Key genes and drug delivery systems to improve the efficiency of chemotherapy

Cancer cells can develop resistance to anticancer drugs, thereby becoming tolerant to treatment through different mechanisms. The biological mechanisms leading to the generation of anticancer treatment resistance include alterations in transmembrane proteins, DNA damage and repair mechanisms, alterations in target molecules, and genetic responses, among others. The most common anti-cancer drugs reported to develop resistance to cancer cells include cisplatin, doxorubicin, paclitaxel, and fluorouracil. These anticancer drugs have different mechanisms of action, and specific cancer types can be affected by different genes. The development of drug resistance is a cellular response which uses differential gene expression, to enable adaptation and survival of the cell to diverse threatening environmental agents. In this review, we briefly look at the key regulatory genes, their expression, as well as the responses and regulation of cancer cells when exposed to anticancer drugs, along with the incorporation of alternative nanocarriers as treatments to overcome anticancer drug resistance

Thiol-maleimide poly(ethylene glycol) crosslinking of L-asparaginase subunits at recombinant cysteine residues introduced by mutagenesis

<div><p>L-Asparaginase is an enzyme successfully being used in the treatment of acute lymphoblastic leukemia, acute myeloid leukemia, and non-Hodgkin’s lymphoma. However, some disadvantages still limit its full application potential, e.g., allergic reactions, pancreatitis, and blood clotting impairment. Therefore, much effort has been directed at improving its performance. A popular strategy is to randomly conjugate L-asparaginase with mono-methoxy polyethylene glycol, which became a commercial FDA approved formulation widely used in recent years. To improve this formulation by PEGylation, herein we performed cysteine-directed conjugation of the L-asparaginase subunits to prevent dissociation-induced loss of activity. The recombinant cysteine conjugation sites were introduced by mutagenesis at surface-exposed positions on the protein to avoid affecting the catalytic activity. Three conjugates were obtained using different linear PEGs of 1000, 2000, and 5000 g/mol, with physical properties ranging from a semi-solid gel to a fully soluble state. The soluble-conjugate exhibited higher catalytic activity than the non-conjugated mutant, and the same activity than the native enzyme. The cysteine-directed crosslinking of the L-asparaginase subunits produced a higher molecular weight conjugate compared to the native tetrameric enzyme. This strategy might improve L-asparaginase efficiency for leukemia treatment by reducing glomerular filtration due to the increase in hydrodynamic size thus extending half-live, while at the same time retaining full catalytic activity.</p></div

Subunit mass of recombinant native and mutant A38C-T263C L-asparaginase.

<p>The <i>m/z</i> peak for the native L-asparaginase subunit (in green) was observed at 34605 g/mol, while the <i>m/z</i> peak for the mutant A38C-T263C subunit (in black) was observed at 34634 g/mol.</p

Catalytic activity of L-asparaginase C77-105S mutant.

<p>Catalytic activity of L-asparaginase C77-105S mutant.</p

Cysteine-directed PEGylation followed by size exclusion chromatography.

<p>(A) Mutant L-asparaginase (A38C-T263C) reduced with TCEP and eluted from the gel filtration column. Fractions 8–13 ml were pooled and used for the PEGylation crosslinking reaction. (B) 5kDa-PEG-Conjugate after crosslinking reaction, reduced with TCEP and eluted from the gel filtration column. The continuous curve represents the relative absorbance at 280 nm and the bars are the relative asparaginase catalytic activity of the fractions.</p

Extracytoplasmic secretion of recombinant L-asparaginase constructs.

<p>Extracytoplasmic secretion of recombinant L-asparaginase constructs.</p

Dependence of L-asparaginase catalytic activity on the degree of PEGylation.

<p>Relative catalytic activity is expressed as percentage compared to the non-conjugated L-asparaginase defined as 100% and reported by each study. Modification degree refers to the average percentage of conjugated sites relative to the available amine groups per L-asparaginase tetramer. The results to generate this figure were extracted from references [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197643#pone.0197643.ref016" target="_blank">16</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197643#pone.0197643.ref022" target="_blank">22</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197643#pone.0197643.ref034" target="_blank">34</a>] and this work.</p

Selection of L-asparaginase PEGylation sites.

<p>(A) L-Asparaginase tetramer with subunits highlighted with different colors, one active site is represented in red spheres, the pre-selected PEGylation positions in blue spheres, and the distances between mutated residues (A38C and T263C) are shown by black dotted lines. (B) L-Asparaginase dimer showing two active sites relatively buried (red spheres) in comparison with the PEGylations sites (blue spheres).</p

Computational analysis of the C77-105S mutation.

<p>(A) Superposition of geometrically optimized structures of the natural disulfide bond and the C77-105S mutation. (B) Formation of a new hydrogen bond in the C77-105S mutant.</p

Reusability of the 1kDa-PEG-conjugate.

<p>Relative catalytic activity at temperatures ranging from 20°C to 80°C. The 1kDa-PEG-conjugate (in green), native L-asparaginase (in blue), and a commercial randomly-PEGylated L-asparaginase formulation (Millipore Sigma, USA) (in black). Relative catalytic activity was calculated by the absorption at 425 nm divided by the maximum signal for each sample and expressed as percentile (%). Reported values are average with error bars representing the 95% confidence interval. For the 1kDa-PEG-conjugate the same sample (semi-solid gel) was used throughout all the experiment.</p