The Prostate 67:955 ^ 967 (2007)
`
`The Establishment of Two Paclitaxel-Resistant
`Prostate Cancer Cell Lines and the Mechanisms
`of Paclitaxel Resistance withTwo Cell Lines
`
`Masashi Takeda,1 Atsushi Mizokami,1* Kiminori Mamiya,1 You Qiang Li,1
`Jian Zhang,2 Evan T. Keller,3 and Mikio Namiki1
`1Department of Integrative CancerTherapyand Urology,Kanazawa University Graduate Schoolof
`Medical Sciences,Kanazawa,Ishikawa, Japan
`2Department of Medicine, Division of Hematology/Oncology,University Drive, Pittsburgh
`3Unitfor Laboratory Animal Medicine and Department of Pathology,Universityof Michigan,
`Ann Arbor, Michigan
`
`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 IC50 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. Prostate 67: 955–967,
`2007. # 2007 Wiley-Liss, Inc.
`
`KEY WORDS:
`
`prostate cancer; paclitaxel resistance; MDR-1; cDNA microarray
`
`INTRODUCTION
`
`Prostate cancer (PCa) is the most common malig-
`nancy and the second most frequent cause of cancer-
`related death of men in the United States [1]. Androgen
`deprivation treatment is very effective for more than
`80% of advanced PCa. More than half of those cases of
`advanced PCa become resistant to deprivation treat-
`ment after several years and then several other
`
`*Correspondence to: Atsushi Mizokami, MD, PhD, Department of
`Integrative Cancer Therapy and Urology, Kanazawa University
`Graduate School of Medical Sciences, 13-1 Takara-machi, Kanazawa,
`Ishikawa 920-8640, Japan. E-mail: mizokami@med.kanazawa-u.ac.jp
`Received 24 November 2006; Accepted 14 February 2007
`DOI 10.1002/pros.20581
`Published online 17 April 2007 in Wiley InterScience
`(www.interscience.wiley.com).
`
`ß 2007 Wiley-Liss, Inc.
`
`AVENTIS EXHIBIT 2091
`Mylan v. Aventis, IPR2016-00712
`
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`956
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`Takeda et al.
`
`palliative treatments, such as estramustine phosphate
`(EMP), steroids, are employed for these patients.
`However, the results are very disappointing because
`a half of those cases lead to death within a year or
`2 years.
`Recently, the taxanes [paclitaxel or docetaxel (DTX)]
`with other agents, such as EMP or predonisone have
`been used for hormone-resistant prostate cancer
`(HRPC) and have shown good response [2–5]. Pacli-
`taxel, which is purified from Taxus brevifolia, stabilize
`microtubule and causes apoptosis [6]. The response
`rates of taxane-based combination therapies are better
`than combination therapies with other anticancer
`agents. However, even HRPC treated with paclitaxel-
`based chemotherapy also relapses as occurred using
`other anticancer agents. Then the prognosis of the
`patients after the relapse is extremely poor.
`In order to investigate the mechanisms of paclitaxel-
`resistance, several paclitaxel-resistance cell lines have
`been generated in ovarian cancer, breast cancer, and
`lung cancer [7,8]. Some of major mechanisms of taxane-
`resistance are overexpression of multiple drug resis-
`tance (MDR1), and multidrug resistance protein (MRP)
`family [9]. Especially accumulation of P-glycoprotein
`(P-gp) encoded from MDR1 might cause resistance of
`several drugs in some cancers. The microtubule
`dynamics may also be important for paclitaxel-resis-
`tance because the target of paclitaxel is the microtubule
`[10]. As for the role of bcl-2 as a modulator of paclitaxel
`sensitivity remains controversial. In human paclitaxel-
`resistant hepatocellular carcinoma cells bcl-2 was
`overexpressed [11]. Whereas bcl-2 expression was
`consistently down-regulated in T47-D breast cancer
`cells [12]. In PCa, although Bcl-2/Bcl-xL bispecific
`antisense oligonucleotide also enhanced paclitaxel
`chemosensitivity in PC-3 and LNCaP cells [13,14],
`involvement to paclitaxel-resistance of Bcl-2/Bcl-xL in
`PCa is not clear. Recently, cDNA microarray analyses
`were performed in order to reveal the key genes that are
`related with paclitaxel resistance. Not only MDR-1
`gene but also Rho guanine dinucleotide phosphate
`dissociation inhibitor beta (RhoGDI) and insulin-like
`growth factor-binding protein 3 (IGFBP-3) were up-
`regulated in paclitaxel-resistant ovarian cancer cell
`lines [15]. Villeneuve et al.[16]described that 1.9% of
`1,728 genes were regulated in paclitaxel-resistant MCF-
`7 breast cancer cells. Thus it is very important to know
`the mechanisms of paclitaxel-resistance in PCa.
`In the present study, we established two paclitaxel-
`resistant cell lines from androgen-independent DU145
`and PC-3 PCa cell lines by increasing concentration of
`paclitaxel gradually. Although both cell lines showed
`resistance to paclitaxel over 30 times more than parents
`cells and cross-resistance to other anticancer drugs, the
`mechanism of resistance was different.
`
`The Prostate DOI 10.1002/pros
`
`MATERIALS AND METHODS
`
`Cell Culture and Cell Proliferation Assay
`
`DU145 and PC-3 cells purchased from American
`type culture collection were cultured in Dulbecco’s
`modified Eagle medium (DMEM) and RPMI1640
`containing 5% fetal calf serum (FCS) and penicillin/
`streptomycin (Invitrogen, CA, USA). Cell growth
`inhibition assay was preformed by plating 1 105 cells
`on 6-well plates. Twenty-four hours later, cells were
`treated with the indicated concentration of anticancer
`agents, and cultured for an additional 48 hr. At the end
`of the culture period, the cells were trypsinized and
`counted with a hemocytometer.
`
`Establishment of Paclitaxel-resistant DU145 and
`PC-3 Cell Lines
`
`Paclitaxel-resistant cancer cells were obtained by
`stepwise increased concentrations of paclitaxel. DU145
`and PC-3 cells maintained as described above were
`incubated with 10 nM paclitaxel for 2 days. Then the
`medium was changed to fresh one without paclitaxel
`and cells were cultured cells grow well. Whenever we
`subcultured, the cells were incubated with gradual
`increasing concentration of paclitaxel for 2 days and
`cultured without paclitaxel until cells grow well. Some
`aliquots of the cells were stored whenever we sub-
`cultured it. When cells were killed by increased
`paclitaxel, the aliquots were subcultured again and
`lower concentration of paclitaxel was used for treat-
`ment. Cells that grew at the maximum concentration of
`paclitaxel were stored for further analyses. For main-
`tenance of paclitaxel-resistant cells, 10 nM paclitaxel
`was added into the normal medium every time.
`
`RNA extraction and RT-PCR. Twenty-four hours
`after plating of 1 106 DU145 or PC-3 cells, total RNA
`was purified with RNeasy mini kit (Qiagen, Maryland,
`USA). Complementary DNA (cDNA) was made by
`reverse-transcription (RT) of 1 mg each total RNA using
`ThermoScript RT-PCR system (Invitrogen). Each
`cDNA sample was amplified with ExTaq (Takara,
`Japan). PCR reactions for indicated genes were carried
`out using the following forward (F) and reverse (R) in
`Table I. Each of the amplified PCR products was
`determined by electrophoresis on an 1.5% agarose gel.
`
`Western blot analysis. Twenty-four hours after plat-
`ing 1 106 DU145, DU145-TxR or PC-3, and PC-3-TxR
`cells on 6 cm dishes in DMEM-5% FBS, the cells were
`lysed with 200 ml hypotonic buffer (20 mM Tris-HCl
`(pH 7.6), 10 mM NaCl, 1 mM MgCl2, and 0.5% NP-40)
`and the membrane and cytosol fractions were collected
`by centrifugation as described previously [17]. To
`
`

`
`The Establishment of Two Paclitaxel-Resistant Prostate Cancer Cell Lines
`
`957
`
`TABLE I. The Primers Used for RT-PCR Analysis
`
`Gene
`
`GAPDH
`MDR-1
`MRP-1
`MRP2
`MRP-3
`MRP-4
`MRP-5
`MRP-6
`MRP-7
`Fas
`Caspase-8
`Bcl-2
`Bax
`c-jun
`YB-1
`Sp-1
`
`Forward
`50-GACCACAGTCCATGCCATCA-30
`50-ATGCTCTGGCCTTCTGG ATG GGA-30
`50-GCATGA TCCCTGAAGACGA-30
`50-TAGAGCTGGCCCTTGTACTC-30
`50-CGCCTGTTTTTCTGGTGGTT-30
`50-GCTGAGAATGACGCACAGAA-30
`50-GTCCTGGGTATAGAAGTGTG-30
`50-TTGGATTCGCCCTCATAGTC-30
`50-CTCCCACTGGATCTCTCAGC-30
`50-CAGGCTAACCCCACTCTATG-30
`50-ACTTCAGACACCAGGCAGGGC T-30
`50-ATGTCCAGCCAGCTGCACCTGAC-30
`50- GCTTCAGGGTTTCATCCAGG-30
`50- GGAAA GACCTTCTATGACGATGC -30
`50-GACTGCCATAGAGAATAACCCCAG-30
`50-GCTGCCGCTCCCAACTTACA-30
`
`Reverse
`50-TCCACCACCCTGTTGCTGTA-30
`50-ATGGCGATCCTCTGCTTCTGCCCA C-30
`50-TAGAGCTGG CCCTTGTACTC-30
`50-TCAACTTCCCAGACATCCTC-30
`50-TCCCCCAGTCACAAAGATG -30
`50-TCCCAGCAAGGCACGATATT-30
`50-CAGAAGATCCACACAACCCT-30
`50-TCTTTTGGTCTCAGTGGCCT-30
`50-TCGCATACACGGTGAGGTAG-30
`50-TGGGGGTGCATTAGGCCATT-30
`50-GCCCCTGCATCCAAGTGTGTTC-30
`50-GCAGAGTCTTCAGAGACAGCCAGG-30
`50-AAAGTAGGAGAGGAGGCCGT-30
`50-GAACCCCTCCTGCTCATCTGT CAC-30
`50-CTCTCTAGGCTGTTTTGGGCGAGGA-30
`50-ATCGTGACTGCCTGAGAGCT-30
`
`extract nuclear protein, the centrifuged pellet after
`fraction was lysed with 50 ml
`separating cytosol
`hypertonic buffer (20 mM Tris-HCl (pH 7.6), 0.42 M
`NaCl, 1 mM EDTA, and 0.5% NP-40) and nuclear
`fraction were collected by centrifugation. To extract
`whole cell protein, cells were lysed with hypertonic
`buffer directly. Fifty micrograms of cytosol protein, 50 mg
`of whole cell protein, or 10 mg of nuclear protein was
`loaded in each lane of 7.5 or 12.5% Ready Gel J (Bio-Rad,
`NY), subjected to electrophoresis, then electrotrans-
`ferred to a PVDF membrane (Bio-Rad). The immobi-
`lized proteins were incubated with primary antibody,
`P-gp (rabbit polyclonal IgG, 200-fold dilution; Santa
`Cruz, CA), YB-1 (goat polyclonal IgG, 200-fold dilution;
`Santa Cruz), or GAPDH (rabbit polyclonal IgG, 1,000-
`fold dilution; Trevigen, MD). The presence of primary
`antibody was visualized by Super signal west pico
`luminol/enhancer solution (Pearce, IL).
`
`Methylation analysis of MDR1 promoter. Genomic
`DNA from PC-3, PC-3-TxR, DU145, and DU145-TxR
`was purified using Blood and cell culture DNA mini kit
`(Quiagen) 24 hr after 5 105 cells were plated on 6 cm
`dish. One microgram of DNA was subjected to sodium
`bisulfite modification kit (BisulFast DNA Modification
`Kit, Toyobo, Osaka, Japan). MDR-1 (223 bp) promoter
`region (183 to þ40 of transcription initiation site) was
`amplified from bisulfite-modified DNA as described
`by Enokida et al. [18,19]. The amplified DNA was
`further amplified using methylation-specific primer
`(MSP) or unmethylation-specific primer (USP) after
`100-fold dilution of the amplified DNA [19]. PCR
`reaction was modified to 948C 15 s, 708C 30 s, 728C and
`20 cycles for MSP primers and 948C 15 s, 688C 30 s, 728C
`and 20 cycles for USP primers. Then DNA sequence
`
`The Prostate DOI 10.1002/pros
`
`analysis was also carried out using the amplified 223 bp
`PCR products.
`
`Small interfering RNA transfection. MDR-1 small
`interfering RNA (siRNA),
`lamina/C siRNA, non-
`targeting siRNA were purchased from Dharmacon
`(Lafayette, CO). After 3 104 DU145-TxR and PC-3-
`TxR cells or 3 105 those cells were cultured on 24-well
`plates or in 6-well plates for total RNA purification or
`for protein extraction, respectively, cells were trans-
`fected with 0, 10, 20, or 30 nM MDR-1 siRNA, 30 nM
`lamina/C siRNA, and 30 nM non-targeting siRNA by
`X-treme GENE siRNA Transfection Reagent (Roche).
`Forty-eight hours after transfection, total RNA and
`protein was extracted. In order to see the effect of siRNA
`on drug resistance, cells were transfected with 30 nM
`MDR-1 siRNA or non-targeting siRNA 24 hr after
`plating on 24-well plates. Twenty-four hours later cells
`were treated with 0, 1, 3, 10, 30, 100, 300, and 1,000 nM
`paclitaxel and cultured for 48 hr. Then the cells were
`trypsinized and counted with a hemocytometer.
`
`cDNA Microarray Analysis
`Twenty-four hours after plating of 5 105 PC-3 cells,
`total RNA was purified with RNeasy mini kit (Qiagen,).
`RNA samples were sent to Hokkaido system science
`(Sapporo, Japan) and analyzed by Agilent technologies
`(human 1A microarray kit).
`
`RESULTS
`
`Establishment of Paclitaxel-resistant Cell Lines
`
`When we examined the sensitivity for paclitaxel
`of parent DU145 and PC-3 cells,
`IC50 values of
`these cells were 11.3 and 5.0 nM,
`respectively
`
`

`
`958
`
`Takeda et al.
`
`(Tables II and III). We established paclitaxel-resistant
`DU145 (DU145-TxR) and PC-3 (PC-3-TxR) cells by
`stepwise exposure method (from 10 nM paclitaxel) for
`9 and 15 months, respectively. Cell growth inhibi-
`tion assay demonstrated that these DU145-TxR and
`PC-3-TxR cells become 34.0-fold (IC50: 384.2 nM) and
`43.4-fold (IC50: 217.1 nM) more paclitaxel resistant than
`parent cells (Tables II and III and Fig. 1). We also
`compared the cross-resistance to other anticancer
`drugs [EMP, vinblastin (VBL), doxorubicin (DOX),
`DTX, VP-16, and cisplatin] between parent and
`paclitaxel-resistant cells (Figs. 2 and 3, Tables II
`and III). Both of DU145-TxR and PC-3-TxR cells
`showed almost same cross-resistance to EMP, VBL,
`DOX, and DTX. However, cross-resistance to cisplatin
`and VP-16 was hardly observed.
`
`Expression of Several Potential
`Chemoresistant Genes
`
`Cellular mechanisms of drug resistance include
`in decreasing intracellular drug concentrations by
`increased efflux or decreased influx. The drug distribu-
`tion in an organism is highly dependent on transporters
`which play a role in absorption and elimination. P-gp
`and MRP which belong to the ATP-binding cassettes
`(ABC) family are well-known typical transporters. We
`evaluated the expression of MDR-1 and MRP1 to MRP7
`of DU145-TxR and PC-3-TxR cells by RT-PCR. Only
`MDR-1 mRNA was overexpressed in both cells
`(Fig. 4A). Since MDR-1 mRNA was overexpressed in
`both cells, we confirmed the expression of P-gp which
`
`was encoded from MDR-1 mRNA. P-gp as well as
`MDR-1 mRNA was overexpressed in DU145-TxR and
`PC-3-TxR cells but not in parent cells (Fig. 4B). More-
`over, the level of P-gp in DU145-TxR cells was more
`expressed than PC-3 cells. Since the cell death by
`paclitaxel is associated with apoptosis, we also com-
`pared the expression of major apoptosis-related genes,
`Bcl-2, Bax, Fas, and Capase-8 in these cells. However,
`expression level of all of these genes was not changed
`between parent and resistant cells (Fig. 4C).
`
`Mechanisms of MDR1Overexpresssion in
`DU145-TxR and PC-3-TxRCells
`
`One of mechanisms by which of MDR-1 is over-
`expressed in paclitaxel-resistant cells is the induction
`by Y-box-binding protein 1 (YB-1). YB-1 is mainly
`located in the cytoplasm [20]. Once cells are exposed to
`UV irradiation and anticancer drugs, such as paclitaxel,
`YB-1 tanslocates into nucleus, bind to a cis-acting
`element of the MDR-1 promoter, and induce MDR-1
`mRNA expression [21]. In order to see the nuclear
`localization of YB-1 protein, we performed Western
`blot analysis. The YB-1 protein level in nucleus was
`about three times higher in DU145-TxR cells than in
`DU145 cells and it was almost at the same level between
`PC-3 and PC-3-TxR cells (Fig. 5A). Nuclear localization
`of YB-1 was less dramatic compared to the MDR-1
`expression in paclitaxel-resistant cells.
`Next, we investigated methylation status of CpG
`sites at the MDR1 promoter region because some
`
`Fig. 1. Establishment of paclitaxel-treated cell lines. DU145 (A), paclitaxel-resistant DU145-TxR (B), PC-3 (C), and paclitaxel-resistant
`PC-3-TxR (D) cellswere exposedwithindicatedconcentrations ofpaclitaxel for 24 hr andcounted 2 days afterexposure.
`
`The Prostate DOI 10.1002/pros
`
`

`
`The Establishment of Two Paclitaxel-Resistant Prostate Cancer Cell Lines
`
`959
`
`Fig. 2. Cross-resistance of DU145 and DU145-TxR cells. DU145
`and DU145-TxR cells were exposed with indicated concentrations
`of EMP, docetaxel (DTX), vinblastin (VBL), doxorubicin (DOX), cis-
`platin (CDDP), and etoposide (VP-16) for 24 hr and counted 2 days
`afterexposure.
`
`Fig. 3. Cross-resistance ofPC-3andPC-3-TxRcells.PC-3andPC-
`3-TxRcellswere exposedwithindicatedconcentrations of EMP,doc-
`etaxel(DTX),vinblastin(VBL),doxorubicin(DOX),cisplatin(CDDP),
`andetoposide(VP-16)for24hr andcounted2days afterexposure.
`
`groups reported inverse correlation between methyla-
`tion and MDR1 expression in [19,22,23]. Since DU145-
`TxR and PC-3-TxR cells overexpressed MDR1 mRNA
`compared to parent cells, we expected that paclitaxel-
`resistance might cause demethylation of CpG sites at
`MDR1 promoter. Although MSP published by Enokida
`et al. detected PCR products from bisulfite-modified
`DNA in both parent cells and paclitaxel-resistant cells,
`USP detected stronger PCR band in DU145-TxR cells
`than in DU145 cells, suggesting that MDR1 promoter in
`
`DU145-TxR cells is less methylated than in DU145 cells.
`However, USP did not detect PCR band in PC-3-TxR
`cells compared to PC-3 (Fig. 5B). To further confirm the
`methylated CpG site at
`the MDR1 promoter, we
`performed DNA sequence analysis using bisulfite-
`modified DNA. The MDR1 promoter region of DU145
`cells was methylated at the CpG sites of 134, 105,
`59, 56, 51, 34, and 29 of the transcription
`initiation site. The MDR1 promoter region of DU145-
`TxR cells was methylated only at the CpG site of -105
`
`TABLE II. IC50 Value of DU145 and DU145-TxRCells
`
`TABLE III. IC50 Value of PC-3 and PC-3 -TxRCells
`
`Drug
`
`DU145
`
`DU145-TxR
`
`Fold difference
`
`Drug
`
`PTX (nM)
`EMP (mM)
`DTX (nM)
`VBL (nM)
`DOX (nM)
`VP-16 (mM)
`CDDP (mM)
`
`11.3
`15.1
`8.30
`14.1
`17.5
`0.83
`1.32
`
`384.2
`49.6
`55.6
`40.8
`61.1
`1.10
`1.97
`
`The Prostate DOI 10.1002/pros
`
`34.0
`3.28
`6.70
`2.89
`3.49
`1.33
`1.49
`
`PTX (nM)
`EMP (mM)
`DTX (nM)
`VBL (nM)
`DOX (nM)
`VP-16 (mM)
`CDDP (mM)
`
`PC-3
`
`5.00
`8.57
`3.67
`8.00
`121.3
`4.40
`1.47
`
`PC-3-TxR
`
`Fold difference
`
`217.1
`33.0
`28.2
`27.4
`1,218.2
`5.95
`1.66
`
`43.4
`3.85
`7.68
`2.43
`10.0
`1.35
`1.13
`
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`Takeda et al.
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`Fig. 4. Expressionofvariousdrug-resistance-relatedgenesinparentandpaclitaxel-resistantcells.(A)RT-PCRofMDRandMRP1-7mRNAin
`DU145,DU145-TxR,PC-3,andPC-3-TxRcells.AftermRNAwaspurifiedfromthesecells,RT-PCRwasperformedusingprimersasdescribedin
`Table1. (B) Expression ofP-gp.Cellswere culturedfor12 hin thepresence ofindicatedconcentration ofDHTor Adiolandharvested.Membrane
`and cytosol protein were extracted as described in Materials and Methods and loaded on an 7.5% SDS-polyacrylamide gel for Western blot
`analysis. After protein was transferred to PVDF-membrane, anti-P-gp antibody and anti-GAPDH antibody were employed for detection of
`170 kDa P-gp and 37 kDa GAPDHprotein, respectively. (C) RT-PCR of bcl-2, Bax, Fas, and capase-8 mRNA in DU145, DU145-TxR,PC-3, and
`PC-3-TxR cells.
`
`(data not sown). Especially, the important region for
`MDR1 transcriptional regulation that included a G-box
`(59, 56, and 51) [24] was demethylated in DU145-
`TxR cells (Fig. 5C). This demethylation of MDR1
`promoter in DU145-TxR cells was coincident with the
`enhanced MDR1 expression. Whereas DNA sequence
`analysis of the amplified PCR product showed that the
`MDR1 promoter regions of PC-3 and PC-3-TxR cells
`were methylated at the CpG sites of 134, 110, 59,
`51, 34, and 29 and at the CpG sites of, 110, 105,
`59, 56, 51, and 29, respectively. Much difference
`was not observed in the methylated sites and the
`number between PC-3 and PC-3-TxR promoter region.
`
`Recovery of Paclitaxel Sensitivity by MDR-1
`Knockdown
`
`In order to investigate if MDR-1 mRNA overexpres-
`sion in TxR cells is the main cause of paclitaxel
`resistance, we knocked-down the MDR-1 mRNA by
`MDR-1 siRNA. Ten to thirty nanometer MDR-1 siRNA
`down-regulated MDR-1 mRNA in DU145-TxR and PC-
`3-TxR cells 48 h after transfection (Fig. 5A and C). Non-
`targeting siRNA and laminin siRNA failed to inhibit
`MDR-1 mRNA expression. MDR-1 mRNA down-
`regulation by MDR-1 siRNA treatment also inhibited
`the expression of P-gp protein.
`Since MDR-1 siRNA down-regulated P-gp, we
`confirmed if MDR-1 down-regulation could restore
`
`The Prostate DOI 10.1002/pros
`
`paclitaxel sensitivity. As shown in Table IV and
`Figure 5B and D, IC50 of in parent DU145 and PC-3
`cells was not changed when non-target (NT) siRNA or
`MDR-1 siRNA was transfected. Transfection with
`MDR-1 siRNA into DU145-TxR cells after 48 hr restored
`paclitaxel sensitivity compared to transfection with NT
`siRNA (Fig. 6B). IC50 of paclitaxel of DU145-TxR was
`reduced from 537.9 nM to 60.8 nM and recovery ratio
`became 88.7% 48 hr after transfection (Table IV).
`Whereas transfection with MDR-1 siRNA into PC-3-
`TxR cells hardly changed paclitaxel sensitivity. IC50 of
`paclitaxel of PC-3-TxR was reduced only from 198.4 nM
`to 140.6 nM and recovery ratio became 229.1% (Table IV
`and Fig. 6D) sensitivity.
`Mechanisms of Paclitaxel Resistance in
`PC-3-TxRCells
`
`Although P-gp overexpression played important
`role on paclitaxel resistance in DU145-TxR cells, this
`was not an important factor in PC-3-TxR cells. There
`should be P-gp-independent pathway to become
`paclitaxel-resistance. In order to identify the genes that
`are associated with on paclitaxel resistance in PC-3-TxR
`cells, we performed cDNA microarray using mRNA
`from parent PC-3 and PC-3-TxR cells and compared
`differentially expressed genes as described in Materials
`and Methods. Approximately 15,000 genes were
`screened by microarray analysis. two hundred and
`one (1.34%) of screened genes were induced more than
`
`

`
`The Establishment of Two Paclitaxel-Resistant Prostate Cancer Cell Lines
`
`961
`
`Fig. 5. Expression of YB-1protein and methylation status of MDR1promoter. (A) Western blotting of YB-1protein.Whole cell protein and
`nuclearproteinwereextractedasdescribedinMaterialsandMethodsandloadedona12.5%SDS-polyacrylamidegelfor Westernblotting.After
`protein was transferred to PVDF membrane, anti-YB-1or GAPDH antibody was employed for detection of 35.4 or 37 kDaYB-1or GAPDH
`protein, respectively. B: Detection of methylated and unmethylated promoter of MDR1 genes. USP and MSP were employed for detection
`of unmethylated and methylated MDR1 promoter after the 223 bp MDR1 promoter region was amplified from bisulfite-modified DNA.
`C: Bisulfite-modified DNA sequence of MDR1promoter.The sequences of bisulfite-modified MDR1promoter regions from DU145, DU145-
`TxR,PC-3, and PC-3-TxR cells were shown from65 to21of transcriptioninitiation site.Underlines anddoubleunderline showmethylated
`CpG sites and G-box, respectively.
`
`two-fold and 218 (1.45%) of genes were reduced more
`than two-fold in PC-3-TxR cell line compared with
`parent PC-3 cell line. Tables V and VI describe the major
`30 genes that showed up-regulated and down-regu-
`lated expression in PC-3-TxR cells compared with PC-3
`cells. As we confirmed in Figure 4, MDR-1 genes was
`
`up-regulated to 6.0-fold in PC-3-TxR cells. Some
`microtubule-related genes, tubuline b6, b2, and b4,
`were up-regulated to 3.5-, 2.2-, and 2.1-fold in PC-3-TxR
`cells, respectively. Calcium is an important factor
`that is associated with microtubule polymerization.
`Calcium-binding protein, S100A9 and S100A8 were
`
`TABLE IV. IC50 Value of Paclitaxel in iMDR-1-Transfected TxRCells
`
`Transfected cells
`
`IC50 (nM)
`
`Relative resistant ratio
`
`Recovery ratio
`
`DU145 (NT siRNA)
`DU145 (iMDR-1)
`DU145-TxR (NT siRNA)
`DU145-TxR (iMDR-1)
`PC-3 (NT siRNA)
`PC-3 (iMDR-1)
`PC-3-TxR (NT siRNA)
`PC-3-TxR (iMDR-1)
`
`9.74
`9.11
`537.9
`60.8
`10.5
`10.0
`198.4
`140.6
`
`1.0
`0.94
`55.2
`6.24
`1.0
`0.95
`18.9
`13.4
`
`6%
`
`88.7%
`
`5%
`
`29.1%
`
`The Prostate DOI 10.1002/pros
`
`

`
`962
`
`Takeda et al.
`
`Fig. 6. PaclitaxeliniMDR-1transfectedTxRcells.Aand C:Forty-eighthoursafter transfectionwith0,10,20,or30nMMDR-1siRNA,30nM
`LaminA/C siRNA (La), and 30 nM non-targeting siRNA (La), total RNA and protein was extracted according to the Materials and Methods.
`B and D:In order to see the effectof siRNAondrugresistance, cellswere transfectedwith 30 nMMDR-1siRNAornon-targeting siRNA 24 hr
`afterplatingon24-wellplates.Twenty-fourhoursafter transfectionwith30nMnon-targetingiRNAoriMDR-1,cellswere treatedwith0,1,3,10,
`30,100, 300, and1,000 nM paclitaxel and cultured for 48 hr.Then the cells were counted with a hemocytometer.The data represent mean of
`triplicate experiments and thebars show SD.The datawere describedinTable1V.
`
`down-regulated to 4.34- and 2.56-fold in PC-3-TxR
`cells, respectively. Other calcium-related genes, tumor-
`associated calcium signal transducer 1 (TACSTD1),
`S100P, and S100A2 mRNA were also down-regulated
`in PC-3-TxR cells. MMP-1 that is related with cancer
`invasion is overexpressed in multiple drug-resistant
`cell lines [25]. We also observed overexpression of
`MMP-1 in PC-3-TxR cells (4.77-fold).
`
`DISCUSSION
`
`In order to elucidate the mechanisms of paclitaxel -
`resistant in hormone refractory PCa, we established
`two paclitaxel-resistant cell
`lines from androgen-
`independent cell lines. Several potential mechanisms
`have been proposed for resistance to taxans. The result
`that cross-resistance to cisplatin and VP-16 was not
`observed in both paclitaxel-resistant cell lines indicates
`that resistance to paclitaxel is resulted from different
`pathways from resistance to cisplatin and VP-16.
`Although paclitaxel induces apoptosis, we could not
`detect differences of expression in apoptosis-related
`genes, such as bcl-2, bax, caspase 8 between parent cells
`
`The Prostate DOI 10.1002/pros
`
`and TxR cells. One of major mechanisms of paclitaxel-
`resistance is overexpression of P-gp [9]. The MDR-1
`overexpression was the important factor as a respon-
`sible gene when DU145 cells became paclitaxel resis-
`tance. Since MDR-1 siRNA almost restored paclitaxel
`sensitivity in DU145-TxR cells, P-gp overexpression is
`the main reason of paclitaxel resistance in this cell line.
`Our results showed that one of main mechanisms by
`which of MDR-1 was overexpressed in paclitaxel-
`resistant DU145 cells was the demethylation of CpG
`sites at the MDR1 promoter region. Originally CpG
`sites at the MDR1 promoter region in parent DU145
`cells were hypermethylated [19]. Because it is rare, as
`for the necessity of MDR1, expression of MDR1 is
`inhibited for cancer cell by methylation of MDR1
`promoter. However, when cells can leave damage by
`paclitaxel, demethylation of MDR1 promoter, espe-
`cially G-box that includes Sp1-binding site and EGR-1-
`binding site and is very important for transcription [24],
`is promoted and induces expression of MDR1 so that
`cell themselves survives it, then cells may be going to
`remove paclitaxel
`from intracellular. However,
`it
`remains unclear why PC-3-TxR cells overexpressed
`
`

`
`The Establishment of Two Paclitaxel-Resistant Prostate Cancer Cell Lines
`
`963
`
`TABLE V. List of Genes which were Overexpressed in PC-3-TxRCells
`
`Systematic
`name
`
`PC-3-TxR
`signal
`
`PC-3
`signal
`
`Fold
`change
`
`Description
`
`704
`5,902
`10,609
`2,586
`22,410
`10,946
`2,786
`821
`4,353
`
`2,058
`1,121
`
`557
`31,443
`9,899
`20,402
`21,336
`
`893
`2,295
`26,713
`25,746
`2,557
`
`24,317
`1,953
`776
`14,523
`776
`32,764
`3,952
`4,652
`
`3,036
`8,897
`1,507
`6,677
`395
`10,368
`540
`3,167
`7,643
`15,949
`530
`2,548
`488
`6,500
`553
`2,554
`874
`10,124
`
`97
`980
`1,991
`486
`4,403
`2,293
`644
`199
`1,068
`
`508
`286
`
`148
`8,611
`2,791
`5,787
`6,056
`
`262
`705
`8,310
`8,094
`815
`
`7,812
`647
`259
`5,115
`274
`11,683
`1,425
`1,685
`
`1,101
`3,302
`565
`2,517
`149
`3,935
`207
`1,228
`2,966
`6,219
`207
`998
`192
`2,560
`218
`1,008
`348
`4,042
`
`7.23
`6.02
`5.33
`5.32
`5.09
`4.77
`4.33
`4.12
`4.08
`
`4.05
`3.92
`
`3.76
`3.65
`3.55
`3.53
`3.52
`
`3.41
`3.26
`3.21
`3.18
`3.14
`
`3.11
`3.02
`2.99
`2.84
`2.83
`2.80
`2.77
`2.76
`
`2.76
`2.69
`2.66
`2.65
`2.64
`2.64
`2.61
`2.58
`2.58
`2.56
`2.56
`2.55
`2.54
`2.54
`2.53
`2.53
`2.51
`2.50
`
`Tensin
`ATP-binding cassette, subfamily B (MDR/TAP)
`Laminin, alpha 4 (LAMA4)
`Immunoglobulin superfamily, member 4
`cDNA FLJ35427 fis, clone SMINT2001731
`Matrix metalloproteinase 1 (interstitial collagenase)
`Tissue inhibitor of metalloproteinase 4
`Autism susceptibility candidate 2
`Phospholipase A2, group VII (platelet-activating
`factor acetylhydrolase, plasma)
`Roundabout homolog 4, magic roundabout (Drosophila)
`Interleukin 1 receptor-like 1 (IL1RL1), transcript
`variant 1
`POU domain, class 4, transcription factor 3
`Solute carrier family 35, member F2
`Frizzled homolog 4 (Drosophila) (FZD4)
`Tubulin beta MGC4083
`cDNA FLJ35180 fis, clone PLACE6014882, similar to
`Tissue Factor pathway inhibitor 2
`Choline kinase
`PFTAIRE protein kinase 1
`BCL2/adenovirus E1B 19 kDa interacting protein 3
`Hypothetical protein FLJ25436
`cDNA FLJ32212 fis, clone PLACE6003399, weakly
`similar to SPIDROIN 1
`Hypothetical protein MGC2574
`Hypothetical protein BC013767
`Hypothetical protein MGC10981
`Protease, serine, 11 (IGF binding)
`Dihydropyrimidinase-like 4
`G protein-coupled receptor 56
`BCL2-associated athanogene 3
`Sterol-C5-desaturase (ERG3 delta-5-desaturase
`homolog, fungal)-like
`FLJ26016 protein (FLJ26016)
`Mouse mammary turmor virus receptor homolog 1
`G protein-coupled receptor 4
`Hypothetical protein DKFZp566J2046
`Hypothetical protein FLJ37078
`Polymerase (DNA-directed), alpha (70kD)
`cDNA FLJ12555 fis
`Hypothetical protein MGC16291
`Endothelial cell-specific molecule 1
`Cell division cycle associated 5
`Diacylglycerol O-acyltransferase homolog 2
`cDNA FLJ46182 fis
`cDNA FLJ12555 fis, clone NT2RM4000764
`Endothelial cell-specific molecule 1
`cDNA FLJ12555 fis, clone NT2RM4000764
`cDNA DKFZp762C186
`Fanconi anemia, complementation group A
`Endothelial cell-specific molecule 1 (ESM1), mRNA
`
`Gene
`name
`
`TNS
`ABCB1
`LAMA4
`IGSF4
`CD33L3
`MMP1
`TIMP4
`AUTS2
`PLA2G7
`
`ROBO4
`IL1RL1
`
`POU4F3
`SLC35F2
`FZD4
`MGC4083
`TFPI2
`
`CHKA
`PFTK1
`BNIP3
`C14orf149
`H19
`
`NM_022648
`NM_000927
`NM_002290
`NM_014333
`AK092746
`NM_002421
`NM_003256
`NM_015570
`NM_005084
`
`NM_019055
`NM_016232
`
`NM_002700
`NM_017515
`NM_012193
`NM_032525
`AK092499
`
`NM_001277
`NM_012395
`NM_004052
`NM_144581
`AK056774
`
`NM_024098
`MGC2574
`LOC114990 NM_138440
`MGC10981
`BC004397
`PRSS11
`NM_002775
`DPYSL4
`NM_006426
`GPR56
`NM_005682
`BAG3
`NM_004281
`SC5DL
`BC012333
`
`C10orf125
`MTVR1
`GPR4
`FAHD1
`FLJ37078
`POLA2
`LOC201194
`MGC16291
`ESM1
`CDCA5
`DGAT2
`SLC35F2
`LOC201194
`ESM1
`LOC201194
`EHBP1L1
`FANCA
`ESM1
`
`NM_198472
`NM_152832
`NM_005282
`NM_031208
`NM_153043
`NM_002689
`AK022617
`NM_032770
`NM_007036
`NM_080668
`NM_032564
`AK128062
`AK022617
`NM_007036
`AK022617
`AL834433
`NM_000135
`NM_007036
`
`The Prostate DOI 10.1002/pros
`
`

`
`964
`
`Takeda et al.
`
`TABLE VI. List of Genes which were Repressed in PC-3-TxR
`
`Systematic
`name
`
`PC-3-TxR
`signal
`
`PC-3
`signal
`
`16,873
`7,509
`6,318
`2,886
`1,957
`4,957
`17,878
`930
`10,288
`7,457
`752
`8,099
`1,037
`3,545
`
`15,173
`7,840
`1,774
`16,181
`1,344
`749
`10,029
`2,674
`1,834
`21,027
`6,811
`2,213
`915
`11,624
`931
`594
`8,503
`6,122
`545
`3,076
`2,783
`785
`584
`9,570
`14,847
`1,875
`16,726
`17,552
`4,461
`
`3,109
`7,908
`3,108
`845
`5,074
`1,591
`4,544
`5,680
`
`Fold
`change
`
`17.07
`15.57
`10.69
`9.95
`9.13
`7.92
`6.93
`6.66
`6.46
`5.84
`5.26
`5.08
`5.07
`5.01
`
`4.91
`4.59
`4.55
`4.54
`4.53
`4.53
`4.50
`4.50
`4.47
`4.45
`4.45
`4.39
`4.35
`4.34
`4.31
`4.27
`4.19
`4.19
`4.16
`4.11
`3.99
`3.96
`3.89
`3.88
`3.77
`3.75
`3.74
`3.71
`3.70
`
`3.68
`3.68
`3.65
`3.64
`3.58
`3.54
`3.51
`3.51
`
`Description
`
`Interleukin 23, alpha subunit p19
`Calbindin 1, 28 kDa
`C-terminal tensin-like
`Placenta-specific 8
`Latexin protein
`Cadherin 1, type 1, E-cadherin (epithelial)
`S100 calcium-binding protein A2
`Kallikrein 6 (neurosin, zyme)
`Interleukin 6 (interferon, beta 2)
`Lipocalin 2 (oncogene 24p3)
`Interleukin 13 receptor, alpha 2
`Colony stimulating factor 2 (granulocyte-macrophage)
`CD33 antigen (gp67)
`Serine (or cysteine) proteinase inhibitor, clade B
`(ovalbumin), member 4
`Cytochrome P450, family 1, subfamily B, polypeptide 1
`NADPH oxidase, EF hand calcium-binding domain 5
`Crumbs homolog 3 (Drosophila) (CRB3)
`Normal mucosa of esophagus specific 1
`Platelet-activating factor receptor
`Thrombomodulin
`Spermidine/spermine N1-acetyltransferase
`FXYD domain containing ion transport regulator 6
`Serum amyloid A1 (SAA1)
`Tumor-associated calcium signal transducer 1
`Interleukin 1 family, member 7 (zeta)
`ADMP
`Interferon, alpha-inducible protein 27
`S100 calcium-binding protein A9 (calgranulin B)
`Clone DNA129549 ALGV3072 (UNQ3072)
`Interferon, alpha-inducible protein 27
`Arachidonate 5-lipoxygenase-activating protein
`Amphiregulin (schwannoma-derived growth factor)
`Ankyrin repeat domain 1 (cardiac muscle)
`Secretory granule, neuroendocrine protein 1 (7B2 protein)
`Int