Impact of small molecules immunosuppressants on P-glycoprotein activity and T-cell function

Purpose. P-glycoprotein (Pgp) is a member of the ABC-transporter family that transports substances across cellular membranes acting as an efflux pump extruding drugs out of the cells. Pgp plays a key role on the pharmacokinetics of several dr ugs. Herein, we have studied the effects of immunosuppressants on Pgp function, assessing rhodamine-123 (Rho123) uptake and efflux in different T- cell subsets. Methods. Different immunosuppressants such as Cyclosporine (CsA), Rapamycin (Rapa) and Tacrolimus (Tac) were used to assess the in vitro effect on Pgp function of main T-cell subsets among healthy volunteers. We measured Rho123 upta ke, efflux and kinetic of extrusion in CD4 + and CD8 + subsets by flow cytometry. Antigen-specific memory T-ce ll responses were assessed by measuring T-cell proliferation and cytokine secretion using an allogeneic mixed lymphocyte reaction. Results. Rho123 uptake in groups treated with CsA and CsA+Rapa was signif icantly decreased compared to non-treated group and the other immunosupressants in both T cells subsets. Pgp activity was also reduced in CsA and CsA+Rapa compared to the other immunosupressants but it was only significant in the CsA group for CD8 + subset. Kinetic extrusion of Rho123 by Pgp in all groups was faster in CD8 + T cells. All immunosuppressants and the specific Pgp inhibitor PSC833 diminished antigen-primed T-cell proliferation, especially CD8 + T-cell subset. Conclusions. Our data indicate that small molecules immunosuppressants, especially CsA, inhibit Pgp activity and T-cell function being the CD8 + T cells more susceptible to this effect. These findings support the importance of Pgp when designing combined immunosuppressive regimens

Impact of small molecules immunosuppressants on P-glycoprotein activity and T-cell function

Purpose. P-glycoprotein (Pgp) is a member of the ABC-transporter family that transports substances across cellular membranes acting as an efflux pump extruding drugs out of the cells. Pgp plays a key role on the pharmacokinetics of several dr ugs. Herein, we have studied the effects of immunosuppressants on Pgp function, assessing rhodamine-123 (Rho123) uptake and efflux in different T- cell subsets. Methods. Different immunosuppressants such as Cyclosporine (CsA), Rapamycin (Rapa) and Tacrolimus (Tac) were used to assess the in vitro effect on Pgp function of main T-cell subsets among healthy volunteers. We measured Rho123 upta ke, efflux and kinetic of extrusion in CD4 + and CD8 + subsets by flow cytometry. Antigen-specific memory T-ce ll responses were assessed by measuring T-cell proliferation and cytokine secretion using an allogeneic mixed lymphocyte reaction. Results. Rho123 uptake in groups treated with CsA and CsA+Rapa was signif icantly decreased compared to non-treated group and the other immunosupressants in both T cells subsets. Pgp activity was also reduced in CsA and CsA+Rapa compared to the other immunosupressants but it was only significant in the CsA group for CD8 + subset. Kinetic extrusion of Rho123 by Pgp in all groups was faster in CD8 + T cells. All immunosuppressants and the specific Pgp inhibitor PSC833 diminished antigen-primed T-cell proliferation, especially CD8 + T-cell subset. Conclusions. Our data indicate that small molecules immunosuppressants, especially CsA, inhibit Pgp activity and T-cell function being the CD8 + T cells more susceptible to this effect. These findings support the importance of Pgp when designing combined immunosuppressive regimens

Impact of small molecules immunosuppressants on P-glycoprotein activity and T-cell function

Purpose. P-glycoprotein (Pgp) is a member of the ABC-transporter family that transports substances across cellular membranes acting as an efflux pump extruding drugs out of the cells. Pgp plays a key role on the pharmacokinetics of several dr ugs. Herein, we have studied the effects of immunosuppressants on Pgp function, assessing rhodamine-123 (Rho123) uptake and efflux in different T- cell subsets. Methods. Different immunosuppressants such as Cyclosporine (CsA), Rapamycin (Rapa) and Tacrolimus (Tac) were used to assess the in vitro effect on Pgp function of main T-cell subsets among healthy volunteers. We measured Rho123 upta ke, efflux and kinetic of extrusion in CD4 + and CD8 + subsets by flow cytometry. Antigen-specific memory T-ce ll responses were assessed by measuring T-cell proliferation and cytokine secretion using an allogeneic mixed lymphocyte reaction. Results. Rho123 uptake in groups treated with CsA and CsA+Rapa was signif icantly decreased compared to non-treated group and the other immunosupressants in both T cells subsets. Pgp activity was also reduced in CsA and CsA+Rapa compared to the other immunosupressants but it was only significant in the CsA group for CD8 + subset. Kinetic extrusion of Rho123 by Pgp in all groups was faster in CD8 + T cells. All immunosuppressants and the specific Pgp inhibitor PSC833 diminished antigen-primed T-cell proliferation, especially CD8 + T-cell subset. Conclusions. Our data indicate that small molecules immunosuppressants, especially CsA, inhibit Pgp activity and T-cell function being the CD8 + T cells more susceptible to this effect. These findings support the importance of Pgp when designing combined immunosuppressive regimens

Multidrug resistance in tumour cells: characterisation of the multidrug resistant cell line K562-Lucena 1

Multidrug resistance to chemotherapy is a major obstacle in the treatment of cancer patients. The best characterised mechanism responsible for multidrug resistance involves the expression of the MDR-1 gene product, P-glycoprotein. However, the resistance process is multifactorial. Studies of multidrug resistance mechanisms have relied on the analysis of cancer cell lines that have been selected and present cross-reactivity to a broad range of anticancer agents. This work characterises a multidrug resistant cell line, originally selected for resistance to the Vinca alkaloid vincristine and derived from the human erythroleukaemia cell K562. This cell line, named Lucena 1, overexpresses P-glycoprotein and have its resistance reversed by the chemosensitisers verapamil, trifluoperazine and cyclosporins A, D and G. Furthermore, we demonstrated that methylene blue was capable of partially reversing the resistance in this cell line. On the contrary, the use of 5-fluorouracil increased the resistance of Lucena 1. In addition to chemotherapics, Lucena 1 cells were resistant to ultraviolet A radiation and hydrogen peroxide and failed to mobilise intracellular calcium when thapsigargin was used. Changes in the cytoskeleton of this cell line were also observed.<br>A resistência a múltiplos fármacos é o principal obstáculo no tratamento de pacientes com câncer. O mecanismo responsável pela resistência múltipla mais bem caracterizado envolve a expressão do produto do gene MDR-1, a glicoproteína P. Entretanto, o processo de resistência tem fatores múltiplos. Estudos de mecanismos de resistência m��ltipla a fármacos têm dependido da análise de linhagens celulares tumorais que foram selecionadas e apresentam reatividade cruzada a uma ampla faixa de agentes anti-tumorais. Este trabalho caracteriza uma linhagem celular com múltipla resistência a fármacos, selecionada originalmente pela resistência ao alcalóide de Vinca vincristina e derivado da linhagem eritro-leucêmica K562. Esta linhagem celular, denominada Lucena 1, super-expressa a glicoproteína P e tem sua resistência revertida pelos quimio-sensibilizantes verapamil, trifluoperazina e ciclosporinas A, D e G. Ademais, demonstramos que o azul de metileno era capaz de reverter parcialmente a resistência nesta linhagem celular. Em contraste, o uso de 5-flúor-uracil aumentava a resistência de Lucena 1. Adicionalmente aos quimioterápicos, células Lucena 1 eram resistentes radiação ultra-violeta A e peróxido de hidrogênio e deixavam de mobilizar o cálcio intra-celular quando se usava tapsigargina. Mudanças no cito-esqueleto desta linhagem foram também observadas