Paclitaxel-2'-Ethylcarbonate prodrug can circumvent P-glycoprotein-mediated cellular efflux to increase drug cytotoxicity.
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.
Tanino
Tadatoshi
T
School of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502, Japan.
Nawa
Akihiro
A
Kondo
Eisaku
E
Kikkawa
Fumitaka
F
Daikoku
Tohru
T
Tsurumi
Tatsuya
T
Luo
Chenhong
C
Nishiyama
Yukihiro
Y
Takayanagi
Yuki
Y
Nishimori
Katuhiko
K
Ichida
Seiji
S
Wada
Tetsuyuki
T
Miki
Yasuyoshi
Y
Iwaki
Masahiro
M
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
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