Eucalyptus globulus Labill. is a widespread evergreen plant belonging to the Myrtaceae family. Several species of Eucalyptus are known to have a plethora of medicinal properties, particularly anti-tumor activity, which prompts the study of the chemical composition and bioactivity of extracts from this plant. Hereby, the main aims of this work were to (i) profile the phenolic compounds in E. globulus extracts prepared by decoction and infusion; (ii) test the cell growth inhibitory activity of E. globulus decoction and infusion, in three human tumor cell line models: colorectal, pancreatic and non-small cell lung cancer (HCT-15, PANC-1 and NCI-H460, respectively); and (iii) study the mechanism of action of the most potent extract in the most sensitive cell line. Our work demonstrated that both the decoction and infusion preparations revealed the presence of phenolic acids, flavonoids and gallotannins, the last group being the most abundant polyphenols found, especially two digalloyl-glucosides. Both extracts inhibited the growth of all the tumor cell lines tested. The decoction extract was the most potent in inhibiting the NCI-H460 cell growth (lower GI₅₀ determined by sulforhodamine B assay), which could be due to its higher content of phenolic compounds. Hence, the effect of the decoction extract on the NCI-H460 cells was further investigated. For this, cell viability (by Trypan blue exclusion assay), the cell cycle profile and apoptosis (by flow cytometry), cell proliferation (by bromodeoxyuridine assay) and protein expression (by western blot) were analyzed. Two different concentrations of the extract (52 μg mL⁻¹ and 104 μg mL⁻¹, corresponding to GI₅₀ and 2 × GI₅₀ concentration) were tested in these studies. Remarkably, the E. globulus decoction extract caused a dose-dependent decrease in the NCI-H460 cell number, which was correlated with a cell cycle arrest in the G0/G1 phase, a decrease in cell proliferation and an increase in the expression of p53, p21 and cyclin D1 proteins. Interestingly, no differences were found in the levels of ds-DNA damage and in the levels of apoptosis. This work highlights the relevance of the Eucalyptus globulus Labill. extract as a source of bioactive compounds with potential anti-tumor activity.

Photobacterium damselae subsp. piscicida (Phdp) is a Gram-negative fish pathogen with worldwide distribution and broad host specificity that causes heavy economic losses in aquaculture. Although Phdp was first identified more than 50 years ago, its pathogenicity mechanisms are not completely understood. In this work, we report that Phdp secretes large amounts of outer membrane vesicles (OMVs) when cultured in vitro and during in vivo infection. These OMVs were morphologically characterized and the most abundant vesicle-associated proteins were identified. We also demonstrate that Phdp OMVs protect Phdp cells from the bactericidal activity of fish antimicrobial peptides, suggesting that secretion of OMVs is part of the strategy used by Phdp to evade host defense mechanisms. Importantly, the vaccination of sea bass (Dicentrarchus labrax) with adjuvant-free crude OMVs induced the production of anti-Phdp antibodies and resulted in partial protection against Phdp infection. These findings reveal new aspects of Phdp biology and may provide a basis for developing new vaccines against this pathogen.

Photobacterium damselae subsp. piscicida (Phdp) is a Gram-negative bacterium that infects a large number of marine fish species in Europe, Asia, and America, both in aquacultures and in the natural environment. Among the affected hosts are economically important cultured fish, such as sea bream (Sparus aurata), sea bass (Dicentrarchus labrax), yellowtail (Seriola quinqueradiata), and cobia (Rachycentron canadum). The best characterized virulence factor of Phdp is the Apoptosis-Inducing Protein of 56 kDa (AIP56), a secreted AB-type toxin that has been shown to induce apoptosis of sea bass phagocytes during infection. AIP56 has an A subunit that displays metalloprotease activity against NF-kB p65 and a B subunit that mediates binding and internalization of the A subunit in susceptible cells. Despite the fact that the aip56 gene is highly prevalent in Phdp isolates from different fish species, the toxicity of AIP56 has only been studied in sea bass. In the present study, the toxicity of AIP56 for sea bream was evaluated. Ex vivo assays showed that sea bream phagocytes are resistant to AIP56 cytotoxicity and that resistance was associated with an inefficient internalization of the toxin by those cells. Accordingly, in vivo intoxication assays revealed that sea bream is much more resistant to AIP56-induced lethality than sea bass. These findings, showing that the effect of AIP56 is different in sea bass and sea bream, set the basis for future studies to characterize the effects of AIP56 and to fully elucidate its virulence role in different Phdp susceptible hosts.