title: Therapeutic targeting of a novel 6-substituted pyrrolo [2,3-d]pyrimidine thienoyl antifolate to human solid tumors based on selective uptake by the proton-coupled folate transporter.
abstract: The proton-coupled folate transporter (PCFT) is a proton-folate symporter with an acidic pH optimum. By real-time reverse transcription-polymerase chain reaction, PCFT was expressed in the majority of 53 human tumor cell lines, with the highest levels in Caco-2 (colorectal adenocarcinoma), SKOV3 (ovarian), and HepG2 (hepatoma) cells. A novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate (compound 1) was used to establish whether PCFT can deliver cytotoxic drug under pH conditions that mimic the tumor microenvironment. Both 1 and pemetrexed (Pmx) inhibited proliferation of R1-11-PCFT4 HeLa cells engineered to express PCFT without the reduced folate carrier (RFC) and of HepG2 cells expressing both PCFT and RFC. Unlike Pmx, 1 did not inhibit proliferation of R1-11-RFC6 HeLa cells, which express RFC without PCFT. Treatment of R1-11-PCFT4 cells at pH 6.8 with 1 or Pmx inhibited colony formation with dose and time dependence. Transport of [(3)H]compound 1 into R1-11-PCFT4 and HepG2 cells was optimal at pH 5.5 but appreciable at pH 6.8. At pH 6.8, [(3)H]compound 1 was metabolized to (3)H-labeled polyglutamates. Glycinamide ribonucleotide formyltransferase (GARFTase) in R1-11-PCFT4 cells was inhibited by 1 at pH 6.8, as measured by an in situ GARFTase assay, and was accompanied by substantially reduced ATP levels. Compound 1 caused S-phase accumulation and a modest level of apoptosis. An in vivo efficacy trial with severe combined immunodeficient mice implanted with subcutaneous HepG2 tumors showed that compound 1 was active. Our findings suggest exciting new therapeutic possibilities to selectively deliver novel antifolate drugs via transport by PCFT over RFC by exploiting the acidic tumor microenvironment.
authors.affiliation: Graduate Program in Cancer Biology, Wayne State University School of Medicine, Detroit, Michigan, USA.
authors.forename: Sita
authors.initials: S
authors.lastname: Kugel Desmoulin
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Eric
authors.initials: E
authors.lastname: Hales
authors.forename: Lisa
authors.initials: L
authors.lastname: Polin
authors.forename: Kathryn
authors.initials: K
authors.lastname: White
authors.forename: Juiwanna
authors.initials: J
authors.lastname: Kushner
authors.forename: Mark
authors.initials: M
authors.lastname: Stout
authors.forename: Zhanjun
authors.initials: Z
authors.lastname: Hou
authors.forename: Christina
authors.initials: C
authors.lastname: Cherian
authors.forename: Aleem
authors.initials: A
authors.lastname: Gangjee
authors.forename: Larry H
authors.initials: LH
authors.lastname: Matherly
mesh_terms: D000818:Animals; D004305:Dose-Response Relationship, Drug; D016503:Drug Delivery Systems / Q000379:methods*; D005260:Female; D005493:Folic Acid Antagonists / Q000008:administration & dosage / Q000378:metabolism*; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D051379:Mice; D008813:Mice, Inbred ICR; D016513:Mice, SCID; D009369:Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D058979:Proton-Coupled Folate Transporter / Q000378:metabolism*; D011743:Pyrimidines / Q000008:administration & dosage / Q000378:metabolism*; D023041:Xenograft Model Antitumor Assays / Q000379:methods
chemical_list: D005493:Folic Acid Antagonists; D058979:Proton-Coupled Folate Transporter; D011743:Pyrimidines; C505058:SLC46A1 protein, human