A fluorescent quantum dot conjugate to probe the interaction of Enterolobium contortisiliquum trypsin inhibitor with cancer cells.

Serine proteases play crucial biological roles and have their activity controlled by inhibitors, such as the EcTI, a serine protease inhibitor purified from Enterolobium contortisiliquum seeds, which has anticancer activity. This study aimed to conjugate EcTI with quantum dots (QDs), fluorophores with outstanding optical properties, and investigate the interaction of QDs-EcTI nanoprobe with cancer cells. The conjugation was evaluated by fluorescence correlation spectroscopy (FCS) and fluorescence microplate assay (FMA). EcTI inhibitory activity after interaction with QDs was also analyzed. From FCS, the conjugate presented a hydrodynamic diameter about 4× greater than bare QDs, suggesting a successful conjugation. This was supported by FMA, which showed a relative fluorescence intensity of ca. 3815% for the nanosystem, concerning bare QDs or EcTI alone. The EcTI inhibitory activity remained intact after its interaction with QDs. From flow cytometry analyses, approximately 62% of MDA-MB-231 and 90% of HeLa cells were labeled with the QD-EcTI conjugate, suggesting that their membranes have different protease levels to which EcTI exhibits an affinity. Concluding, the QD-EcTI represents a valuable nanotool to study the interaction of this inhibitor with cancer cells using fluorescence-based techniques with the potential to unravel the intricate dynamics of interplays between proteases and inhibitors in cancer biology.

A Trypsin Inhibitor from Tecoma stans Leaves Inhibits Growth and Promotes ATP Depletion and Lipid Peroxidation in Candida albicans and Candida krusei.

Tecoma stans (yellow elder) has shown medicinal properties and antimicrobial activity. Previous reports on antifungal activity of T. stans preparations and presence of trypsin inhibitor activity from T. stans leaves stimulated the investigation reported here. In this work, we proceeded to the purification and characterization of a trypsin inhibitor (TesTI), which was investigated for anti-Candida activity. Finally, in order to determine the potential of TesTI as a new natural chemotherapeutic product, its cytotoxicity to human peripheral blood mononuclear cells (PBMCs) was evaluated. TesTI was isolated from saline extract by ammonium sulfate fractionation followed by ion exchange and gel filtration chromatographies. Antifungal activity was evaluated by determining the minimal inhibitory (MIC) and fungicide (MFC) concentrations using fungal cultures containing only yeast form or both yeast and hyphal forms. Candida cells treated with TesTI were evaluated for intracellular ATP levels and lipid peroxidation. Cytotoxicity of TesTI to PBMCs was evaluated by MTT assay. TesTI (39.8 kDa, pI 3.41, K i 43 nM) inhibited similarly the growth of both C. albicans and C. krusei culture types at MIC of 100 µg/mL. The MFCs were 200 µg/mL for C. albicans and C. krusei. Time-response curves revealed that TesTI (at MIC) was more effective at inhibiting the replication of C. albicans cells. At MIC, TesTI promoted reduction of ATP levels and lipid peroxidation in the Candida cells, being not cytotoxic to PBMCs. In conclusion, TesTI is an antifungal agent against C. albicans and C. krusei, without toxicity to human cells.