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Background/aim: High expression level of Wilm's tumor gene (WT1) in several types of tumors appears to confer disruption of apoptosis and resistance to chemotherapeutic drugs, and correlate with poor outcome. The aim of this work was to determine if down-regulation of WT1 expression results in decreased cell proliferation and the increased action of different types of drugs, both in vitro in B16F10 cells, and in vivo in C57BL/6 mice., Materials and Methods: Inhibition of cell proliferation by short hairpin RNA against WT1 (shRNA-WT1), cisplatin, and gemcitabine in B16F10 cells in vitro was determined by the MTT assay and analysis of clonogenic survival. The apoptosis rate was determined by flow cytometry for annexin-V- fluorescein isothiocyante and propidium iodide., Results: Compared to treatment with shRNA-WT1 alone, treatment with shRNA-WT1 in combination with drugs had a synergistic inhibitory effect on B16F10 cell proliferation, particularly for the combination of cisplatin and gemcitabine at their 25% cytotoxic concentrations in vitro. Furthermore, mice treated with shRNA-WT1 in combination with cisplatin and gemcitabine were protected in the same way as those treated with the drugs alone, but were in better physical condition., Conclusion: Decreased WT1 expression induces cell death and potentiates the action of anticancer drugs by inducing synergistic effects both in vitro and in vivo, which may be an attractive strategy in lung cancer therapy., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Although preventable by vaccination, Measles still causes thousands of deaths among young children worldwide. The discovery of new antivirals is a good approach to control new outbreaks that cause such death. In this study, we tested the antiviral activity against Measles virus (MeV) of Polyphenol-rich extracts (PPs) coming from five seaweeds collected and cultivated in Mexico. An MTT assay was performed to determine cytotoxicity effect, and antiviral activity was measured by syncytia reduction assay and confirmed by qPCR. PPs from Ecklonia arborea (formerly Eisenia arborea , Phaeophyceae) and Solieria filiformis (Rhodophyta) showed the highest Selectivity Index (SI), >3750 and >576.9 respectively. Both PPs extracts were selected to the subsequent experiments owing to their high efficacy and low cytotoxicity compared with ribavirin (SI of 11.57). The combinational effect of PPs with sulphated polysaccharides (SPs) and ribavirin were calculated by using Compusyn software. Synergistic activity was observed by combining both PPs with low concentrations of Solieria filiformis SPs (0.01 µg/mL). The antiviral activity of the best combinations was confirmed by qPCR. Virucidal assay, time of addition, and viral penetration evaluations suggested that PPs act mainly by inactivating the viral particle. To our knowledge, this is the first report of the virucidal effect of Polyphenol-rich extracts of seaweeds.

This study evaluated the potential toxicity and antiviral activity of fucoidan from Cladosiphon okamuranus against Newcastle disease virus (NDV), one of the most serious threats to the poultry industry in the world. Toxicity was assayed on chicken embryo fibroblast (CEF) secondary cultures at concentrations ranging from 0.1 to 1500 μg per mL culture medium, assessing the cell viability by the yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay, and on 9-day-old embryonated chicken eggs by inoculation of 2 to 500 μg doses in the allantoic cavity, assessing the embryos morphology and liver histology. At 48 h post-inoculation, viability of CEF exposed to concentrations up to 10 μg/mL was not significantly affected, and the 50% cytotoxic concentration was estimated as of 1062 μg/mL; after exposure in ovo, some chick embryos showed liver steatosis when treated with fucoidan doses over 20 μg per egg (15 to 28% at 200 μg, 27 to 56% at 500 μg), but no change was detected in their size or aspect. Antiviral activity was tested by treating 9-day-old embryos via the allantoic route with 0.25 to 16 μg fucoidan doses that were applied at different times (-1, 0 and +1 h) relative to the inoculation of 10,000 folds the 50% Tissue Culture Infective Dose (TCID 50 ) of the NDV, La Sota strain. At 72 h post infection, virus titration in the allantoic fluid by hemagglutination assay (HA) showed a considerable and significant inhibition of infectivity for all doses, the best result (a 90% decrease) being obtained in embryos treated with 1 μg fucoidan one hour before infection. Viral RNA semi-quantification in pooled liver and small intestine of embryos that had been treated with 4 and 16 μg fucoidan 1 h before the infection showed reductions of the virus replication by 60 and 99.8%, respectively. Since this high anti-NDV activity in ovo was obtained with quite innocuous doses, fucoidan from C. okamuranus could be a potential low-toxicity antiviral compound to be used in areas exposed to NDV., (© 2016 Poultry Science Association Inc.)

Sulfated polysaccharides (SPs) extracted from five seaweed samples collected or cultivated in Mexico (Macrocystis pyrifera, Eisenia arborea, Pelvetia compressa, Ulva intestinalis, and Solieria filiformis) were tested in this study in order to evaluate their effect on measles virus in vitro. All polysaccharides showed antiviral activity (as measured by the reduction of syncytia formation) and low cytotoxicity (MTT assay) at inhibitory concentrations. SPs from Eisenia arborea and Solieria filiformis showed the highest antiviral activities (confirmed by qPCR) and were selected to determine their combined effect. Their synergistic effect was observed at low concentrations (0.0274 μg/mL and 0.011 μg/mL of E. arborea and S. filiformis SPs, resp.), which exhibited by far a higher inhibitory effect (96% syncytia reduction) in comparison to the individual SP effects (50% inhibition with 0.275 μg/mL and 0.985 μg/mL of E. arborea and S. filiformis, resp.). Time of addition experiments and viral penetration assays suggest that best activities of these SPs occur at different stages of infection. The synergistic effect would allow reducing the treatment dose and toxicity and minimizing or delaying the induction of antiviral resistance; sulfated polysaccharides of the tested seaweed species thus appear as promising candidates for the development of natural antiviral agents.

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.

Canine distemper virus (CDV) is a morbillivirus related to measles virus that infects dogs and other carnivores. CDV has a significant global impact on animal health; however, there is no current antiviral treatment for CDV infection. In recent years, it has been demonstrated that sulfated polysaccharides exhibit antiviral properties both in vivo and in vitro, despite their low cytotoxicity to host cells. Fucoidan is a sulfated polysaccharide found in the cell wall matrix of brown algae. In this study, we evaluated in vitro anti-CDV activity of fucoidan, which was derived from Cladosiphon okamuranus. Fucoidan actively inhibited CDV replication in Vero cells at a 50 % inhibitory concentration (IC50) of 0.1 µg/ml. The derived selectivity index (SI50) was >20,000. This polysaccharide likely inhibits viral infection by interference in the early steps and by inhibiting CDV-mediated cell fusion. Fucoidan may be useful in development of pharmacological strategies to treat and control CDV infection.

Background: Newcastle 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., Methods: The 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., Results: Fucoidan 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., Conclusion: The 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.

The Wilm's tumor gene (WT1), encoding a transcription factor that modulates the expression of certain genes that are involved in proliferation and apoptosis, is overexpressed in numerous solid tumors. WT1 is important for cell proliferation and in the diagnosis of melanoma. The objectives of this study were to investigate whether WT1 silencing is capable of synergizing with chemotherapeutic agents and whether this silencing is capable of sensitizing cancer cells to doxorubicin and cisplatin in the B16F10 murine melanoma cell line. In the present study, B16F10 cells were simultaneously treated with median lethal doses (LD50s) of WT1-1 or WT1-2 small hairpin RNAs (shRNAs) and chemotherapeutic agents. A total of 24 h post-transfection, a [3-(4,5-dimethylthiazol-2yl)-2,5- diphenyl tetrazolium bromide assay] MTT assay was performed. To determine whether shRNA interference (shRNAi) is capable of sensitizing B16F10 cells to chemotherapeutic agents, cells were transfected with an LD50 of each of the recombinant plasmids, treated with varying concentrations of doxorubicin or cisplatin 24 h post-transfection, and analyzed 48 h later for inhibition of cell proliferation using the MTT assay. We observed that WT1-RNAi and the two chemotherapeutic agents acted synergistically to inhibit B16F10 cell proliferation. The greatest inhibition of cell proliferation was observed with the WT1-2/cisplatin (91%) and WT1-1/cisplatin combinations (85%). WT1 silencing using shRNAi induced the chemosensitization of cells to doxorubicin and cisplatin, with the greatest inhibition (85%) of cell proliferation being observed in the cells treated with the WT1-2/cisplatin 6 ng/µl combination. Our results provide direct evidence that WT1 gene silencing has a synergistic effect with chemotherapeutic drugs and sensitizes B16F10 melanoma cells to doxorubicin and cisplatin. This suggests that these combination strategies are potentially utilized in melanoma therapy.

Background: Previous studies have reported on the presence of Murine Mammary Tumor Virus (MMTV)-like gene sequences in human cancer tissue specimens. Here, we search for MMTV-like gene sequences in lung diseases including carcinomas specimens from a Mexican population. This study was based on our previous study reporting that the INER51 lung cancer cell line, from a pleural effusion of a Mexican patient, contains MMTV-like env gene sequences., Results: The MMTV-like env gene sequences have been detected in three out of 18 specimens studied, by PCR using a specific set of MMTV-like primers. The three identified MMTV-like gene sequences, which were assigned as INER6, HZ101, and HZ14, were 99%, 98%, and 97% homologous, respectively, as compared to GenBank sequence accession number AY161347. The INER6 and HZ-101 samples were isolated from lung cancer specimens, and the HZ-14 was isolated from an acute inflammatory lung infiltrate sample. Two of the env sequences exhibited disruption of the reading frame due to mutations., Conclusion: In summary, we identified the presence of MMTV-like gene sequences in 2 out of 11 (18%) of the lung carcinomas and 1 out of 7 (14%) of acute inflamatory lung infiltrate specimens studied of a Mexican Population.

The Wilms' tumor gene 1 (WT1) is a universal tumor antigen and consequently a good therapeutic target for the development of gene therapy strategies. Earlier, we reported the in vitro efficacy of WT1 RNAi in the inhibition of B16F10 murine melanoma cell line growth. In this study, we used an aerosol system to deliver WT1 RNAi complexes, polyethyleneimine (PEI)-WT1-1 or PEI-WT1-2, to the lungs of mice with B16F10 lung metastasis. This treatment produced a statistically significant (P=0.020) reduction in the number and size of lung tumor foci, resulting in decreased lung weight and tumor index in treated mice compared with controls. The WT1 RNAi treatment also reduced the number and size of tumor blood vessels, suggesting decreased angiogenesis. Furthermore, the treated lung tissue showed cells in the tumor infiltrations undergoing apoptosis and elevated expression of the proapoptotic genes Bcl-xS and Bax, suggesting an activation of the intrinsic apoptotic pathway. Overall, WT1-1 treatment prolonged the mean survival time of tumor-bearing mice in comparison with the control and WT1-2-treated mice. Our data show that WT1 gene silencing in vivo by aerosol delivery of PEI-WT1 RNAi complexes is an effective therapeutic strategy for the treatment of lung metastases.

The Wilms' tumor protein 1 (WT1) is essential for tumor cell proliferation and is highly expressed in various hematological and solid malignancies including human malignant melanoma. We investigated whether WT1 expression is essential for growth in the B16F10 murine melanoma cell line. Toward this end, we examined WT1 protein expression and WT1 isoforms (17AA+/17AA-, KTS+/KTS-) in this cell line. WT1 was silenced by two RNA interference constructs, designated WT1-1 and WT1-2. RNA interference-mediated reduction of WT1 protein expression significantly inhibited B16F10 cell viability. Loss of WT1 also increased caspase-3 and poly-ADP-ribose polymerase activation, as well as apoptotic body formation, chromatin condensation, and DNA fragmentation. Together, these findings implicate decreased WT1 protein levels in the induction of apoptosis. These results imply that WT1 plays a distinct role in B16F10 melanoma growth.

The prevalence of antibodies against human T-cell lymphotrophic virus (HTLV-I/II) in blood donors from the city of Monterrey, Mexico was investigated. We found that 4 out of 1017 sera (0.39%) reacted against HTLV-I/II, as determined by a passive agglutination test (PA). However, none of PA-positive sera reacted to HTLV-I/II specific polypeptides as demonstrated by Western blot. These findings indicate that the population of Monterrey has very low or no seroprevalence for HTLV-I/II.

HIV and HTLV-1 are retrovirus that can produce human disease. It is known that HTLV-1 is associated to the adult T cell leukemia and to the spastic tropical paraparesis. AIDS is now a pandemic infection and HTLV-1 has a high endemicity in the Caribbean region and Japan, whereas the south of the United States has a low endemicity. In Mexico there is little information on HTLV-1 incidence. In the present work we looked for anti HTLV-1 antibodies in one hundred persons that belong to the high risks AIDS population in the city of Monterrey, Mexico. We found that 93 sera were positive for anti HIV antibodies in a ELISA test and seven were negative. All 93 sera were also positive in the Western Blot assay. In the confirmatory test two out of the seven negative sera were classified as indeterminate and five as negative. We also included in this study 50 sera from healthy control volunteers that did not belong to the high risk AIDS population and resulted negative in the HIV and HTLV-1 test. Anti HTLV-1 antibodies were determined by using an agglutination test with gelatin particles covered with HTLV-1 and confirmed by a Western Blot assay. We found that only three sera resulted positive in this agglutination test, but were negative by the Western Blot technique.