Modulation of reactive oxygen levels and gene expression in sensitive and resistant tumoral cells by C-phyocyanin.

In this work we investigated the possible cellular changes induced by C-phycocyanin (C-PC) in erythroleukemic cells with (K562-Lucena and FEPS) and without (K562) the multidrug resistance (MDR) phenotype. The reactive oxygen levels (ROS) (evaluated by fluorimetry) were increased relative to the control in K562 and K562-Lucena cells treated with 100 µg/mL and were not increased in FEPS cells. The expression of the following genes related to resistance was evaluated by real-time PCR: COX2, ALOX5, ABCB1 and ABCC1. Treatment with 100 µg/mL of C-PC increased COX2 and ABCB1 expression in K562-Lucena cells and reduced expression of ALOX5 in K562-Lucena and FEPS cells. ROS levels appear to be involved in biological responses to C-PC in K562 and K562-Lucena cells, although expression of the genes studied here was only modified by C-PC in the K562-Lucena cell line. Thus, it is possible to suggest that C-PC modulates the expression of COX2 and ABCB1 for the K562-Lucena in a ROS-dependent manner and the expression of ALOX5 for the FEPS in a ROS-independent manner; however, more studies are needed to elucidate these mechanisms.

C-phycocyanin to overcome the multidrug resistance phenotype in human erythroleukemias with or without interaction with ABC transporters.

The phenotype of multidrug resistance (MDR) is one of the main causes of chemotherapy failure. Our study investigated the effect of C-phycocyanin (C-PC) in three human erythroleukemia cell lines with or without the MDR phenotype: K562 (non-MDR; no overexpression of drug efflux proteins), K562-Lucena (MDR; overexpression of ATP-binding cassette, sub-family B/ABCB1), and FEPS (MDR; overexpression of ABCB1 and ATP-binding cassette, sub-family C/ABCC1). Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, we showed that 20 and 200 µg/mL C-PC decreased K562 viable cells after 24 h and 200 µg/mL C-PC decreased K562-Lucena cell proliferation after 48 h. C-PC did not decrease viable cells of FEPS cells. On the other hand, the MTT assay showed that exposure of 2, 20, and 200 µg/mL C-PC for 24 or 48 h was not cytotoxic to peritoneal macrophages. At 72 h, the trypan blue exclusion assay showed that 20 µg/mL C-PC decreased K562 and K562-Lucena cell proliferation and in FEPS cells, only 200 µg/mL C-PC decreased proliferation. In addition, protein-protein docking showed differences in energy and binding sites of ABCB1 and ABCC1 for C-PC, and these results were confirmed by the efflux protein activity assay. Only ABCC1 activity was altered in the presence of C-PC and FEPS cells showed lower C-PC accumulation, suggesting C-PC extrusion by ABCC1, conferring C-PC resistance. In combination with chemotherapy (vincristine [VCR] and daunorubicin [DNR]), the sensitivity of K562-Lucena cells for C-PC + VCR did not increase, whereas FEPS cell sensitivity for C-PC + DNR was increased. In molecular docking experiments, the estimated free energies of binding for C-PC associated with chemotherapy were similar (VCR: -6.9 kcal/mol and DNR: -7.2 kcal/mol) and these drugs were located within the C-PC cavity. However, C-PC exhibited specificity for tumor cells and K562 cells were more sensitive than K562-Lucena cells, followed by FEPS cells. Thus, C-PC is a possible chemotherapeutic agent for cells with the MDR phenotype, both alone in K562-Lucena cells (resistance due to ABCB1), or in combination with other drugs for cells similar to FEPS (resistance due to ABCC1). Moreover, C-PC did not damage healthy cells (peritoneal macrophages of Mus musculus).

The production of rhamnolipid by a Pseudomonas aeruginosa strain isolated from a southern coastal zone in Brazil.

The production and properties of a rhamnolipid-type biosurfactant, synthesized by the Pseudomonas aeruginosa LBM10 strain, isolated from a southern coastal zone in Brazil, were investigated. The assays were conducted in a rotary shaker at 30 degrees C and 180 rpm for a period of 96 h. Soybean oil and sodium nitrate were the best sources of carbon and nitrogen, respectively. A nitrogen-limiting condition (C/N ratio of 100) was favorable to biosurfactant production. The formation of stable emulsions was better in saline concentrations below 0.5%, pH values in the range from 6 to 9 and temperatures in the range from 35 to 40 degrees C, maintaining about 80% of its original activity for salinity up to 3% and 120 min of exposure at 100 degrees C. The biosurfactant may be produced with this microorganism using renewable substrates that are readily available, reaching values of 1.42 g l(-1) measured as rhamnose. This biosurfactant has interesting and useful properties for many industrial applications.

Optimization of phycocyanin extraction from Spirulina platensis using factorial design.

Phycocyanin extraction from cyanobacteria Spirulina platensis was optimized using factorial design and response surface techniques. The effects of temperature and biomass-solvent ratio on phycocyanin concentration and extract purity were evaluated to determine the optimum conditions for phycocyanin extraction. The optimum conditions for the extraction of phycocyanin from S. platensis were the highest biomass-solvent ratio, 0.08 gmL(-1), and 25 degrees C. Under these conditions it's possible to obtain an extract of phycocyanin with a concentration of 3.68 mgmL(-1) and purity ratio (A(615)/A(280)) of 0.46.

Optimization of inulinase production by Kluyveromyces marxianus using factorial design.

Factorial design and response surface techniques were used to optimize the culture medium for the production of inulinase by Kluyveromyces marxianus. Sucrose was used as the carbon source instead of inulin. Initially, a fractional factorial design (2(5-1)) was used in order to determine the most relevant variables for enzyme production. Five parameters were studied (sucrose, peptone, yeast extract, pH, and K2HPO4), and all were shown to be significant. Sucrose concentration and pH had negative effects on inulinase production, whereas peptone, yeast extract, and K2HPO4 had positive ones. The pH was shown to be the most significant variable and should be preferentially maintained at 3.5. According to the results from the first factorial design, sucrose, peptone, and yeast extract concentrations were selected to be utilized in a full factorial design. The optimum conditions for a higher enzymatic activity were then determined: 14 g/L of sucrose, 10 g/L of yeast extract, 20 g/L of peptone, 1 g/L of K2HPO4. The enzymatic activity in the culture conditions was 127 U/mL, about six times higher than before the optimization.