Profiling SLCO and SLC22 genes in the NCI-60 cancer cell lines to identify drug uptake transporters

Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
Molecular Cancer Therapeutics (Impact Factor: 6.11). 10/2008; 7(9):3081-91. DOI: 10.1158/1535-7163.MCT-08-0539
Source: PubMed

ABSTRACT Molecular and pharmacologic profiling of the NCI-60 cell panel offers the possibility of identifying pathways involved in drug resistance or sensitivity. Of these, decreased uptake of anticancer drugs mediated by efflux transporters represents one of the best studied mechanisms. Previous studies have also shown that uptake transporters can influence cytotoxicity by altering the cellular uptake of anticancer drugs. Using quantitative real-time PCR, we measured the mRNA expression of two solute carrier (SLC) families, the organic cation/zwitterion transporters (SLC22 family) and the organic anion transporters (SLCO family), totaling 23 genes in normal tissues and the NCI-60 cell panel. By correlating the mRNA expression pattern of the SLCO and SLC22 family member gene products with the growth-inhibitory profiles of 1,429 anticancer drugs and drug candidate compounds tested on the NCI-60 cell lines, we identified SLC proteins that are likely to play a dominant role in drug sensitivity. To substantiate some of the SLC-drug pairs for which the SLC member was predicted to be sensitizing, follow-up experiments were performed using engineered and characterized cell lines overexpressing SLC22A4 (OCTN1). As predicted by the statistical correlations, expression of SLC22A4 resulted in increased cellular uptake and heightened sensitivity to mitoxantrone and doxorubicin. Our results indicate that the gene expression database can be used to identify SLCO and SLC22 family members that confer sensitivity to cancer cells.

  • 01/2014; 2(1):15. DOI:10.1186/2052-8426-2-15
  • [Show abstract] [Hide abstract]
    ABSTRACT: Increased expression of transporters-mediating uptake of antineoplastic drugs could render renal cell carcinoma (RCC) more sensitive to chemotherapy. Here, we studied the effect of hepatocyte nuclear factor 4α (HNF4α) on the expression of selected uptake transporters in RCC lines. Organic cation transporters (OCTs) and organic anion transporters (OATs) mRNA levels in HNF4α-transfected RCCs were measured by real-time PCR. Expression of HNF4α, β-catenin, N-cadherin, and E-cadherin was detected by immunofluorescence. OCT1, OAT2, and concentrative nucleoside transporter 3 (CNT3) were tested using tritium-labeled substrates and an apoptosis assay. Most RCC did not express uptake transporters in the absence or presence of HNF4α. In RCCNG1 cells, HNF4α-expression increased the chemosensitivity to oxaliplatin and enhanced the accumulation of methyl-4-phenylpyridinium acetate, a model substrate for OCT1. Furthermore, HNF4α enhanced OAT2 mRNA and increased caspase-3 activity upon incubation with a purported OAT2 substrate, 5-fluorouracil (5-FU). However, functional OAT2 protein was not upregulated. CNT3 mRNA was significantly elevated by HNF4α. Inhibition of CNT3-mediated uridine uptake by 5-FU metabolite 5-fluoro-2′-deoxyuridine suggested the involvement of CNT3 in increased caspase-3 activity. Our data suggest that HNF4α increases the expression of OCT1 and CNT3 in RCCNG1 cells, thereby increasing the chemosensitivity of tumor cells to oxaliplatin and 5-FU. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci
    Journal of Pharmaceutical Sciences 10/2014; 103(10). DOI:10.1002/jps.24128 · 3.01 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The main goal of this study is to investigate the expression of sodium dependent vitamin C transport system (SVCT2). Moreover this investigation has been carried out to define uptake mechanism and intracellular regulation of ascorbic acid (AA) in human breast cancer cells (MDA-MB231, T47D and ZR-75-1). Uptake of [(14)C] AA was studied in MDA-MB231, T47D and ZR-75-1cells. Functional parameters of [(14)C] AA uptake were delineated in the presence of different concentrations of unlabeled AA, pH, temperature, metabolic inhibitors, substrates and structural analogs. Molecular identification of SVCT2 was carried out with reverse transcription-polymerase chain reaction (RT-PCR). Uptake of [(14)C] AA was studied and found to be sodium, chloride, temperature, pH and energy dependent in all breast cancer cell lines. [(14)C] AA uptake was found to be saturable, with Km values of 53.85±6.24, 49.69±2.83 and 45.44±3.16μM and Vmax values of 18.45±0.50, 32.50±0.43 and 33.25±0.53pmol/min/mg protein, across MDA-MB231, T47D and ZR-75-1, respectively. The process is inhibited by structural analogs (L-AA and D-Iso AA) but not by structurally unrelated substrates (glucose and PAHA). Ca(++)/calmodulin and protein kinase pathways appeared to play a crucial role in modulating AA uptake. A 626bp band corresponding to a vitamin C transporter (SVCT2) based on the primer design was detected by RT-PCR analysis in all breast cancer cell lines. This research article describes AA uptake mechanism, kinetics, and regulation by sodium dependent vitamin C transporter (SVCT2) in MDA-MB231, T47D and ZR-75-1cells. Also, MDA-MB231, T47D and ZR-75-1 cell lines can be utilized as a valuable in vitro model to investigate absorption and permeability of AA-conjugated chemotherapeutics.
    International Journal of Pharmaceutics 08/2014; 474(1-2). DOI:10.1016/j.ijpharm.2014.07.056 · 3.79 Impact Factor

Full-text (2 Sources)

Available from
May 19, 2014