P‐glycoprotein activity in human Caucasian male lymphocytes does not follow its increased expression during aging

Abstract

P-glycoprotein (P-gp) is a transmembrane protein that mediates the efflux of innumerous structurally unrelated compounds. It was initially found over-expressed in tumor cells, associated to a multidrug resistance phenotype (MDR). Then, P-gp was found constitutively expressed in excretory cells/tissues and in circulating cells, such as lymphocytes. Considering the importance of this transporter in the establishment of therapeutic protocols and the existence of contradictory results, this study aimed at evaluating the influence of aging in the expression and function of P-gp in human lymphocytes, comparing two different methodologies to assess both parameters. P-gp activity and expression were evaluated in lymphocytes isolated from whole blood samples of 65 healthy caucasian male donors, divided into two groups according to age (group 1: under 30-years old; group 2: above 60-years old). P-gp expression was assessed using the anti-P-gp monoclonal antibody, UIC2, in the presence and in absence of vinblastine (Vbl). P-gp activity was evaluated measuring the efflux rate of the fluorescent P-gp substrate rhodamine 123 (Rho 123) and also using UIC2 shift assay. Flow cytometric analysis was performed to assess all the proceedings. Furthermore, Pgp expression and each of the P-gp activity determination methods were compared, through correlation analysis and linear regression models. We observed a significant age-dependent increase in mean P-gp expression (p ¼ 0.029), which was not reflected in the transporter's activity (p > 0.050). Statistical analysis allowed selection of UIC2 shift assay over Rho 123 efflux assay as a more selective method to assess P-gp activity. Despite the significant correlation between P-gp expression and P-gp activity found in lymphocytes (Gp1(group 1)À Àr ¼ 0.609, p < 0.001; Gp2À Àr ¼ 0.461, p ¼ 0.012), using UIC2 shift assay, these data reinforce the need for P-gp activity assessment, rather than P-gp expression determination alone, when starting new therapeutic regimens with P-gp substrates, especially in men older than 60 years of age. ' 2011 International Society for Advancement of Cytometry Key terms P-glycoprotein; aging; UIC2 shift assay; Rho 123 efflux; flow cytometry P-GLYCOPROTEIN (P-gp), the product of multidrug-resistance 1 (MDR-1) gene in humans, is one of the most important and interesting members of the numerous, highly conserved, widespread membrane transporters of the ATP-binding cassette superfamily. It was first detected as a surface phosphoglycoprotein over-expressed in Chinese hamster ovary cultured cells selected for MDR, where it mediated resistance to many amphipathic drugs (1). Then, P-gp was also found constitutively expressed in epithelial cells from tissues with excretory function such as intestine, liver, kidneys, lungs, blood-brain barrier, and placenta, and also in blood cells such as lymphocytes (2), where P-gp expression may limit tissue penetration of its substrates. Thus, P-gp plays an important role in pharmacokinetics and pharmacodynamics of many

Expression of multidrug resistance protein 1 and multidrug resistance-associated protein 1 in peripheral blood lymphocytes amongst children and young adults

“…The study by Vilas-Boas et al (2011) revealed a significant correlation between the lymphocyte MDR1 expression and MDR1 activity using sensitive methods amongst young adults up to 30 years of age [22]. Our evidence of increased expression in younger children, generally concurs with the similar results of those studies which assessed the highest activity amongst the youngest children by Machado et al and Giraud et al [20,23].…”

“…In contrast to the previous one, Brenner et al (2004) in young and elderly adults as well as, Cortada et al (2009) in adults, did not detect the relation between activity and age in PBMCs [48,49]. In addition, Vilas-Boas et al (2011) demonstrated a significant age-dependent increase in the MDR1 expression in adults after 60 years of age, although, this increase did not correspond to MDR1 activity [22].…”

“…In addition, a link between age and expression and activity of MDR1 in PBLs was also demonstrated [18][19][20][21][22][23]. The results of these studies are conflicting, however.…”

“…If the relationship between physiological MDR1 or MRP1 expression (and thus the activity as well [22]) in peripheral blood lymphocytes and age or selected polymorphisms is demonstrated, it should lead to the modification of therapy with MDR1 or MRP1-transported immunomodulatory drugs and antivirals, based on the identified dependencies.…”

P-glycoprotein induction in Caco-2 cells by newly synthetized thioxanthones prevents paraquat cytotoxicity

“…2b, c). P-glycoprotein transport activity RHO 123 is a P-gp fluorescent substrate widely used for the evaluation of P-gp activity (Palmeira et al 2011;Silva et al 2011Silva et al , 2014bVilas-Boas et al 2011). In the present study, two different approaches for the evaluation of transport activity were performed.…”