title: Molecular expression and functional activity of vitamin C specific transport system (SVCT2) in human breast cancer cells.
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-75-1 cells. 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 ± 3.16 μM and Vmax values of 18.45 ± 0.50, 32.50 ± 0.43 and 33.25 ± 0.53 pmol/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 626 bp 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-75-1 cells. 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.
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, 2464 Charlotte Street, Kansas City, MO 64108-2718, USA; INSYS Therapeutics Inc., 444 South Ellis Road, Chandler, AZ 85224, USA.
authors.forename: Varun
authors.initials: V
authors.lastname: Khurana
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, 2464 Charlotte Street, Kansas City, MO 64108-2718, USA; Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.
authors.forename: Deep
authors.initials: D
authors.lastname: Kwatra
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, 2464 Charlotte Street, Kansas City, MO 64108-2718, USA.
authors.forename: Dhananjay
authors.initials: D
authors.lastname: Pal
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, 2464 Charlotte Street, Kansas City, MO 64108-2718, USA. Electronic address: mitraa@umkc.edu.
authors.forename: Ashim K
authors.initials: AK
authors.lastname: Mitra
mesh_terms: D001205:Ascorbic Acid / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D060347:Sodium-Coupled Vitamin C Transporters / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors; D014407:Tumor Cells, Cultured
chemical_list: C555163:SLC23A2 protein, human; D060347:Sodium-Coupled Vitamin C Transporters; D001205:Ascorbic Acid
keywords: Ascorbic acid kinetics; In vitro cell culture models; MDA-MB231; Nutrient transporter; T47D and ZR-75-1 cells; Targeted drug delivery
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