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Therapeutic targeting malignant mesothelioma with a novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate via its selective uptake by the proton-coupled folate transporter

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Abstract

Purpose

We examined whether the novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate, compound 2, might be an effective treatment for malignant pleural mesothelioma (MPM), reflecting its selective membrane transport by the proton-coupled folate transport (PCFT) over the reduced folate carrier (RFC).

Methods

HeLa sublines expressing exclusively PCFT (R1-11-PCFT4) or RFC (R1-11-RFC6) and H2452 MPM cells were assayed for transport with [3H]compound 2. [3H]Polyglutamate metabolites of compound 2 were measured in R1-11-PCFT4 and H2452 cells. In vitro cell proliferation assays and colony formation assays were performed. Inhibition of glycinamide ribonucleotide formyltransferase (GARFTase) was assayed by nucleoside protection assays and in situ GARFTase assays with [14C]glycine. In vivo efficacy was established with early- and advanced-stage H2452 xenografts in severe-combined immunodeficient (SCID) mice administered intravenous compound 2.

Results

[3H]Compound 2 was selectively transported by PCFT and was metabolized to polyglutamates. Compound 2 selectively inhibited proliferation of R1-11-PCFT4 cells over R1-11-RFC6 cells. H2452 human MPM cells were sensitive to the antiproliferative effects of compound 2. By colony-forming assays with H2452 cells, compound 2 was cytotoxic. Compound 2 inhibited GARFTase in de novo purine biosynthesis. In vivo efficacy was confirmed toward early- and advanced-stage H2452 xenografts in SCID mice administered compound 2.

Conclusions

Our results demonstrate potent antitumor efficacy of compound 2 toward H2452 MPM cells in vitro and in vivo, reflecting its efficient membrane transport by PCFT, synthesis of polyglutamates, and inhibition of GARFTase. Selectivity for non-RFC cellular uptake processes by tumor-targeted antifolates such as compound 2 presents an exciting new opportunity for treating solid tumors.

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Abbreviations

AICA:

5-Amino-4-imidazolecarboxamide

AICARFTase:

AICA ribonucleotide formyltransferase

CHO:

Chinese hamster ovary

DPBS:

Dulbecco’s phosphate-buffered saline

FR:

Folate receptor

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

GAR:

β-Glycinamide ribonucleotide

GARFTase:

Glycinamide ribonucleotide formyltransferase

HA:

Hemagglutinin

HBS:

HEPES-buffered saline

HEPES:

4-(2-Hydroxyethyl)-1-piperazine ethanesulfonic acid

hPCFT:

Human PCFT

HPLC:

High-performance liquid chromatography

hRFC:

Human RFC

IC50 :

Fifty percent inhibitory concentration

LCV:

Leucovorin

MBS:

MES-buffered saline

MEM:

Minimal essential media

MES:

4-Morphilinopropane sulfonic

MPM:

Malignant pleural mesothelioma

Mtx:

Methotrexate

PCFT:

Proton-coupled folate transporter

PIPES:

Piperazine-N,N′-bis(2-ethanesulfonic acid)

Pmx:

Pemetrexed

PVDF:

Polyvinylidene difluoride

RFC:

Reduced folate carrier

SC:

Subcutaneous

SCID:

Severe-combined immunodeficient

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

TCA:

Trichloroacetic acid

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Acknowledgments

We thank Dr. Anil Wali (National Cancer Institute, Bethesda, MD) for providing the H2452 mesothelioma cell line. We thank Dr. I. David Goldman (Bronx, NY) for his generous gifts of the R1-11 HeLa cell line series (R1-11-mock, R1-11-RFC6, and R1-11-PCFT4). This study was supported by grants from the National Cancer Institute, National Institutes of health [CA53535, CA125153, and CA152316], a pilot grant from the Barbara Ann Karmanos Cancer Institute, and a grant from the Mesothelioma Applied Research Foundation. Ms. Kugel Desmoulin was supported by a Doctoral Research Award from the Canadian Institutes of Health Research (CIHR).

Conflict of interest

None.

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Authors and Affiliations

  1. Graduate Program in Cancer Biology, Wayne State University School of Medicine, Detroit, MI, 48201, USA

    Sita Kugel Desmoulin & Larry H. Matherly

  2. Department of Oncology, Wayne State University School of Medicine, Detroit, MI, 48201, USA

    Christina Cherian, Sita Kugel Desmoulin, Lisa Polin, Kathryn White, Juiwanna Kushner, Zhanjun Hou & Larry H. Matherly

  3. Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, 110 E. Warren Ave, Detroit, MI, 48201, USA

    Christina Cherian, Lisa Polin, Zhanjun Hou & Larry H. Matherly

  4. Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI, 48201, USA

    Larry H. Matherly

  5. Department of Pediatrics, Children’s Hospital of Michigan, Detroit, MI, 48201, USA

    Mark Stout

  6. Division of Medicinal Chemistry, Graduate School of Pharmaceutical Science, Duquesne University, 600 Forbes Avenue, Pittsburgh, PA, 15282, USA

    Lei Wang & Aleem Gangjee

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  1. Christina Cherian
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  2. Sita Kugel Desmoulin
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  3. Lei Wang
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  4. Lisa Polin
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  8. Zhanjun Hou
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  9. Aleem Gangjee
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  10. Larry H. Matherly
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Corresponding authors

Correspondence to Aleem Gangjee or Larry H. Matherly.

Additional information

Larry H. Matherly and Aleem Gangjee contributed equally to this work.

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Cherian, C., Kugel Desmoulin, S., Wang, L. et al. Therapeutic targeting malignant mesothelioma with a novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate via its selective uptake by the proton-coupled folate transporter. Cancer Chemother Pharmacol 71, 999–1011 (2013). https://doi.org/10.1007/s00280-013-2094-0

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