title: Biotin uptake by T47D breast cancer cells: functional and molecular evidence of sodium-dependent multivitamin transporter (SMVT).
abstract: The objective of this study was to investigate functional and molecular evidence of carrier mediated system responsible for biotin uptake in breast cancer (T47D) cells and to delineate mechanism of intracellular regulation of this transporter. Cellular accumulation of [3H] biotin was studied in T47D and normal mammary epithelial (MCF-12A) cells. Reverse transcription polymerase chain reaction (RT-PCR) was carried out to confirm the molecular expression of sodium dependent multivitamin transporter (SMVT) in T47D cells. Quantitative real time PCR analysis was also performed to compare the relative expression of SMVT in T47D and MCF-12A cells. [3H] biotin uptake by T47D cells was found to be concentration dependent with K(m) of 9.24 μM and V(max) of 27.34 pmol/mg protein/min. Uptake of [3H] biotin on MCF-12A cells was also found to be concentration dependent and saturable, but with a relatively higher K(m) (53.10 μM) indicating a decrease in affinity of biotin uptake in normal breast cells compared to breast cancer cells. [3H] biotin uptake appears to be time-, temperature-, pH- and sodium ion-dependent but independent of energy and chloride ions. [3H] biotin uptake was significantly inhibited in the presence of biotin, its structural analog desthiobiotin, pantothenic acid and lipoic acid. Concentration dependent inhibition of biotin uptake was evident in the presence of valeric acid which possesses free carboxyl group and biocytin and NHS biotin which are devoid of free carboxyl group. No significant inhibition was observed in the presence of structurally unrelated vitamins (ascorbic acid, folic acid, nicotinic acid, thiamine, pyridoxine and riboflavin). Modulators of PTK, PKC and PKA mediated pathways had no effect, but uptake in presence of calmidazolium (calcium-calmodulin inhibitor) was significantly inhibited. [3H] biotin uptake in the presence of calmidazolium was found to be saturable with a K(m) and V(max) values of 13.49 μM and 11.20 pmol/mg protein/min, respectively. A band of SMVT mRNA at 774 bp was identified by RT-PCR analysis. Quantitative real time PCR confirmed higher expression of SMVT in T47D cells relative to MCF-12A cells. All these studies demonstrated for the first time the functional and molecular expression of sodium dependent multivitamin transporter (SMVT), a specific carrier-mediated system for biotin uptake, in human derived breast cancer (T47D) cells. The present study also indicated that cancer cells could import more vitamin compared to normal breast cells possibly for maintaining high proliferative status. We investigated the likelihood of selecting this cell line (T47D) as an in vitro cell culture model to study biotin-conjugated anti-cancer drugs/drug delivery systems.
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: Aswani Dutt
authors.initials: AD
authors.lastname: Vadlapudi
authors.forename: Ramya Krishna
authors.initials: RK
authors.lastname: Vadlapatla
authors.forename: Dhananjay
authors.initials: D
authors.lastname: Pal
authors.forename: Ashim K
authors.initials: AK
authors.lastname: Mitra
mesh_terms: D001710:Biotin / Q000378:metabolism*; D001940:Breast / Q000166:cytology / Q000378:metabolism; D001943:Breast Neoplasms / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D006863:Hydrogen-Ion Concentration; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012964:Sodium / Q000378:metabolism; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors
chemical_list: D012333:RNA, Messenger; D027981:Symporters; C119981:biotin transporter; D001710:Biotin; D012964:Sodium