Simultaneous activity of two different mechanisms of folate transport in ovarian carcinoma cell lines

Silvia Miotti, Marina Bagnoli, Francesca Ottone, Antonella Tomassetti, Maria I. Colnaghi, Silvana Canevari

Research output: Contribution to journalArticlepeer-review

Abstract

We investigated whether the folate receptor α-isoform (FRα), which is overexpressed on ovarian carcinoma cells, is functionally active in internalizing the physiological form of folate, 5-methyl tetrahydrofolate (THF). Six ovarian tumor cell lines, expressing different levels of FRα (COR >> OVCAR3 > IGROV1 > OVCAR4 > SKOV3 > OVCAR5), were maintained in folate- depleted medium and internalization of 10 nM evaluated as acid-resistant radioactivity at 0°and 37°C. The amount of 5-methyl{3H}THF present in this fraction was not strictly related to the number of membrane receptors, since even cell lines with low FRα expression, e.g., OVCAR4, showed efficient internalization. Time course studies indicated that, whereas no uptake was detected at 0°C, at 37°C the internalized fraction showed a slow and constant increase, until 4 h. At this time, the internalized radioactivity represented 3H]THF, which best ensures receptors saturation on cells with highest FR levels (COR and OVCAR3), had slight effect on surface binding of all the tested cell lines, including IGROV1 and SKOV3. In contrast, the increase of the uptake was more pronounced, particularly in SKOV3 cells. These results, together with the accumulation curves of folic acid (FA) and 5-methyl THF at 37°C, suggested the presence of a molecule on ovarian carcinoma cells with high affinity for reduced folates, possibly a reduced folate carrier (RFC). Measurement of radioactivity present in the supernatant of IGROV1 and SKOV3 cells, subjected to hypotonic lysis and cell fractionation, further indicated that 5 methyl[3H] THF was translocated to the cytosol and, despite differences in membrane levels of FRα expression this internalized fraction was similar in both cell lines. Inhibition experiments to selectively block FRα or RFC activity showed a differential sensitivity of the two pathways depending on the cell line examined. Internalization was more consistently inhibited on IGROV1 than on SKOV3 cells by treatments that disrupt FRα activity, e.g., incubation with excess FA and phosphatidylinositol specific phospholipase C, whereas Probenecid, which preferentially inhibits the carrier-mediated pathway, showed a strong inhibitory effect on both cell lines. These findings suggest that the internalization of 5-methyl THF in these tumor cells depends not only on the level of overexpressed FRα, but another transport route, with features characteristic for RFC, is functional and participates in folate uptake.

Original languageEnglish
Pages (from-to)479-491
Number of pages13
JournalJournal of Cellular Biochemistry
Volume65
Issue number4
DOIs
Publication statusPublished - Jun 15 1997

Keywords

  • folate receptor
  • folate uptake
  • ovarian carcinoma cells
  • reduced folate carrier

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

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