Curcumin induces apoptosis in breast cancer cell lines and delays the growth of mammary tumors in neu transgenic mice.

Breast cancer is a leading cancer in women and despite the benefits of the current therapies a significant number of patients with this tumor is at risk of relapse. Some of the alterations taking place in breast cancer cells are currently exploited by molecularly targeted drugs. Different drugs have been developed which target a single molecule but, given that the tumor originates from the dysregulation of many genes, there is the need to find new drugs that have more than one molecular target. Curcumin [1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] (CUR), a polyphenolic compound found in the spice turmeric, is a pleiotropic molecule able to interact with a variety of molecular targets and has antitumor, anti-inflammatory, antioxidant, immunomodulatory and antimicrobial activities. Here we demonstrate that CUR inhibits the growth of breast cancer cell lines in a dose dependent manner, with IC50 values in the micromolar range, and induces an increase in the percentage of cells in sub-G0 phase, representing the apoptotic cell population. The activation of apoptosis was confirmed by PARP-1 cleavage and by the increased ratio between the pro-apoptotic Bax and the anti-apoptotic Bcl-2 protein. In addition, in CUR-treated cells the activity of ERK1/ERK2 MAP kinases was down-regulated. The cytotoxic effects of CUR were observed in breast cancer cells expressing either high or low levels of ErbB2/neu. The in vivo antitumor activity of CUR was tested in BALB-neuT mice transgenic for the neu oncogene, which develop atypical hyperplasia of the mammary gland at 6 weeks of age and invasive carcinoma at 16 weeks of age. CUR, administered to mice both early and in an advanced stage of mammary carcinogenesis, induced a significant prolongation of tumor-free survival and a reduction of tumor multiplicity. In addition, CUR administration was safe, since no modification of hematological and clinical chemistry parameters could be observed in BALB-neuT and BALB/c mice treated with this compound for several weeks. These findings support further studies on the therapeutic potential of CUR in combination with standard therapies in breast cancer patients.

Hyaluronan-estradiol nanogels as potential drug carriers to target ER+ breast cancer cell line.

An innovative hyaluronan-based nano-delivery system is proposed for the active targeting towards ER+ breast cancer. Hyaluronic acid (HA), an endogenous and bioactive anionic polysaccharide, is functionalized with estradiol (ES), a sexual hormone involved in the development of some hormone-dependent tumors, to give an amphiphilic derivative (HA-ES) able to spontaneously self-assemble in water to form soft nanoparticles or nanogels (NHs). The synthetic strategy used to obtain the polymer derivatives and the physico-chemical properties of the obtained nanogels (ES-NHs) are reported. ES-NHs ability to entrap hydrophobic molecules has also been investigated, by loading curcumin (CUR) and docetaxel (DTX), both able to inhibit the growth of ER+ breast cancer. The formulations are studied for their capability to inhibit the growth of the MCF-7 cell line, thus evaluating their efficacy and potential as a selective drug delivery systems. Our results demonstrate that ES-NHs have not toxic effects on the cell line, and that both ES-NHs/CUR and ES-NHs/DTX treatments inhibit MCF-7 cell growth, with ES-NHs/DTX effect higher than that of free DTX. Our findings support the use of ES-NHs to deliver drugs to ER+ breast cancer cells, assuming a receptor-dependent targeting.

Changes in serum levels of TNF-alpha, IL-6, OPG, RANKL and their correlation with radiographic and clinical assessment in fragility fractures and high energy fractures.

Stages of bone turnover during fracture repair can be assessed employing serum markers of osteoblastic and osteoclastic activity, inflammatory cytokines, clinical evaluation and imaging instruments. Our study compare the fracture healing process in fragility fractures and high energy fractures by evaluating serum changes of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), osteoprotegerin (OPG) and receptor activator of the nuclear factor-kB ligand (RANKL) in combination with radiographic (Radiographic Union Scale for Tibial fractures, RUST) and clinical (Lower extremity measure, LEM) assessments. We enrolled 56 patients divided into four corresponding groups: group A with high energy trauma fracture (tibial/femoral shaft); group B with low energy trauma fracture (femoral fractures); healthy (control A) and osteoporotic subjects (control B). Blood samples were collected before surgery (T0) and after 10 weeks (T10). Serum concentrations of IL-6, TNF-alpha, RANKL and OPG were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits. Our results show that RANKL values are significantly higher at T10 than at T0 in low energy trauma fractures (group B). OPG is significantly lower in each control group than that of the respective fractured group and its concentration at T0 and at T10 is significantly lower in high than in low energy fractures. RANKL/OPG ratio is significantly higher in both controls than in fractured groups, and significantly increases after 10 weeks. IL-6 and TNF-alpha concentrations significantly decrease during fracture healing and are higher in high (group A) than in low energy fractures (group B). Significant differences were also found in both RUST score and LEM between groups A and B. Changes in TNF-alpha and IL-6 levels correlate with RUST and LEM in fragility and high energy fractures, while RANKL/OPG ratio is associated with these clinical parameters only in fragility fractures. These findings suggest that serum levels of IL-6, TNF-alpha, RANKL and OPG might be used to monitor the stages of fracture repair. Further studies will be needed to confirm the role of these cytokines in fracture repair.

Identification and characterization of the product encoded by ORF69 of Kaposi's sarcoma-associated herpesvirus.

We report the identification and characterization of p33, the product of Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 69 (ORF69), a positional homolog of the conserved herpesvirus protein UL31. p33 is expressed upon induction of viral lytic cycle with early kinetics. Immunofluorescence analysis revealed that in infected cell lines, the protein is localized in the nucleus, both in dotted spots and along the nuclear membrane. Nuclear fractionation experiments showed that p33 partitions with the nuclear matrix, and both immunoblotting of purified virions and immunoelectron microscopy indicated that the novel protein is not a component of the mature virus. Following ectopic expression in KSHV-negative cells, the protein was never associated with the nuclear membrane, suggesting that p33 needs to interact with additional viral proteins to reach the nuclear rim. In fact, after cotransfection with the ORF67 gene, the KSHV positional homolog of UL34, the p33 intranuclear signal changed and the two proteins colocalized on the nuclear membrane. A similar result was obtained when ORF69 was cotransfected with BFRF1, the Epstein-Barr virus (EBV) positional homolog of UL34 and ORF67. Finally, upon cotransfection, ORF69 significantly increased nuclear membrane reduplications induced by BFRF1. The above results indicate that KSHV p33 shares many similarities with its EBV homolog BFLF2 and suggest that functional cross-complementation is possible between members of the gammaherpesvirus subfamily.

Placenta growth factor is a survival factor for human malignant mesothelioma cells.

Placenta growth factor (PlGF) is a key regulator of pathological angiogenesis and its overexpression has been linked to neoplastic progression. To assess whether PlGF could have a role in malignant mesothelioma (MM), we analyzed the expression of PlGF, VEGF, and their cognate receptors (VEGF-R1 and VEGF-R2) and co-receptors (neuropilin-1 and neuropilin-2) in MM cell lines as well as in resected MM tissues, hyperplastic/reactive mesothelium and normal mesothelium. MM cell cultures expressed both ligands and the associated receptors to a variable extent and released different amounts of PlGF. As assessed by immunohistochemistry, PlGF expression was switched on in hyperplastic/reactive compared to normal mesothelium. Moreover, 74 and 94 percent of MM tissues overexpressed PlGF and VEGF-R1, respectively (p<0.05 MM vs normal mesothelium). Administration of recombinant PlGF-2 did not elicit a significant stimulation of MM cell growth, while it was associated with a transient phosphorylation of Akt, suggesting that PlGF-2 could activate downstream effectors of proliferative and cytoprotective signals via VEGF-R1 in MM cells. Indeed, the administration of an anti-PlGF antibody was found to cause a significant reduction of MM cell survival. In conclusion, our data demonstrate that, by acting as a survival factor, PlGF can play a role which goes beyond the stimulation of angiogenesis in MM. This evidence could help the rational design of new therapeutic interventions for this aggressive tumor.

Chloroquine supplementation increases the cytotoxic effect of curcumin against Her2/neu overexpressing breast cancer cells in vitro and in vivo in nude mice while counteracts it in immune competent mice.

Autophagy is usually a pro-survival mechanism in cancer cells, especially in the course of chemotherapy, thus autophagy inhibition may enhance the chemotherapy-mediated anti-cancer effect. However, since autophagy is strongly involved in the immunogenicity of cell death by promoting ATP release, its inhibition may reduce the immune response against tumors, negatively influencing the overall outcome of chemotherapy. In this study, we evaluated the in vitro and in vivo anti-cancer effect of curcumin (CUR) against Her2/neu overexpressing breast cancer cells (TUBO) in the presence or in the absence of the autophagy inhibitor chloroquine (CQ). We found that TUBO cell death induced by CUR was increased in vitro by CQ and slightly in vivo in nude mice. Conversely, CQ counteracted the Cur cytotoxic effect in immune competent mice, as demonstrated by the lack of in vivo tumor regression and the reduction of overall mice survival as compared with CUR-treated mice. Immunohistochemistry analysis revealed the presence of a remarkable FoxP3 T cell infiltrate within the tumors in CUR/CQ treated mice and a reduction of T cytotoxic cells, as compared with single CUR treatment. These findings suggest that autophagy is important to elicit anti-tumor immune response and that autophagy inhibition by CQ reduces such response also by recruiting T regulatory (Treg) cells in the tumor microenvironment that may be pro-tumorigenic and might counteract CUR-mediated anti-cancer effects.

Immunity to extracellular matrix antigens is associated with ultrastructural alterations of the stroma and stratified epithelium basement membrane in the skin of Hashimotos thyroiditis patients.

Employing purified extracellular matrix (ECM) proteins, i.e. type I, III, IV and V collagens (CI, CIII, CIV, CV), laminin (LM) and fibronectin (FN), as antigen sources we detected autoantibodies to conformational and/or denatured ECM antigens among 34 of 50 sera obtained from Hashimotos thyroiditis (HT) patients and 6 of 51 control sera obtained from non-autoimmune thyroid disease patients and healthy donors (HT sera vs. control sera p=4 x 10-9). Reactivity to conformational antigens, mostly due to autoantibodies of the IgG isotype, was observed in 30/50 HT sera and in 6/51 control sera (p=3.5 x 10-7) and was not always concomitant with that to linear antigens, found in 23/50 HT and in 6/51 control sera (p=1.6 x 10-4). Ultrastructural analysis of skin biopsies obtained from 18 HT patients without symptomatic cutaneous diseases revealed defects of the stratified squamous epithelium basement membrane in 11/18, alterations of the stroma in 13/18 and both basement membrane and stromal defects in 9/18. Interestingly, 13/13 (p=0.012) and 9/11 (p=0.012) patients with stromal and basement membrane defects respectively, exhibited serum antibodies to at least one ECM antigen involved in the organization of the altered tissue compartment. Lastly, 10/18 skin biopsies presented immunoglobulin (Ig) and/or complement (C3) deposits along the cutaneous basement membrane zone (BMZ) or in the papillary dermis and 9/10 sera from the same patients simultaneously showed antibodies to at least one ECM antigen involved in the organization of these two skin compartments. Besides, 8/11 HT patients with basement membrane defects exhibited Ig or C3 deposits along the BMZ. Our findings suggest that autoantibodies to ECM molecules might contribute to the development of asymptomatic extra-thyroid skin diseases in HT patients.

Baculovirus recombinants expressing the human carcinoembryonic antigen gene.

Carcinoembryonic antigen (CEA), one of the most extensively studied human tumor-associated antigens, represents a potential target for passive as well as active immunotherapy. We describe here the first baculovirus recombinants expressing the human CEA gene. Eight baculovirus clones were isolated which expressed products of varying molecular weights; one clone, termed BVCEA-140, was shown to contain multiple CEA epitopes by reactivity to a panel of anti-CEA monoclonal antibodies. When purified protein isolated from this clone was deglycosylated, immunoreactive species ranging from M(r) 50,000 to M(r) 110,000 were found. Results of Southern blot analysis carried out on BVCEA-140 DNA were consistent with the hypothesis that these products result from the stable expression of variants which have recombined within the repeated domains of CEA. Other baculovirus recombinants expressing products comprising different portions of the CEA gene were also derived. One, termed BVCEA-35, was shown to be a recombination between the first 87 bases of domains I and III of the CEA gene. A variant, termed BVCEA-16, contained only the NH2-terminal domain of the CEA gene. Moreover, a recombinant expressing the closely related molecule nonspecific cross-reactive antigen was also derived. As shown here, commercially available preparations of CEA, which are derived from tumor biopsies or cell line supernatants, may contain nonspecific cross-reacting antigens and other contaminants. Thus, the recombinant CEA molecules described should have numerous uses including validation of the use of monoclonal antibodies as standards in CEA serum assays, the characterization of immune responses to CEA, the use as immunogen, and the study of structure function relationships.

Cryptic epitopes on alpha-fetoprotein induce spontaneous immune responses in hepatocellular carcinoma, liver cirrhosis, and chronic hepatitis patients.

To determine alpha-fetoprotein (AFP) immunogenicity in vivo, the presence of antibodies in sera of 60 hepatocellular carcinoma, 15 liver cirrhosis, and 15 chronic hepatitis patients was evaluated by Western blotting and immunoprecipitation analyses using purified human AFP. High titers of anti-AFP immunoglobulins were detected in 14 hepatocellular carcinomas (P = 0.0006), 3 liver cirrhosis (P = 0.0173), and 1 chronic hepatitis patient, but they were not detected in 40 healthy individuals. Therefore, spontaneous immune responses to AFP are significantly associated to liver diseases (P = 0.0015). Patient immunoglobulins recognized proteic linear epitopes that were cryptic in the native protein, as demonstrated by their restricted reactivity with denatured deglycosylated AFP. Thus, in pathological liver conditions, tolerance to this self-molecule is circumvented. The identification of AFP immunogenic epitopes may contribute to defining novel immunotherapeutic strategies targeting this antigen.

Regional heterogeneity and complementation in the expression of the tumor-associated glycoprotein 72 epitopes in colorectal cancer.

We analyzed the immunohistochemical expression of three epitopes of the tumor-associated glycoprotein 72 (TAG-72) in whole cross-sections of primary colorectal carcinomas and in regional lymph node metastases using monoclonal antibodies (MAbs) B72.3, CC-49, and CC-83, which recognize distinct carbohydrate antigenic determinants. B72.3, CC-49, and CC-83 reacted with 13 of 27 (48%), 25 of 27 (92%), and 21 of 27 (77%) carcinomas, respectively. The immunoreactivity with lymph node metastases followed a similar pattern; MAb CC-49 was again the most reactive of the three antibodies, since it labeled 13 of 15 metastatic lesions. Positive reactions of the MAbs with the primary tumors were not always predictive of the immunorecognition of their metastases. Distinct areas within whole cross-sections of TAG-72-positive primary carcinomas demonstrated marked differences in the expression of the three epitopes. CC-49 tended to react with the highest number of areas and with the highest percentages of carcinoma cells within each area. In no instances did B72.3 demonstrate reactivity superior to that of either CC-49 or CC-83. Tumors negative for the CC-49 epitope in any area also did not express the other two TAG-72 epitopes. However, the comparison of the immunostaining obtained with each MAb in TAG-72-positive primary lesions revealed areas where CC-83 was clearly more reactive than CC-49. Moreover, one lymph node metastasis, negative for CC-49, was recognized by CC-83. Thus, the combined use of MAbs CC-49 and CC-83 resulted in additive immunostaining of primary and metastatic colorectal carcinoma cells. The study provides evidence of intratumoral heterogeneity in the glycosylation pattern of the TAG-72 antigen in colorectal cancer and emphasizes the advantages of cocktails of anti-tumor-associated antigen MAbs in the immunodetection of colorectal tumor cells.

Immune responses to all ErbB family receptors detectable in serum of cancer patients.

Employing NIH3T3 transfectants with individual human ErbB receptor coding sequences as recombinant antigen sources, we detected by immunoblot analysis specific immunoreactivity against all four ErbB receptors among 13 of 41 sera obtained from patients with different types of epithelial malignancies. Overall, serum positivity was most frequently directed against ErbB2 followed by EGFR, ErbB3 and ErbB4. Specificity patterns comprised tumor patients with unique serum reactivity against ErbB2 or ErbB4. Moreover, approximately half of the positive sera exhibited concomitant reactivity with multiple ErbB receptors including EGFR and ErbB2, EGFR and ErbB4, ErbB2 and ErbB3 or EGFR, ErbB2 and ErbB3. Serum reactivity was confirmed for the respective ErbB receptors expressed by human tumor cells and corroborated on receptor-specific immunoprecipitates. Positive sera contained ErbB-specific antibodies of the IgG isotype. Representative immunohistochemical analysis of tumor tissues suggested overexpression of ErbB receptors for which serum antibodies were detectable in five of six patients. These findings implicate multiple ErbB receptors including ErbB3 and ErbB4 in addition to EGFR and ErbB2 in primary human cancer. Heterogeneity of natural ErbB-specific responses in cancer patients warrants their evaluation in light of immunotherapeutic approaches targeting these receptors.

Serological and biochemical characterization of recombinant baculovirus carcinoembryonic antigen.

Carcinoembryonic antigen (CEA), a glycosylated protein of M(r) 180 kDa, is one of the most widely used human tumor markers. A majority of gastrointestinal cancers as well as breast and nonsmall cell lung carcinomas express CEA. We have previously described a recombinant baculovirus BVCEA-140 expressing the full-length human CEA and a variant, BVCEA-16, that encodes only the NH2-terminal domain, as well as a recombinant (BVNCA) expressing the closely related molecule nonspecific cross-reactive antigen (NCA). We have now compared a panel of 24 anti-CEA and anti-NCA monoclonal antibodies (MAbs) for their ability to bind to these recombinant CEA and NCA proteins, as well as with a new 60 kDa subgenomic form designated BVCEA-60. The epitope mapping studies indicate that all the CEA specific MAbs can recognize BVCEA-140. We also compared the sugar composition of BVCEA-140 to native CEA, using a lectin-linked immunoradiometric assay. The results demonstrated that both the native and recombinant baculovirus CEA contain simple high-mannose carbohydrates as well as biantennary and biantennary hybrid complexes. However, native CEA also contains triantennary and tetraantennary complex sugars, while the recombinant CEA molecule does not. Immunogenicity of the recombinant CEA molecules was demonstrated in mice. ELISA and Western blot analyses were used to determine the cross-reactivity of the anti-CEA sera. Mice immunized with BVCEA-140 elicit antibodies that are reactive to native CEA. When the BVCEA-16 was used as an immunogen, the antisera failed to detect native CEA or BVCEA-140. These studies demonstrate that minor sugar differences exist between native and baculovirus-derived CEA. However, epitope mapping with a panel of 24 anti-CEA MAbs (recognizing at least 10 CEA epitopes) stowed virtual immunologic identity between these two molecules. Moreover, BVCEA-140 appears to be a more potent humoral immunogen in mice than native CEA. These purified recombinant proteins can thus serve as standards in CEA serum assays for the possible detection and characterization of cell-mediated immune responses to CEA and as a potential source of immunogen (primary or for boosting) for active specific immunotherapy protocols of human carcinomas.

CDR substitutions of a humanized monoclonal antibody (CC49): contributions of individual CDRs to antigen binding and immunogenicity.

One of the major obstacles in the successful clinical application of monoclonal antibodies has been the development of host immune responses to murine Ig constant and variable regions. While the CDR grafting of MAbs may alleviate many of these problems, the potential remains that one or more murine CDRs on the human Ig backbone of a "humanized" MAb may still be immunogenic. Studies were undertaken employing a MAb of potential clinical utility, CC49, to define those CDRs that are essential for antigen binding and those that may be immunogenic in humans. We previously developed a humanized CC49 (HuCC49) by grafting the MAb CC49 hypervariable regions onto frameworks of human MAbs. To identify those CDRs essential for binding, a panel of variant HuCC49 MAbs was generated here by systematically replacing each of the murine CDRs with their human counterparts. The relative affinity constant of each variant was determined. Serum from a patient who received murine CC49 was used to determine the potential immunogenicity of each CDR in humans. The serum was shown to react with the anti-CC49 variable region. Results showed that patients' anti-idiotypic responses are directed mainly against LCDR3 and moderately against LCDR1 and HCDR2. These studies demonstrate for the first time that variants containing individual CDR substitutions of a humanized MAb can be constructed, and each CDR can be defined for the two most important properties for potential clinical utility: antigen binding and immunogenicity.

Baculovirus expression of a functional single-chain immunoglobulin and its IL-2 fusion protein.

The baculovirus expression system has been used for the production of a variety of proteins, including antibodies. Two single-gene constructs encoding single-chain immunoglobulins have recently been developed. The antibody employed was monoclonal antibody (MAb) CC49 which reacts with the pancarcinoma antigen, tumor associated glycoprotein, TAG-72. One, single-chain construct designated SCA delta CLCH1 (SCIg), consists of the CC49 sFv covalently joined to the human Fc (gamma 1) through the hinge region. The other, SCA delta CLCH1-IL-2 (SCIg-IL-2), has a human IL-2 molecule attached to the carboxyl end of the SCIg. These constructs have been used to test the feasibility of producing biologically active antibodies using the baculovirus expression system. Both constructs have been successfully expressed in insect cells and purified. The baculovirus recombinant single-chain antibodies have been designated, bV-SCA delta CLCH1 (bV-SCIg) and bV-SCA delta CLCH1-IL-2 (bV-SCIg-IL-2) they have been shown to be secreted in the culture supernatant as dimeric molecules of approximately 115 kDa and 140 kDa, respectively. The specificity and antibody dependent cellular cytolytic activity of the baculovirus recombinant single-chain antibodies were shown to be similar to that of the myeloma derived molecules. Glycosylation analysis showed that baculovirus derived proteins were N-glycosylated, but carried few if any high mannose residues. The biological activity of the IL-2 moiety was retained in bV-SCIg-IL-2, as evidenced by its stimulatory effect on the proliferation of the IL-2 dependent cell line HT-2. The observation that a significantly shorter time is required to develop baculovirus recombinant molecules as compared to myeloma derived molecules and that insect cells express single chain MAbs at acceptable levels may have implications for the production of these molecules for clinical use.

Cell mediated cytotoxicity of human colon carcinoma cells by a monoclonal antibody (R4) recognizing the carcinoembryonic antigen (CEA) and CEA-related molecules.

We report a novel anti-CEA monoclonal antibody (MAb) designated R4, which mediates antibody-dependent cell-mediated cytotoxicity (ADCC) of human colon carcinoma cells and displays differential reactivity for human carcinomas versus the normal counterparts. R4 (IgG1) reacted with the cell surface of 6 colon carcinoma cell lines expressing CEA. Western blot analysis and epitope mapping using native and baculovirus recombinant CEA and non specific cross-reacting antigen (NCA) demonstrated that MAb R4 recognizes a proteinic epitope located on the 3' end of the domain I shared by CEA and NCA molecules. Immunohistochemical analysis demonstrated an intense staining of MAb R4 with the majority of the neoplastic tissues tested, including colon (13/13), stomach (2/2), breast (9/10), lung (7/10) and endometrial (2/4) carcinomas, whereas no reactivity with the correspondent normal tissues was observed. Using human PBLs from healthy donors as effector cells, we have shown that MAb R4 mediated antibody dependent-cell mediated cytotoxicity (ADCC) of human carcinoma cells LS-174T, CBS and WiDr. This activity was enhanced after PBLs activation with interleukin-2 (IL-2). The specificity of MAb R4 for an epitope shared by two tumor overexpressed antigens, CEA and NCA, resulting in an intense reactivity with neoplastic cells and the peculiar property to mediate ADCC, indicate that MAb R4 might be a novel powerful reagent for diagnostic and immunotherapy of carcinoma patients.

Immunohistochemical analysis of epidermal growth factor receptor (EGF-R) and c-erbB-2 and correlation with pathological prognostic variables in endometrial carcinomas.

In this study the immunohistochemical expressions of epidermal growth factor receptor (EGF-R) and erbB-2 in endometrial carcinomas at different stages of disease progression were evaluated and correlated with clinicopathological prognostic variables. Our results indicate that EGF-R and erbB-2 molecules are expressed in normal endometrium as well as in the majority of the carcinomas evaluated (83% and 92% respectively). The intensity of the immunostaining varied greatly (1+ to 3+) in the different tumors. Within these tumors we focused on those characterized by high levels of expression (i.e. overexpression). Expression and overexpression of EGF-R has been associated with poorly differentiated tumors and with a 3.3 times higher risk of a more rapid disease relapse. Overexpression of the erbB-2 oncoprotein was significatively correlated with depth of myometrial invasion (M0-M1 vs M2-M3 p=0.05), pathological stage (stage I vs II-IV p=0.02) and grade of tumor differentiation (G1 vs G2-G3 p=0.001). In particular, c-erbB-2 overexpression was able to discriminate between the different subsets of stage I carcinomas. None of the IA tumors overexpressed the oncoprotein, as compared to IB and IC (41% and 100% respectively, p=0.04). This finding highlights a role for erbB-2 protein as a prognostic parameter in stage I endometrial carcinoma patients.

Alpha fetoprotein is more than a hepatocellular cancer biomarker: from spontaneous immune response in cancer patients to the development of an AFP-based cancer vaccine.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, with a poor prognosis and limited therapeutic options. Due to its overexpression in the majority of HCCs, alpha-fetoprotein (AFP) represents one of the most useful markers for hepatocarcinomas and for monitoring patients' response to therapy. Although it was earlier reported that AFP has immunosuppressive properties, it has been recently demonstrated that AFP induces spontaneous T and B cells responses in HCC patients. The characterization of AFP-immunogenic epitopes gives the opportunity to design AFP-based cancer vaccines for human HCC. The activity of AFP-based vaccines has been investigated in HCC mouse models in order to develop novel strategies to treat patients with HCC. This review will discuss the rationale for using the AFP-based vaccination strategy and recent results corroborating the usefulness of AFP vaccines as a potential tool for cancer therapy.

A common repertoire of autoantibodies is shared by cancer and autoimmune disease patients: Inflammation in their induction and impact on tumor growth.

The repertoire of autoantibodies found in cancer patients partly overlaps with that typical of patients with autoimmune diseases. Beside the biochemical and immunological properties of the target antigens and their altered expression in tumor tissues, the intratumoral inflammatory context can play a key role in the induction of autoimmune disease-associated autoantibodies in cancer patients. Furthermore, the impact of such antibodies on cancer growth and progression can be deeply influenced by the interplay with inflammation. The characterization of the spontaneous humoral responses occurring in cancer patients, of the mechanisms that trigger and sustain the autoantibody response and of the biological effects of such autoantibodies may help the rational design of anti-cancer immunotherapeutic protocols.

Murine red blood cell fragility is not affected by either vitamin E depletion or supplementation.

Male ICR mice were Pair-fed semipurified diets containing 0, 55 (control), and 500 IU/kg of vitamin E. Plasma and hepatic concentrations of vitamin E were determined and found to parallel the vitamin E levels in the diet. Even though plasma vitamin E levels were virtually zero in mice fed the depleted vitamin E diet for up to 304 days, there was no statistical difference in the red blood cell fragility between these animals and controls, as determined by a hypoosmotic fragility test. The diet with enriched vitamin E concentrations also did not affect the fragility of the red blood cell (RBC). Even after 300 days of zero dietary vitamin E, mice appeared healthy, demonstrating neither neurologic dysfunction nor failure to thrive. The data indicates that mice, unlike several other species, are more resistant to vitamin E depletion and may have other mechanisms to compensate for loss of this important antioxidant.

Generation, purification, and characterization of a recombinant source of human prostate-specific antigen.

Human prostate-specific antigen (PSA), a 33- to 34-kDa serine proteinase with extensive homology to glandular kallikrein, is a single-chain glycoprotein that contains 7% carbohydrate. The presence of PSA in the serum of patients with prostatic cancer is widely employed as a marker of disease status. PSA has also been thought of as a possible target for use in active specific immunotherapy protocols. To date, the source of PSA employed has been seminal fluid from different individuals; this has raised concerns about differences among PSA batches for standardization of assays. This report is the first description of the production and the purification of a recombinant source of PSA using a baculovirus expression system. A baculovirus recombinant of the cDNA encoding the full length PSA was expressed in insect cells yielding two major immunoreactive products of 31 and 29 kDa. The latter size conforms to the molecular weight of a core preprotein deduced from the sequence of the cDNA insert. The larger protein represents the N-linked glycosylated form of the preprotein. Western blot analysis showed that both the glycosylated and aglycosylated forms of PSA reacted with a polyclonal and two different monoclonal antibodies specific for PSA. bV-PSA, like commercially available PSA, showed also low-molecular-weight immunoreactive products when culture supernatants were concentrated or taken through steps of purification. bV-PSA was purified to a final product consisting of a major 29-kDa protein and a minor 31-kDa protein.(ABSTRACT TRUNCATED AT 250 WORDS)