RESUMO
Fighting cancer still relies on chemo- and radiation therapy, which is a trade-off between effective clearance of malignant cells and severe side effects on healthy tissue. Targeted cancer treatment on the other hand is a promising and refined strategy with less systemic interference. The enzyme horseradish peroxidase (HRP) exhibits cytotoxic effects on cancer cells in combination with indole-3-acetic acid (IAA). However, the plant-derived enzyme is out of bounds for medical purposes due to its foreign glycosylation pattern and resulting rapid clearance and immunogenicity. In this study, we generated recombinant, unglycosylated HRP variants in Escherichia coli using random mutagenesis and investigated their biochemical properties and suitability for cancer treatment. The cytotoxicity of the HRP-IAA enzyme prodrug system was assessed in vitro with HCT-116 human colon, FaDu human nasopharyngeal squamous cell carcinoma and murine colon adenocarcinoma cells (MC38). Extensive cytotoxicity was shown in all three cancer cell lines: the cell viability of HCT-116 and MC38 cells treated with HRP-IAA was below 1% after 24 h incubation and the surviving fraction of FaDu cells was ≤ 10% after 72 h. However, no cytotoxic effect was observed upon in vivo intratumoral application of HRP-IAA on a MC38 tumor model in C57BL/6J mice. However, we expect that targeting of HRP to the tumor by conjugation to specific antibodies or antibody fragments will reduce HRP clearance and thereby enhance therapy efficacy.
Assuntos
Antineoplásicos/farmacologia , Peroxidase do Rábano Silvestre/farmacologia , Ácidos Indolacéticos/química , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Animais , Antineoplásicos/química , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Feminino , Células HCT116 , Peroxidase do Rábano Silvestre/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Nasofaríngeas/tratamento farmacológico , Neoplasias Nasofaríngeas/patologia , Pró-FármacosRESUMO
Ellagic acid (EA), a natural polyphenol abundant in fruits and common in our diet, is under intense investigation for its chemopreventive activity resulting from multiple effects. EA inhibits topoisomerase II, but the effects on the human enzyme of urolithins, its monolactone metabolites, are not known. Therefore, the action of several synthetic urolithins toward topoisomerases II was evaluated, showing that polyhydroxylated urolithins, EA, and EA-related compounds are potent inhibitors of the α and ß isoforms of human topoisomerase II at submicromolar concentrations. Competition tests demonstrate a dose-dependent relationship between ATP and the inhibition of the enzyme. Docking experiments show that the active compounds bind the ATP pocket of the human enzyme, thus supporting the hypothesis that EA and polyhydroxylated urolithins act as ATP-competitive inhibitors of human topoisomerase II.