Anti-proliferative effects of a blueberry extract on a panel of tumor cell lines of different origin.

BACKGROUND: Blueberries are among the fruits with the highest antioxidant activity and have been recognized by their health promoting properties. AIM: In vitro study of the anti-proliferative effects of a blueberry extract on a panel of cancer cells from different origin. MATERIALS AND METHODS: A blueberry extract was produced using ethanol as extracting solvent. The anti-proliferative activity of the extract was evaluated against seven tumor cell lines. The properties of blueberry extract to decrease cell adhesion and migration were also investigated. RESULTS: Blueberry extract showed a dose-dependent inhibitory effect on cell proliferation for all cell lines. Non-cytotoxic concentrations of the extract decreased cell adhesion in five of seven cell lines studied and inhibited the migration of MDA-MB-231 and PC-3 tumor cells. CONCLUSION: This work provides additional evidence regarding the ability of blueberry extract to inhibit the growth and decrease cell adhesion and migration of different cancer cell lines in vitro.

Relevance of small GTPase Rac1 pathway in drug and radio-resistance mechanisms: Opportunities in cancer therapeutics.

Rac1 GTPase signaling pathway has a critical role in the regulation of a plethora of cellular functions governing cancer cell behavior. Recently, it has been shown a critical role of Rac1 in the emergence of resistance mechanisms to cancer therapy. This review describes the current knowledge regarding Rac1 pathway deregulation and its association with chemoresistance, radioresistance, resistance to targeted therapies and immune evasion. This supports the idea that interfering Rac1 signaling pathway could be an interesting approach to tackle cancer resistance.

Cigarette smoking significantly alters sperm DNA methylation patterns.

Numerous health consequences of tobacco smoke exposure have been characterized, and the effects of smoking on traditional measures of male fertility are well described. However, a growing body of data indicates that pre-conception paternal smoking also confers increased risk for a number of morbidities on offspring. The mechanism for this increased risk has not been elucidated, but it is likely mediated, at least in part, through epigenetic modifications transmitted through spermatozoa. In this study, we investigated the impact of cigarette smoke exposure on sperm DNA methylation patterns in 78 men who smoke and 78 never-smokers using the Infinium Human Methylation 450 beadchip. We investigated two models of DNA methylation alterations: (i) consistently altered methylation at specific CpGs or within specific genomic regions and (ii) stochastic DNA methylation alterations manifest as increased variability in genome-wide methylation patterns in men who smoke. We identified 141 significantly differentially methylated CpGs associated with smoking. In addition, we identified a trend toward increased variance in methylation patterns genome-wide in sperm DNA from men who smoke compared with never-smokers. These findings of widespread DNA methylation alterations are consistent with the broad range of offspring heath disparities associated with pre-conception paternal smoke exposure and warrant further investigation to identify the specific mechanism by which sperm DNA methylation perturbation confers risk to offspring health and whether these changes can be transmitted to offspring and transgenerationally.

Pharmacological inhibition of Rac1-PAK1 axis restores tamoxifen sensitivity in human resistant breast cancer cells.

Tamoxifen is a standard endocrine therapy for estrogen receptor positive breast cancer patients. Despite its success, development of resistance mechanisms is still a serious clinical problem. Deregulation of survival signaling pathways play a key role in tamoxifen resistance, being upregulation of Rac1-PAK1 signaling pathway one of the most important. Here, we report the development of the breast cancer cell model MCF7::C1199 having Rac1 enhanced activity with the aim of evaluating the role of Rac1 in acquired endocrine resistance. These cells not only showed distinctive features of Rac1-regulated process as increased migration and proliferation rates, but also showed that upregulation of Rac1 activity triggered a hormonal-independent and tamoxifen resistant phenotype. We also demonstrated that PAK1 activity increases in response to Tamoxifen, increasing phosphorylation levels of estrogen receptor at Ser305, a key phosphorylation site involved in tamoxifen resistance. Finally, we evaluated the effect of 1A-116, a specific Rac1 inhibitor developed by our group, in tamoxifen-resistant cells. 1A-116 effectively restored tamoxifen anti-proliferative effects, switched off PAK1 activity and decreased estrogen receptor phospho-Ser305 levels. Since combination schemes of novel targeted agents with endocrine therapy could be potential new strategies to restore tamoxifen sensibility, these results show that inhibition of Rac1-PAK1 signaling pathway may provides benefits to revert resistance mechanisms in endocrine therapies.

RAC3 more than a nuclear receptor coactivator: a key inhibitor of senescence that is downregulated in aging.

Receptor-associated coactivator 3 (RAC3) is a nuclear receptor coactivator usually overexpressed in tumors that exerts oncogenic functions in the cytoplasm and the nucleus. Although as part of its oncogenic actions it was previously identified as an inhibitor of apoptosis and autophagy, its expression is required in order to preserve the pluripotency and embryonic stem cell self-renewal. In this work we investigated its role in cellular senescence. We found that RAC3 overexpression in the nontumoral HEK293 cells inhibits the premature senescence induced by hydrogen peroxide or rapamycin. The mechanism involves not only the inhibition of autophagy early induced by these stimuli in the pathway to senescence, but also the increase in levels and nuclear localization of both the cell cycle suppressors p53/p21 and the longevity promoters FOXO1A, FOXO3A and SIRT1. Furthermore, we found that RAC3 overexpression is required in order to maintain the telomerase activity. In tumoral HeLa cells its activity was inhibited by depletion of RAC3 inducing replicative senescence. Moreover, we demonstrated that in vivo, levels of RAC3 are downregulated in the liver from aged as compared with young rats, whereas the levels of p21 are increased, correlating with the expected senescent cell contents in aged tissues. A similar downregulation of RAC3 was observed in the premature and replicative senescence of human fetal WI-38 cells and premature senescence of hepatocyte HepG2 cell line. Taken together, all these results demonstrate that RAC3 is an inhibitor of senescence whose downregulation in aged individuals could be probably a tumor suppressor mechanism, avoiding the clonal expansion of risky old cells having damaged DNA.

Optimizing CIGB-300 intralesional delivery in locally advanced cervical cancer.

BACKGROUND: We conducted a phase 1 trial in patients with locally advanced cervical cancer by injecting 0.5 ml of the CK2-antagonist CIGB-300 in two different sites on tumours to assess tumour uptake, safety, pharmacodynamic activity and identify the recommended dose. METHODS: Fourteen patients were treated with intralesional injections containing 35 or 70 mg of CIGB-300 in three alternate cycles of three consecutive days each before standard chemoradiotherapy. Tumour uptake was determined using (99)Tc-radiolabelled peptide. In situ B23/nucleophosmin was determined by immunohistochemistry. RESULTS: Maximum tumour uptake for CIGB-300 70-mg dose was significantly higher than the one observed for 35 mg: 16.1 ± 8.9 vs 31.3 ± 12.9 mg (P = 0.01). Both, AUC24h and biological half-life were also significantly higher using 70 mg of CIGB-300 (P < 0.001). Unincorporated CIGB-300 diffused rapidly to blood and was mainly distributed towards kidneys, and marginally in liver, lungs, heart and spleen. There was no DLT and moderate allergic-like reactions were the most common systemic side effect with strong correlation between unincorporated CIGB-300 and histamine levels in blood. CIGB-300, 70 mg, downregulated B23/nucleophosmin (P = 0.03) in tumour specimens. CONCLUSION: Intralesional injections of 70 mg CIGB-300 in two sites (0.5 ml per injection) and this treatment plan are recommended to be evaluated in phase 2 studies.

Metastasis: recent discoveries and novel perioperative treatment strategies with particular interest in the hemostatic compound desmopressin.

Metastatic disease is responsible for most of cancer lethality. A main obstacle for therapy of advanced cancers is that the outcome of metastasis depends on a complex interplay between malignant and host cells. The perioperative period represents an underutilized window of opportunity for cancer treatment where tumor-host interactions can be modulated, reducing the risk of local recurrences and distant metastases. Blood-saving agents are attractive compounds to be administered during tumor surgery. Desmopressin (DDAVP) is a safe and convenient hemostatic peptide with proved antimetastastic properties in experimental models and veterinary clinical trials. The compound seems to induce a dual angiostatic and antimetastatic effect, breaking the cooperative function of cancer cells and endothelial cells during residual tumor progression. DDAVP is therefore an interesting lead compound to develop novel synthetic peptide analogs with enhanced antitumor properties.

Tissue factor as a novel marker for detection of circulating cancer cells.

Tissue factor (TF) is a molecular marker that is up-regulated in cancer cells and aids tumoral dissemination. Our purpose was to develop a nested RT-PCR strategy against TF for detecting blood-borne tumour cells. Our method detected TF expression in a minimum of 1.5 pg total RNA from MCF7 cells. A preliminary study in blood samples from 16 advanced breast carcinoma patients showed that 80% of patients with high TF load progressed and died, while only 18% with low TF load showed the same behaviour. Kaplan-Meier analysis confirmed worse overall survival in patients with high TF load.

Combined therapeutic effect of a monoclonal anti-idiotype tumor vaccine against NeuGc-containing gangliosides with chemotherapy in a breast carcinoma model.

Anti-idiotypic monoclonal antibodies (mAb) have been evaluated for actively induced immunotherapy with encouraging results. However, rational combination of cancer vaccines with chemotherapy may improve the therapeutic efficacy of these two approaches used separately. The main objective of this study was to evaluate the antitumor effect of the co-administration of 1E10 (Racotumomab), a monoclonal anti-idiotype tumor vaccine against an IgM mAb, named P3 that reacts specifically with NeuGc-containing gangliosides and low-dose Cyclophosphamide in a mammary carcinoma model. F3II tumor-bearing mice were immunized subcutaneously with 100 microg of 1E10 mAb in Alum or with 150 mg/m(2) of Cyclophosphamide intravenously 7 days after the tumor inoculation. While a limited antitumor effect was induced by a single 1E10 mAb immunization; its co-administration with low-dose Cyclophosphamide reduced significantly the F3II mammary carcinoma growth. That response was comparable with the co-administration of the standard high-dose chemotherapy for breast cancer based on 60 mg/m(2) of Doxorubicin and 600 mg/m(2) of Cyclophosphamide, without toxicity signs. Combinatorial chemo-immunotherapy promoted the CD8(+) lymphocytes tumor infiltration and enhanced tumor apoptosis. Furthermore, 1E10 mAb immunization potentiated the antiangiogenic effect of low-dose Cyclophosphamide. Additionally, splenic myeloid cells Gr1(+)/CD11b(+) associated with a suppressor phenotype were significantly reduced in F3II tumor-bearing mice immunized with 1E10 mAb alone or in combination with low-dose Cyclophosphamide. This data may provide a rational for chemo-immunotherapy combinations with potential medical implications in breast cancer.

A purified GM3 ganglioside conjugated vaccine induces specific, adjuvant-dependent and non-transient antitumour activity against B16 mouse melanoma in vitro and in vivo.

The presence of substantial amounts of GM3 ganglioside on human melanomas and other tumours, together with its peculiar biological properties, makes this glycolipid a unique target for cancer immunotherapy. B16 mouse melanoma expresses GM3 and constitutes an appropriate model for the development of novel GM3-based vaccines. Recently, we hydrophobically incorporated purified GM3 into the outer membrane protein complex from Neisseria meningitidis to form very small size proteoliposomes (GM3/VSSP). We have examined the antitumour properties of GM3/VSSP vaccine and compared it with GM3 incorporated in very low density serum lipoproteins (GM3/VLDL). Immunization with four doses of GM3/VSSP vaccine (120 microg of ganglioside) plus Freund's adjuvant or Montanide ISA 51 significantly increased the overall survival of mice inoculated in the subcutis with 103 B16-F1 cells, whereas the GM3/VLDL immunogen was ineffective. The non-transient character of tumour protection was confirmed in animals surviving the first challenge and re-inoculated with 5 x 103 cells. GM3/VSSP vaccine also reduced the subcutaneous growth of highly aggressive B16-F10 cells. The importance of ganglioside structure in the tumour-protective effect of GM3/VSSP vaccine was confirmed using GM3 containing N-glycolylneuraminic acid, a ganglioside absent in melanoma cells. Immunostaining and enzyme-linked immunosorbent assay (ELISA) experiments showed a high specificity of immune sera against GM3 and the presence of all four IgG subclasses, with a preponderance of IgG2b and IgG3. In addition, a strong anti-B16 complement-mediated cytotoxicity was induced by vaccination with GM3/VSSP. The present data indicate the molecular specificity of GM3/VSSP vaccine as well as the adjuvant-dependent and non-transient character of tumour protection in the B16 mouse model. These findings suggest that an appropriate GM3 vaccine may be capable of inducing prolonged tumour protection in melanoma patients.

In vitro activity of a Solanum tuberosum extract against mammary carcinoma cells.

We investigated the antitumor properties of a Solanum tuberosum extract (STE) on F3II mouse mammary carcinoma cells. STE significantly inhibited adhesion on fibronectin-coated surfaces and blocked migration of tumor cells in vitro. A major gelatinolytic activity (gelatinase) of 82 kD was identified in STE by zymographic analysis and characterized by exposure to different experimental conditions. Proteolytic activity of STE may be responsible, at least in part, for the in vitro effects on mammary carcinoma cells.

Chronic in vitro exposure to 3'-azido-2', 3'-dideoxythymidine induces senescence and apoptosis and reduces tumorigenicity of metastatic mouse mammary tumor cells.

Normal cells in culture divide a certain amount of times and undergo a process termed replicative senescence. Telomere loss is thought to control entry into senescence. Activation of telomerase in tumors bypasses cellular senescence and is thus a requirement for tumor progression. We reported previously the preferential incorporation of 3'-azido-2', 3'-dideoxythymidine (AZT) in telomeric sequences of immortalized cells in culture. In this work, we have investigated the effects of chronic in vitro AZT exposure on F3II mouse mammary carcinoma cells. We demonstrate, for the first time, that AZT-treated tumor cells have a reduced tumorigenicity in syngeneic BALB/c mice. Tumor incidence was reduced and survival was prolonged in animals inoculated with AZT-treated cells when comparing with control counterparts. The number and size of spontaneous metastases were also decreased in animals inoculated with AZT-treated cells. In addition, we present evidence of morphological and biochemical signs of senescence, as shown by the staining for senescence associated beta-galactosidase activity, and induction of programmed cell death, as demonstrated by an increase of caspase-3 activity, in tumor cells exposed to AZT. These data indicate that chronic exposure of mammary carcinoma cells to AZT may be sufficient to induce a senescent phenotype and to reduce tumorigenicity.

Evaluation of the models available for the prediction of pressure drop in venturi scrubbers.

The major running cost derived from the operation of venturi scrubbers is pressure drop. In the present study, the predictions of different models are compared to experimental data from venturi scrubbers of different sizes (throat diameter from 1.9 to 16cm), geometries, operating variables and liquid injection arrangements. As a result, it is concluded that most of the models must be used with caution. Much attention must be paid to the validity of the assumptions employed in the mathematical models. The equations proposed by Calvert [Scrubbing, Air Pollution, 3rd Edition, Vol. IV, Academic Press, New York, 1982], Yung et al. [JAPCA 27 (1977) 348] or Hesketh [Atomization and cloud behaviour in wet scrubbers, in: Proceedings of the US-USSR Symposium Control Fine Particulate Emissions 1974, San Francisco, 15-18 January 1974] produce good results only in very specific situations. The model proposed by Boll [Ind. Eng. Chem. Fundam. 12 (1973) 40] is simple, easy to compute and agrees reasonably well with the experimental data. Unfortunately, it cannot predict the effect of different liquid injection arrangements. The model by Azzopardi and coworkers [Filtr. Sep. 21 (1984) 196; Trans. IchemE. 69B (1991) 237; Chem Eng. J. 67 (1997) 9] was the only one to give good predictions for all the range of variables studied. On the other hand, this model is not simple and requires from the engineer an additional effort in terms of computation. In order to apply this model to the rectangular geometry, the concept of hydraulic equivalent diameter was used.

Antitumor properties of an anti-idiotypic monoclonal antibody in relation to N-glycolyl-containing gangliosides.

We examined the antitumor effects of 1E10 monoclonal antibody, an anti-idiotypic IgG to an IgM monoclonal antibody, named P3, that reacts specifically with N-glycolyl-containing gangliosides and also recognizes antigens in human breast and melanoma tumors. Two murine tumor cell lines positive for the P3 antibody, F3II mammary carcinoma (BALB/c) and B16 melanoma (C57BL/6), were employed. In BALB/c mice, vaccination with several i.p. doses at 14-day intervals of 50 microgram of 1E10 coupled to keyhole limpet hemocyanin in Freund's adjuvant, significantly reduced s.c. tumor growth of F3II carcinoma cells and the number of spontaneous lung metastases. Also, the effect of 1E10 as a biological response modifier on tumor lung colonization was evaluated in C57BL/6 mice injected i.v. with B16 melanoma cells. Interestingly, i.v. administration of 10 microgram of uncoupled 1E10 antibody, 10-14 days after inoculation of B16 cells, dramatically reduced the number of experimental metastases in comparison with lungs from mice treated with an irrelevant IgG. The present data suggest that this 'non-internal image' anti-idiotypic monoclonal antibody may activate more than one mechanism of antitumor response against melanoma and mammary tumor cells.

Nafoxidine modulates the expression of matrix-metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in endothelial cells.

During angiogenesis, proteases and their inhibitors interact in the remodelling of the basement membrane. It has been demonstrated that nafoxidine has antiangiogenic activity in the chick egg chorioallantoic membrane assay, but the precise mechanism of action is unknown. We have analyzed the effect of the partial estrogen antagonist nafoxidine on human umbilical vein endothelial cells (HUVEC). Our data indicated that in nafoxidine-treated endothelial cells MMP-2 was activated. Nafoxidine upregulated, in a dose-dependent manner, the secretion of a 66 kDa TIMP-1 dimer, that lacks anti-MMP activity and inhibited angiogenesis in the endothelial cord formation assay. We can postulate that nafoxidine induces an increase in TIMP-1, which has antiangiogenic activity in the late stages of tube formation, independent of its capacity to inhibit MMPs.

The role of protein kinase C and novel phorbol ester receptors in tumor cell invasion and metastasis (Review).

Phorbol ester tumor promoters activate protein kinase C (PKC) isozymes and novel non-kinase receptors, suggesting a high degree of complexity in the signaling mechanisms of tumorigenesis. Many studies have shown that PKC isozymes contribute to the progression of malignant phenotype. We review the emerging understanding of the roles of PKC isozymes in the three sequential cellular processes of tumor invasion and metastasis: attachment to extracellular matrix or basement membrane components, matrix degradation by proteolytic enzymes, and migration through the digested matrix. In addition, we discuss the potential role of chimaerins, novel non-kinase phorbol ester receptors, in carcinogenesis.

Modulation of urokinase-type plasminogen activator and metalloproteinase activities in cultured mouse mammary-carcinoma cells: enhancement by paclitaxel and inhibition by nocodazole.

Paclitaxel is a potent anti-tumor drug used in the treatment of breast cancer. It induces de-centralization of the microtubular system in tumor cells, blocking cell division. In the search for dissemination to a secondary site, cancer cells are capable of degrading most components of the extracellular matrix via an extracellular proteolytic cascade, including urokinase-type plasminogen activator (uPA) and the matrix metalloproteinases (MMPs). In the present study, the effects of paclitaxel and nocodazole, 2 drugs known to affect microtubules with opposite mechanisms of action, have been tested for their effect on the secretion of uPA and MMPs in cultures of F3II mouse mammary-tumor cells. Tumor-derived uPA activity significantly increased after pre-treatment of tumor cells for 24 hr with micromolar concentrations of paclitaxel (4 microM), while decreasing after pre-treatment with nocodazole (1 microM). A similar modulation was found for MMP-9 by zymographic analysis. Immunofluorescence and Western-blot analysis confirmed the formation of parallel microtubule fragments in paclitaxel-treated cells and almost complete de-polymerization of microtubules in nocodazole-treated ones. Our data suggest that, through opposite actions on microtubule organization and dynamics, paclitaxel and nocodazole exert inverse modulation of tumor-derived proteolytic activity in mammary tumor cells.

Expression of metalloproteinases (MMP-1, MMP-2, and MMP-9) and their inhibitors (TIMP-1 and TIMP-2) in schistosomal portal fibrosis.

Focal extracellular matrix degradation morphologically identified in human portal pipestem fibrosis due to Schistosoma mansoni did not express immunohistochemical reactivity for metalloproteinases (MMP-1, MMP-2, and MMP-9) and their inhibitors (TIMP-1 and TIMP-2). However, when active schistosomal periovular granulomas were present, a strong reactivity for MMP-1, MMP-2, TIMP-1, and TIMP-2 was observed. No reactivity was ever observed for MMP-9. However, the positive pattern of immunohistochemical expression was not seen in old fibrotic periovular granulomas, which were sometimes situated in other areas of the same microscopic section. Positive staining for MMPs and TIMPs was observed at the same time in hepatocytes and within the apical portion of bile duct epithelium. These findings are consistent with the concept that matrix degradation in recent and old fibroses, in addition to differing at the ultrastructural level, also differs in immunohistochemical expression of metalloproteinases and their inhibitors.

Deregulation of the signaling pathways controlling urokinase production. Its relationship with the invasive phenotype.

We review the evidence in support of the notion that, upon experimental oncogenic transformation or in spontaneous human cancers, mitogenesis and expression of urokinase (uPA) and its receptor (uPAR) are activated through common signaling complexes and pathways. It is well documented that uPA, uPAR or metalloproteinases (MMPs) are overexpressed in tumor cells of mesenchymal or epithelial origin and these molecules are required for tumor invasion and metastasis. Furthermore, oncogenic stimuli, which may render the transformed cells tumorigenic and metastatic in vivo, activate, in a constitutive fashion, the extracellular-regulated kinases (Erk 1 and 2) classical mitogenic pathway and others such as the NH(2)-Jun-kinase (Jnk). Cells from human tumors or oncogene-transformed cells overexpress uPA and uPAR, and also show a sustained activation of the above-mentioned signaling modules. In this paper we show that the classical mitogenic pathway involving Ras-Erk, PKC-Erk or Rac-JNK, among others, is activated by growth factors or endogenously by oncogenes, and constitutively activates uPA and uPAR expression. All the data obtained from human tumors or experimental systems, incorporated into a general model, indicate that oncogenic stimuli lead to the constitutive activation of mitogenesis and uPA and its receptor expression, through the activation of the same classical and nonclassical signaling complexes and pathways that regulate cell proliferation. We also discuss contrasting points of view. For instance, what governs the differential regulation of mitogenesis and the signal that leads to protease overexpression in a way that allows normal cells during physiological events to respond to growth factors, and proliferate without overexpressing extracellular matrix (ECM) proteases? Or how can cells remodel their microenvironment without proliferating? What restrains benign tumors from overexpressing tumor-associated proteases when they certainly have the mitogenic signal fully activated? This may occur by the differential regulation of transcriptional programs and recent reports reviewed in this paper may provide an insight into how this occurs at the signaling and transcriptional levels.

A novel hydrophobized GM3 ganglioside/Neisseria meningitidis outer-membrane-protein complex vaccine induces tumor protection in B16 murine melanoma.

Gangliosides are sialic acid-containing glycosphingolipids that have increased surface membrane expression on cancers of neuroectodermal origin. The present study was designed to investigate at a preclinical level the therapeutic usefulness of a consistently immunogenic and safe conjugate vaccine in melanoma. We have examined a novel vaccine of GM3 monosialoganglioside hydrophobically conjugated with the outer-membrane-protein complex from Neisseria meningitidis plus Montanide ISA 51 in the B16 melanoma mouse model. B16 cell line is characterized by the predominant presence of ganglioside GM3 on the cell surface. Vaccines were administered i.m. in the quadriceps at 14-day intervals and B16 cells were injected in the subcutis of the right flank of C57BL/6 mice, 7 days after the fourth dose. Significant suppression of tumor growth and prolongation of survival were seen by immunization with GM3 vaccine in animals challenged with 5x10(3) or 10(3) live melanoma cells. In addition, vaccination reduced tumor growth in animals challenged with 5x10(4) cells. The reactivity of serum IgG from vaccinated mice was examined by a sensitive immunoperoxidase assay on B16 tumor specimens. Most melanoma cells displayed a distinct positive staining associated with both cell membrane and cytoplasm. In accordance with the immunohistochemical stainings, the antisera of immunized mice reacted brightly against B16 melanoma cells in flow cytometry studies. Anti-sera also mediated complement-mediated cytotoxicity and specific response could be totally ascribed to antibodies of the IgG2b subclass. The present data suggest that GM3 vaccine may provide a useful immunotherapeutic strategy for melanoma.