No abstract

The use of carboplatin in cancer chemotherapy is limited by the emergence of drug resistance. To understand the molecular basis for this resistance, a chemogenomic screen was performed in 53 yeast mutants that had previously presented strong sensitivity to this widely used anticancer agent. Thirty-four mutants were responsive to carboplatin, and from these, 21 genes were selected for further studies because they have human homologues. Sixty percent of these yeast genes possessed human homologues which encoded proteins that interact with cullin scaffolds of ubiquitin ligases, or whose mRNA are under the regulation of Human antigen R (HuR) protein. Both HuR and cullin proteins are regulated through NEDDylation post-translational modification, and so our results indicate that inhibition of this process should sensitise resistant tumour cells to carboplatin. We showed that treatment of a tumour cell line with MLN4924, a NEDDylation inhibitor, overcame the resistance to carboplatin. Our data suggest that inhibition of NEDDylation may be a useful strategy to resensitise tumour cells in patients that have acquired carboplatin resistance.

Crisantaspase is an asparaginase enzyme produced by Erwinia chrysanthemi and used to treat acute lymphoblastic leukemia (ALL) in case of hypersensitivity to Escherichia coli L-asparaginase (ASNase). The main disadvantages of crisantaspase are the short half-life (10 H) and immunogenicity. In this sense, its PEGylated form (PEG-crisantaspase) could not only reduce immunogenicity but also improve plasma half-life. In this work, we developed a process to obtain a site-specific N-terminal PEGylated crisantaspase (PEG-crisantaspase). Crisantaspase was recombinantly expressed in E. coli BL21(DE3) strain cultivated in a shaker and in a 2-L bioreactor. Volumetric productivity in bioreactor increased 37% compared to shaker conditions (460 and 335 U L −1 H −1 , respectively). Crisantaspase was extracted by osmotic shock and purified by cation exchange chromatography, presenting specific activity of 694 U mg −1 , 21.7 purification fold, and yield of 69%. Purified crisantaspase was PEGylated with 10 kDa methoxy polyethylene glycol-N-hydroxysuccinimidyl (mPEG-NHS) at different pH values (6.5-9.0). The highest N-terminal pegylation yield (50%) was at pH 7.5 with the lowest poly-PEGylation ratio (7%). PEG-crisantaspase was purified by size exclusion chromatography and presented a K M value three times higher than crisantaspase (150 and 48.5 µM, respectively). Nonetheless, PEG-crisantaspase was found to be more stable at high temperatures and over longer periods of time. In 2 weeks, crisantaspase lost 93% of its specific activity, whereas PEG-crisantaspase was stable for 20 days. Therefore, the novel PEG-crisantaspase enzyme represents a promising biobetter alternative for the treatment of ALL.

The scientific production on the lifestyle and cardiometabolic risk factors among the immigrants in Spain is quite recent and scarce. Thus, it does not allow for characterizing the risk profile of this population.