Edgar Mendoza-Gamboa

Antitumor activity of colloidal silver on MCF-7 human breast cancer cells

BackgroundColloidal silver has been used as an antimicrobial and disinfectant agent. However, there is scarce information on its antitumor potential. The aim of this study was to determine if colloidal silver had cytotoxic effects on MCF-7 breast cancer cells and its mechanism of cell death.MethodsMCF-7 breast cancer cells were treated with colloidal silver (ranged from 1.75 to 17.5 ng/mL) for 5 h at 37°C and 5% CO2 atmosphere. Cell Viability was evaluated by trypan blue exclusion method and the mechanism of cell death through detection of mono-oligonucleosomes using an ELISA kit and TUNEL assay. The production of NO, LDH, and Gpx, SOD, CAT, and Total antioxidant activities were evaluated by colorimetric assays.ResultsColloidal silver had dose-dependent cytotoxic effect in MCF-7 breast cancer cells through induction of apoptosis, shown an LD50 (3.5 ng/mL) and LD100 (14 ng/mL) (*P < 0.05), significantly decreased LDH (*P < 0.05) and significantly increased SOD (*P < 0.05) activities. However, the NO production, and Gpx, CAT, and Total antioxidant activities were not affected in MCF-7 breast cancer cells. PBMC were not altered by colloidal silver.ConclusionsThe present results showed that colloidal silver might be a potential alternative agent for human breast cancer therapy.

In vitro characterization of the antiviral activity of fucoidan from Cladosiphon okamuranus against Newcastle Disease Virus

BackgroundNewcastle Disease Virus (NDV) causes a serious infectious disease in birds that results in severe losses in the worldwide poultry industry. Despite vaccination, NDV outbreaks have increased the necessity of alternative prevention and control measures. Several recent studies focused on antiviral compounds obtained from natural resources. Many extracts from marine organisms have been isolated and tested for pharmacological purposes, and their antiviral activity has been demonstrated in vitro and in vivo. Fucoidan is a sulfated polysaccharide present in the cell wall matrix of brown algae that has been demonstrated to inhibit certain enveloped viruses with low toxicity. This study evaluated the potential antiviral activity and the mechanism of action of fucoidan from Cladosiphon okamuranus against NDV in the Vero cell line.MethodsThe cytotoxicity of fucoidan was determined by the MTT assay. To study its antiviral activity, fusion and plaque-forming unit (PFU) inhibition assays were conducted. The mechanism of action was determined by time of addition, fusion inhibition, and penetration assays. The NDV vaccine strain (La Sota) was used in the fusion inhibition assays. PFU and Western blot experiments were performed using a wild-type lentogenic NDV strain.ResultsFucoidan exhibited antiviral activity against NDV La Sota, with an obtained IS50 >2000. In time of addition studies, we observed viral inhibition in the early stages of infection (0–60 min post-infection). The inhibition of viral penetration experiments with a wild-type NDV strain supported this result, as these experiments demonstrated a 48% decrease in viral infection as well as reduced HN protein expression. Ribavirin, which was used as an antiviral control, exhibited lower antiviral activity than fucoidan and high toxicity at active doses. In the fusion assays, the number of syncytia was significantly reduced (70% inhibition) when fucoidan was added before cleavage of the fusion protein, perhaps indicating a specific interaction between fucoidan and the F0 protein.ConclusionThe results of this study suggest that fucoidan from C. okamuranus represents a potential low-toxicity antiviral compound for the poultry industry, and our findings provide a better understanding of the mode of action of sulfated polysaccharides.

Sulphated Polysaccharides from Ulva clathrata and Cladosiphon okamuranus Seaweeds both Inhibit Viral Attachment/Entry and Cell-Cell Fusion, in NDV Infection

Sulphated polysaccharides (SP) extracted from seaweeds have antiviral properties and are much less cytotoxic than conventional drugs, but little is known about their mode of action. Combination antiviral chemotherapy may offer advantages over single agent therapy, increasing efficiency, potency and delaying the emergence of resistant virus. The paramyxoviridae family includes pathogens causing morbidity and mortality worldwide in humans and animals, such as the Newcastle Disease Virus (NDV) in poultry. This study aims at determining the antiviral activity and mechanism of action in vitro of an ulvan (SP from the green seaweed Ulva clathrata), and of its mixture with a fucoidan (SP from Cladosiphon okamuranus), against La Sota NDV strain. The ulvan antiviral activity was tested using syncytia formation, exhibiting an IC50 of 0.1 μg/mL; ulvan had a better anti cell-cell spread effect than that previously shown for fucoidan, and inhibited cell-cell fusion via a direct effect on the F0 protein, but did not show any virucidal effect. The mixture of ulvan and fucoidan showed a greater anti-spread effect than SPs alone, but ulvan antagonizes the effect of fucoidan on the viral attachment/entry. Both SPs may be promising antivirals against paramyxovirus infection but their mixture has no clear synergistic advantage.

In vitro effects of bovine dialyzable leukocyte extract (bDLE) in cancer cells

BACKGROUND Bovine dialyzable leukocyte extract (bDLE) is a dialyzate of a heterogeneous mixture of low molecular weight substances released from disintegrated blood leukocytes or lymphoid tissue obtained from homogenized bovine spleen. The purpose of this study was to determine if bDLE had cytotoxic effects and modulated apoptosis gene expression in breast cancer cells. METHODS The MCF-7, BT-474, MDA-MB-453, A-427, Calu-1, U937 and L5178Y cancer cell lines and PBMC human cells were treated with bDLE (0-0.66 U/mL) for 72 h. The bDLE effect on cell growth proliferation was evaluated by MTT assay, and the MCF-7 was evaluated by ethidium bromide-acridine orange staining; total DNA was evaluated for DNA fragmentation, and total RNA was isolated for p53, bag-1, c-myc, bim, bax, bcl-2 and bad mRNA expression. RESULTS The bDLE had dose-dependent cytotoxic effects and demonstrated an IC50 at a dosage of 0.06 U/mL (P<0.05). The bDLE did not affect the viability of normal human PBMC. The bDLE induced DNA fragmentation at doses of 0.06 and 0.13 U/mL in MCF-7 breast cancer cells. The bDLE induced cytotoxic effects and suppressed the p53, bag-1, c-myc, bax, bcl-2, and bad mRNA expression that influences apoptosis in MCF-7 breast cancer cells. Bim mRNA expression was not detected. DISCUSSION This may open up interesting prospects for the treatment of human breast cancer.

Antiangiogenic and antitumor effects of IMMUNEPOTENT CRP in murine melanoma.

Background: Skin cancers are common, and there has recently been a dramatic increase in their incidence, particularly in the occurrence of melanoma. Furthermore, relapse after curative surgical treatment of melanoma remains a significant clinical challenge and accounts for most of the mortality of this disease. Objective: The aim of this study was to determine whether IMMUNEPOTENT CRP affects B16F10 melanoma cells and tumors growth and vascular endothelial growth factor (VEGF) production in vivo and in vitro. Methods: B16F10 cells and B16F10-inoculated mice were treated with different concentrations of IMMUNEPOTENT CRP. Outcomes were then evaluated using MTT, TUNEL, Caspase-3, senescence, ELISA and colorimetric assays. Parameters related to survival and tumor weight were also assessed. Results: IMMUNEPOTENT CRP decreased the viability of B16F10 cells by increasing apoptosis of the treated cells, and VEGF production was decreased both in vitro and in vivo. Furthermore, treatment prevented metastasis, delayed the appearance of tumors, decreased tumor weight and improved the survival of tumor-bearing mice. Discussion: These observations suggest that IMMUNEPOTENT CRP can be used to suppress growth and metastasis by using targeting proteins such as VEGF.

Bovine dialyzable leukocyte extract modulates AP-1 DNA-binding activity and nuclear transcription factor expression in MCF-7 breast cancer cells

BACKGROUND We have previously demonstrated that bovine dialyzable leukocyte extract (bDLE) induces death through an apoptosis mechanism in MCF-7 breast cancer cells. Depending on the cell type and stimulus, activating protein-1 (AP-1) has been shown to regulate cell proliferation and differentiation, the stress response, apoptosis and survival. It remains unknown whether AP-1 and other transcription factors are mechanisms by which bDLE induces cell death. METHODS To determine whether bDLE modulates the AP-1 DNA binding and gene expression, MCF-7 breast cancer cells were treated with bDLE (0, 1, 5, 10 U) for 72 h and evaluated by electrophoretic mobility shift assay, reverse transcriptase-polymerase chain reaction and Western blot assays. RESULTS bDLE induced inhibition of cell growth, suppressed the AP-1 DNA-binding activity, decreased c-Jun protein expression and modulated NFATx, NFATc, NFkappaB, c-Jun and c-Fos transcription factor gene expression in MCF-7 breast cancer cells. DISCUSSION The present data indicate that bDLE can block the AP-1 DNA-binding activity and expression of several transcriptions factors in breast cancer cells, which will have great potential in improving cancer therapy.

Expression of Foxp3, CD25 and IL-2 in the B16F10 cancer cell line and melanoma is correlated with tumor growth in mice

The forkhead box P3 (Foxp3) transcription factor is one of the most studied markers used to identify CD4+CD25+ regulatory T cells (Tregs), and has been identified as a key regulator in the development and function of Tregs. Foxp3 expression has been reported in a variety of solid human tumors, including melanoma. The aims of the present study were to analyze Foxp3 expression in B16F10 melanoma cells in vitro, to determine whether this expression was affected during tumor growth in a murine melanoma model and to correlate Foxp3 expression with CD25 expression, interleukin (IL)-2 production and tumor weight. Foxp3 expression was analyzed with quantitative (q)PCR, flow cytometry and confocal microscopy. CD25 expression was analyzed by flow cytometry, and cytokine production was measured by ELISA [IL-2, interferon (IFN)-γ, transforming growth factor (TGF)-β and IL-10] and flow cytometry (IL-2, IFN-γ, IL-4 and IL-5). Foxp3 and CD25 expression was detected in the B16F10 cells in culture and in the intratumoral B16F10 cells. An increase in Foxp3 and CD25 expression was observed in a time-dependent manner during tumor growth at 7, 14 and 21 days. The production of the IL-2, IL-10, IFN-γ and TGF-β cytokines was observed in the B16F10 cells and also detected in the tumoral microenvironment during tumor growth (7, 14 and 21 days). An increase in IL-2 and IL-10 production was observed, whereas IFN-γ production decreased in a time-dependent manner. The production of tumor necrosis factor (TNF)-α was not observed in culture, but was detected during tumor growth, whereas the production of IL-4 and IL-5 was not detected. These data showed a positive correlation between the expression of Foxp3, CD25 and IL-2 and tumor weight in murine melanoma. From these data, it may be suggested that Foxp3 participates in melanoma growth, the modulation of the IL-2, IFN-γ and TNF-α cytokines and CD25 expression, and that it also plays a possible role in immunosuppression.

In Vitro Antibacterial Activity of Bovine Dialyzable Leukocyte Extract

The rapidly developing resistance of many infectious pathogenic organisms to modern drugs has spurred scientists to search for new sources of antibacterial compounds. One potential candidate, bDLE (dialysis at 10 to 12 kDa cut-off) and its fractions (“S” and “L” by 3.5 kDa cut-off and I, II, III, and IV by molecular exclusion chromatography), was evaluated for antibacterial activity against pathogenic bacterial strains (Staphylococcus aureus, Streptococcus pyogenes, Lysteria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhi) using standard antimicrobial assays. A minimum inhibitory concentration (MIC) of bDLE and its fractions was determined by agar and broth dilutions methods. Only bDLE and its “S” fraction had an effect upon all bacteria evaluated (MIC ranging from 0.29 to 0.62 U/ml), and the bactericidal and bacteriostatic effects (evaluated by MTT assay) were bacterial species-dependent. These results showed a remarkable in vitro antibacterial property of bDLE against several pathogenic bacteria.

Silencing of Foxp3 delays the growth of murine melanomas and modifies the tumor immunosuppressive environment

Forkhead box p3 (Foxp3) expression was believed to be specific for T-regulatory cells but has recently been described in non-hematopoietic cells from different tissue origins and in tumor cells from both epithelial and non-epithelial tissues. The aim of this study was to elucidate the role of Foxp3 in murine melanoma. The B16F10 cell line Foxp3 silenced with small interference Foxp3 plasmid transfection was established and named B16F10.1. These cells had lower levels of Foxp3 mRNA (quantitative real-time reverse transcription-polymerase chain reaction [0.235-fold]), protein (flow cytometry [0.02%]), CD25+ expression (0.06%), cellular proliferation (trypan blue staining), and interleukin (IL)-2 production (enzyme-linked immunosorbent assay [72.35 pg/mL]) than those in B16F10 wild-type (WT) cells (P<0.05). Subcutaneous inoculation of the B16F10.1 cell line into C57BL/6 mice delayed the time of visible tumor appearance, increased the time of survival, and affected the weight of tumors, and also decreased the production of IL-10, IL-2, and transforming growth factor beta compared with mice inoculated with the B16F10 WT cell line. The B16F10.1 cells derived from tumors and free of T-cells (isolated by Dynabeads and plastic attachment) expressed relatively lower levels of Foxp3 and CD25+ than B16F10 WT cells (P<0.05) in a time-dependent manner. The population of tumor-infiltrating lymphocytes of T CD4+ cells (CD4+, CD4+CD25+, and CD4+CD25+Foxp3+) increased in a time-dependent manner (P<0.05) in tumors derived from B16F10 WT cells and decreased in tumors derived from B16F10.1 cells. Similar data were obtained from spleen cells. These results suggest that, in melanomas, Foxp3 partly induces tumor growth by modifying the immune system at the local and peripheral level, shifting the environment toward an immunosuppressive profile. Therapies incorporating this transcription factor could be strategies for cancer treatment.

Bovine dialyzable leukocyte extract modulates cytokines and nitric oxide production in lipopolysaccharide-stimulated human blood cells

BACKGROUND In the current study, we determined whether bovine dialyzable leukocyte extract (bDLE) modulates lipopolysaccharide (LPS)-induced nitric oxide and cytokine overproduction. METHODS Human whole blood cells were treated with LPS (50 ng) + bDLE (1 U). RESULTS The bDLE treatment decreased nitric oxide as well as TNF-alpha, IL-6 and IL-10 (P <0.01) cytokine production. In addition, it decreased TNF-alpha, IL-1beta and IL-6 mRNA expression and suppressed IL-10 and IL-12p40 mRNA expression, but did not modulate IL-8 mRNA expression in LPS-stimulated human blood cells. DISCUSSION Our results suggest that bDLE may effectively modulate the fatal symptoms of hypotensive shock associated with endotoxin (LPS)-induced nitric oxide and cytokine production, and this may offer therapeutic potential for the treatment of endotoxic shock.