Dicoumarol enhances doxorubicin-induced cytotoxicity in p53 wild-type urothelial cancer cells through p38 activation.
OBJECTIVE
To investigate the effectiveness of a combined treatment of 3-30-methylene-bis[4-hydroxycoumarin] (dicoumarol) with doxorubicin for the treatment of urothelial cancer, as doxorubicin is a common chemotherapeutic agent but its therapeutic efficacy is limited.


METHODS
The synergistic effect of dicoumarol with chemotherapeutic agents such as cisplatin, doxorubicin and paclitaxel was evaluated in RT112 urothelial cancer cells. Then, dicoumarol-mediated enhancement of doxorubicin-induced cytotoxicity was screened in urothelial cancer cell lines with different p53 statuses or RT112 stable transfectants with a dominant-negative mutant of p53 (p53DN). To clarify the importance of the modification of p53 function by dicoumarol to enhance doxorubicin toxicity, the change in the p53-p21 pathway and mitogen-activated protein kinase (MAPK)-mitochondria pathway by the combined treatment were elucidated by Western blot analysis. Finally, the effect of p21 knockdown in the susceptibility to doxorubicin was examined with RT112 stable transfectants with short hairpin RNA (shRNA) of p21.


RESULTS
Dicoumarol significantly increased the susceptibility of RT112 cells to cisplatin and doxorubicin, but not to paclitaxel in RT112 cells. Dicoumarol (100 microm) also enhanced the cytotoxicity of doxorubicin in other bladder cancer cell lines with wild-type p53 (wt-p53; three times in 253J and 13 times in KK47), but not in those with mutant-type p53 (TCCsup, J82 and EJ) or in RT112 p53DN. The combined treatment with dicoumarol suppressed p53/p21 induction by doxorubicin and resulted in sequential p38 MAPK activation, myeloid cell leukaemia 1 suppression and caspase cleavage. The synergistic effect of doxorubicin/dicoumarol was suppressed by the p38 MAPK inhibitor SB202190 and, furthermore, p21 knockdown with shRNA transfection made RT112 cells six times more susceptible to doxorubicin with p38 MAPK activation.


CONCLUSIONS
These results suggest that concomitant use of dicoumarol could enhance the cytotoxicity of doxorubicin in urothelial cancer cells with wt-p53 through the p53/p21/p38 MAPK pathways. This combined treatment may provide a new therapeutic option to overcome chemoresistance in bladder cancer.
Matsui
Yoshiyuki
Y
Department of Urology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Watanabe
Jun
J
Ding
Sentai
S
Nishizawa
Koji
K
Kajita
Yoichiro
Y
Ichioka
Kentaro
K
Saito
Ryoichi
R
Kobayashi
Takashi
T
Ogawa
Osamu
O
Nishiyama
Hiroyuki
H
D000971:Antineoplastic Combined Chemotherapy Protocols / Q000378:metabolism / Q000627:therapeutic use*; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000008:administration & dosage; D001728:Dicumarol / Q000008:administration & dosage; D004317:Doxorubicin / Q000008:administration & dosage; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D004357:Drug Synergism; D006801:Humans; D015151:Immunoblotting; D017239:Paclitaxel / Q000008:administration & dosage; D015398:Signal Transduction / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy*; D019459:Urothelium; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D016159:Tumor Suppressor Protein p53; D001728:Dicumarol; D004317:Doxorubicin; D048051:p38 Mitogen-Activated Protein Kinases; D017239:Paclitaxel; D002945:Cisplatin