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Link to original content: http://pubmed.ncbi.nlm.nih.gov/24249723/
Enhanced receptor-mediated endocytosis and cytotoxicity of a folic acid-desacetylvinblastine monohydrazide conjugate in a pemetrexed-resistant cell line lacking folate-specific facilitative carriers but with increased folate receptor expression - PubMed Skip to main page content
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. 2014 Feb;85(2):310-21.
doi: 10.1124/mol.113.089110. Epub 2013 Nov 18.

Enhanced receptor-mediated endocytosis and cytotoxicity of a folic acid-desacetylvinblastine monohydrazide conjugate in a pemetrexed-resistant cell line lacking folate-specific facilitative carriers but with increased folate receptor expression

Rongbao Zhao  1 , Ndeye Diop-Bove, I David Goldman
Affiliations

Enhanced receptor-mediated endocytosis and cytotoxicity of a folic acid-desacetylvinblastine monohydrazide conjugate in a pemetrexed-resistant cell line lacking folate-specific facilitative carriers but with increased folate receptor expression

Rongbao Zhao et al. Mol Pharmacol. 2014 Feb.

Abstract

The reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), and folate receptors (FR) are folate-specific transporters. Antifolates currently in the clinic, such as pemetrexed, methotrexate, and pralatrexate, are transported into tumor cells primarily via RFC. Folic acid conjugated to cytotoxics, a new class of antineoplastics, are transported into cells via FR-mediated endocytosis. To better define the role of PCFT in antifolate resistance, a methotrexate-resistant cell line, M160-8, was selected from a HeLa subline in which the RFC gene was deleted and PCFT was highly overexpressed. These cells were cross-resistant to pemetrexed. PCFT function and the PCFT mRNA level in M160-8 cells were barely detectable, and FR-α function and mRNA level were increased as compared with the parent cells. While pemetrexed rapidly associated with FR and was internalized within endosomes in M160-8 cells, consistent with FR-mediated transport, subsequent pemetrexed and (6S)-5-formyltetrahydrofolate export into the cytosol was markedly impaired. In contrast, M160-8 cells were collaterally sensitive to EC0905, a folic acid-desacetylvinblastine monohydrazide conjugate also transported by FR-mediated endocytosis. However, in this case a sulfhydryl bond is cleaved to release the lipophilic cytotoxic moiety into the endosome, which passively diffuses out of the endosome into the cytosol. Hence, resistance to pemetrexed in M160-8 cells was due to entrapment of the drug within the endosome due to the absence of PCFT under conditions in which the FR cycling function was intact.

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Figures

Fig. 1.
Fig. 1.
Growth inhibition by methotrexate (A), trimetrexate (B), PT523 (C), pemetrexed (D), and raltitrexed (E). Cells were seeded in 96-well plates before a spectrum of antifolate concentrations was added the next day and exposure was continued for 5 days. Growth inhibition is expressed as percentages of control growth in the absence of drugs. Data are the mean ± S.E.M. from three independent experiments for all panels.
Fig. 2.
Fig. 2.
Influx of (A) methotrexate, (B) 5-CHO-THF, and (C) pemetrexed. Influx was determined at pH 5.5 at a substrate concentration of 0.5 µM. Influx in PCFT-h cells was measured over 1 minute, and influx in the other cells was measured over 2 minutes. The interrupted y-axes are used to facilitate visualization of influx comparisons among the different cell lines. The numbers above the bars are P values of t test comparisons of the influx between R1-11-PCFT-4 and M160-8 cells or between M160-8 and R1-11 cells. Data in all panels are the mean ± S.E.M. from three independent experiments.
Fig. 3.
Fig. 3.
(A) Folic acid surface binding, (B) pemetrexed uptake in the presence or absence of 1 µM unlabeled folic acid, (C) (6S)-5-CHO-THF accumulation, and (D) (6S)-5-CHO-THF growth requirement. (A) Folic acid bound to the cell surface. Cells were prewashed with acidic buffer (pH 3.5) to remove folates bound to FR. After exposure of cells to 5 nM [3H]folic acid in HBS (pH 7.4) in the presence or absence 1 µM folic acid on ice for 20 minutes, unbound folic acid was washed away, and then folic acid bound to the cell surface was released with the acid buffer (pH 3.5). (B) Pemetrexed uptake in folate-free RPMI medium (pH 7.3) in a 5% CO2 incubator. Cells were washed once with the acid buffer at 4°C to remove folates bound to FR. After washing with HBS (pH 7.4) and a 20-minute preincubation in folate-free medium, cells were exposed for 2 hours to [3H]pemetrexed (100 nM) in folate-free PPMI medium (pH 7.3) in the presence or absence of 1 µM folic acid in a 5% CO2 incubator at 37°C. Both unbound and surface-bound pemetrexed was washed away, and the values shown in the figure represent pemetrexed internalized in the cells. *P = 0.028 for the difference between pemetrexed uptake in the presence or absence of 1 µM folic acid. (C) Accumulation of [3H](6S)-CHO-THF. Intracellular folates pools were depleted by growing cells in folate-free medium supplemented with GAT for 1 to 2 weeks. Cells were then exposed to 25 nM [3H](6S)-5-CHO-THF for 3 days. (D) Growth requirement for (6S)-5-CHO-THF. Intracellular folate pools were depleted by growing cells in folate-free medium containing GAT for 1 to 2 weeks. Cells were then exposed to different concentrations of (6S)-5-CHO-THF for 4 hours in the presence of GAT to provide nutrients during the interval in which cellular folate repletion was initiated, and then 5 days in the absence of GAT. Data in all panels are the mean ± S.E.M. from three independent experiments.
Fig. 4.
Fig. 4.
Comparison of cell surface-bound and internalized pemetrexed after exposure to tritiated pemetrexed for various intervals (A). Confluent M160-8 and PCFT-4 cells were exposed to 100 nM [3H]pemetrexed in folate-free RPMI medium (pH 7.3, 37°C) in a 5% CO2 incubator for 0.5, 1, 2, 4, 6, and 16 hours before the cells were washed 3 times with HBS (pH 7.4) to remove unbound pemetrexed. Both the cell surface–bound pemetrexed, which was released by 0.5 ml of acid buffer (pH 3.5) at 0°C, and pemetrexed uptake into cells were assessed. (B) Amplification of the y-axis to facilitate comparison between cell surface–bound pemetrexed among M160-8, R1-11-PCFT-4 cells and pemetrexed uptake in M160-8 cells. Data are the mean ± S.E.M. from three independent experiments.
Fig. 5.
Fig. 5.
Comparison of folates or antifolates accumulated in the cytosolic and membrane fractions, and inhibitory effects of pemetrexed, (6S)-5-CHO-THF and folic acid on binding of [3H]folic acid to FR at the cell surface at pH 7.4 and 6.5. (A) Cells were exposed to 50 nM tritiated pemetrexed, 5-CHO-THF, folic acid, or methotrexate for 2 days in folate-free RPMI-medium in a 5% CO2 incubator. GAT was added to cell cultures during exposure to pemetrexed and methotrexate to prevent cytotoxicity mediated by these agents. The data for PCFT-4 cells are indicated on the left side of the graph, and the values for M160-8 cells are shown on the right side of the graph. (B) [3H]Folic acid bound to the surface of M160-8 cells was assessed with 20 nM [3H]folic acid in the presence or absence of unlabeled pemetrexed, (6S)-5-CHO-THF, or folic acid. Data are the mean ± S.E.M. from three independent experiments in both panels. (C) Comparison of 20 nM [3H]folic acid bound to the surface of M160-8 cells at pH 7.4 and pH 6.5 in the presence or absence of various concentrations of unlabeled folic acid, pemetrexed, or (6S)-5-CHO-THF; *P < 0.05 for the comparison in A and C.
Fig. 6.
Fig. 6.
The structure of EC0905. The molecule consists of DAVLBH (a lipophilic microtubule inhibitor) connected to folic acid with a glycosylated linker containing a sulfhydryl bond.
Fig. 7.
Fig. 7.
Growth inhibition by EC0905 or DAVLBH in the presence or absence of 20 µM folic acid. (A) Growth inhibition by EC0905 in the absence of 20 µM folic acid. (B) Growth inhibition by EC0905 in the presence of 20 µM folic acid. (C) Growth inhibition by DAVLBH in the absence of 20 µM folic acid. (D) Growth inhibition by DAVLBH in the presence of 20 µM folic acid. M160-8, PCFT-h and PCFT-4 cells were detached from culture plates with 0.5 mM EDTA in phosphate-buffered saline and seeded in 96-well plates (0.1 ml) at a density of 2000 cells/well. An equal volume of drug solutions in the presence or absence of folic acid, diluted in the growth medium, was added to cells the next day, after which the cells were grown for an additional 5 days. Data are the mean ± S.E.M. from three independent experiments.
Fig. 8.
Fig. 8.
A diagram illustrating FR-mediated endocytosis, the role of PCFT, and the transport of pemetrexed (PMX) and EC0905 in M160-8 cells. In wild-type cells, pemetrexed is exported from endosomes via PCFT. It can also enter cells by this mechanism even at neutral pH. In M160-8 cells, pemetrexed is endocytosed by the receptor, but, upon dissociation, it is trapped within the endosome in the absence of PCFT. This is the case also for (6S)5-CHO-THF (not shown). In contrast, there is a high level of FR-mediated transport of EC0905. In this case, the disulfide bond linking folic acid and DAVLBH is reduced within the endosome, releasing the lipophilic DAVLBH, which exits the endosome by passive diffusion, a PCFT-independent process.
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