title: Role of folate receptor and reduced folate carrier in the transport of 5-methyltetrahydrofolic acid in human ovarian carcinoma cells.
abstract: Folate receptor-alpha (FR-alpha) is generally over-expressed in non-mucinous human ovarian carcinomas. The meaning of FR-alpha over-expression and its role in the 5-methyltetrahydrofolic acid (N5-CH3-H4PteGlu) transport in such tumors is not clear, especially compared with the reduced folate carrier (RFC), the other known folate transporter. In this study, we analyzed molecular FR-alpha and RFC expression in 16 ovarian carcinoma tissues and in 5 ovarian carcinoma cell lines using competitive PCR. Co-expression of the 2 transporters was found both in vivo and in vitro. FR-alpha mRNA expression in the cell lines was in good agreement with the corresponding protein expression evaluated by cellular folic acid binding and immunofluorescence analysis, using a specific monoclonal antibody (MAb) (MOv18). Moreover, RFC mRNA expression levels were consistent with the selective cellular binding of N-hydroxysuccinimide of [3H]-methotrexate (NHS-MTX). The 5 ovarian carcinoma cell lines (IGROV-1, SW-626, SKOV-3, OVCAR-3 and OAW-42), grown at physiological N5-CH3-H4PteGlu concentrations (20 nM) and expressing FR-alpha and RFC levels superimposable to those observed in vivo, were used as in vitro cellular model to evaluate the different contribution of FR-alpha and RFC to the transport of N5-CH3-H4PteGlu. The cytoplasmic N5-CH3-[3H]H4PteGlu accumulation observed in each cell line was approximately linear over 4 hr of incubation, but there was no correlation between the rate of folate internalization and FR-alpha and RFC expression levels. Furthermore, the selective inhibition of FR-alpha and RFC functionality allowed us to distinguish their differential role on the overall N5-CH3-[3H]H4PteGlu intracellular delivery. Treatment with the N-hydroxysuccinimide of folic acid, which blocks FR-alpha activity, showed only a partial inhibition (about 20%) of folate internalization in all the cell lines. In contrast, the inhibition of RFC by NHS-MTX, under conditions that did not affect FR-alpha functionality, generally reduced folate accumulation by more than 70%. Only one cell line (IGROV-1) showed a comparable contribution of the 2 transport systems. Our findings suggest that in ovarian carcinomas, in spite of its over-expression, FR-alpha generally plays a minor role in N5-CH3-H4PteGlu transport compared with RFC.
authors.affiliation: Division of Experimental Oncology 1, Centro di Riferimento Oncologico, Aviano, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Corona
authors.forename: F
authors.initials: F
authors.lastname: Giannini
authors.forename: M
authors.initials: M
authors.lastname: Fabris
authors.forename: G
authors.initials: G
authors.lastname: Toffoli
authors.forename: M
authors.initials: M
authors.lastname: Boiocchi
mesh_terms: D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D005260:Female; D058973:Folate Receptors, GPI-Anchored; D006801:Humans; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010051:Ovarian Neoplasms / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism / Q000502:physiology*; D058978:Reduced Folate Carrier Protein; D013763:Tetrahydrofolates / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; C120043:SLC19A2 protein, human; D013763:Tetrahydrofolates; C005984:5-methyltetrahydrofolate