Defensin c-thionin from Capsicum chinense improves butyrate cytotoxicity on human colon adenocarcinoma cell line Caco-2

BACKGROUND: Butyrate is a histone deacetylase inhibitor that induces apoptosis and inhibits cell proliferation of colorectal cancer cells. To improve its anticancer activity, butyrate has been evaluated mixed with drugs and different molecules. Plant antimicrobial peptides are attractive anticancer alternative molecules because they show selective cytotoxic activity against different cancer cell lines. In this work, we explore if the plant defensin c-thionin (Capsicum chinense) can improve butyrate activity on Caco-2 cell line and we also determined the mechanism of death activated. RESULTS: The combined treatment of c-thionin (3.5 mM) and butyrate (50 mM) showed higher cytotoxicity on Caco-2 cells with respect to single treatments. Also, the combined treatment reduced cell proliferation and exhibited a higher rate of apoptosis than single treatments. Combined treatment induced caspases 8 and 9 activation to an extent comparable with that of butyrate while c-thionin did not activate caspases. Additionally, reactive oxygen species generation preceded the onset of apoptosis, and superoxide anion production was higher in cells treated with the combined treatment. CONCLUSIONS: The c-thionin from Habanero chili pepper improved the butyrate cytotoxicity on Caco-2 cells. This effect occurred through apoptosis induction associated with reactive oxygen species production. Therefore, the combination of butyrate with cytotoxic antimicrobial peptides could be an attractive strategy for cancer therapy.

Different resistance patterns of reference and field strains of Brucella abortus

The aim of this study was to evaluate the growth of the B. abortus reference strains and field isolates on media containing different inhibitor agents. Reference strains were seeded on tryptose agar containing: i-erythritol (1.0 mg/mL), fuchsin (20 μg/mL and 80 μg/mL), thionin (2.5 μg/mL and 10 μg/mL), rifampicin (200 μg/mL) and safranin O (200 μg/mL). Field isolates were tested only on media containing i-erythritol, rifampicin and thionin. Furthermore, each suspension was also inoculated on tryptose agar incubated in air, to test its ability to grow without CO2. Sensitivity to fuchsin was similar among reference strains evaluated. Growth of S19, 544 and 2308 but not RB51 were inhibited on media containing rifampicin. Medium with safranin O showed no inhibition for RB51, 544 and 2308, but it partially inhibited the S19 growth as well as medium containing i-erythritol. Treatment/control growth ratio for 2308 on tryptose agar containing thionin (2.5 μg/mL) was approximatelly 1.0, whereas S19 and RB51 showed 0.85 and 0.89 ratios, respectively. Growth of 544, S19 and RB51 but not 2308 was completely inhibited on medium with thionin (10 μg/mL). All field strains grew on medium containing i-erythritol, but were completelly inhibited by rifampicin. With exception of A1 (B. abortus biovar 3) all field isolates grew on medium with thionin, although some strains showed a treatment/control growth ratio of 0.75–0.80 (10 μg/mL). These results showed that tryptose agar with thionin, i-erythritol or rifampicin could be useful for differentiating vaccine, challenge and field strains of B. abortus.

Antimicrobial effects of plant defence peptides expressed by bovine endothelial cells on intracellular pathogens

Background: The actions of plant antimicrobial peptides (PAP) against intracellular pathogens are poorly known. It has been reported that wheat puroindolines show antibacterial activity against Staphylococcus epidermidis endocyted by macrophages. In this work, we evaluated the intracellular antimicrobial activity of PAP gamma-thionin and thionin Thi2.1 produced by bovine endothelial cells against intracellular Staphylococcus aureus and Candida albicans. We used three host-pathogen models: 1) bovine mammary epithelial cells (BMEC)-S. aureus, 2) bovine endothelial cells (BEC)-S. aureus and 3) BEC-C. albicans, and evaluated the effect of conditioned media from BEC producers of PAP (gamma-thionin and thionin Thi2.1). Results: In the first model, conditioned medium (CM) containing Thi2.1 completely inhibited S. aureus intracellular after 24 hrs treatment. In the second model, CM from BEC containing gamma-thionin has a better effect killing intracellular S. aureus for 12-24 hrs incubations than CM from endothelial cells producers of Thi2.1; this was related with an increase of nitric oxide production (~2 times) in BEC infected and treated for 12 hrs with CM containing gamma-thionin, which negatively correlates with bacterial viability. In the third model, CM containing Thi2.1 showed a more potent intracellular fungicidal activity (~85 percent of inhibition) at 24 hrs treatment than CM containing gamma-thionin (~35 percent of inhibition). Conclusions: This work shows new effects of PAP to control intracellular bacterial or fungal infections.