RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a lethal aggressive cancer, in part due to elements of the microenvironment (hypoxia, hypoglycemia) that cause metabolic network alterations. The FDA-approved antihelminthic pyrvinium pamoate (PP) has previously been shown to cause PDAC cell death, although the mechanism has not been fully determined. We demonstrated that PP effectively inhibited PDAC cell viability with nanomolar IC50 values (9-93 nmol/L) against a panel of PDAC, patient-derived, and murine organoid cell lines. In vivo, we demonstrated that PP inhibited PDAC xenograft tumor growth with both intraperitoneal (IP; P < 0.0001) and oral administration (PO; P = 0.0023) of human-grade drug. Metabolomic and phosphoproteomic data identified that PP potently inhibited PDAC mitochondrial pathways including oxidative phosphorylation and fatty acid metabolism. As PP treatment reduced oxidative phosphorylation (P < 0.001), leading to an increase in glycolysis (P < 0.001), PP was 16.2-fold more effective in hypoglycemic conditions similar to those seen in PDAC tumors. RNA sequencing demonstrated that PP caused a decrease in mitochondrial RNA expression, an effect that was not observed with established mitochondrial inhibitors rotenone and oligomycin. Mechanistically, we determined that PP selectively bound mitochondrial G-quadruplexes and inhibited mitochondrial RNA transcription in a G-quadruplex-dependent manner. This subsequently led to a 90% reduction in mitochondrial encoded gene expression. We are preparing to evaluate the efficacy of PP in PDAC in an IRB-approved window-of-opportunity trial (IND:144822).
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antihelmínticos/uso terapéutico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Metabolómica/métodos , Compuestos de Pirvinio/uso terapéutico , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Animales , Antihelmínticos/farmacología , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/patología , Humanos , Ratones , Compuestos de Pirvinio/farmacología , Análisis de Supervivencia , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Grapevine-shoot extracts (GSE), containing trans-resveratrol and resveratrol oligomers, are commercially available as food supplements. Considering the topoisomerase-targeting properties of trans-resveratrol, the question of whether GSE affect these enzymes, thereby potentially causing DNA damage, was addressed. In a decatenation assay, GSE potently suppressed the catalytic activity of topoisomerase IIα (≥5 µg/mL). The resveratrol oligomers ε-viniferin, r2-viniferin, and hopeaphenol, isolated from GSE, were also identified as topoisomerase IIα inhibitors. In the in vivo complexes of enzyme to DNA (ICE) bioassay, neither GSE, r2-viniferin, nor hopeaphenol affected the level of enzyme-DNA intermediates in A431 cells, thus representing catalytic inhibitors rather than topoisomerase poisons. GSE caused moderate DNA strand breaks (≥25 µg/mL) in the comet assay. Taken together, GSE presumably acts as a catalytic inhibitor of topoisomerase II with r2-viniferin and hopeaphenol as potentially contributing constituents. However, the increase of FPG-sensitive sites points to an additional mechanism that may contribute to the DNA-damaging properties of GSE constituents.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Extractos Vegetales/química , Brotes de la Planta/química , Estilbenos/química , Vitis/química , Antígenos de Neoplasias/metabolismo , Biocatálisis/efectos de los fármacos , Línea Celular Tumoral , Ensayo Cometa , Daño del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Extractos Vegetales/farmacología , Resveratrol , Estilbenos/farmacologíaRESUMEN
In the present study, the question was addressed whether anthocyanins interfere with the topoisomerase I poison irinotecan in vivo. In vivo complexes of enzyme to DNA bioassay was used to detect irinotecan-induced stabilization of topoisomerase I/DNA complexes and single cell gel electrophoresis to determine DNA-strand-break induction in the colon of male Wistar rats. Furthermore, analysis of anthocyanin concentrations in rat plasma and rat colon was included in the testing, demonstrating that anthocyanins reach the colon and the concentrations do not differ between rats that only received anthocyanins and the anthocyanin/irinotecan group. Blackberry extract was found to significantly reduce irinotecan-mediated topoisomerase I/DNA cleavable complex formation. Overall, anthocyanins did not notably increase cleavable complex formation. However, a significant increase of DNA damage was shown after a single dose of irinotecan as well as the single compounds cyanidin (cy) and cyanidin-3-glucoside (cy-3-g). Furthermore, a significant reduction of irinotecan-induced DNA-strand breaks after a pretreatment with cy, cy-3-g and blackberry extract was observed. Thus, the question arises whether anthocyanin-rich preparations might interfere with chemotherapy or whether, due to low systemic bioavailability, the preparations might provide protective potential in the gastrointestinal tract.
Asunto(s)
Antocianinas/farmacología , Camptotecina/análogos & derivados , Colon/efectos de los fármacos , Roturas del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo I/metabolismo , Animales , Antocianinas/análisis , Antocianinas/sangre , Camptotecina/farmacología , Colon/citología , Colon/metabolismo , Daño del ADN/efectos de los fármacos , Frutas , Glucósidos/farmacología , Irinotecán , Masculino , Extractos Vegetales/farmacología , Ratas , Ratas WistarRESUMEN
SCOPE: The effect of two anthocyanin-rich berry extracts (A, bilberry; B, red grape) on topoisomerases was investigated in a cell-free system and in human HT29 colon carcinoma cells. In parallel, their impact on DNA integrity was determined. METHODS AND RESULTS: The berry extracts suppressed the activity of topoisomerase I at concentrations ≥50 µg/mL. The activity of the topoisomerase II isoform was preferentially diminished (≥1 µg/mL). Within HT29 cells, the extracts were found to act as catalytic inhibitors without stabilizing the cleavable complex. Although topoisomerase activity was inhibited, none of the extracts induced DNA strand breaks up to 50 µg/mL. Moreover, pre- and coincubation of HT29 cells with A (≥1 µg/mL) significantly suppressed (p-value ≤0.001) the strand-breaking effects of camptothecin, whereas B was found to be less effective (1 µg/mL; p-value ≤0.05). Both extracts were found to significantly diminish doxorubicin-mediated DNA strand breaks at concentrations ≥1 µg/mL (p-value ≤0.001). Consistent with these results, the extracts suppressed doxorubicin-mediated enhancement of levels of topoisomerase II covalently linked to DNA in HT29 cells. CONCLUSION: These results raise the possibility that high intake of berry extracts may protect DNA and thus counteract the therapeutic effectiveness of orally applied topoisomerase poisons during chemotherapy.