An effective placental cotyledons proteins extraction method for 2D gel electrophoresis.

Effective protein extraction is essential especially in producing a well-resolved proteome on 2D gels. A well-resolved placental cotyledon proteome, with good reproducibility, have allowed researchers to study the proteins underlying the physiology and pathophysiology of pregnancy. The aim of this study is to determine the best protein extraction protocol for the extraction of protein from placental cotyledons tissues for a two-dimensional gel electrophoresis (2D-GE). Based on widely used protein extraction strategies, 12 different extraction methodologies were carefully selected, which included one chemical extraction, two mechanical extraction coupled protein precipitations, and nine chemical extraction coupled protein precipitations. Extracted proteins were resolved in a one-dimensional gel electrophoresis and 2D-GE; then, it was compared with set criteria: extraction efficacy, protein resolution, reproducibility, and recovery efficiency. Our results revealed that a better profile was obtained by chemical extraction in comparison to mechanical extraction. We further compared chemical extraction coupled protein precipitation methodologies, where the DNase/lithium chloride-dense sucrose homogenization coupled dichloromethane-methanol precipitation (DNase/LiCl-DSH-D/MPE) method showed good protein extraction efficiency. This, however, was carried out with the best protein resolution and proteome reproducibility on 2D-gels. DNase/LiCl-DSH-D/MPE was efficient in the extraction of proteins from placental cotyledons tissues. In addition, this methodology could hypothetically allow the protein extraction of any tissue that contains highly abundant lipid and glycogen.

Identification of dual DNA-PK MDR1 inhibitors for the potentiation of cytotoxic drug activity.

Inhibition of DNA repair is an attractive therapeutic approach to enhance the activity of DNA-damaging anticancer chemotherapeutic agents. Similarly, blockade of the multidrug-resistance protein 1 (MDR1) can overcome efflux-mediated resistance. DNA-dependent protein kinase (DNA-PK) is essential for the non-homologous end-joining DNA repair pathway. NU7441 is a potent DNA-PK inhibitor (IC50=14nM) that is used widely to study the effects of DNA-PK inhibition in vitro. In growth inhibition studies, 1µM NU7441 sensitised vincristine-resistant CCRF-CEM VCR/R leukaemia cells (1200-fold resistant) to a range of MDR1 substrates, including doxorubicin (8-fold, p=0.03), vincristine (14-fold, p=0.01) and etoposide (63-fold, p=0.02), compared with 1.4-fold (p=0.02), 2.2-fold (p=0.04) and 3.6-fold (p=0.01) sensitisation, respectively, in parental CCRF-CEM cells. This difference in NU7441 sensitivity was confirmed in another two parental and MDR1-overexpressing cell line pairs. A doxorubicin fluorescence assay showed that in MDR1-overexpressing canine kidney MDCKII-MDR1 cells, 1µM NU7441 increased doxorubicin nuclear fluorescence 16-fold. NU7441 and 3 structurally related compounds (NU7742 (an NU7441 analogue that does not inhibit DNA-PK - IC50>10µM), DRN1 (DNA-PK-inhibitory atropisomeric NU7441 derivative - IC50=2nM) and DRN2 (DNA-PK non-inhibitory atropisomeric NU7441 derivative - IC50=7µM)) all increased intracellular vincristine accumulation in the CCRF-CEM VCR/R cells to a level similar to verapamil, as measured by LC-MS. This paper demonstrates that NU7441 is a dual DNA-PK and MDR1 inhibitor, and this extends the therapeutic potential of the compound when used in combination with MDR substrates.