Modulation of breast cancer resistance protein (BCRP/ABCG2) gene expression using RNA interference
University of Maryland, Baltimore, Baltimore, Maryland, United States Molecular Cancer Therapeutics
(Impact Factor: 5.68).
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.
Available from: PubMed Central
- "RNA interference (RNAi) technology provides a novel therapeutic approach for the treatment of drug-resistant tumors. Various RNAi strategies have been applied to reverse MDR in different tumor models in vitro and in vivo by down-regulating genes associated with MDR, such as multidrug resistance 1(MDR1), multidrug resistance-associated protein(MRP) and breast cancer resistance protein (BCRP) [7-9]. Recently, it was reported that Erythroblastosis virus E26 oncogene homolog 1 (ETS1) gene is over-expressed in drug-resistant human breast cancer cell lines . "
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ABSTRACT: Clinical resistance to chemotherapeutic agents is one of the major hindrances in the treatment of human cancers. Erythroblastosis virus E26 oncogene homolog 1 (ETS1) is involved in the drug resistance of various cancer cells, and is overexpressed in drug-resistant human breast cancer cell lines. In this study, we investigated the effects of ETS1 on adriamycin resistance in MCF-7/ADR cells.
siRNAs against ETS1 or negative control siRNAs was transfected to MCF-7/ADR breast cancer cells. Reverse transcription-PCR and Western blotting were used to determine the mRNA and protein expression of ETS1 and MDR1. The cytotoxicity of adriamycin was assessed using the MTT assay. Drug efflux was investigated by flow cytometry using the Rhodamine 123 intracellular accumulation assay.
ETS1 mRNA and protein was significantly overexpressed in MCF-7/ADR cells, compared to MCF-7 cells. ETS1 siRNA successfully silenced ETS1 mRNA and protein expression. Silencing of ETS1 also significantly reduced the mRNA and protein expression levels of MDR1 (multidrug resistance 1; also known as ABCB1, P-glycoprotein/P-gp), which is a major ATP-binding cassette (ABC) transporter linked to multi-drug resistance in cancer cells. Silencing of ETS1 significantly increased the sensitivity of MCF-7/ADR cells to adriamycin, compared to cells transfected with negative control siRNA. In addition, intracellular accumulation of Rhodamine 123 significantly increased in MCF-7/ADR cells transfected with ETS1 siRNA, indicating that silencing of ETS1 may reduce drug efflux.
This study demonstrates that drug resistance can be effectively reversed in adriamycin-resistant breast carcinoma cells through delivery of siRNAs targeting ETS1.
Available from: Susana Genti-Raimondi
- "ABCG2 is an integral plasma membrane glycoprotein distributed in normal human tissues and particularly highly expressed in those with barrier function, including the placenta, testes, liver, kidney, intestine and brain , . ABCG2 is overexpressed in tumors, cancer cell lines and in a subpopulation of stem cells: the side populations, conferring multidrug resistance , . Alterations in ABCG2 expression linked with changes in cell proliferation, migration and invasion have been reported –. "
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ABSTRACT: StAR-related lipid transfer domain containing 7 (StarD7) is a member of the START-domain protein family whose function still remains unclear. Our data from an explorative microarray assay performed with mRNAs from StarD7 siRNA-transfected JEG-3 cells indicated that ABCG2 (ATP-binding cassette sub-family G member 2) was one of the most abundantly downregulated mRNAs.
Here, we have confirmed that knocking down StarD7 mRNA lead to a decrease in the xenobiotic/lipid transporter ABCG2 at both the mRNA and protein levels (-26.4% and -41%, p<0.05, at 48 h of culture, respectively). Also a concomitant reduction in phospholipid synthesis, bromodeoxyuridine (BrdU) uptake and (3)H-thymidine incorporation was detected. Wound healing and transwell assays revealed that JEG-3 cell migration was significantly diminished (p<0.05). Conversely, biochemical differentiation markers such as human chorionic gonadotrophin β-subunit (βhCG) protein synthesis and secretion as well as βhCG and syncytin-1 mRNAs were increased approximately 2-fold. In addition, desmoplakin immunostaining suggested that there was a reduction of intercellular desmosomes between adjacent JEG-3 cells after knocking down StarD7.
Altogether these findings provide evidence for a role of StarD7 in cell physiology indicating that StarD7 modulates ABCG2 multidrug transporter level, cell migration, proliferation, and biochemical and morphological differentiation marker expression in a human trophoblast cell model.
Available from: Ezharul Hoque Chowdhury
- "A pioneering study using exogenous siRNA demonstrated suppression of ABCB1 expression in conjunction with reversal of doxorubicin and paclitaxel resistance in human breast cancer cells.33 Reversal of multidrug-resistant ABCG2 phenotype was also investigated with a siRNA-mediated knockdown study in several human cancer cells.34 "
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ABSTRACT: Multidrug resistance, a major impediment to successful cancer chemotherapy, is the result of overexpression of ATP-binding cassette (ABC) transporters extruding internalized drugs. Silencing of ABC transporter gene expression with small interfering RNA (siRNA) could be an attractive approach to overcome multidrug resistance of cancer, although delivery of siRNA remains a major hurdle to fully exploit the potential of siRNA-based therapeutics. Recently, we have developed pH-sensitive carbonate apatite nanoparticles to efficiently carry and transport siRNA across the cell membrane, enabling knockdown of the cyclin B1 gene and consequential induction of apoptosis in synergy with anti-cancer drugs.
We report that carbonate apatite-mediated delivery of the siRNAs targeting ABCG2 and ABCB1 gene transcripts in human breast cancer cells which constitutively express both of the transporter genes dose-dependently enhanced chemosensitivity to doxorubicin, paclitaxel and cisplatin, the traditionally used chemotherapeutic agents. Moreover, codelivery of two specific siRNAs targeting ABCB1 and ABCG2 transcripts resulted in a more robust increase of chemosensitivity in the cancer cells, indicating the reversal of ABC transporter-mediated multidrug resistance.
The delivery concept of multiple siRNAs against ABC transporter genes is highly promising for preclinical and clinical investigation in reversing the multidrug resistance phenotype of breast cancer.
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