Caveolin-1 overexpression is associated with simultaneous abnormal expression of the E-cadherin/α-ß catenins complex and multiple ErbB receptors and with lymph nodes metastasis in head and neck squamous cell carcinomas.

The presence of lymph node metastases is one of the most important prognostic indicators in head and neck squamous cell carcinomas (HNSCCs). An alteration of the E-cadherin-catenins complex and EGFR is essential for the invasiveness of cancer cells. Caveolin-1, the major structural protein of the caveolae, represents a scaffolding molecule for several signaling proteins including EGFR. Although caveolin-1 has been shown to play a role in inducing the invasive phenotype of cancer cells, its role appears to be cell-type specific and for some tumors it has not been defined yet. In this study we used 57 HNSCC specimens to investigate whether the abnormal expression of caveolin-1 was associated with the derangement of the E-cadherin-catenins complex and with the overexpression of ErbB receptors. We demonstrate that in HNSCCs caveolin-1 overexpression is associated with the simultaneous abnormal expression of at least one member of the E-cadherin/α-ß catenins complex and multiple ErbB receptors as well as with lymph node metastases. We also demonstrate that chronic stimulation of a human hypopharyngeal carcinoma cell line (FaDu) with EGF induced the internalization of ß-catenin and caveolin-1 and their co-localization with EGFR. Moreover, EGF treatment induced an increased physical interaction between EGFR/ß-catenin/caveolin-1 and between E-cadherin/ß-catenin/caveolin-1. These molecular events were associated with an increased directional motility of FaDu cells in vitro. These findings may provide new insight into the biology of HNSCC progression and help to identify subgroups of primary HNSCCs with a more aggressive behavior.

West Nile alternative open reading frame (N-NS4B/WARF4) is produced in infected West Nile Virus (WNV) cells and induces humoral response in WNV infected individuals.

BACKGROUND: West Nile Virus (WNV) is a flavivirus that requires an efficient humoral and cellular host response for the control of neuroinvasive infection. We previously reported the existence of six alternative open reading frame proteins in WNV genome, one of which entitled WARF4 is exclusively restricted to the lineage I of the virus. WARF4 is able to elicit antibodies in WNV infected horses; however, there was no direct experimental proof of the existence of this novel protein. The purpose of this study was to demonstrate the in vitro production of WARF4 protein following WNV infection of cultured VERO cells and its immunity in WNV infected individuals. RESULTS: We produced a monoclonal antibody against WARF4 protein (MAb 3A12) which detected the novel protein in WNV lineage I-infected, cultured VERO cells while it did not react with WNV lineage II infected cells. MAb 3A12 specificity to WARF4 protein was confirmed by its reactivity to only one peptide among four analyzed that cover the full WARF4 amino acids sequence. In addition, WARF4 protein was expressed in the late phase of WNV lineage I infection. Western blotting and bioinformatics analyses strongly suggest that the protein could be translated by programmed -1 ribosomal frameshifting process. Since WARF4 is embedded in the NS4B gene, we rename this novel protein N-NS4B/WARF4. Furthermore, serological analysis shows that N-NS4B/WARF4 is able to elicit antibodies in WNV infected individuals. CONCLUSIONS: N-NS4B/WARF4 is the second Alternative Reading Frame (ARF) protein that has been demonstrated to be produced following WNV infection and might represent a novel tool for a better characterization of immune response in WNV infected individuals. Further serological as well as functional studies are required to characterize the function of the N-NS4B/WARF4 protein. Since the virus might actually make an extensive use of ARFs, it appears important to investigate the novel six ARF putative proteins of WNV.

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.

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.

Intercalated disk remodeling in delta-sarcoglycan-deficient hamsters fed with an alpha-linolenic acid-enriched diet.

Cardiomyocyte intercalated disks of delta-sarcoglycan-deficient cardiomyopathic hamsters (CMPHs) exhibit a pathological accumulation of the N-cadherin/catenin complex. CMPHs fed with an alpha-linolenic acid (ALA)-enriched diet (CMPH/FS) display an extended longevity compared to those fed with a standard diet (CMPH/PT) owing to, among others, the amelioration of both cardiac tissue structure and myocardial function. The present investigation was aimed at evaluating whether and to what extent the ALA-enriched diet affects the remodeling of CMPH cardiomyocyte intercalated disks and the expression of molecules, including N-cadherin, catenins and connexin 43 (CX43), involved in their organization. Western blot and immunohistochemical analysis demonstrated that the expression of N-cadherin, alpha- and beta-catenin is significantly reduced in cardiomyocyte intercalated disks of CMPH/FS vs. CMPH/PT and is lowered to levels similar to those found in healthy hamsters (GSH/PT), as well as transmission electron microscopy indicated that the cardiomyocyte intercalated disk ultrastructure is also re-established in CMPH/FS. In addition, the delocalization of CX43 as well as the presence of gap junctions were detectable at the lateral plasmamembrane of CMPH/FS cardiomyocytes, while the expression of myocardial CX43 was markedly reduced in both CMPH/PT and CMPH/FS, as compared to GSH/PT. Collectively, the present results demonstrate a substantial effect of an ALA-enriched diet on cardiomyocyte intercalated disk structure and molecular composition and further supports the beneficial effects of omega-3 polyunsaturated fatty acids in the prevention of potentially dangerous arrhythmias in cardiac diseases.

The ribosomal P0 protein induces a spontaneous immune response in patients with head and neck advanced stage carcinoma that is not dependent on its overexpression in carcinomas.

A typical feature in systemic lupus erythemathosus patients is the presence of autoantibodies to the carboxyl-terminal homologous P proteins (P0, P1, P2) domain (C-22 P0 epitope). In this report we provide evidence for the in vivo immunogenicity of the P0 protein in head and neck cancer patients as well as overexpression by immunohistochemistry of the C-22 P0 epitope in invasive carcinomas (55/57). Overexpression of this epitope was also significantly associated with a number of pathological lesions arising in the head and neck stratified epithelium including acanthosis (8/8), benign tumors (11/11), dysplasia (23/25) and in situ carcinomas (9/9). Intermediate cell layer restricted epitope overexpression was observed in well differentiated carcinomas, while undifferentiated tumors showed overexpression throughout the cell layers. Employing recombinant P proteins, sera from 40 of the 57 carcinoma patients and 39 normal donors, were subjected to immunoblot analysis. Immunity to P0 protein (7/40) was associated with malignancy and with advanced disease stage, but it was not dependent on the C-22 P0 epitope overexpression, although it was the preferential autoantibody target in 4 patients. Increased expression of the C-22 P0 epitope on the surface of pharynx cancer cells following cellular stress in vitro, may imply P0 protein presentation as an in vivo autoantibody target in cancer patients. Evidence for immunity to the P0 protein, as well as overexpression in patients with head and neck carcinoma may be relevant for monitoring cancer progression, in planning immunotherapeutic strategies, and contribute to the understanding of immuno-biological behaviour of head and neck carcinomas.

Resveratrol potentiates the in vitro and in vivo anti-tumoral effects of curcumin in head and neck carcinomas.

The survival rate of head and neck squamous cell carcinomas (HNSCC) patients has not considerably changed over the last two decades. Polyphenols inhibit the growth of cancer cells. We determined whether the combination of Resveratrol (RES) and Curcumin (CUR) enhanced their in vitro and in vivo antitumor activities on HNSCC cell lines compared to the single compounds. We provide evidence that RES potentiated the apoptotic effect and reduced the IC50 of CUR on HNSCC cell lines. The model of compounds interaction indicated the onset of an additive effect of the two compounds compared to the single treatment after decrease of their concentrations. RES+CUR compared to CUR increased the PARP-1 cleavage, the Bax/Bcl-2 ratio, the inhibition of ERK1 and ERK2 phosphorylation, and the expression of LC3 II simultaneously with the formation of autophagic vacuoles. RES and CUR induced cytoplasmic NF-κB accumulation. RES+CUR administrations were safe in BALB/c mice and reduced the growth of transplanted salivary gland cancer cells (SALTO) more efficiently than CUR. Overall, combinations of CUR and RES was more effective in inhibiting in vivo and in vitro cancer growth than the treatment with CUR. Additional studies will be needed to define the therapeutic potential of these compounds in combination.

Autophagy induction extends lifespan and reduces lipid content in response to frataxin silencing in C. elegans.

Severe mitochondria deficiency leads to a number of devastating degenerative disorders, yet, mild mitochondrial dysfunction in different species, including the nematode Caenorhabditis elegans, can have pro-longevity effects. This apparent paradox indicates that cellular adaptation to partial mitochondrial stress can induce beneficial responses, but how this is achieved is largely unknown. Complete absence of frataxin, the mitochondrial protein defective in patients with Friedreich's ataxia, is lethal in C. elegans, while its partial deficiency extends animal lifespan in a p53 dependent manner. In this paper we provide further insight into frataxin control of C. elegans longevity by showing that a substantial reduction of frataxin protein expression is required to extend lifespan, affect sensory neurons functionality, remodel lipid metabolism and trigger autophagy. We find that Beclin and p53 genes are required to induce autophagy and concurrently reduce lipid storages and extend animal lifespan in response to frataxin suppression. Reciprocally, frataxin expression modulates autophagy in the absence of p53. Human Friedreich ataxia-derived lymphoblasts also display increased autophagy, indicating an evolutionarily conserved response to reduced frataxin expression. In sum, we demonstrate a causal connection between induction of autophagy and lifespan extension following reduced frataxin expression, thus providing the rationale for investigating autophagy in the pathogenesis and treatment of Friedreich's ataxia and possibly other human mitochondria-associated disorders.

Resveratrol and diallyl disulfide enhance curcumin-induced sarcoma cell apoptosis.

Malignant tumors of mesenchimal origin such as rhabdomyosarcoma and osteosarcoma are highly aggressive pedriatic malignancies with a poor prognosis. Indeed, the initial response to chemotherapy is followed by chemoresistance. Diallyl disulfide (DADS), resveratrol (RES) and curcumin (CUR) are dietary chemopreventive phytochemicals which have been reported to have antineoplastic activity on rhabdomyosarcoma and osteosarcoma cells as single drugs. In this study we evaluated whether, as compared to the single compounds, the combination of DADS+RES, DADS+CUR and RES+CUR resulted in an enhancement of their antitumor potential on malignant rhabdoid (SJ-RH4, RD/18) or osteosarcoma (Saos-2) cell lines. Through FACS analysis and activated caspase-3 labeling we demonstrate that CUR induces apoptosis of rabdomyosarcoma and osteosarcoma cells and that this effect is potentiated when CUR is combined with RES or DADS. Further, we explored the effects of the compounds, alone or in combination, on signal transduction pathways involved in apoptosis and growth of cancer cells and show that in rhabdomyosarcoma cells the apoptotic effect of CUR, either alone or in combination, is independent of p53 activity. Our findings suggest that CUR and CUR-based combinations may have relevance for the treatment of p53-deficient cancers, which are often unaffected by conventional chemotherapies or radiotherapy.

Dietary flavonoids: molecular mechanisms of action as anti- inflammatory agents.

Flavonoids are a large group of polyphenolic compounds, which are ubiquitously expressed in plants. They are grouped according to their chemical structure and function into flavonols, flavones, flavan-3-ols, anthocyanins, flavanones and isoflavones. Many of flavonoids are found in fruits, vegetables and beverages. Flavonoids have been demonstrated to have advantageous effects on human health because their anti-allergic, anti-inflammatory, anti-platelet aggregation, anti-tumor and anti-oxidant behavior. This report reviews the current knowledge on the molecular mechanisms of action of flavonoids as anti-inflammatory agents and also discusses the relevant patents.

Apigenin induces apoptosis and impairs head and neck carcinomas EGFR/ErbB2 signaling.

The development of head and neck squamous cell carcinomas (HNSCCs) is a multistep process progressing from precancerous lesions to highly malignant tumors. A critical role in HNSCCs development and progression is played by EGFR family members including EGFR and ErbB2. The aim of this study was to investigate the effect of apigenin, a low molecular weight flavonoid contained in fruits and vegetables, on growth and survival and on EGFR/ErbB2 signaling in cell lines derived from HNSCCs of the tongue (CAL-27, SCC-15) or pharynx (FaDu). Using sulforhodamine B assay, FACS analysis and activated caspase-3 detection by immunofluorescence, we here demonstrate that apigenin dose-dependently inhibits survival and induces apoptosis of HNSCC cells. Further, by performing western blotting with antibodies specific for phosphorylated EGFR, ErbB2, Erk1/2 and Akt we demonstrate that apigenin reduces ligand-induced phosphorylation of EGFR and ErbB2 and impairs their downstream signaling. On the whole, our results suggest that apigenin properties might be exploited for chemoprevention and/or therapy of head and neck carcinomas.

Effects of omega-3 polyunsaturated fatty acids on cardiac myocyte protection.

Many epidemiologic, observational and randomized human clinical trials have demonstrated beyond doubt the protective cardiovascular effects of omega-3 polyunsaturated fatty acids (PUFAs). Cardiac myocytes protection by omega-3 PUFAs involves several mechanisms which might have a synergistic effect. This review provides a summary of the in vitro and in vivo effects of omega-3 PUFAs on cardiac myocytes health and reports the outcome of a number of clinical trials in patients consuming omega-3 PUFAs.

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.