Arsenic and Benzo[a]pyrene Co-exposure Effects on MDA-MB-231 Cell Viability and Migration.

Although humans are frequently exposed to multiple pollutants simultaneously, research on their harmful effects on health has typically focused on studying each pollutant individually. Inorganic arsenic (As) and benzo[a]pyrene (BaP) are well-known pollutants with carcinogenic potential, but their co-exposure effects on breast cancer cell progression remain incompletely understood. This study aimed to assess the combined impact of BaP and As on the viability and migration of MDA-MB-231 cells. The results indicated that even at low levels, both inorganic As (0.01 µM, 0.1 µM, and 1 µM) and BaP (1 µM, 2.5 µM), individually or in combination, enhanced the viability and migration of the cells. However, the cell cycle analysis revealed no significant differences between the control group and the cells exposed to BaP and As. Specifically, exposure to BaP alone or in combination with As (As 0.01 µM + BaP 1 µM) for 24 h led to a significant increase in vimentin gene expression. Interestingly, short-term exposure to As not only did not induce EMT but also modulated the effects of BaP on vimentin gene expression. However, there were no observable changes in the expression of E-cadherin mRNA. Consequently, additional research is required to evaluate the prolonged effects of co-exposure to As and BaP on the initiation of EMT and the progression of breast cancer.

Design, modeling, and expression of erythropoietin cysteine analogs in Pichia pastoris: improvement of mean residence times and in vivo activities through cysteine-specific PEGylation.

In this study, the low-cost production of recombinant human erythropoietin cysteine analogs (Cys-rhEPOs) from Pichia pastoris and the potential to increase their serum residency and in vivo activity through cysteine-specific PEGylation were investigated. Three-dimensional structures of several Cys-rhEPOs were generated using homology modeling, and three stable Cys-rhEPOs were selected on the basis of model stability in molecular dynamics simulation and surface accessibility of the inserted cysteine. cDNAs encoding Cys-rhEPOs were constructed by site-directed mutagenesis and expressed as secreted proteins in flask cultures of P. pastoris. The selection of highly expressing clones and the optimization of certain culture parameters resulted in protein expression levels of 100-170 mg/l. Purified Cys-rhEPOs were cysteine-specifically PEGylated using 20 kDa and 30 kDa mPEG-maleimides (methoxy polyethylene glycol-maleimides). The E89CEPO analog with the highest (96.6%) cysteine accessibility was conjugated to PEG-polymers with the largest yields (about 80%). In comparison with rhEPO, 30 kDa PEG-E89CEPO demonstrated a significant (approximately 30%) increase in the mean residence time. Whereas the in vitro activities of 30 kDa PEG-E89CEPO were comparable to those of rhEPO, the in vivo activity of this conjugate was more prolonged compared to rhEPO (12 days vs. 7 days). Our results demonstrate that the site-specific PEGylation of Pichia-expressed EPO analogs may be considered as a promising approach for generating cost-effective and long-acting erythropoiesis-stimulating agents.

Evaluation of bioactivity and pharmacokinetic characteristics of PEGylated P.pastoris-expressed erythropoietin.

High costs of production and relatively short serum half-life of mammalian cell-derived recombinant human erythropoietin (rHuEpo) necessitate finding and developing superior hosts/technologies for more efficient production of longer-acting erythropoietic agents. With these aims, we provide the first report on reductive alkylation of low-cost P.pastoris-expressed rHuEpo (PPEpo) with PEG-aldehyde. The PCR-amplified cDNA of native rHuEpo was cloned into the pPICZαA vector and transformed into the yeast Pichia pastoris. The best expressing transformant was selected and employed for secreted-expression of PPEpo using the standard protocols. Purified PPEpo was N-terminally PEGylated with 20-kDa mPEG-propionaldehyde in a low pH (5) condition. The in vitro and in vivo biological activities of purified mono-PEGylated PPEpo was evaluated by the UT-7 cells proliferation assay and normocythaemic mice assay, respectively. Pharmacokinetic parameters were determined following intravenous administration of Epo proteins in rabbits. While PPEpo showed a higher in vitro bioactivity compared to rHuEpo, no in vivo efficiency was determined for PPEpo. However, the in vivo activity of PEG-PPEpo conjugate was comparable to that of rHuEpo. Pharmacokinetic studies showed that the terminal half-life and mean residence time of PEG-PPEpo were increased approximately 4-fold and 6.5-fold respectively, compared with those of PPEpo. The results indicate that N-terminal PEGylation of Pichia-expressed Epo could be considered as a promising approach for generating cost-effective and long-acting erythropoiesis-stimulating agents.

High Expression of Methylotrophic Yeast-Derived Recombinant Human Erythropoietin in a pH-Controlled Batch System.

To accomplish the worldwide demand for recombinant human erythropoietin (rHuEpo) as a therapeutic, application of cost-efficient expression system of methylotrophic yeast Pichia pastoris (P. pastoris) rather than mammalian cells is indispensable. Herein, a report on high levels secreted-expression of Pichia-derived rHuEpo by batch fermentation in a pH stabilized format is presented. The full length cDNA of rHuEpo was inserted into pPICZαA vector under control of AOX1 promoter, downstream of the secretion-α-factor and electroporated into P. pastoris strain X33. The highest expression transformant was selected by screening among the colonies surviving high concentration of Zeocin (1.0 mg/ml), followed by comparative small scale expression analysis by ELISA. Stabilization of pH around 6.0 by adding phosphoric acid into the culture media during induction period, improved the yield of expression to 150 mg/l of the media. Single-step Nickel-affinity chromatography was employed for purification of rHuEpo-6xHis to 80% purity. Analyses by SDS-PAGE, Western blot and N-terminal protein sequencing confirmed the authenticity of the 33 kDa expressed rHuEpo with a native N-terminal indicating the proper cleavage of secretion-signal. Results of this study, further confirmed the possibility of employing methylotrophic yeast for scaled up production aims of rHuEpo as a cost-efficient expression system when provided evidence for higher expression yields through application of pH-controlled systems.