Investigation of anti-inflammatory and toxicity effects of mangrove-derived Streptomyces rochei strain VITGAP173.

Mangrove ecosystems generate the major biodiversity hotspots of actinobacteria. Among the actinobacteria, Streptomyces species are the prolific producers of bioactive natural products. In this study, with research efforts to discover biopotential compounds from marine actinobacteria, 41 actinobacterial strains were isolated from sediment soil sample of Indian mangrove regions. The phylogeny prediction using the 16S rRNA gene sequences revealed that the isolates were related to Streptomyces. Isolates were further screened based on a two-step process wherein the first step, around nine strains, unveiled the presence of type 1 polyketide synthase gene and dTDP-glucose 4,6-dehydratase gene through polymerase chain reaction. As the second step of the screening process, cell viability assay was performed in RAW264.7 cells to assess the toxicity of extracts. Among all the isolates, Streptomyces rochei strain VITGAP173 was subjected to further analysis. To explore the bioactivities, the organic solvent extraction method was utilized to extract the broth culture of VITGAP173. Inhibition of nitric oxide and cyclooxygenase enzymes upon lipopolysaccharide-induced inflammation were utilized to evaluate the anti-inflammatory efficacy, and the results showed the potency of VITGAP173 in a dose-dependent manner. The extract significantly suppressed the messenger RNA levels of the inflammatory mediators such as tumor necrosis factor-α and interleukin-6 induced by lipopolysaccharide in RAW264.7 macrophages. The presence of several chemical constituents was identified through gas chromatography-mass spectrometry analysis of VITGAP173 extract. To achieve the toxicity analysis, oral administration of VITGAP173 extract in Wistar albino rats was carried out to investigate the biochemical parameters, histopathology which revealed its nontoxic nature.

Bioactivity Assessment of Indian Origin-Mangrove Actinobacteria against Candida albicans.

Actinobacteria is found to have a potent metabolic activity against pathogens. The present study reveals the assessment of potent antifungal secondary metabolites from actinobacteria isolated from Indian marine mangrove sediments. The samples were collected from the coastal regions of Muthupet, Andaman and the Nicobar Islands. Identification was carried out using 16S rRNA analysis and biosynthetic genes (Polyketide synthase type I/II and Non-ribosomal peptide synthase) were screened. Actinobacteria were assayed for their antifungal activity against 16 clinical Candida albicans and the compound analysis was performed using gas chromatography-mass spectrometry GC-MS. The 31 actinobacterial strains were isolated and 16S rRNA gene sequencing revealed that this ecosystem is rich on actinobacteria, with Streptomyces as the predominant genus. The PCR based screening of biosynthetic genes revealed the presence of PKS-I in six strains, PKS-II in four strains and NRPS in 11 strains. The isolated actinobacteria VITGAP240 and VITGAP241 (two isolates) were found to have a potential antifungal activity against all the tested C. albicans. GC-MS results revealed that the actinobacterial compounds were belonging to heterocyclic, polyketides and peptides. Overall, the strains possess a wide spectrum of antifungal properties which affords the production of significant bioactive metabolites as potential antibiotics.

Oral Administration of Carotenoid-Rich Dunaliella salina Powder Inhibits Colon Carcinogenesis via Modulation of Wnt/ß-catenin Signaling Cascades in a Rat Model.

The present study aims to investigate the oral therapeutic and molecular role of carotenoid-rich Dunaliella salina powder (DSP) against 1,2-dimethylhydrazine (DMH)-triggered colon carcinogenesis. In this study, thirty six male Wistar rats were categorized into six distinct groups (G1-G6): G1 group with no intervention, G2 group received only DSP (1000 mg/kg), G3 group received only DMH carcinogen (20 mg/kg), and G4-G6 group received both DMH and DSP at various phases (pre-initiation, post-initiation and entire phases) for 32 weeks. Body weight, tumor incidence, tumor volume, histopathological examination, antioxidants, and detoxification enzymes activities were analyzed in the experimental rats. In addition, the protein expression profile of components involved in the Wnt/ß-catenin signaling pathway was determined by western blot analysis. Matrix metalloproteinases (MMP-7 and MMP-9), proliferation marker (PCNA), and pro-apoptotic (Bcl-2 and Bax) proteins were analyzed using immunohistochemistry. Colorimetric assay was used to determine the levels of anti-inflammatory (iNOS and COX-2) and apoptotic proteins (Caspase-3 and Caspase-9). Results showed that concomitant administration of DSP with DMH significantly reduced tumor progression and prevented colon carcinogenesis in rats. However, treatment with DSP before or after DMH exposure did not significantly prevent colon carcinogenesis. DMH and DSP treatment group showed increased activities of antioxidant enzymes with significant reduction in the oxidative stress. Additionally, the detoxification enzymes and colonic histopathology of those rats were restored to that of control rats. The administration of DSP to rats exposed to DMH exhibited antitumor effects via inhibition of the Wnt/ß-catenin signaling pathway with induced apoptosis through the Bcl-2/Bax/caspases signaling cascades. Moreover, the same group also showed significant anti-inflammatory activity via regulating iNOS and COX-2 biomarkers. Our findings revealed molecular chemopreventive activity of carotenoid-rich DSP through regulating Wnt/beta-catenin and intrinsic apoptotic pathways. Thus, DSP is propound to function as a potent antioxidant, anti-proliferative, and anti-inflammatory therapeutic agent against colon carcinogenesis.

Apoptosis inducing metabolite from marine mangrove actinobacteria VITGAP173.

BACKGROUND: Marine actinobacteria have proven to be a remarkable source of bioactive metabolites. METHODS: The present study focused on the isolation of anticancer metabolites from marine actinobacteria. Streptomyces sp. VITGAP173 was found to have promising anticancer activity against breast cancer cell lines (MCF-7). RESULTS: Bioassay-guided fractionation was followed to identify the bioactive metabolites from crude ethyl acetate extract of VITGAP173, which yielded four fractions. Among the four fractions, fraction B exhibited the highest cytotoxic activity against MCF-7 cell lines. Further structural characterization of the fraction was done by FTIR and NMR spectroscopy. The fraction-2 induced cytotoxicity against MCF-7 cell lines and the half maximal inhibition (IC50) value was calculated as 4.7µg/ml. To elucidate the possible mechanism of cell death, MCF-7 cells were treated with fraction-2 for 24 hours and the morphological changes were examined using acridine orange - ethidium bromide (AO/EB) staining. The fraction also increased the reactive oxygen species (ROS) generation (Flow cytometry, DCFH-DA). The molecular mechanism of fraction-induced cell death was analysed by real-time PCR, which revealed that the fraction promotes apoptosis through the CHOP-ATF-4 pathway which is involved in ER stress signalling. CONCLUSION: The present findings suggest the apoptosis inducing potential of fraction-2 in breast cancer therapy.

Recombinant engineered phage-derived enzybiotic in Pichia pastoris X-33 as whole cell biocatalyst for effective biocontrol of Vibrio parahaemolyticus in aquaculture.

Vibrio parahaemolyticus is a major seafood-borne pathogen causing significant economic losses in aquaculture systems. Therefore, application of phage encoded enzymes, particularly endolysin, as a new strategy for effective biocontrol and therapeutic agent against bacterial diseases. In the present study, we synthesized endolysin gene (Vplys60) of bacteriophage qdv001 and biochemically characterized by expressing in Pichia pastoris X-33. In addition to, we also investigated the anti-biofilm and anti-vibriosis activity of Pichia-expressing Vplys60 against vibrio challenged in vivo aquaculture model, Artemia franciscana. The result indicated that the predicted molecular size of Pichia expressed Vplys60 was approximately 28 kDa as verified by SDS-PAGE and zymogram. Vplys60 manifested stable activity over broad range of pH (6-10), temperatures (37-75 °C) and salinity (100-600 mM NaCl). Biochemical and in silico analysis revealed that addition of calcium ion (Ca2+) enhanced the lytic activity of Vplys60 whereas other metal ions inhibited the activity. Additionally, calcium-dependent Vplys60 has showed a strong amidase activity by cleaving the peptidoglycan of V. parahaemolyticus. Our data also showed that Vplys60 (75 µg/ml) significantly inhibits biofilm formation (91.6%) and significantly reduced the bacterial population. The in vivo challenge study showed enhanced survival rate in combination with reduced vibrio load in Artemia after administration of Pichia-expressing Vplys60.

Oral administration of lyophilized Dunaliella salina, a carotenoid-rich marine alga, reduces tumor progression in mammary cancer induced rats.

Dunaliella salina is a photosynthetic cell factory used for the commercial production of food additives, cattle stock feed and cosmetics as well as active ingredients for pharmaceutical industries. The investigation of the in vivo antitumor activity of D. salina lyophilized powder (DSLP) against 7,12-dimethylbenz(a)anthracene (DMBA) induced mammary carcinogenesis in female Wistar rats indicated a dose-dependent effect of DSLP. We studied the effect of DSLP at two different dosages of 500 and 1000 mg per kg bw on DMBA induced mammary cancer in rats by measuring the status of antioxidant enzymes, phase I and phase II detoxification enzymes, lipid peroxidation, and glycoconjugated proteins and by investigating the expression pattern of cell proliferation (Ki-67), hormonal receptor (ER, PR and HER2) status by immunohistochemical analysis, and apoptotic (caspase-3 and -9) and pro-inflammatory (COX-2) markers by colorimetric analysis. After 16 weeks of the study, we observed 100% tumor formation (including high tumor incidence and tumor volume) and a significant increase in the level of hormonal receptors, cell proliferation, and pro-inflammatory and apoptosis markers in tumor-bearing animals compared to the control. The oral administration of DSLP (1000 mg per kg bw) to the DMBA treated animals showed up to 83.4% reduction of tumors and effectively restored the levels of biochemical markers in the mammary tissues in addition to the downregulation of the expression of molecular markers. In conclusion, DSLP was found to show a chemopreventive effect against breast cancer induced in rats through the suppression of cell proliferation and the induction of apoptosis.

Ocean Dwelling Actinobacteria as Source of Antitumor Compounds

ABSTRACT Extending over decades, research has been of great focus and enormous progress on exploring the ocean for natural products from marine actinobacteria. Attraction towards alternative medicine has led to improvements in natural product discovery. With great potential to survive in extreme environments, marine actinobacteria, efficiently produce an array of metabolites with diverse bioactivity by evolving the secondary metabolic pathways. Exploiting the secondary metabolite producing potential of actinobacteria, many compounds with antitumor, antibacterial, antifungal, antimalarial, antiprotozoal, antiparasitic, antiviral, anti-parasitic, anti-inflammatory activities has been discovered. Efforts in bioprospecting alternative sources of natural products have thus led to several explorations and improvements in technologies which has decreased the bottle neck difficulties in the drug discovery process. Emphasizing on the recent advancements in bioactive compound production in actinobacteria, this paper comprises a review of the available literature, compiles the antitumor compounds from marine actinobacteria with brief discussions and the perspectives to develop better antitumor compounds which would stimulate further research.