Molecular expression and functional activity of vitamin C specific transport system (SVCT2) in human breast cancer cells

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 [14C] AA was studied in MDA-MB231, T47D and ZR-75-1 cells. Functional parameters of [14C] 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 [14C] AA was studied and found to be sodium, chloride, temperature, pH and energy dependent in all breast cancer cell lines. [14C] 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.

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