Tamires Aparecida Bitencourt

Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest

Chalcones are precursors of flavonoids that exhibit structural heterogeneity and potential antitumor activity. The objective of this study was to characterize the cytotoxicity of trans-chalcone and licochalcone A (LicoA1) against a breast cancer cell line (MCF-7) and normal murine fibroblasts (3T3). Also the mechanisms of the anti-cancer activity of these two compounds were studied. The alkaline comet assay revealed dose-dependent genotoxicity, which was more responsive against the tumor cell line, compared to the 3T3 mouse fibroblast cell line. Flow cytometry showed that the two chalcones caused the cell cycle arrest in the G1 phase and induced apoptosis in MCF-7 cells. Using PCR Array, we found that trans-chalcone and LicoA trigger apoptosis mediated by the intrinsic pathway as demonstrated by the inhibition of Bcl-2 and induction of Bax. In western blot assay, the two chalcones reduced the expression of cell death-related proteins such as Bcl-2 and cyclin D1 and promoted the cleavage of PARP. However, only trans-chalcone induced the expression of the CIDEA gene and protein in these two experiments. Furthermore, transient transfections of MCF-7 using a construction of a promoter-luciferase vector showed that trans-chalcone induced the expression of the CIDEA promoter activity in 24 and 48h. In conclusion, the results showed that trans-chalcone promoted high induction of the CIDEA promoter gene and protein, which is related to DNA fragmentation during apoptosis.

Transcription profile of Trichophyton rubrum conidia grown on keratin reveals the induction of an adhesin-like protein gene with a tandem repeat pattern

BackgroundTrichophyton rubrum is a cosmopolitan filamentous fungus that can infect human keratinized tissue (skin, nails and, rarely, hair) and is the major agent of all chronic and recurrent dermatophytoses. The dermatophyte infection process is initiated through the release of arthroconidial adhesin, which binds to the host stratum corneum. The conidia then germinate, and fungal hyphae invade keratinized skin structures through the secretion of proteases. Although arthroconidia play a central role in pathogenesis, little is known about the dormancy and germination of T. rubrum conidia and the initiation of infection. The objective of this study was to evaluate the transcriptional gene expression profile of T. rubrum conidia during growth on keratin- or elastin-containing medium, mimicking superficial and deep dermatophytosis, respectively.ResultsA transcriptional profiling analysis was conducted using a custom oligonucleotide-based microarray by comparing T. rubrum conidia grown on elastin and keratin substrates. This comparison shows differences according to protein source used, but consisted of a very small set of genes, which could be attributed to the quiescent status of conidia. The modulated genes were related to the dormancy, survival and germination of conidia, including genes involved in the respiratory chain, signal transduction and lipid metabolism. However, an induction of a great number of proteases occurred when T. rubrum was grown in the presence of keratin such as the subtilisin family of proteases (Sub 1 and Sub 3) and leucine aminopeptidase (Lap 1 and Lap 2). Interestingly, keratin also promoted the up-regulation of a gene encoding an adhesin-like protein with a tandem repeat sequence. In silico analysis showed that the protein contains a domain related to adhesin that may play a role in host-pathogen interactions. The expression of this adhesin-like gene was also induced during the co-culture of T. rubrum with a human keratinocyte cell line, confirming its role in fungal-host interactions.ConclusionThese results contribute to the discovery of new targets involved in the adhesion of conidia and the maintenance of conidial dormancy, which are essential for triggering the process of infection and the chronicity of dermatophytosis.

Gene Expression Response of Trichophyton rubrum during Coculture on Keratinocytes Exposed to Antifungal Agents

Trichophyton rubrum is the most common causative agent of dermatomycoses worldwide, causing infection in the stratum corneum, nails, and hair. Despite the high prevalence of these infections, little is known about the molecular mechanisms involved in the fungal-host interaction, particularly during antifungal treatment. The aim of this work was to evaluate the gene expression of T. rubrum cocultured with keratinocytes and treated with the flavonoid trans-chalcone and the glycoalkaloid α-solanine. Both substances showed a marked antifungal activity against T. rubrum strain CBS (MIC = 1.15 and 17.8 µg/mL, resp.). Cytotoxicity assay against HaCaT cells produced IC50 values of 44.18 to trans-chalcone and 61.60 µM to α-solanine. The interaction of keratinocytes with T. rubrum conidia upregulated the expression of genes involved in the glyoxylate cycle, ergosterol synthesis, and genes encoding proteases but downregulated the ABC transporter TruMDR2 gene. However, both antifungals downregulated the ERG1 and ERG11, metalloprotease 4, serine proteinase, and TruMDR2 genes. Furthermore, the trans-chalcone downregulated the genes involved in the glyoxylate pathway, isocitrate lyase, and citrate synthase. Considering the urgent need for more efficient and safer antifungals, these results contribute to a better understanding of fungal-host interactions and to the discovery of new antifungal targets.

Caffeic acid and licochalcone A interfere with the glyoxylate cycle of Trichophyton rubrum

Trichophyton rubrum is the most common causative agent of dermatomycoses worldwide. Despite the increasing incidence of fungal infections, the number of commercially available antifungal drugs is limited, mainly because of the biochemical similarities between fungal and mammalian cells. Biomolecules of different origins might lead to the discovery of new pharmacological targets that are more specific to the fungal cell. In this respect, caffeic acid (CA) and licochalcone A (LicoA) exhibit activity against some human pathogenic fungi by acting on important fungal molecular targets. The glyoxylate cycle is involved in the adaptation of fungal cells inside the human cell and is well established for some fungi of clinical interest. Activation of this cycle is related to the survival of fungi in nutrient-limited environments. However, little is known about the involvement of the glyoxylate cycle in this process in dermatophytes. The objective of this study was to evaluate the antifungal activity of CA and LicoA against T. rubrum, investigating specifically the effect of these compounds on important antifungal targets such as ergosterol synthesis, cell wall and glyoxylate cycle. The minimum inhibitory concentration was 86.59 μM for CA and 11.52 μM for LicoA. Plasma membrane damage and a reduction in ergosterol levels were observed after the exposure of T. rubrum to CA, but not to LicoA. Evaluation of gene expression in T. rubrum co-cultured with human keratinocytes (HaCat) in the absence of the antifungal compounds showed induction of genes related to the ergosterol biosynthesis pathway and genes encoding enzymes involved in cell wall synthesis and in the glyoxylate cycle. The same genes were significantly repressed after exposure of the co-culture to subinhibitory concentrations of CA and LicoA. The enzymatic activity of isocitrate lyase was reduced in the presence of LicoA and a moderate reduction was observed in the presence of CA. These results indicate that CA and LicoA act on targets that play important roles in pathogen-host interactions, in antifungal activity and, especially, in the glyoxylate cycle.

Antifungal activity of flavonoids and modulation of expression of genes of fatty acid synthesis in the dermatophyte Trichophyton rubrum

Background Dermatophytosis are fungal infections caused by keratinophilic fungi known as dermatophytes and classified in three genera: Trichophyton, Epidermophyton and Microsporum. Trichophyton rubrum is the most frequent species associated to dermatophytosis worldwide [1]. The infections caused by dermatophytes are not lethal, but are difficult to treat and uncomfortable. In the case of T. rubrum, they tend to be chronic, and although the superficial infections are more common, cases of deep infection have been reported in immunocompromised patients [2][3]. The number of antifungal drugs are still limited, and the acquired resistance for some of clinical antifungal have been shown as well as the side effects that have been promoted by them. Reasons for the challenge in development of new antifungal drugs are the similarities shared by fungal and mammalian cells and the lack of knowledge about the biology of these pathogens. Recent evidences have shown that the fatty acid sinthase (FAS) is an interesting antifungal target [4] because of marked differences between human and fungal cells. The aim of this study was to evaluate the antifungal activity of four flavonoids described as FAS inhibitors and verify the modulation of genes in the pathway of fatty acid synthesis in T. rubrum growth in presence of the most effective one as FAS inhibitor.

Evaluation of antifungal activity of glycoalkaloids from the Solanum lycocarpum St. Hil (lobeira) in the cell membrane of dermatophyte of Trichophyton rubrum

Background The dermatophytes belong to one of the main groups of pathogenic fungus, characterized by the use of the host’s keratin for its nutrition, which are the most common cause of fungal infection in the world, affecting millions of individuals annually, causing a huge economic impact [1]. Therefore, there was an increase in the search for new antifungal agents from natural sources, because the majority of the available drugs in the market presents a restricted number of cellular targets and there are reports of resistant fungal strain to these utilized drugs [2]. Glycoalkaloids from the Solanum lycocarpum plant (lobeira) presents several biological activities, such as cytotoxic and antimicrobial activities [3]. The goal of this work was determination the Minimum Inhibitory Concentration (MIC) of the solanine, solamargine and solasodine from the S. lycocarpum in addition to evaluate the effect of these alkaloids in the regeneration of the Trichophyton rubrum cell wall.

Chalcones Repressed the AURKA and MDR Proteins Involved in Metastasis and Multiple Drug Resistance in Breast Cancer Cell Lines

In the present investigation, trans-chalcone and licochalcone A were tested against MCF-7 and BT-20 breast cancer cell lines for anti-tumor activity. We found that both chalcones down regulated important genes associated to cancer development and inhibited cell migration of metastatic cells (BT-20). Finally, we observed that licochalcone A reduces the MDR-1 protein, while both chalcones suppress the AURKA protein in a dose-dependent manner. In conclusion, we observed the trans-chalcone and licochalcone A affected the cell viability of breast cancer cell lines MCF-7 and BT-20 and presents anti-metastatic and anti-resistance potential, by the repression of AUKA and MDR-1 proteins.

In silico characterization of tandem repeats in Trichophyton rubrum and related dermatophytes provides new insights into their role in pathogenesis

Abstract Trichophyton rubrum is the most common etiological agent of dermatophytoses worldwide, which is able to degrade keratinized tissues. The sequencing of the genome of different dermatophyte species has provided a large amount of data, including tandem repeats that may play a role in genetic variability and in the pathogenesis of these fungi. Tandem repeats are adjacent DNA sequences of 2–200 nucleotides in length, which exert regulatory and adaptive functions. These repetitive DNA sequences are found in different classes of fungal proteins, especially those involved in cell adhesion, a determinant factor for the establishment of fungal infection. The objective of this study was to develop a Dermatophyte Tandem Repeat Database (DTRDB) for the storage and identification of tandem repeats in T. rubrum and six other dermatophyte species. The current version of the database contains 35 577 tandem repeats detected in 16 173 coding sequences. The repeats can be searched using entry parameters such as repeat unit length (nt—nucleotide), repeat number, variability score, and repeat sequence motif. These data were used to study the relative frequency and distribution of repeats in the sequences, as well as their possible functions in dermatophytes. A search of the database revealed that these repeats occur in 22–33% of genes transcribed in dermatophytes where they could be involved in the success of adaptation to the host tissue and establishment of infection. The repeats were detected in transcripts that are mainly related to three biological processes: regulation, adhesion, and metabolism. The database developed enables users to identify and analyse tandem repeat regions in target genes related to pathogenicity and fungal–host interactions in dermatophytes and may contribute to the discovery of new targets for the development of antifungal agents. Database URL: http://comp.mch.ifsuldeminas.edu.br/dtrdb/

Marine natural compounds can be efficient toward aflatoxigenic Aspergillus flavus strain

Background The presence of high levels of aflatoxin is a serious problem to the production of raw peanuts and peanut crumbs. The high incidence of aflatoxin in peanuts in our country is mainly due to problems in primary production. High humidity and temperature conditions increase the likelihood of Aspergillus development and aflatoxins production, which is worsened during rainy weather [1]. Aflatoxins may remain in the food after the death of fungus without visible alterations [2]. The effects of aflatoxins on human and animal health, besides resistance of fungi to conventional antifungal agents has motived the search for new inhibitors. The research of marine natural products from sponges has been considered as a promising source for the development of new antifungal agents in order to discover compounds more effective and less toxic [3]. The objective of this study was to evaluate the antifungal activity of 21 marine natural compounds toward an aflatoxigenic A.flavus ATCC strains.

Evaluation of antifungal and cytotoxic activity of trans -Chalcone and α-Solanine

Background Dermatophytes are adapted to grow in keratinized tissues such as skin, nail and hair. Trichophyton rubrum is the most frequent cause of dermatophytosis in Brazil and in the world [1]. Despite its incidence there are only a limited number of antifungal drugs available for clinical use and some drugs are highly toxic to humans. In this regard, chalcones and alkaloids are phytochemical products which provide a rich source of chemical diversity for the development of new antifungals. Chalcones inhibit the biosynthesis of the cell wall and activity of fatty acid synthase in yeast [2,3]. The glycoalkaloid a-Solanine purified from potate sprout presents antifungal activity by altering cell membrane integrity and inhibition of sporulation [4]. The aim of the present study was to evaluate the minimum inhibitory concentration (MIC) and cytotoxicity (by MTT) of trans-Chalcone and a-Solanine toward strain MYA3108 of T.rubrum and the keratinocyte cell line HaCat, in order to evaluate the potential use of these phytochemicals against fungal skin infection.