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.