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PLoS One
2013 Jan 01;82:e56423. doi: 10.1371/journal.pone.0056423.
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Human concentrative nucleoside transporter 3 transfection with ultrasound and microbubbles in nucleoside transport deficient HEK293 cells greatly increases gemcitabine uptake.
Paproski RJ
,
Yao SY
,
Favis N
,
Evans D
,
Young JD
,
Cass CE
,
Zemp RJ
.
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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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Figure 2. Fluorescence microscopy image of two LSM containing fluorescent water soluble pyranine.Distance between major grid lines is one micrometer. Perfluoropropane gas bubbles in LSM reduces aqueous volume.
Figure 3. In vitro and in vivo transfection of cells with ultrasound and LSM.(A) pIRES2-EGFP-hCNT3 was transfected into cultured HEK293 cells using the setup in Figure 2. (B, left) A mouse was injected with LSM and DNA encoding luciferase into both back legs. Ultrasound was only applied to the back right leg and bioluminescence imaging was performed 3 days later. Mice bearing subcutaneous CEM/araC tumors had luciferase encoding DNA with (right) or without (middle) LSM injected into the tumors. Ultrasound was applied to both tumors and bioluminescence imaging was performed 5 days later. Four tumor-bearing mice were imaged with bioluminescence imaging with consistent results. In vivo images suggest efficient transfection only occurs in the presence of both LSM and ultrasound.
Figure 4. Nucleoside transporter mRNA levels in HEK293, AsPC-1, and MIA PaCa-2 cells with or without transfection of pIRES2-EGFP-hCNT3 DNA.Cultured HEK293 cells were transfected with pIRES2-EGFP-hCNT3 using ultrasound and LSM while AsPC-1 and MIA PaCa-2 cells were transfected using lipofectamine 2000. The following day cells were harvested and analyzed for mRNA levels using real-time PCR with TaqMan® probes and primers. Bars are mean values from three different experiments.
Figure 5. Transfection efficiency corrected 3H-gemcitabine uptake in HEK293 (A), AsPC-1 (B), and MIA PaCa-2 (C) cells with or without transfection of hCNT3 cDNA.HEK293 cells in 12-well plates were incubated with buffer containing LSM with or without pIRES2-EGFP-hCNT3 DNA and exposed to ultrasound. AsPC-1 and MIA PaCa-2 cells were transfected with lipofectamine 2000. Uptake assays were performed the following day using 50 nM 3H-gemcitabine in the presence or absence of 100 µM dilazep. For all cell lines, hENT inhibition by dilazep significantly decreased 3H-gemcitabine uptake whereas cells transfected with hCNT3 cDNA exhibited significantly increased 3H-gemcitabine uptake. Uptake values were corrected for transfection efficiency as described in the methods section. Bars represent mean values from three separate experiments (each performed in triplicate).
Figure 6. 3H-Gemcitabine efflux from HEK293, AsPC-1, and MIA PaCa-2 cells.(A) HEK293 cells in 12-well plates were incubated with 3H-gemcitabine for 60 min and were washed with PBS to remove extracellular 3H-gemcitabine. Cells were incubated in PBS with or without 100 µM dilazep and PBS aliquots were taken over 60 min and analyzed for 3H-gemcitabine. Shown is a representative experiment. (B) 3H-Gemcitabine efflux rates for AsPC-1, MIA PaCa-2, and HEK293 cells with or without 100 µM dilazep. Bars represent mean values from three different experiments.
Figure 7. Gemcitabine toxicity in HEK293 cells with or without transfection of hCNT3 cDNA.(A) Inhibiting endogenous hENT proteins with 10 µM dilazep significantly increased gemcitabine resistance (340±43 and 26±2 µM EC50 values with and without dilazep, respectively). (B) Transfection of hCNT3 in HEK293 cells not incubated with dilazep had no affect on gemcitabine sensitivity. (C) Cells transfected with hCNT3 cDNA and subsequently incubated with dilazep exhibited significantly increased sensitivity to gemcitabine (35±10 EC50 value, P<0.005). Representative experiments shown with each experiment performed using 6 replicates. EC50 values shown above were determined from averaging the EC50 values from three different experiments.
Figure 8. 3H-gemcitabine uptake in Xenopus oocytes with or without production of recombinant hENT1 and hCNT3.(A) Time courses of 3H-gemcitabine uptake in Xenopus oocytes over one hour. Oocytes were microinjected with hENT1/hCNT3 mRNA and uptake assays were performed four days later using 10 µM 3H-gemcitabine. (B) Oocytes were incubated with or without hENT1 inhibitor NBMPR (1 µM) and 3H-gemcitabine uptake was analyzed 60 minutes after 3H-gemcitabine incubation. Oocytes producing hCNT3 alone demonstrate the greatest levels of 3H-gemcitabine uptake although uptake was greatly reduced upon co-expression of active hENT1. Uptake values for each time point is the mean from 12 oocytes. Error bars not shown when smaller than symbols.
Figure 9. Model of gemcitabine uptake by nucleoside transporters.Gemcitabine is a hydrophilic drug that requires nucleoside transporters (hENT1/2 or hCNT1/3) for efficient uptake. hENTs can equilibrate gemcitabine levels across membranes but cannot actively accumulate the drug within cells. hCNT3 can use the Na+ (or H+ for hCNT3) cation transmembrane gradient to accumulate greater levels of gemcitabine within cells. For cells with significant levels of both hENTs and hCNTs, hCNTs actively transport gemcitabine within cells but hENTs will primarily efflux gemcitabine to equilibrate drug levels across membranes, causing reduced gemcitabine uptake compared to cells with only hCNTs. hENT1-negative cancer cells (which correlate with gemcitabine resistance) are presumed to have relatively low hENT activity such that transfection of these cells with a hCNT would be ideal for increasing gemcitabine uptake.
Figure 1. Ultrasound/LSM transfection setup.The bottom of the 12-well plate with cells was submerged in a 37°C water bath. The SP100 ultrasound transducer was placed underneath the plate and ultrasound was emitted upwards into the bottom of each well.
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