title: Paclitaxel-2'-Ethylcarbonate prodrug can circumvent P-glycoprotein-mediated cellular efflux to increase drug cytotoxicity.
abstract: OBJECTIVE
  The aim of the study was to investigate whether 2'-ethylcarbonate-linked paclitaxel (TAX-2'-Et) circumvents P-glycoprotein (P-gp)-mediated cellular efflux and cytotoxicity enhanced by TAX-2'-Et activation within human culture cells transfected with a rabbit liver carboxylesterase (Ra-CES) cDNA.
  
  
  METHODS
  TAX-2'-Et transport was characterized in a human colon carcinoma cell line (Caco-2) and paclitaxel (TAX)-resistant ovarian carcinoma cells (SKOV3/TAX60). Expression of P-gp, multidrug resistance protein (MRP) 2 and Ra-CES was detected by Western blotting. Cytotoxicity against Ra-CES-expressing cells and cellular amount of TAX produced were determined by MTT assay and using HPLC, respectively.
  
  
  RESULTS
  Unlike rhodamine123 and TAX, TAX-2'-Et did not exhibit polarized transport in the Caco-2 cells in the absence or presence of verapamil. P-gp levels were expressed much higher in the SKOV3/ TAX60 cells than in the Caco-2 cells. MRP2 protein was not detectable in the SKOV3/TAX60 cells. Uptake by the SKOV3/TAX60 cells was similar in quantity to the amount internalized by P-gp-negative SKOV3 cells. In the SKOV3/TAX60 cells, cellular uptake of TAX-2'-Et was not altered regardless of the absence or presence of verapamil. The cytotoxicity to the untransfected SKOV3 cells induced by TAX-2'-Et was significantly lower than that induced by TAX. In the Ra-CES-expressing SKOV3 line, the EC50 value of TAX (10.6 nM) was approximately four-fold higher than that of TAX-2'-Et (2.5 nM). Transfection of Ra-CES into another TAX-resistant ovarian carcinoma cells (KOC-7c) conferred a high level of TAX-2'-Et cytotoxicity via prodrug activation. The intracellular levels of TAX produced from TAX-2'-Et in the Ra-CES-positive KOC-7c cells significantly increased compared with the levels seen in exposure of the untransfected KOC-7c cells to TAX.
  
  
  CONCLUSIONS
  TAX-2'-Et can circumvent P-gp-associated cellular efflux of TAX. TAX-2'-Et is converted into TAX by the Ra-CES, supporting its potential use as a theoretical GDEPT strategy for cancer cells expressing high levels of P-gp. The TAX-2'-Et prodrug efficiently increased the amount of intracellular TAX, which mediates tumor cell death.
authors.affiliation: School of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502, Japan.
authors.forename: Tadatoshi
authors.initials: T
authors.lastname: Tanino
authors.forename: Akihiro
authors.initials: A
authors.lastname: Nawa
authors.forename: Eisaku
authors.initials: E
authors.lastname: Kondo
authors.forename: Fumitaka
authors.initials: F
authors.lastname: Kikkawa
authors.forename: Tohru
authors.initials: T
authors.lastname: Daikoku
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Tsurumi
authors.forename: Chenhong
authors.initials: C
authors.lastname: Luo
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Nishiyama
authors.forename: Yuki
authors.initials: Y
authors.lastname: Takayanagi
authors.forename: Katuhiko
authors.initials: K
authors.lastname: Nishimori
authors.forename: Seiji
authors.initials: S
authors.lastname: Ichida
authors.forename: Tetsuyuki
authors.initials: T
authors.lastname: Wada
authors.forename: Yasuyoshi
authors.initials: Y
authors.lastname: Miki
authors.forename: Masahiro
authors.initials: M
authors.lastname: Iwaki
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology; D001692:Biological Transport; D015153:Blotting, Western; D018938:Caco-2 Cells; D043182:Carboxylesterase / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D005260:Female; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D015394:Molecular Structure; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism; D017239:Paclitaxel / Q000737:chemistry / Q000493:pharmacokinetics* / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011355:Prodrugs / Q000737:chemistry / Q000493:pharmacokinetics* / Q000494:pharmacology; D011817:Rabbits; D014162:Transfection
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000972:Antineoplastic Agents, Phytogenic; D043182:Carboxylesterase; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D017239:Paclitaxel; D011355:Prodrugs; C000715087:ABCC2 protein, human