Reactive oxygen species participate in mdr1b mRNA and P-glycoprotein overexpression in primary rat hepatocyte cultures

Abstract

P-glycoproteins encoded by multidrug resistance type 1 (mdr1) genes mediate ATP-dependent efflux of numerous lipophilic xenobiotics, including several anticancer drugs, from cells. Overexpression of mdr1-type transporters in tumour cells contributes to a multidrug resistance phenotype. Several factors shown to induce mdr1 overexpression (UV irradiation, epidermal growth factor, tumour necrosis factor alpha, doxorubicin) have been associated with the generation of reactive oxygen species (ROS). In the present study, primary rat hepatocyte cultures that exhibit time-dependent overexpression of the mdr1b gene were used as a model system to investigate whether ROS might participate in the regulation of intrinsic mdr1b overexpression. Addition of H2O2 to the culture medium resulted in a significant increase in mdrlb mRNA and P-glycoprotein after 3 days of culture, with maximal (approximately 2-fold) induction being observed with 0.5-1 mM H2O2. Furthermore, H2O2 led to activation of poly(ADP-ribose) polymerase, a nuclear enzyme activated by DNA strand breaks, indicating that ROS reached the nuclear compartment. Thus, extracellularly applied H2O2 elicited intracellular effects. Treatment of rat hepatocytes with the catalase inhibitor 3-amino-1,2,4-triazole (2-4 mM for 72 h or 10 mM for 1 h following the hepatocyte attachment period) also led to an up-regulation of mdrlb mRNA and P-glycoprotein expression. Conversely, antioxidants (1 mM ascorbate, 10 mM mannitol, 2% dimethyl sulphoxide, 10 mM N-acetylcysteine) markedly suppressed intrinsic mdr1b mRNA and P-glycoprotein overexpression. Intracellular steady-state levels of the mdrl substrate rhodamine 123, determined as parameter of mdr1-type transport activity, indicated that mdr1-dependent efflux was increased in hepatocytes pretreated with H2O2 or aminotriazole and decreased in antioxidant-treated cells. The induction of mdr1b mRNA and of functionally active mdr1-type P-glycoproteins by elevation in intracellular ROS levels and the repression of intrinsic mdrlb mRNA and P-glycoprotein overexpression by antioxidant compounds support the conclusion that the expression of the mdr1b P-glycoprotein is regulated in a redox-sensitive manner.

Post-transcriptional Regulation of P-Glycoprotein Expression in Cancer Cell Lines

“…In cultured cells, constitutive overexpression of Pgp is mediated by changes in gene dosage or transcription. Pgp can also be transiently induced in cultured cells by a variety of stimuli, such as heat shock, UV radiation, and chemotherapeutic agents (8)(9)(10)(11). The regulation of Pgp expression has been mostly related to transcriptional control of the mdr1 gene expression (8)(9)(10)(11).…”

“…Pgp can also be transiently induced in cultured cells by a variety of stimuli, such as heat shock, UV radiation, and chemotherapeutic agents (8)(9)(10)(11). The regulation of Pgp expression has been mostly related to transcriptional control of the mdr1 gene expression (8)(9)(10)(11). The proximal promoter of mdr1 contains several regulatory regions, such as an inverted CCAAT box and a GC element, both of which are required for constitutive promoter activity in several cell lines (12)(13)(14)(15)(16).…”

Mdr1b facilitates p53-mediated cell death and p53 is required for Mdr1b upregulation in vivo

“…The basis for the switch is a presumed transcriptional upregulation of mdr1a. Although, the mechanism accounting for this conversion is unknown, it may be hypothesized that high levels of mdr1b are deleterious to cell survival, a concept that is consistent with studies showing that cytotoxic drugs that are not mdr1 substrates as well as oxidative stress induce mdr1b (Ziemann et al, 1999;Thevenod et al, 2000) and that transcriptional upregulation of mdr1b has been correlated with decreased viability (Schrenk et al, 1996).…”

“…As mdr1b is the more primitive of the mdr1 genes (as determined by phylogenetic analysis (Growtree) (Furuya et al, 1997a)), one interpretation of the current studies is that increased mdr1b decreases cell survival in a p53 and mdr1b dependent fashion. This idea is circumstantially supported by studies showing mdr1b is readily upregulated by cytotoxic agents that are, for the most part, not mdr1 substrates (e.g., 3-methylcholanthracene, a¯atoxinB1, methylmethanesulfonate, mitoxantrone or reactive oxygen (Fardel et al, 1998;Ziemann et al, 1999;Thevenod et al, 2000)) and mdr1b is transcriptionally upregulated in cells undergoing cell death (Schrenk et al, 1996). However, given that di erent levels of p53 are linked to di erent cellular outcomes, i.e., low levels of p53 arrest cell growth and high levels induce apoptosis (Levine, 1997); mdr1b's role in apoptosis may be dramatically in¯uenced by the cell context.…”

The network of P-glycoprotein and microRNAs interactions

“…2 Indeed, several signalling pathways and transcription factors have been described as being involved in the regulation of P-gp expression, such as Wnt=b-catenin, Ras=Raf, MAPK=ERK, p53, NF-jB and PKC. [71][72][73][74][75][76][77] Moreover, several extracellular stimuli such as heat shock, 78 UV radiation, 79 reactive oxygen species 80,81 and cytotoxic drugs 82,83 induce P-gp expression ( Fig. 1-Panel a).…”