additional suppressive functions on critical tumor biological properties. These results speak in favor of a potential role of resveratrol in chemoprevention/chemotherapy of breast cancer.

Plasma ghrelin in anorexia, bulimia, and binge-eating disorder: relations with eating patterns and circulating concentrations of cortisol and thyroid hormones.

The present study was designed to investigate the relations between plasma ghrelin concentrations, eating patterns, and circulating concentrations of cortisol and thyroid hormones in women with anorexia nervosa, bulimia nervosa, and binge-eating disorder. The patterns of disordered eating behavior were assessed using the Eating Attitudes Test (EAT-26) and the Bulimia Test-Revised (BULIT-R). In women with eating disorders, but not in healthy control women, plasma ghrelin concentrations were negatively correlated with body mass index (BMI) and plasma concentrations of thyreotropin (TSH), free T3 and free T4, and positively correlated with plasma concentrations of cortisol. The ghrelin concentrations of women with binge-eating and purging behavior were significantly lower than those of women with anorexia nervosa, restricting type, and there was a negative relation between the frequency and severity of binge-eating and purging behavior, as measured by the BULIT-R total score, and ghrelin concentrations. In a multivariate regression model controlling for the confounding effects of body mass index (BMI) and age, higher ghrelin concentrations were correlated with lower BULIT-R total scores. The results of this study did not confirm the hypothesis advanced in previous studies that ghrelin concentrations are higher in patients with binge-eating/purging forms of eating disorders. Based on these data, we suggest that, in women with eating disorders, ghrelin concentrations best reflect nutritional status rather than specific patterns of disordered eating behavior.

Combined effects of protein kinase inhibitors and 5-fluorouracil on CEA expression in human colon cancer cells.

Previous studies showed that 5-fluorouracil (5-FU) and Staurosporine (ST), a protein kinase inhibitor (PKI), were able to increase the expression of carcinoembryonic antigen (CEA) in human colon cancer cells. In the present study, we examined the in vitro effects of five PKIs, i.e. ST, 1-5-isoquinolinyl-sulfonyl-2-methylpiperazine (H-7), bisindolylmaleimide-I (BIS), Genistein (GEN), and Herbimycin A (HERB) alone or in combination with 5-FU on CEA expression. C22-20, a clonal subline, derived from colon cancer HT-29 line, selected for low expression of CEA, was used in our experimental model. Among the PKIs tested, only ST, at non-toxic concentrations of 5 nM, was capable of increasing the level of CEA. The other PKIs did not modify CEA expression when used either alone or in combination with 5-FU. Flow cytometric analysis showed that treatment of cells with 5-FU + ST resulted in a synergistic increase of CEA expression, being higher than that obtainable with both agents alone. Moreover, the increase of CEA expression occurred not only in membrane fractions but also in cytosolic compartments, as indicated by Western blot analysis. The present study suggests that ST-mediated induction of CEA expression in cancer cells is PKC independent and could be of potential clinical interest for the development of new diagnostic and/or immunotherapeutic approaches.

Drug-induced increase of carcinoembryonic antigen expression in cancer cells.

Most of gastrointestinal, breast and lung cancer cells express carcinoembryonic antigen (CEA). Therefore, this protein represents a suitable target for innovative diagnostic and immunotherapeutic strategies of various tumours. Presently CEA can be involved in three main approaches concerning cancer detection and therapy, i.e. (a) detection of tumour cells in the peripheral blood, bone marrow or lymph node using reverse transcriptase-polymerase chain reaction (RT-PCR)-based measurement of CEA mRNA; (b) targeting of anticancer agents or radionuclides by tumour-selective anti-CEA monoclonal antibodies (mAbs); (c) use of antitumour vaccines capable of eliciting major histocompatibility complex (MHC)-restricted immune responses against CEA-derived peptides. Actually, it has been shown that the expression of CEA can be up-regulated by pharmacological agents including, antineoplastic drugs (i.e. 5-fluorouracil), cytokines (i.e. interferons or interleukin-6), differentiating agents (i.e. sodium butyrate) and protein kinase inhibitors (i.e. staurosporine). Therefore, the use of drugs capable of increasing CEA expression, could amplify the sensitivity of diagnostic procedures that rely on CEA determination. Moreover, the same agents could increase the efficacy of vaccines based on immunogenic CEA-derived peptides restricted by the MHC. The purpose of this review is to describe several agents that are able to increase CEA expression and to discuss the rational bases for new strategies in cancer detection and therapy aimed at increasing the expression of tumour-associated antigens.

Treatment of colon and breast carcinoma cells with 5-fluorouracil enhances expression of carcinoembryonic antigen and susceptibility to HLA-A(*)02.01 restricted, CEA-peptide-specific cytotoxic T cells in vitro.

Cancer vaccines directed against tumor associate antigen (TAA) have produced encouraging results in preclinical models but not in cancer patients. A major limitation of this strategy is the relative degree of tolerance to these antigens and the low and heterogeneous tumor cell expression of TAA and major histocompatibility complex (MHC). Previous studies have shown that 5-fluorouracil (5-FU) can upregulate the expression of membrane-associated carcino-embryonic antigen (CEA), and MHC molecules in colon and breast carcinoma cell lines. We have investigated whether this drug can also enhance their sensitivity to the lytic effects of CEA-peptide specific Cytotoxic T cell lymphocytes (CTL). The CEA peptide-specific CTLs generated in our laboratory from normal HLA-A(*)02.01(+) donor PBMCs, were able to kill HLA-A(*)02.01(+)/CEA(+) breast (MCF-7-T103) and colon (HLA-A(*)02.01 gene-transfected HT-29 and C22.20) carcinoma cells in HLA-A(*)02.01 restricted manner. The treatment of target cells with 5-FU, enhanced their CEA expression and susceptibility to CTL-mediated lysis. Cold competition assays confirmed these results, thus supporting the hypothesis that immune target cell lysis and 5-FU mediated enhancement were dependent on CEA peptide presentation by cancer cells. 5-FU treatment of functionally "mature" CTL after in vitro expansion, did not reduce their cytolytic activity against MT-2 target cells but, when the anti-metabolite was added during the immune-sensitization phase, CTL generation was significantly inhibited. These results provide a rationale for investigating a possible new role of 5-FU as an immuno targeting amplifier agent in breast and colorectal cancer patients immunized with CEA-directed cancer vaccines.

Residual telomerase activity: a marker of cell survival after exposure to gamma radiation in vitro.

BACKGROUND: Residual telomerase activity (TA) could be used as a marker of malignant or normal cell survival after exposure to cytotoxic agents. Therefore TA after treatment with ionizing radiation was used in a radiosensitivity assay. MATERIALS AND METHODS: Radiosensitive MOLT-4 and relatively radioresistant Jurkat leukemia cells or normal peripheral blood mononuclear cells (MNC) were irradiated with 5-160 Gy. Cell count, MTT assay and telomerase activity were evaluated on day 3 and clonogenic assay on day 10. RESULTS: In Jurkat cells, irradiation inhibited tumor growth and total TA per culture (TA/culture), but up-regulated TA per viable cell (TA/cell). TA/culture and TA/cell were profoundly depressed by irradiation of MOLT-4 or phytohemagglutinin-activated MNC. CONCLUSION: Susceptibility of normal or neoplastic cells to high-dose radiation can be monitored by evaluating residual TA/culture. In some cases, however, difficulties in the interpretation of the results could stem from radiation-induced increase of TA/cell, as observed for the Jurkat cell line.