SANIST: a rapid mass spectrometric SACI/ESI data acquisition and elaboration platform for verifying potential candidate biomarkers.

RATIONALE: Surface-Activated Chemical Ionization/Electrospray Ionization mass spectrometry (SACI/ESI-MS) is a technique with high sensitivity and low noise that allows accurate biomarker discovery studies. We developed a dedicated SACI/ESI software, named SANIST, for both biomarker fingerprint data acquisition and as a diagnostic tool, using prostate cancer (PCa) as the disease of interest. METHODS: Liquid chromatography (LC)/SACI/ESI-MS technology was employed to detect a potential biomarker panel for PCa disease prediction. Serum from patients with histologically confirmed or negative prostate biopsies for PCa was employed. The biomarker data (m/z or Thompson value, retention time and extraction mass chromatogram peak area) were stored in an ascii database. SANIST software allowed identification of potential biomarkers. A Bayesian scoring algorithm developed in house allowed sample separation based on comparison with samples in the database. RESULTS: Biomarker candidates from the carnitine family were detected at significantly lower levels in patients showing histologically confirmed PCa. Using these biomarkers, the SANIST scoring algorithm allowed separation of patients with PCa from biopsy negative subjects with high accuracy and sensitivity. CONCLUSIONS: SANIST was able to rapidly identify and perform a preliminary evaluation of the potential diagnostic efficiency of potential biomarkers for PCa.

A PSA-guided approach for a better diagnosis of prostatic adenocarcinoma based on MALDI profiling and peptide identification.

BACKGROUND: Prostate cancer (PCa) is the second cause of mortality in men worldwide. The prostate-specific antigen (PSA) test is routinely adopted in diagnosis; nevertheless more reliable biomarkers are continuously under investigation by monitoring the release of molecules into the bloodstream. The serum protein profiles appear to provide cancer-specific fingerprints that help to discriminate patients (especially with low PSA level) from controls, improving the performance of existing clinical tests. METHODS: Samples from healthy controls and PCa patients with low (≤4 ng/mL) and high PSA (>4 ng/mL) levels were analyzed by MALDI profiling, and by a multi fractionation approach coupled to ESI-MS for peaks identification. RESULTS: MALDI profiling achieved to detect 10 and 14 changed peaks (p-value <0.05), respectively, in PCa patients with low and high PSA versus controls. In particular, a peak identified as C3f fragment, resulted overexpressed in low PSA PCa patients. CONCLUSIONS: PSA test, coupled to MALDI profiling, is able to detect changes, specifically related to PCa, in low molecular weight protein range. Furthermore, for the first time in prostate cancer research, the identification and quantification of the small peptide C3f appears to be relevant for the detection of false negatives, providing an additive diagnostic power to PSA (p<0.01) and suggesting its use in clinical tests.

Surface-activated chemical ionization-electrospray ionization source improves biomarker discovery with mass spectrometry.

RATIONALE: Mass spectrometry (MS) is increasingly employed for the discovery of clinical biomarkers. However, due to sensitivity limitations related to in-source ionization yield, many potential biomarkers are not detected by standard mass spectrometers. Therefore, more efficient ion-source technologies are needed to improve MS applications in biomarker discovery. METHODS: Among novel ion-source technologies, Surface-Activated Chemical Ionization (SACI), although endowed with high sensitivity linked to its ability to reduce chemical noise in mass spectra, has seen limited application in biomarker discovery to date, due to its selectivity for highly polar compounds. However, in combination with an Electrospray Ionization (ESI) source, SACI selectivity can be enlarged in the range of less polar compounds. To validate the new SACI-ESI approach in biomarker discovery, we applied it to a translational setting in oncology. We performed MS profiles of 101 human serum samples from a male population, aged 40 or older, coming to the clinic for prostate cancer evaluation based on multiple PSA exams, digital rectal examination and echography. The SACI-ESI MS spectra were analyzed and classified with an innovative bioinformatic approach based on the MS-search freeware developed in house. RESULTS: Here we demonstrate that the SACI-ESI combination can produce MS spectra with greater sensitivity and lower noise than those obtained with the common ESI alone. We found that the SACI-ESI combination increased the number of detectable compounds and produced better quality of profiles in liquid chromatography (LC) coupled with MS (LC/MS) analysis of human serum samples, improving disease prediction potential. CONCLUSIONS: SACI-ESI can facilitate MS-based discovery of potential biomarkers in human serum. Combined with the proposed bioinformatic approach (based on XCMS and NIST data elaboration) for the analysis of the MS spectra obtained, the potential for developing biomarkers with diagnostic capabilities are demonstrated in a prostate cancer diagnosis clinical setting.

Interactions of single-wall carbon nanotubes with endothelial cells.

Single-wall carbon nanotubes (SWCNTs) could be promising delivery vehicles for cancer therapy. These carriers are generally introduced intravenously, however, little is known of their interactions with endothelial cells, the cells lining vessels and mediating clearance of nanoparticles. Here we show that SWCNTs of 1 to 5 microm in length, both "pristine" and functionalized by oxidation, had limited toxicity for endothelial cells in vitro as determined by growth, migration morphogenesis, and survival assays. Endothelial cells transiently took up SWCNTs, and several lines of data indicated that they were associated with an enhanced acidic vesicle compartment within the endothelial cells. Our findings of SWCNT interactions with endothelial cells suggest these may be optimal vehicles for targeting the vasculature and potential carriers of anti-angiogenic drugs. The implications on their biological activity must be taken into account when considering the use of these nanoparticles for therapeutic delivery of drugs. FROM THE CLINICAL EDITOR: Interactions of single walled carbon nanotubes (SWCNTs) with endothelial cells following IV administration remains unclear. Functionalized and naïve SWCNTs of 1-5 mm in length had limited toxicity to endothelial cells in vitro. Endothelial cells transiently took up SWCNTs and were associated with an enhanced acidic vesicle compartment within the cells. These findings suggest that SWCNTs may be promising vehicles for targeting the vasculature and potential carriers of anti-angiogenic drugs.

Angioprevention with fenretinide: targeting angiogenesis in prevention and therapeutic strategies.

Clinical trials have revealed that N-(4-hydroxyphenyl) retinamide (4HPR; fenretinide), a synthetic retinoic acid derivative, is a highly active and promising therapeutic and chemopreventive agent. Fenretinide shows biological activity against numerous cancer types in vitro and in preclinical studies. Clinical trials have shown that fenretinide induces a significant reduction of second breast cancer in premenopausal women. Several studies on different neoplasms are ongoing, such as breast and ovarian cancer, neuroblastoma, glioblastoma, head and neck and skin cancers and others. It has minimal side effects in humans, so that trials in young women at high-risk of breast cancer and ovarian and for the prevention of other tumor types such as lung cancer could be envisaged. Here we review some ongoing clinical trials and evaluate the possible mechanisms underlying the secondary chemopreventive effects of 4HPR. In particular we report basic and translational data on the anti-angiogenic "angiopreventive" properties of fenretinide, its anti-invasive activity, its ability to induce apoptosis and to generate or enhance production of reactive oxygen species as possible molecular bases for a chemopreventive action in patients.

Anti-angiogenic properties of chemopreventive drugs: fenretinide as a prototype.

Several cancer chemopreventive agents have been demonstrated to exert antiangiogenic effects. Blocking tumor angiogenesis, a process critical for tumor mass expansion and metastasis, represents an intriguing approach not only to cancer therapy, but also to cancer chemoprevention. We found that angiogenesis is a common and key target of many chemopreventive molecules, where they most likely suppress the angiogenic switch in premalignant tumors, a concept we termed "angioprevention." In this manuscript we use as an example the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR), a molecule with confirmed clinical applications in breast cancer adjuvant therapy to prevent cancer recurrence and under evaluation in neuroblastoma and glioblastoma treatment.

Anti-angiogenic activity of a novel class of chemopreventive compounds: oleanic acid terpenoids.

Angiogenesis is the base for solid tumour growth and dissemination, and anti-angiogenic drugs have been demonstrated to be active in clinical trials. In addition, it has become increasingly clear that inflammation is a key component in tumour insurgence. Chemoprevention focuses on the primary or secondary prevention of cancer using natural or synthetic agents that usually show mild or no collateral effects. We have noted that angiogenesis, particularly 'inflammatory angiogenesis', is a common target of many chemopreventive molecules, where they most likely suppress the angiogenic switch in pre-malignant tumours, a concept we have termed 'angioprevention'. We have shown that various molecules, such as flavonoids, antioxidants and retinoids, act in the tumour microenvironment inhibiting the recruitment and/or activation of endothelial cells and phagocytes of the innate immunity. We have recently assessed the activity of novel compounds derived from the oleanolic acid triterpenoid, called CDDO-Me and CDDO-Imm. These compounds show a potent anti-angiogenic activity at low dosages. In vivo they inhibit angiogenesis in the Matrigel sponge assay and in KS-Imm (an immortalized Kaposi's sarcoma cell line) tumour growth. In vitro they are able to prevent endothelial cell tubulogenesis when cultured on Matrigel. In human umbilical vein endothelial (HUVE) cells these compounds can inhibit the activation of the extracellular signal-regulated kinase ERK1/2 pathway after stimulation with vascular endothelial growth factor (VEGF). Moreover, from immunofluorescence experiments we observed that treatment with these triterpenoids prevents nuclear factor NF-kappaB translocation into the nucleus and thereby the activation of downstream pathways. The particularly potent anti-angiogenic activity seen in vivo suggest that CDDO-Me may be interacting with an important network of molecular and cellular targets, on endothelial cells, and could be employed for 'angioprevention'. These substances are being assessed in phase I trials in humans in the United States.

Mechanisms of Hyperforin as an anti-angiogenic angioprevention agent.

Hyperforin, the major lipophilic compound contained in extracts of Hypericum perforatum, is responsible for the antidepressant activity associated with the extract. Recently, several other biological properties of Hyperforin have been unveiled including inhibition of tumour invasion and angiogenesis. The mechanism of the anti-angiogenic activity of Hyperforin remains to be fully elucidated. We show that treatment with non-cytotoxic concentrations of Hyperforin restrains, in a dose-dependent manner, the capacity of endothelial cells to migrate towards relevant chemotactic stimuli. Hyperforin inhibits the organisation of HUVE endothelial cells in capillary-like structures in vitro, and potently represses angiogenesis in vivo in the Matrigel sponge assay in response to diverse angiogenic agents. Immunofluorescent staining shows that in cytokine-activated endothelial HUVE cells Hyperforin prevents translocation to the nucleus of NF-kappaB, a transcription factor regulating numerous genes involved in cell growth, survival, angiogenesis and invasion. Under Hyperforin treatment in vivo, the growth of Kaposi's sarcoma - a highly angiogenic tumour - is strongly inhibited, with the resultant tumours remarkably reduced in size and in vascularisation as compared with controls. Hyperforin has also been reported to have anti-inflammatory properties. Here we show that Hyperforin inhibits neutrophil and monocyte chemotaxis in vitro and angiogenesis in vivo induced by angiogenic chemokines (CXCL8 or CCL2). These results highlight the potential for Hyperforin as an anti-inflammatory angioprevention agent, acting as a strong inhibitor of inflammation- or tumour-triggered angiogenesis, and provide new therapeutic approaches to halting pathology-associated angiogenesis.

Prevention and treatment of experimental estrogen receptor-negative mammary carcinogenesis by the synthetic triterpenoid CDDO-methyl Ester and the rexinoid LG100268.

PURPOSE: To test whether the triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me) and the rexinoid LG100268 (268) prevent the formation of estrogen receptor (ER)-negative mammary tumors or either arrest the growth or cause regression of established tumors in MMTV-neu mice. EXPERIMENTAL DESIGN: For prevention, mice were fed control diet, CDDO-Me (60 mg/kg diet), 268 (20 mg/kg diet), or the combination for 45 weeks. For treatment, mice with established tumors at least 4 mm in diameter were fed control diet, CDDO-Me (100 mg/kg diet), 268 (60 mg/kg diet), or the combination for 4 weeks. RESULTS: CDDO-Me and 268 significantly delayed the development of ER-negative tumors, with a 14- and 24-week delay, respectively, compared with the control group for the time required to reach 50% tumor incidence. The combination of CDDO-Me and 268 was significantly more potent than the individual drugs, as only one tumor was found in the combination group, after 45 weeks on diet, at which time all control animals had tumors. Treating established tumors with CDDO-Me arrested the growth of 86% of the tumors, and 268 induced tumor regression in 85% of tumors. CDDO-Me and 268 target different signaling pathways and cell types. CDDO-Me inhibited constitutive STAT3 phosphorylation and the degradation of IKBalpha in ER-negative breast cancer cells, whereas 268 blocked IKBalpha degradation and the release of interleukin-6 in RAW264.7 macrophage-like cells, inhibited the ability of endothelial cells to organize into networks, and blocked angiogenesis in vivo. CONCLUSIONS: CDDO-Me and 268 are useful as individual drugs to prevent ER-negative mammary tumorigenesis and to treat established tumors. They synergize when used in combination for prevention.

Natural and synthetic agents targeting inflammation and angiogenesis for chemoprevention of prostate cancer.

Prostate cancer is the most common cancer in men and one of the leading causes of cancer-related deaths in Western countries. The extraordinary biological heterogeneity, the increasing incidence of this disease, and the presence of putative premalignant conditions make prostate cancer a crucial pathology to study and test pharmacological or nutritional chemopreventive strategies. It has been demonstrated that the incidence of prostate cancer is lower in Asian people, and that it increases in Asian men living in Western countries; these data point to a pivotal role of diet in the onset of prostate cancer. A large amount of work has been done in investigating chemopreventive properties of dietary compounds widely used in Asian countries (i.e. soy, soybeans, green tea, fish) in respect of the oxidants- and meat-rich diet typical of Western people, particularly of central and northern Europe. Some dietary products appear promising as chemo-preventive agents for prostate cancer, because they display both anti-oxidant and anti-inflammatory activity - and inflammation is crucial for the aetiology of adeno-carcinoma of the prostate. There is increasing evidence for close correlation between inflammation, the microenvironment and tumour-associated neo-angiogenesis causing the adverse outcomes of prostate cancer. It may thus be useful to develop new strategies to couple the treatment of inflammation-related prostate cancer and the generation of angiopreventive or antiinflammatory molecules to prevent this disease. The search for compounds with few or no adverse effects - particularly cardiovascular - as compared with the agents currently in use is therefore of greatest relevance.