Search

Simona Dostalova,1,2 Katerina Vasickova,1 David Hynek,1,2 Sona Krizkova,1,2 Lukas Richtera,1,2 Marketa Vaculovicova,1,2 Tomas Eckschlager,3 Marie Stiborova,4 Zbynek Heger,1,2 Vojtech Adam1,2 1Department of Chemistry and Biochemistry, Mendel University in Brno, 2Central European Institute of Technology, Brno University of Technology, Brno, 3Department of Pediatric Hematology and Oncology, Second Faculty of Medicine, University Hospital Motol, Charles University, 4Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic Abstract: Due to many adverse effects of conventional chemotherapy, novel methods of targeting drugs to cancer cells are being investigated. Nanosize carriers are a suitable platform for this specific delivery. Herein, we evaluated the long-term stability of the naturally found protein nanocarrier apoferritin (Apo) with encapsulated doxorubicin (Dox). The encapsulation was performed using Apo’s ability to disassemble reversibly into its subunits at low pH (2.7) and reassemble in neutral pH (7.2), physically entrapping drug molecules in its cavity (creating ApoDox). In this study, ApoDox was prepared in water and phosphate-buffered saline and stored for 12 weeks in various conditions (-20°C, 4°C, 20°C, and 37°C in dark, and 4°C and 20°C under ambient light). During storage, a very low amount of prematurely released drug molecules were detected (maximum of 7.5% for ApoDox prepared in PBS and 4.4% for ApoDox prepared in water). Fourier-transform infrared spectra revealed no significant differences in any of the samples after storage. Most of the ApoDox prepared in phosphate-buffered saline and ApoDox prepared in water and stored at -20°C formed very large aggregates (up to 487% of original size). Only ApoDox prepared in water and stored at 4°C showed no significant increase in size or shape. Although this storage caused slower internalization to LNCaP prostate cancer cells, ApoDox (2.5 µM of Dox) still retained its ability to inhibit completely the growth of 1.5×104 LNCaP cells after 72 hours. ApoDox stored at 20°C and 37°C in water was not able to deliver Dox inside the nucleus, and thus did not inhibit the growth of the LNCaP cells. Overall, our study demonstrates that ApoDox has very good stability over the course of 12 weeks when stored properly (at 4°C), and is thus suitable for use as a nanocarrier in the specific delivery of anticancer drugs to patients. Keywords: anticancer therapy, doxorubicin-loaded apoferritin, encapsulation, long-term stability, protein nanocarriers

Recently, we reported that aristolochic acid (AA) a naturally occurring nephrotoxin and carcinogen is implicated in a unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN). Indeed, we identified the principal aristolochic acid-DNA adduct in the kidney of five such patients. We now extend these observations and demonstrate the presence of additional AA-DNA adducts by the 32P-post-labelling method not only in the kidneys, but also in a ureter obtained after renal transplantation. Using the nuclease P1 version of the assay not only the major DNA adduct of aristolochic acid, 7-(deoxyadenosin-N6-yl)-aristolactam I (dA-AAI), but also the minor adducts, 7-(deoxyguanosin-N2-yl)-aristolactam I (dG-AAI) and 7-(deoxyadenosin-N6-yl)-aristolactam II (dA-AAII) were detected, and identified by cochromatographic analyses with TLC and HPLC. Quantitative analyses of six kidneys revealed relative adduct levels from 0.7 to 5.3/10(7) for dA-AAI, from 0.02 to 0.12/10(7) for dG-AAI and 0.06 to 0.24/ 10(7) nucleotides for dA-AAII. The detection of the dA-AAII adduct is consistent with the occurrence of aristolochic acid II (AAII) in the herb powder imported under the name of Stephania tetrandra and confirms that the patients had indeed ingested the natural mixture of AAI and AAII. 32P-post-labelling analyses of further biopsy samples of one patient showed the known adduct pattern of AA exposure not only in the kidney, but also in the ureter, whereas in skin and muscle tissue no adduct spots were detectable. In an attempt to explain the higher level of the dA-AAI adduct compared to the dG-AAI adduct level in renal tissue even 44 months after the end of regimen, the persistence of these two purine adducts was investigated in the kidney of rats given a single oral dose of pure AAI. In contrast to the dG-AAI adduct, the dA-AAI adduct exhibited a lifelong persistence in the kidney of rats. Our data demonstrate that AA forms DNA adducts in human tissue by the same activation mechanism(s) reported from animal studies. Thus, the carcinogenic/mutagenic activity of AA observed in animals could also be responsible for the urothelial cancers observed in two of the CHN patients. [ABSTRACT FROM PUBLISHER]

Aristolochic acid I (AAI) and aristolochic acid II (AAII), the two major components of the carcinogenic plant extract aristolochic acid (AA), are known to be mutagenic and to form DNA adducts in vivo. According to the structures of the major DNA adducts identified in animals and humans, nitroreduction is the crucial pathway in the metabolic activation of these naturally occurring nitroarenes to their ultimate carcinogenic species. Using the nuclease P1-enhanced version of the 32P-post-labelling assay we investigated the formation of DNA adducts by AAI and AAII in different in vitro activation systems in order to determine the most suitable in vitro system mimicking target tissue activation. Although DNA adducts resulting from oxidative activation of AAs have not yet been identified both reductive and oxidative in vitro systems were employed. In vitro incubations were conducted under standardized conditions (0.3 mM AAs; 4 mM dNp as calf thymus DNA) using rat liver microsomes, xanthine oxidase (a mammalian nitroreductase), horseradish peroxidase, lactoperoxidase and chemical reduction by zinc. Enzymatic incubations were performed under aerobic and anaerobic conditions. A combination of two independent chromatographic systems (ion-exchange chromatography and reversed-phase HPLC) with reference compounds was used for the identification of DNA adducts detected by the 32P-post-labelling assay. The two known major adducts of AAI or AAII found in vivo were generated by all in vitro systems except for incubations with AAII and horseradish peroxidase where two unknown adducts predominated. Irrespective of the in vitro activation system used, the majority of adduct spots obtained were identified as the previously characterized four AA-DNA adducts: dA-AAI, dA-AAII, dG-AAI and dG-AAII. This indicates that both reductive and peroxidative activation of AAI or AAII resulted in chromatographically indistinguishable DNA adducts. Thus, peroxidase mediated activation of AAs led to the formation of the same adducts that had been observed in vivo and upon reductive activation in several in vitro systems. Quantitative analyses of individual adducts formed in the various in vitro systems revealed relative adduct labelling (RAL) values over a 100,000-fold range from 4 in 10(3) for activation of AAII to deoxyadenosine adducts by zinc to only 3 in 10(8) for activation of AAII by lactoperoxidase. The extent of DNA modification by AAI was higher than by AAII in all enzymatic in vitro systems. Only activation by zinc resulted in higher total binding to exogenous DNA by AAII than by AAI. Aerobic incubations with rat liver microsomes generated AAI- and AAII-DNA adduct profiles reproducing profiles in target tissue (forestomach) of rats, thus providing the most appropriate activation among the in vitro systems tested. [ABSTRACT FROM PUBLISHER]

Protein–protein interaction was investigated using a protein nanoprobe capable of photo-initiated cross-linking in combination with high-resolution and tandem mass spectrometry. This emerging experimental approach introduces photo-analogs of amino acids within a protein sequence during its recombinant expression, preserves native protein structure and is suitable for mapping the contact between two proteins. The contact surface regions involved in the well-characterized interaction between two molecules of human 14-3-3ζ regulatory protein were used as a model. The employed photo-initiated cross-linking techniques extend the number of residues shown to be within interaction distance in the contact surface of the 14-3-3ζ dimer (Gln8–Met78). The results of this study are in agreement with our previously published data from molecular dynamic calculations based on high-resolution chemical cross-linking data and Hydrogen/Deuterium exchange mass spectrometry. The observed contact is also in accord with the 14-3-3ζ X-ray crystal structure (PDB 3dhr). The results of the present work are relevant to the structural biology of transient interaction in the 14-3-3ζ protein, and demonstrate the ability of the chosen methodology (the combination of photo-initiated cross-linking protein nanoprobes and mass spectrometry analysis) to map the protein-protein interface or regions with a flexible structure.