Abstract
BackgroundThe BCRP/ABCG2 transporter, which mediates drug resistance in many types of cells, depends on energy provided by ATP hydrolysis. Here, a retrovirus encoding a shRNA targeting the ATP-binding domain of this protein was used to screen for highly efficient agents that could reverse drug resistance and improve cell sensitivity to drugs, thus laying the foundation for further studies and applications.Methodology/Principal FindingsTo target the ATP-binding domain of BCRP/ABCG2, pLenti6/BCRPsi shRNA recombinant retroviruses, with 20 bp target sequences starting from the 270th, 745th and 939th bps of the 6th exon, were constructed and packaged. The pLenti6/BCRPsi retroviruses (V-BCRPi) that conferred significant knockdown effects were screened using a drug-sensitivity experiment and flow cytometry. The human choriocarcinoma cell line JAR, which highly expresses endogenous BCRP/ABCG2, was injected under the dorsal skin of a hairless mouse to initiate a JAR cytoma. After injecting V-BCRPi-infected JAR tumor cells into the dorsal skin of hairless mice, BCRP/ABCG2 expression in the tumor tissue was determined using immunohistochemistry, fluorescent quantitative RT-PCR and Western blot analyses. After intraperitoneal injection of BCRP/ABCG2-tolerant 5-FU, the tumor volume, weight change, and apoptosis rate of the tumor tissue were determined using in situ hybridization. V-BCRPi increased the sensitivity of the tumor histiocytes to 5-FU and improved the cell apoptosis-promoting effects of 5-FU in the tumor.Conclusions/SignificanceThe goal of the in vivo and in vitro studies was to screen for an RNA interference recombinant retrovirus capable of stably targeting the ATP-binding domain of BCRP/ABCG2 (V-BCRPi) to inhibit its function. A new method to improve the chemo-sensitivity of breast cancer and other tumor cells was discovered, and this method could be used for gene therapy and functional studies of malignant tumors.
Highlights
The ultimate goals of oncology drug resistance mechanism research are to find targets of drug resistance and screen for specific agents that can reverse these phenotypes, to improve the curative effects of chemotherapy for prevention and clinical treatment, and to reduce drug toxicity
The results indicated that, after infection with the recombinant V-BCRpi retroviruses, the amount of mitoxantrone that was retained by the cells was increased but was similar to that of the blank control group
Results of tumor growth after V-BCRPi and 5-FU injection On the 10th and 12th days of inoculation, we observed a grainsized node at the injection site of the JAR cells
Summary
The ultimate goals of oncology drug resistance mechanism research are to find targets of drug resistance and screen for specific agents that can reverse these phenotypes, to improve the curative effects of chemotherapy for prevention and clinical treatment, and to reduce drug toxicity. Reversal-agent studies are performed for specific targets and unitary target structures. These studies have unclear clinical application prospects and weak feasibility. Studying drug-resistance mechanisms and reversal agents with the same inhibitory effects is both valuable and important for understanding the multidrug-resistance phenotype. A retrovirus encoding a shRNA targeting the ATP-binding domain of this protein was used to screen for highly efficient agents that could reverse drug resistance and improve cell sensitivity to drugs, laying the foundation for further studies and applications
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