The establishment of two paclitaxel-resistant prostate cancer cell lines and the mechanisms of paclitaxel resistance with two cell lines.
BACKGROUND
Although paclitaxel is used for hormone-resistant prostate cancer, relapse definitely occurs later. Details of the molecular mechanism responsible for paclitaxel- resistance remain unclear.


METHODS
We established paclitaxel-resistant cells, DU145-TxR and PC-3-TxR from parent DU145 and PC-3. To characterize these cells, we examined cross-resistance to other anticancer drugs. Expression of several potential genes that had been related to drug-resistance was compared with parent cells by RT-PCR and Western blotting. Methylation analysis of multiple drug resistance (MDR1) promoter was carried out using bisulfite-modified DNA from cell lines. Knockdown experiments using small interfering RNA (siRNA) were also performed to confirm responsibility of drug-resistance. Finally, cDNA microarray was performed to quantify gene expression in PC-3 and PC-3-TxR cells.


RESULTS
The IC(50) for paclitaxel in DU145-TxR and PC-3-TxR was 34.0- and 43.4-fold higher than that in both parent cells, respectively. Both cells showed cross-resistance to some drugs, but not to VP-16 and cisplatin. Methylation analysis revealed that methylated CpG sites of MDR1 promoter in DU145 and PC-3 cells were demethylated in DU145-TxR cells, but not in PC-3-TxR cells. Knockdown of P-glycoprotein (P-gp), which was up-regulated in resistant cells, by MDR-1 siRNA restored paclitaxel sensitivity in DU145-TxR but not in PC-3-TxR, indicating that up-regulation of P-gp was not always main cause of paclitaxel-resistance. Microarray analysis identified 201 (1.34%) up-regulated genes and 218 (1.45%) out of screened genes in PC-3-TxR.


CONCLUSIONS
Our data will provide molecular mechanisms of paclitaxel-resistance and be useful for screening target genes to diagnose paclitaxel sensitivity.
Takeda
Masashi
M
Department of Integrative Cancer Therapy and Urology, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Ishikawa, Japan.
Mizokami
Atsushi
A
Mamiya
Kiminori
K
Li
You Qiang
YQ
Zhang
Jian
J
Keller
Evan T
ET
Namiki
Mikio
M
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000187:drug effects / Q000235:genetics; D000970:Antineoplastic Agents / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D002460:Cell Line; D019008:Drug Resistance, Neoplasm; D006801:Humans; D008297:Male; D017239:Paclitaxel / Q000494:pharmacology*; D011471:Prostatic Neoplasms / Q000235:genetics / Q000473:pathology; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D000972:Antineoplastic Agents, Phytogenic; D034741:RNA, Small Interfering; D017239:Paclitaxel