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Modulating drug resistance by targeting BCRP/ABCG2 using retrovirus-mediated RNA interference.

Xie N, Mou L, Yuan J, Liu W, Deng T, Li Z, Jing Y, Jin Y, Hu Z - PLoS ONE (2014)

Bottom Line: To 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.

View Article: PubMed Central - PubMed

Affiliation: Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong, China.

ABSTRACT

Background: The 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 findings: To 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/significance: The 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.

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Immunohistochemical staining of tumor bodies in hairless mice bearing JAR cancer cells after injection with V-BCRPi and 5-FU (×100).The results indicate that V-BCRPi inhibits BCRP/ABCG2 expression and improves drug sensitivity to 5-FU in tumors. The red arrows indicate the dead cells.
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pone-0103463-g006: Immunohistochemical staining of tumor bodies in hairless mice bearing JAR cancer cells after injection with V-BCRPi and 5-FU (×100).The results indicate that V-BCRPi inhibits BCRP/ABCG2 expression and improves drug sensitivity to 5-FU in tumors. The red arrows indicate the dead cells.

Mentions: The expression of BCRP/ABCG2 in tumors injected with V-BCRPi was lower than that in tumors not injected with V-BCRPi (including tumors injected with PBS and 5-FU alone). The membranes of the BCRP/ABCG2-positive histiocytes were brown, and the nuclei were blue, with larger karyoplasm and altered morphologies in different cells. Of the groups injected with 5-FU, more dying cells were evident in the groups injected with V-BCRPi than in the uninjected group. Specifically, cellular morphology was lost, the karyoplasm was large, and necrosis was increased in the virus-infected cells (Fig. 6). These results indicate that V-BCRPi inhibits BCRP/ABCG2 expression in tumor cells and improves their sensitivity to 5-FU.


Modulating drug resistance by targeting BCRP/ABCG2 using retrovirus-mediated RNA interference.

Xie N, Mou L, Yuan J, Liu W, Deng T, Li Z, Jing Y, Jin Y, Hu Z - PLoS ONE (2014)

Immunohistochemical staining of tumor bodies in hairless mice bearing JAR cancer cells after injection with V-BCRPi and 5-FU (×100).The results indicate that V-BCRPi inhibits BCRP/ABCG2 expression and improves drug sensitivity to 5-FU in tumors. The red arrows indicate the dead cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4116202&req=5

pone-0103463-g006: Immunohistochemical staining of tumor bodies in hairless mice bearing JAR cancer cells after injection with V-BCRPi and 5-FU (×100).The results indicate that V-BCRPi inhibits BCRP/ABCG2 expression and improves drug sensitivity to 5-FU in tumors. The red arrows indicate the dead cells.
Mentions: The expression of BCRP/ABCG2 in tumors injected with V-BCRPi was lower than that in tumors not injected with V-BCRPi (including tumors injected with PBS and 5-FU alone). The membranes of the BCRP/ABCG2-positive histiocytes were brown, and the nuclei were blue, with larger karyoplasm and altered morphologies in different cells. Of the groups injected with 5-FU, more dying cells were evident in the groups injected with V-BCRPi than in the uninjected group. Specifically, cellular morphology was lost, the karyoplasm was large, and necrosis was increased in the virus-infected cells (Fig. 6). These results indicate that V-BCRPi inhibits BCRP/ABCG2 expression in tumor cells and improves their sensitivity to 5-FU.

Bottom Line: To 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.

View Article: PubMed Central - PubMed

Affiliation: Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong, China.

ABSTRACT

Background: The 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 findings: To 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/significance: The 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.

Show MeSH
Related in: MedlinePlus