BACKGROUNDRNA interference using short hairpin RNA (shRNA) can mediate sequence-specific inhibition of gene expression in mammalian cells. A vector-based approach for synthesizing shRNA has been developed recently. Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers multidrug resistance (MDR) to cancer cells. In this study, we reversed MDR using shRNA expression vectors in a multidrug-resistant human breast cancer cell line (MCF-7/AdrR).

METHODSThe two shRNA expression vectors were constructed and introduced into MCF-7/AdrR cells. Expression of MDR1 mRNA was assessed by RT-PCR, and P-gp expression was determined by Western Blot and immunocytochemistry. Apoptosis and sensitization of the breast cancer cells to doxorubicin were quantified by flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. Cellular daunorubicin accumulation was assayed by laser confocal scanning microscopy (LCSM). Statistical significance of differences in mean values was evaluated by Student's t tests. P < 0.05 was considered statistically significant.

RESULTSIn MCF-7/AdrA cells transfected with MDR1-A and MDR1-B shRNA expression vectors, RT-PCR showed that MDR1 mRNA expression was reduced by 40.9% (P < 0.05), 30.1% (P < 0.01) (transient transfection) and 37.6% (P < 0.05), 28.0% (P < 0.01) (stable transfection), respectively. Western Blot and immunocytochemistry showed that P-gp expression was significantly and specifically inhibited. Resistance against doxorubicin was decreased from 162-fold to 109-fold (P < 0.05), 54-fold (P < 0.01) (transient transfection) and to 108-fold (P < 0.05), 50-fold (P < 0.01) (stable transfection). Furthermore, shRNA vectors significantly enhanced the cellular daunorubicin accumulation. The combination of shRNA vectors and doxorubicin significantly induced apoptosis in MCF-7/AdrR cells.

CONCLUSIONSshRNA expression vectors effectively reduce MDR expression in a sustained fashion and can restore the sensitivity of drug-resistant cancer cells to conventional chemotherapeutic agents.

ATP-Binding Cassette, Sub-Family B, Member 1 ; analysis ; antagonists & inhibitors ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; Daunorubicin ; pharmacokinetics ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Flow Cytometry ; Genes, MDR ; Genetic Vectors ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

Aim To investigate the efficacy of arctigenin on airway inflammation in a mouse model of asthma and the mechanism related to the SIRT1/NLRP3 signaling pathway. Methods Forty female BALB/c mice of clean grade were selected and divided into control group, OVA model group and ATG group (5, 10 and 20 mg · kg

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