Modulation of breast cancer resistance protein (BCRP/ABCG2) gene expression using RNA interference

ArticleinMolecular Cancer Therapeutics 3(12):1577-83 · January 2005with21 Reads
Source: PubMed
Overexpression of the breast cancer resistance protein (BCRP/ABCG2) confers multidrug resistance (MDR) to tumor cells and often limits the efficacy of chemotherapy. To circumvent BCRP-mediated MDR, a common approach is the use of potent and specific inhibitors of BCRP transport such as fumitremorgin C, novobiocin, and GF120918. Here, we evaluated a new approach using RNA interference for the specific knockdown of BCRP. We designed and synthesized small interfering RNA (siRNA) using T7 RNA polymerase and showed that siRNAs markedly down-regulated both exogenous and endogenous expression of BCRP. As a functional consequence, knockdown of BCRP by siRNAs increased the sensitivity of human choriocarcinoma BeWo cells to mitoxantrone and topotecan by 10.5- and 8.2-fold, respectively. Using flow cytometry, we found that introduction of siRNAs also enhanced the intracellular accumulation of topotecan. We have previously identified an estrogen response element in the BCRP promoter and have shown that 17beta-estradiol increased BCRP mRNA expression. Furthermore, in the present study, we found that expression of BCRP protein was inducible by 17beta-estradiol and that this effect was ameliorated by the introduction of siRNAs. These studies indicate that siRNAs could modulate MDR in vitro and may present a new approach to overcome BCRP-mediated drug resistance.
    • "Selective knock down of endogenous transporters or silencing expression of various genes can lead to the development of new cell lines that can be selected for studying role of particular transporters and enzymes on drug transport and interaction studies. For instance, RNAitriggered mRNA destruction to inhibit MDR1 [110] and BCRP [111] expression has shown that RNAi could prove to be a valuable tool for the downregulation of transporter genes. Cloned transport studies may be implemented which can not only improve our knowledge of molecular enzymology of these transport processes but will also provide details of binding and SARs. "
    [Show abstract] [Hide abstract] ABSTRACT: Introduction: The role of drug transporters as one of the determinants of cellular drug permeability has become increasingly evident. Despite the lipophilicity of a drug molecule as rate-limiting factor for passive diffusion across biological membranes, carrier-mediated and active transport have gained attention over the years. A better understanding of the effects and roles of these influx transporters towards transmembrane permeability of a drug molecule need to be delineated for drug development and delivery. Areas covered: This review focuses on findings relative to role of transporters in drug absorption and bioavailability. Particularly the areas demanding further research have been emphasized. This review will also highlight various transporters expressed on vital organs and their effects on drug pharmacokinetics. Expert opinion: Significant efforts have been devoted to understand the role of transporters, their iterative interplay with metabolizing enzymes through molecular enzymology, binding and structure-activity relationship studies. A few assays such as parallel artificial membrane permeation assay (PAMPA) have been developed to analyze drug transport across phospholipid membranes. Although large web-accessible databases on tissue selective expression profiles at transcriptomic as well as proteomic are available, there is a need to collocate the scattered literature on the role of transporters in drug development and delivery.
    Full-text · Article · Jul 2016
    • "Furthermore, 17β-estradiol has been shown to down-regulate BCRP protein expression in various cells and tissues including placental BeWo cells (Imai, et al., 2005, Wang, et al., 2006, Mahringer and Fricker, 2010), which express both ERα and ERβ (Gambino, et al., 2012). It should be noted, however, that other studies have observed an up-regulation of BCRP protein by 17β-estradiol in BeWo cells (Ee, et al., 2004a, Prouillac, et al., 2009) and cytotrophoblasts (Evseenko, et al., 2006). Nonetheless, we aimed to explore the involvement of the ER signaling pathway in the genistein-mediated downregulation of BCRP expression in placental BeWo cells, the ERα antagonist ICI 182,780 was used (Van Den Bemd, et al., 1999, Peekhaus, et al., 2004). "
    [Show abstract] [Hide abstract] ABSTRACT: Glyburide is frequently used to treat gestational diabetes due to its low fetal accumulation resulting from placental efflux by the BCRP/ABCG2 transporter. Here we sought to determine how exposure to the dietary phytoestrogen genistein and expression of a loss-of-function polymorphism in the ABCG2 gene (C421A) impacted the transport of glyburide by BCRP using stably-transfected human embryonic kidney 293 (HEK) cells, human placental choriocarcinoma BeWo cells and human placental explants. Genistein competitively inhibited the BCRP-mediated transport of (3)H-glyburide in both wild-type (WT) and C421A-BCRP HEK-expressing cells, with greater accumulation of (3)H-glyburide in cells expressing the C421A variant. In BeWo cells, exposure to genistein for 60 min increased the accumulation of (3)H-glyburide 30-70% at concentrations relevant to dietary exposure (IC50 ~180 nM). Continuous exposure of BeWo cells to genistein for 48 h reduced the expression of BCRP mRNA and protein by up to 40%, which impaired BCRP transport activity. Pharmacological antagonism of the estrogen receptor attenuated the genistein-mediated downregulation of BCRP expression, suggesting that phytoestrogens may reduce BCRP levels through this hormone receptor pathway in BeWo cells. Interestingly, genistein treatment for 48 h did not alter BCRP protein expression in explants dissected from healthy term placentas. These data suggest that while genistein can act as a competitive inhibitor of BCRP-mediated transport, its ability to down-regulate placental BCRP expression may only occur in choriocarcinoma cells. Overall, this research provides important mechanistic data regarding how the environment (dietary genistein) and a frequent genetic variant (ABCG2, C421A) may alter the maternal-fetal disposition of glyburide.
    Full-text · Article · Feb 2016
    • "The concept of inhibiting ABC transporter expression is not new; Hiroyuki Kobayashi and colleagues first reported on hammerhead ribozymes that were able to target ABCG2 mRNA in 1994 [94]. More recently, RNA interference (RNAi) has been used to knock down ABCG2 expression in cell culture and restore therapeutic benefit to anti-cancer agents that are ABCG2 substrates95969798. While there was some doubt for a number of years about the practicality of RNAi translating to clinical use [99, 100], the reports in the last five years of successful cleavage of targeted sequences in humans using nanoparticle delivery of siRNA particles [101, 102] opens the door for renewed interest in the potential application of RNAi as a means of disrupting multi-drug resistance mediated by ABC transporters. "
    [Show abstract] [Hide abstract] ABSTRACT: ATP-binding cassette (ABC) transporters make up a superfamily of transmembrane proteins that play a critical role in the development of drug resistance. This phenomenon is especially important in oncology, where superfamily member ABCG2 (also called BCRP - breast cancer resistance protein) is known to interact with dozens of anti-cancer agents that are ABCG2 substrates. In addition to the well-studied and well-reviewed list of cytotoxic and targeted agents that are substrates for the ABCG2 transporter, a growing body of work links ABCG2 to multiple photodynamic therapy (PDT) agents, and there is a limited body of evidence suggesting that ABCG2 may also play a role in resistance to radiation therapy. In addition, the focus of ABC transporter research in regards to therapeutic development has begun to shift in the past few years. The shift has been away from using pump inhibitors for reversing resistance, toward the development of therapeutic agents that are poor substrates for these efflux pump proteins. This approach may result in the development of drug regimens that circumvent ABC transporter-mediated resistance entirely. Here, it is our intention to review: 1) recent discoveries that further characterize the role of ABCG2 in oncology, and 2) advances in reversing and circumventing ABC transporter-mediated resistance to anti-cancer therapies.
    Full-text · Article · Dec 2015
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