Human concentrative nucleoside transporter 3 transfection with ultrasound and microbubbles in nucleoside transport deficient HEK293 cells greatly increases gemcitabine uptake.
Gemcitabine is a hydrophilic clinical anticancer drug that requires nucleoside transporters to cross plasma membranes and enter cells. Pancreatic adenocarcinomas with low levels of nucleoside transporters are generally resistant to gemcitabine and are currently a clinical problem. We tested whether...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2013-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3575408?pdf=render |
| _version_ | 1811288859905884160 |
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| author | Robert J Paproski Sylvia Y M Yao Nicole Favis David Evans James D Young Carol E Cass Roger J Zemp |
| author_facet | Robert J Paproski Sylvia Y M Yao Nicole Favis David Evans James D Young Carol E Cass Roger J Zemp |
| author_sort | Robert J Paproski |
| collection | DOAJ |
| description | Gemcitabine is a hydrophilic clinical anticancer drug that requires nucleoside transporters to cross plasma membranes and enter cells. Pancreatic adenocarcinomas with low levels of nucleoside transporters are generally resistant to gemcitabine and are currently a clinical problem. We tested whether transfection of human concentrative nucleoside transporter 3 (hCNT3) using ultrasound and lipid stabilized microbubbles could increase gemcitabine uptake and sensitivity in HEK293 cells made nucleoside transport deficient by pharmacologic treatment with dilazep. To our knowledge, no published data exists regarding the utility of using hCNT3 as a therapeutic gene to reverse gemcitabine resistance. Our ultrasound transfection system--capable of transfection of cell cultures, mouse muscle and xenograft CEM/araC tumors--increased hCNT3 mRNA and (3)H-gemcitabine uptake by >2,000- and 3,400-fold, respectively, in dilazep-treated HEK293 cells. Interestingly, HEK293 cells with both functional human equilibrative nucleoside transporters and hCNT3 displayed 5% of (3)H-gemcitabine uptake observed in cells with only functional hCNT3, suggesting that equilibrative nucleoside transporters caused significant efflux of (3)H-gemcitabine. Efflux assays confirmed that dilazep could inhibit the majority of (3)H-gemcitabine efflux from HEK293 cells, suggesting that hENTs were responsible for the majority of efflux from the tested cells. Oocyte uptake transport assays were also performed and provided support for our hypothesis. Gemcitabine uptake and efflux assays were also performed on pancreatic cancer AsPC-1 and MIA PaCa-2 cells with similar results to that of HEK293 cells. Using the MTS proliferation assay, dilazep-treated HEK293 cells demonstrated 13-fold greater resistance to gemcitabine compared to dilazep-untreated HEK293 cells and this resistance could be reversed by transfection of hCNT3 cDNA. We propose that transfection of hCNT3 cDNA using ultrasound and microbubbles may be a method to reverse gemcitabine resistance in pancreatic tumors that have little nucleoside transport activity which are resistant to almost all current anticancer therapies. |
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| id | doaj.art-82517bf8aa514ea498cbf14dd0559750 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-13T03:44:42Z |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
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| spelling | doaj.art-82517bf8aa514ea498cbf14dd05597502022-12-22T03:04:03ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0182e5642310.1371/journal.pone.0056423Human concentrative nucleoside transporter 3 transfection with ultrasound and microbubbles in nucleoside transport deficient HEK293 cells greatly increases gemcitabine uptake.Robert J PaproskiSylvia Y M YaoNicole FavisDavid EvansJames D YoungCarol E CassRoger J ZempGemcitabine is a hydrophilic clinical anticancer drug that requires nucleoside transporters to cross plasma membranes and enter cells. Pancreatic adenocarcinomas with low levels of nucleoside transporters are generally resistant to gemcitabine and are currently a clinical problem. We tested whether transfection of human concentrative nucleoside transporter 3 (hCNT3) using ultrasound and lipid stabilized microbubbles could increase gemcitabine uptake and sensitivity in HEK293 cells made nucleoside transport deficient by pharmacologic treatment with dilazep. To our knowledge, no published data exists regarding the utility of using hCNT3 as a therapeutic gene to reverse gemcitabine resistance. Our ultrasound transfection system--capable of transfection of cell cultures, mouse muscle and xenograft CEM/araC tumors--increased hCNT3 mRNA and (3)H-gemcitabine uptake by >2,000- and 3,400-fold, respectively, in dilazep-treated HEK293 cells. Interestingly, HEK293 cells with both functional human equilibrative nucleoside transporters and hCNT3 displayed 5% of (3)H-gemcitabine uptake observed in cells with only functional hCNT3, suggesting that equilibrative nucleoside transporters caused significant efflux of (3)H-gemcitabine. Efflux assays confirmed that dilazep could inhibit the majority of (3)H-gemcitabine efflux from HEK293 cells, suggesting that hENTs were responsible for the majority of efflux from the tested cells. Oocyte uptake transport assays were also performed and provided support for our hypothesis. Gemcitabine uptake and efflux assays were also performed on pancreatic cancer AsPC-1 and MIA PaCa-2 cells with similar results to that of HEK293 cells. Using the MTS proliferation assay, dilazep-treated HEK293 cells demonstrated 13-fold greater resistance to gemcitabine compared to dilazep-untreated HEK293 cells and this resistance could be reversed by transfection of hCNT3 cDNA. We propose that transfection of hCNT3 cDNA using ultrasound and microbubbles may be a method to reverse gemcitabine resistance in pancreatic tumors that have little nucleoside transport activity which are resistant to almost all current anticancer therapies.http://europepmc.org/articles/PMC3575408?pdf=render |
| spellingShingle | Robert J Paproski Sylvia Y M Yao Nicole Favis David Evans James D Young Carol E Cass Roger J Zemp Human concentrative nucleoside transporter 3 transfection with ultrasound and microbubbles in nucleoside transport deficient HEK293 cells greatly increases gemcitabine uptake. PLoS ONE |
| title | Human concentrative nucleoside transporter 3 transfection with ultrasound and microbubbles in nucleoside transport deficient HEK293 cells greatly increases gemcitabine uptake. |
| title_full | Human concentrative nucleoside transporter 3 transfection with ultrasound and microbubbles in nucleoside transport deficient HEK293 cells greatly increases gemcitabine uptake. |
| title_fullStr | Human concentrative nucleoside transporter 3 transfection with ultrasound and microbubbles in nucleoside transport deficient HEK293 cells greatly increases gemcitabine uptake. |
| title_full_unstemmed | Human concentrative nucleoside transporter 3 transfection with ultrasound and microbubbles in nucleoside transport deficient HEK293 cells greatly increases gemcitabine uptake. |
| title_short | Human concentrative nucleoside transporter 3 transfection with ultrasound and microbubbles in nucleoside transport deficient HEK293 cells greatly increases gemcitabine uptake. |
| title_sort | human concentrative nucleoside transporter 3 transfection with ultrasound and microbubbles in nucleoside transport deficient hek293 cells greatly increases gemcitabine uptake |
| url | http://europepmc.org/articles/PMC3575408?pdf=render |
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