Docetaxel maintains its cytotoxic activity under hypoxic conditions in prostate cancer cells

Abstract

Objective:
The efficacy of docetaxel has recently been shown to be increased under hypoxic conditions through the down-regulation of hypoxia-inducible-factor 1α (HIF1A). Overexpression of the hypoxia-responsive gene class III β-tubulin (TUBB3) has been associated with docetaxel resistance in a number of cancer models. We propose that administration of docetaxel to prostate patients has the potential to reduce the hypoxic response through HIF1A down-regulation and that TUBB3 down-regulation participates in sensitivity to docetaxel.

Methods:
The cytotoxic effect of docetaxel was determined in both 22Rv1 and DU145 prostate cancer cell lines and correlated with HIF1A expression levels under aerobic and hypoxic conditions. Hypoxia-induced chemoresistance was investigated in a pair of isogenic docetaxel-resistant PC3 cell lines. Basal and hypoxia-induced TUBB3 gene expression levels were determined and correlated with methylation status at the HIF1A binding site.

Results:
Prostate cancer cells were sensitive to docetaxel under both aerobic and hypoxic conditions. Hypoxic cytotoxicity of docetaxel was consistent with a reduction in detected HIF1A levels. Sensitivity correlated with reduced basal and hypoxia-induced HIF1A and TUBB3 expression levels. The TUBB3 HIF1A binding site was hypermethylated in prostate cell lines and tumor specimens, which may exclude transcription factor binding and induction of TUBB3 expression. However, acquired docetaxel resistance was not associated with TUBB3 overexpression.

Conclusion:
These data suggest that the hypoxic nature of a tumor may have relevance as regard to their response to docetaxel. Further investigation into the nature of this relationship may allow identification of novel targets to improve tumor control in prostate cancer patients.

Full text

Original article
Docetaxel maintains its cytotoxic activity under hypoxic conditions in
prostate cancer cells
夡
James C. Forde, M.D.
a,b
, Antoinette S. Perry, Ph.D.
a
, Kevin Brennan, B.Sc.
a
,
Lynn M. Martin, B.Sc.
a
, Mark P. Lawler, Ph.D.
c
, Thomas H. Lynch, M.D.
b
,
Donal Hollywood, Ph.D.
a
, Laure Marignol, Ph.D.
a,
*
a
Prostate Molecular Oncology Research Group, Academic Unit of Clinical and Molecular Oncology, Institute of Molecular Medicine,
St. James’s Hospital and Trinity College Dublin, Dublin, Ireland
b
Department of Urology, St. James’s Hospital, Dublin, Ireland
c
Department of Haematology and Academic Unit of Clinical and Molecular Oncology, Institute of Molecular Medicine,
St. James’s Hospital and Trinity College Dublin, Dublin, Ireland
Received 25 June 2010; received in revised form 17 August 2010; accepted 18 August 2010
Abstract
Objective: The efficacy of docetaxel has recently been shown to be increased under hypoxic conditions through the down-regulation of
hypoxia-inducible-factor 1
␣
(HIF1A). Overexpression of the hypoxia-responsive gene class III
␤
-tubulin (TUBB3) has been associated with
docetaxel resistance in a number of cancer models. We propose that administration of docetaxel to prostate patients has the potential to
reduce the hypoxic response through HIF1A down-regulation and that TUBB3 down-regulation participates in sensitivity to docetaxel.
Methods: The cytotoxic effect of docetaxel was determined in both 22Rv1 and DU145 prostate cancer cell lines and correlated with
HIF1A expression levels under aerobic and hypoxic conditions. Hypoxia-induced chemoresistance was investigated in a pair of isogenic
docetaxel-resistant PC3 cell lines. Basal and hypoxia-induced TUBB3 gene expression levels were determined and correlated with
methylation status at the HIF1A binding site.
Results: Prostate cancer cells were sensitive to docetaxel under both aerobic and hypoxic conditions. Hypoxic cytotoxicity of docetaxel was consistent
with a reduction in detected HIF1A levels. Sensitivity correlated with reduced basal and hypoxia-induced HIF1A and TUBB3 expression levels. The
TUBB3 HIF1A binding site was hypermethylated in prostate cell lines and tumor specimens, which may exclude transcription factor binding and induction
of TUBB3 expression. However, acquired docetaxel resistance was not associated with TUBB3 overexpression.
Conclusion: These data suggest that the hypoxic nature of a tumor may have relevance as regard to their response to docetaxel. Further
investigation into the nature of this relationship may allow identification of novel targets to improve tumor control in prostate cancer
patients. © 2010 Elsevier Inc. All rights reserved.
Keywords: Hypoxia; Prostate cancer; Docetaxel; TUBB3; HIF1A
1. Introduction
Hypoxia is progressively emerging as a common feature
of prostate tumors. Evidence of tumor hypoxia in the pros-
tate gland has been documented through detection of mo-
lecular markers of hypoxia by immunomolecular imaging
and physical measurements [1– 4]. Tumor hypoxia is pro-
gressively associated with reduced oxidative defense,
genomic instability, apoptosis resistance, and may be asso-
ciated with the transition to androgen-independence in pros-
tate cancer [5]. Many conventional anticancer drugs require
oxygen for maximal activity [6]. However, changes in cel-
lular phenotype following hypoxic shock may also partici-
pate in the reduced cytotoxic properties of anti-cancer
agents. While hypoxia activates a variety of cellular mes-
sengers, hypoxia-inducible-factor-1 (HIF1A) is the only
transcription factor truly regulated by oxygen. It is the
binding of this heterodimer (HIF1A, HIF-1
␤
) to hypoxia
response elements located in the promoter region of target
genes, along with a variety of transcription factors (e.g.,
夡
The following organizations are acknowledged for support: Cancer
Research Ireland, Higher Education Authority Program for Research in
Third Level Institutions, Trinity College Dublin, St. Luke’s Institute for
Cancer Research, and the Prostate Cancer Research Consortium.
* Corresponding author. Tel.: ⫹353-1-896-3255; fax: ⫹353-1-896-3246.
E-mail address: marignol@tcd.ie (L. Marignol).
Urologic Oncology: Seminars and Original Investigations xx (2010) xxx
1078-1439/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.urolonc.2010.08.015

p300/CBP, STAT3) that dictates the hypoxia-induced cel-
lular response [7].
HIF1A overexpression has been reported to occur in almost
70% of all human tumors including primary and metastatic pros-
tate cancer and their metastases [8]. While HIF1A overexpression
has been associated with increased drug-resistance [9], clinically
relevant microtubule-targeting agents (MTA) were recently
shown to down-regulate HIF1A protein levels and activity [10],
increasing the sensitivity of tumor cells to these agents [11–13].
Docetaxel is a MTA currently used for the standard of care first
line chemotherapeutic agent for the treatment of hormone refrac-
tory prostate cancer [14]. Its effect is however limited by intoler-
ance and the development of taxane-refractory tumors [15]. The
mechanism of preferential sensitivity of prostate tumors to do-
cetaxel and this associated acquired docetaxel-resistance remains
poorly understood.
Overexpression of Class III
␤
-tubulin (TUBB3) has been
associated with taxane resistance in melanoma [16], pan-
creatic [17], ovarian [18] and head and neck cancers [19].
This naturally occurring mutant form of tubulin prevents
pro-assembly activity of taxanes on microtubules, thereby
reducing their cytotoxic activity. Up-regulation of TUBB3
has been reported in response to hypoxic exposure via
HIF1A [20]. Recently it was reported that CpG methylation
within the HIF1A response element of the TUBB3 gene
blocked transcription factor binding and resulted in down-
regulation in gene expression [20]. Hypomethylation of the
site was reported in breast and ovarian cancer cell lines,
which permitted binding of HIF1A and up-regulation of
gene expression. However, paclitaxel resistant cells were
largely hypermethylated at this site, suggesting that an in-
crease in TUBB3 expression upon hypoxia is abolished
through hypermethylation of the 3=enhancer [20].
We propose that changes in phenotype following hypoxic
shock may participate in preferential sensitivity of prostate cancer
cells to docetaxel. We first determined the sensitivity of 2 prostate
cancer cell lines in vitro to docetaxel. Activity was correlated with
HIF1A gene and protein expression. We next investigated the
potential role of TUBB3 down-regulation in sensitivity to do-
cetaxel and the mechanism of acquired docetaxel resistance. Our
results indicate that prostate cancer cells are sensitive to docetaxel
under both aerobic and hypoxic conditions. This intrinsic sensi-
tivity to docetaxel appears to correlate with HIF1A down-regula-
tion in hypoxic tumors cells and reduced TUBB3 basal expression
levels. Finally, we report for the first time evidence of TUBB3
hypermethylation in prostate cancer cell lines and prostate tumor
specimens.
2. Materials and methods
2.1. Cell culture and growth conditions
Normal human prostate cell lines PWR-IE and RPWE-1
and human prostate cancer cell lines DU145, 22Rv1, and
LnCaP were obtained from the ATCC (Teddington, UK).
Frozen stocks were prepared within 2 wk of growth and to
ensure authenticity of the lines. Age-matched docetaxel-
sensitive (PC3) and docetaxel-resistant (PC3-D12) cell lines
were kindly provided by Professor Watson, University Col-
lege Dublin, Ireland. The lines were maintained in RPMI
1640 medium (Gibco, Paisley, UK) supplemented with 10%
fetal calf serum (Globepharm, Guildford, UK) and 1%
streptomycin-penicillin (Gibco). Human RC58/T prostate
cancer cells were kindly provided by Professor Rhim, Cen-
ter for Prostate Disease, Bethesda, MD. These cells, along
with normal human prostate cell lines PWR-IE and
RPWE-1, were routinely maintained in keratinocyte SFM
medium (Gibco) supplemented with bovine pituitary ex-
tract, recombinant epidermal growth factor, and 1% strep-
tomycin-penicillin (Gibco). DNA was extracted from cell
lines using a QIAamp DNA mini kit (Qiagen, Crawley,
UK). The lines were grown for a maximum of 10 week and
monitored for mycoplasma on a regular basis. The “pros-
tatic” nature of the lines was confirmed by measuring PSA
expression (data not shown). Hypoxia (0.5% O
2
,pO
2
⬍2
mmHq) was achieved by exposing cells in a 1000 in vivo
hypoxic chamber (BioTrace, Bracknell, UK). The cells
were exposed to a mixture of nitrogen, CO
2
(5%) and
compressed air to achieve a 0.5% oxygen concentration.
pO
2
was monitored with an oxygen probe (OxyLab pO
2
;
Oxford Optronix, Abingdon, UK). Docetaxel, paclitaxel, and
vincristine (Sigma-Aldrich, Poole, UK) were dissolved in
ethanol to a concentration of 1 mM/mL.
2.2. Cell viability assays
Human prostate cancer cells (2.5 ⫻10
4
cells/well) were
seeded into 96-well plates prior to a 48 h treatment with
increasing docetaxel concentrations (0.1, 1, 10, 100, nM).
The sensitivity of the cells was determined using the 96-non
radioactive MTT reagent (Promega, UK) according to man-
ufacturer’s instructions. Cell viability in the treated plates
was compared with that measured in untreated cells to
calculate the surviving fraction.
2.3. Clonogenic assays
Cell survival was evaluated using a standard colony-
forming assay. 1,000 –10,000 cells/well were plated onto
6-well plates prior to a 48 h chemotherapeutic treatment
under aerobic or hypoxic conditions. Two weeks later, the
plates were stained (70% ethanol, Cristal violet; Sigma-
Aldrich, Poole, UK) and the colonies were counted. The
response of aerobic cells was used as a control. The plating
efficiency was calculated as the ratio of the number of
colonies counted over the number cells. The surviving frac-
tion in the treated wells was subsequently calculated as the
ratio of the number of clones counted over the number of
cells plated corrected with the appropriate plating effi-
ciency.
2J.C. Forde et al. / Urologic Oncology: Seminars and Original Investigations xx (2010) xxx

2.4. RNA isolation and quantitative RT-PCR analysis
Quantification of HIF1A and TUBB3 mRNA levels was
performed in triplicate by a real-time fluorescence detection
method as described previously [21]. In brief, after RNA
isolation with an RNeasy Mini Kit (Qiagen, Valencia, CA),
2
␮
g of total RNA was converted to cDNA with a first
strand high capacity cDNA reverse transcription kit (Ap-
plied Biosystems Ltd., Warrington, Cheshire, UK). The
HIF1A and TUBB3 genes and endogenous control gene
(PGK1) were -amplified separately using TaqMan real-time
PCR (Applied Biosystems). Relative gene expression was
determined by applying the arithmetic formula 2-⌬⌬ CT.
2.5. Western blot analysis
HIF1A protein expression was determined in whole cell
lysates of aerobic and hypoxic 22Rv1 and DU145 cells
treated with docetaxel or 5-fluorouracil. The cells were
scraped under hypoxic conditions and stored on ice. The
pellet was resuspended in lysis buffer [22]. Protein was
extracted, subjected to polyacrylamide gel electrophoresis,
and transferred to nylon/nitrocellulose membranes. The
membranes (Amersham, Little Chalfont, UK) were then
probed with anti-HIF1A primary antibody (Cell Signaling
Technologies, Hitchin, UK, 1:1000 dilution) and a second-
ary antibody; polyclonal goat anti-rabbit IgG HRP-linked
antibody (Cell Signaling. 1:1,000 dilution). The Pierce Lu-
minal kit (Pierce, Northumberland, UK) was used for pro-
tein detection. Membranes were stripped prior to reprobing
with a mouse monoclonal anti-actin antibody (1:10,000,
Sigma-Aldrich, Poole, UK).
2.6. Prostate tissue specimens
CaP tissue specimens (n⫽8) from men undergoing radical
prostatectomy for primary prostate cancer were obtained through
the histopathology archive dating from 1999 to 2006 at the Ad-
elaide and Meath incorporating the National Children’s Hospital,
as described previously [23]. Genomic DNA was extracted using
a RecoverAll Total Nucleic Acid Isolation Kit (Ambion Inc.,
Austin, TX) according to manufacturer’s instructions and quanti-
fied using a Nanodrop-1000 spectrophotometer (Labtech Interna-
tional, Ringmer, UK).
2.7. TUBB3 methylation
Hypermethylation of the putative HIF1
␣
response ele-
ment located within a 3=enhancer region of the TUBB3
gene was analyzed by pyrosequencing. Genomic DNA iso-
lated from tissue specimens (50 ng) and cell lines (500 ng;
by use of a QIAamp DNA blood minikit (Qiagen)) was
bisulfite modified using the EZ DNA methylation kit (Zymo
Research, Orange, CA) and eluted into 50
␮
l1⫻TE buffer.
The CpGenome Universal Methylated DNA (Chemicon In-
ternational, Temecula, CA) was employed as a positive
methylated control. The EpiTect unmethylated DNA
(Qiagen, UK) was used as an unmethylated DNA control.
Bisulfite-treated DNA was PCR amplified for 45 cycles
with a biotinylated primer using the PyroMark PCR kit
(Qiagen, UK) in a final volume of 25
␮
l. Forward primer:
5=-BIOTIN-agggtttttttgtCGtttttttgtagtat-3=; reverse primer:
5=-aaatatcccctaaaatataaacacaaaccaat-3=. The PCR prod-
uct (20
␮
l) was immobilized on streptavidin sepharose
beads (GE Healthcare, Little Chalfont, UK), washed, and
denatured using the pyrosequencing vacuum prep tool
(Qiagen, UK), according to the manufacturer’s guidelines.
Then, 0.3
␮
⌴pyrosequencing primer (5=-tgtgagttgtttttgt-3=)
was annealed to the purified single-stranded PCR product
and pyrosequencing was performed using the PyroMark
Q24 system (Qiagen, UK). The degree of methylation was
calculated using the PSQ HS 96A 1.2 software, under the
CpG mode (Qiagen, UK).
2.8. Statistical analysis
All experiments were performed in triplicate. Statistical
analysis was calculated using SPSS software ver. 14.0,
(SPSS Inc., Chicago, IL). Differences in surviving fraction
and relative gene expression were compared using Student’s
t-tests or an analysis of variance (ANOVA). A Pvalue ⬍
0.05 was considered statistically significant. Data are pre-
sented as mean ⫾standard error of the mean.
3. Results
3.1. Sensitivity of prostate cancer cells to docetaxel
We initially generated dose response curves of both
22Rv1 and DU145 cells treated with increasing concentra-
tions of docetaxel. Survival was measured using an MTT
assay (Fig. 1A). The sensitivity of DU145 and 22Rv1 cells
to docetaxel was similar. We next chose a concentration of
1nM for more specific determination of the sensitivity of
each cell line to docetaxel using clonogenic assays (Fig.
1B). DU145 cells were significantly more sensitive to do-
cetaxel than 22Rv1 cells (P⫽0.014).
3.2. Docetaxel maintains its activity in hypoxic prostate
cancer cells
To determine whether docetaxel preferentially main-
tained their cytotoxicity under hypoxic conditions, both cell
lines were treated with 1nM docetaxel for 48 h under aer-
obic or hypoxic conditions. The response of cells to treat-
ment with a non-MTA agent, 5-fluorouracil (5-FU, 100 nM)
was in addition used as a control to determine whether the
response was specific to docetaxel (Fig. 2). Hypoxia-in-
duced chemoresistance was evident in 22Rv1 cells treated
with 5-FU only (P⫽0.04). Hypoxic 22Rv1 and DU145
cells were as sensitive to docetaxel as aerobic controls.
3J.C. Forde et al. / Urologic Oncology: Seminars and Original Investigations xx (2010) xxx

3.3. Sensitivity to docetaxel in hypoxia correlates with
HIF1A down-regulation
We next investigated whether increased sensitivity of
prostate cancer to docetaxel under hypoxic conditions cor-
relates with deregulation of HIF1A protein and gene expres-
sion. 22Rv1 and DU145 cells were treated with either do-
cetaxel (1 nM) or 5-FU (100 nM) for 48 h under both
conditions prior to total protein or mRNA extraction. The
response of aerobic untreated cells was used as a control.
Hypoxia-selective stabilization of the HIF1A protein was
evident in hypoxic cell lysates of both 22Rv1 and DU145
cells (Fig. 3). Docetaxel appeared to down-regulate HIF1A
protein levels in both hypoxic cell lines. The 5-FU treatment
resulted in undetectable HIF1A levels in 22Rv1 cells but
appeared to increase HIF1A stabilization under hypoxic
conditions. The HIF1A gene was down-regulated 5- and
16-fold, respectively, in 22Rv1 and DU145 hypoxic un-
treated cells, compared with aerobic controls. Treatment
with docetaxel did not modify the reduction in HIF1A
expression in both hypoxic lines. In aerobic controls, HIF1A
expression appeared to be up-regulated in response to 5-FU
treatment in 22Rv1 cells and down-regulated in DU145
cells (Fig. 4A, B). Time course experiments confirmed
down-regulation of HIF1A within4hofhypoxic exposure
in both cell lines (Fig. 4C, D). At this time point, the
reduction in HIF1A expression levels was again not modi-
fied in response to both docetaxel and 5-FU treatment in
these hypoxic cells (Fig. 3E, F). At this 4 h time point, in
aerobic controls, both docetaxel and 5-FU appeared to in-
duced an up-regulation of HIF1A expression in 22Rv1 and
down-regulation in DU145 cells.
3.4. Acquired docetaxel resistance is not associated with
hypoxia-induced chemoresistance
We next investigated whether transition to docetaxel
resistance is associated with increased chemoresistance
under hypoxic conditions in an isogenic pair of age-
Fig. 1. Sensitivity of Prostate cancer cells to Docetaxel. Sensitivity of (A)
22Rv1 and (B) DU145 cells treated with increasing concentrations of
docetaxel for 48 h. n⫽3; mean ⫾SEM; *P⬍0.05.
Fig. 2. Docetaxel maintains its cytotoxic activity in hypoxia. Clonogenic
survival of (A) 22Rv1 and (B) DU145 cells treated with 1 nM docetaxel
and 5-FU for 48 h under aerobic or hypoxic conditions. n⫽3; mean ⫾
SEM; *P⬍0.05.
Fig. 3. Treatment with docetaxel down-regulates HIF1A protein expres-
sion. Representative HIF1A and
␤
-actin immunoblots of (A) 22Rv1 and
(B) DU145 cells both treated with 1nM of either docetaxel or 100 nM of
5-FU for 6 h under aerobic and hypoxic conditions, relative to untreated
aerobic and hypoxic controls.
4J.C. Forde et al. / Urologic Oncology: Seminars and Original Investigations xx (2010) xxx

matched PC3 and docetaxel-refractory PC3 lines (PC3-
D12). Both cell lines were treated with docetaxel under
aerobic and hypoxic conditions for 48 h. Clonogenic
survival was measured and compared with that obtained
in cells treated with 2 other MTAs (vincristine and pac-
litaxel) and to a non-MTA, 5-FU (Fig. 5). Age-matched
PC3 cells were significantly more sensitive to docetaxel
than vincristine, paclitaxel, and 5-FU under both aerobic
(ANOVA, P⫽0.0022) and hypoxic (ANOVA, P⬍
0.001) conditions. PC3-D12 cells were significantly more
sensitive to docetaxel than any of the three other drugs
tested under aerobic conditions only (ANOVA P⫽0.04).
Hypoxic treatment was not associated with increased
docetaxel resistance in either of these cell lines (age-
matched PC3, P⫽0.44); PC3-D12, P⫽0.08). PC3-D12
were significantly more resistant to docetaxel than PC3
cells under both aerobic (P⫽0.01) and hypoxic (P⫽
0.001) conditions.
3.5. Sensitivity to docetaxel correlates with
down-regulation of TUBB3
To determine whether basal TUBB3 gene expression cor-
relates with increased intrinsic sensitivity to docetaxel, relative
TUBB3 mRNA levels were measured in aerobic and hypoxic
cells (4 h) treated with docetaxel (1 nM). The response of
aerobic untreated cells was used as a control. Basal TUBB3
expression was down-regulated (2.1-fold) in DU145 compared
with 22Rv1 (Fig. 6A). TUBB3 expression was down-regulated
(2-fold) in response to hypoxic exposure (4 h) in both do-
cetaxel-treated and untreated cells (Fig. 6B, C). To determine
whether this response was docetaxel-specific, TUBB3 expres-
sion was also examined in 22Rv1 and DU145 cells treated with
5-FU (100 nM). Relative TUBB3 mRNA levels were similar to
that of docetaxel treated cells under both aerobic and hypoxic
conditions. To investigate a potential role for TUBB3 in ac-
quired chemoresistance, TUBB3 mRNA levels were next mea-
Fig. 4. Relative quantification of HIF1A mRNA levels. HIF1A gene expression levels were initially measured in (A) 22Rv1 and (B) DU145 cells treated with 1 nM
of either docetaxel or 5-FU for 48 h under aerobic and hypoxic conditions, relative to untreated aerobic controls. Next, the time course of HIF1A gene expression
under hypoxic conditions is presented for (C) 22Rv1 and (D) DU145 cells. Finally, HIF1A gene expression levels were measured in (E) 22Rv1 and (F) DU145 cells
treated with 1nM of either docetaxel or 5-FU for 4 h under aerobic and hypoxic conditions, relative to untreated aerobic controls. n⫽3.
5J.C. Forde et al. / Urologic Oncology: Seminars and Original Investigations xx (2010) xxx

sured in docetaxel resistant (PC3-D12) and age-matched PC3
cells. TUBB3 expression levels appeared elevated in docetaxel-
resistant cells compared with the age-matched control (Fig.
6D).
3.6. Down-regulation of TUBB3 in hypoxia correlates
with hypermethylation of the putative HIF1A response
element
Finally, hypermethylation of a putative HIF1A response ele-
ment within the 3=UTR of the TUBB3 gene was determined in a
panel of normal (PWR1E, RWPE1) and malignant (LNCaP,
PC-3, DU145, 22RV1, and RC58) prostate cell lines (Fig. 7). All
lines tested displayed evidence of hypermethylation, however,
amounts of 5-methylcytosine detected were higher in the cancer
(80% in 22RV1, DU145, and PC-3) than in both normal lines
(50%). The amount of 5-methylcytosine detected in LNCaP
(32.61%) was lower than the other cell lines. Evidence of hyper-
methylation was also observed in all 8 prostate patient tumor
specimens sequenced, although there was variation in the amount
of methylation. Two tumors (T5 and T8) displayed levels similar
to the LNCaP cell line. Culturing of the cells in acute and chronic
hypoxia did not alter the methylation status (results not shown).
4. Discussion
Hypoxia and the stabilization of HIF1A is a known cause
of treatment resistance in solid tumors [24]. Our data con-
firmed that 2 prostate tumor cell lines were sensitive to
treatment with docetaxel. This sensitivity was maintained
under conditions of hypoxia and correlated with taxane-
independent down-regulation of the HIF1A gene in both cell
lines during hypoxic exposure. Relative HIF1A mRNA lev-
els were reduced within4hofhypoxic exposure and were
undetectable at 24 h. This rapid degradation may indicate
the importance of the HIF1A gene in the initial phases of the
hypoxic response. The mechanism behind this rapid down-
regulation was not investigated but may be dependent on
oxygen availability.
We previously reported that HIF1A protein expression in
22Rv1 and DU145 cell peaks between 4 and8hofhypoxic
exposure, and is undetectable following 48 h of exposure
[25]. We now show that after6hinhypoxia, treatment with
docetaxel appears to reduce HIF1A expression in metastatic
DU145 cells to a greater extent than in primary 22Rv1 cells.
Fig. 5. Docetaxel resistance is not associated with hypoxia-induced che-
moresistance. Clonogenic survival of (A) age-matched PC3 and (B) iso-
genic docetaxel-resistant (PC3-D12) cells treated with 1 nM vincristine,
paclitaxel, docetaxel, and 5-FU under aerobic or hypoxic conditions for
48 h. n⫽3; mean ⫾SEM; *P⬍0.05.
Fig. 6. Relative quantification of TUBB3 mRNA levels. Basal TUBB3 gene expression levels were (A) initially measured in DU145 cells, relative to that of
22Rv1 cells; next determined in (B) 22Rv1 and (C) DU145 cells treated with 1 nM of either docetaxel or 5-FU for 4 h under aerobic and hypoxic conditions,
relative to untreated aerobic controls and finally (D) in aged-matched PC3 and isogenic docetaxel-resistant PC3 cells. n⫽3.
6J.C. Forde et al. / Urologic Oncology: Seminars and Original Investigations xx (2010) xxx

This may be due to the mitotic fraction of hormone refrac-
tory prostate cancer cell lines such as DU145, which has
been shown to be less than 2% [10]. Docetaxel has indeed
been proposed to exert a greater effect in metastases of
prostate cancer through inhibition of HIF1A, rather than
anti-mitotic effects in primary tumor systems [10].
Although various authors have described the mecha-
nisms of taxane resistance pathways, there is little evidence
regarding clinical studies of taxane resistance in prostate
cancer [15]. We sought to investigate whether the transition
to docetaxel resistance was associated with increased
chemo-resistance in hypoxia. We used a docetaxel resistant
metastatic cell line (PC3-12) and a docetaxel sensitive age
matched control (PC3). The PC3-D12 cell line was signif-
icantly more resistant to treatment with docetaxel than the
control PC3 line under both aerobic and hypoxic conditions.
Hypoxia was not associated with increased docetaxel resis-
tance. Age matched control PC3 cells were significantly
more sensitive to docetaxel than other MTAs (vincristine
and paclitaxel) as well as the non-taxane control 5-FU in
both aerobic and hypoxic conditions. The mechanisms of
this docetaxel-specific resistance were not investigated. Do-
cetaxel is a synthetic analogue of paclitaxel. The com-
pounds differ in their chemical structure [26] but both com-
pounds exhibit similar mechanisms of action [27].
Docetaxel was demonstrated to be a more potent anti-cancer
drug than paclitaxel in a number of cancer models in vitro
and in vivo (reviewed in [28,29]); possibly via prolonged
intracellular retention [30]. Yet resistance to docetaxel does
not necessarily induce resistance to paclitaxel [31]. Further
research is required to characterize this phenomenon.
Class III
␤
-tubulin (TUBB3) overexpression was pro-
posed to induce taxane resistance by preventing the ability
of the compounds to destabilize microtubules [16 –19].
Down-regulation of TUBB3 expression was associated with
increased taxane sensitivity [16,21].TUBB3 expression was
2-fold lower in the aerobic untreated DU145 cell line com-
pared with the aerobic primary untreated 22Rv1 cells. The
reduced expression of TUBB3 observed in DU145 cells may
contribute to our observation of a greater sensitivity to
treatment with docetaxel in metastatic DU145 cells in clo-
nogenic survival assays. We subsequently measured TUBB3
mRNA levels in cells exposed to4hofhypoxia. Hypoxia
was indeed proposed as a possible inducer of TUBB3 ex-
pression in ovarian and cervical cancer cell lines [20]. Hy-
poxic exposure down-regulated the gene in both untreated
and treated cells and to a greater extent (2-fold) in the
primary 22Rv1 cell line. This down-regulation of TUBB3
expression in primary prostate cell lines is consistent with
the continued sensitivity of hypoxic prostate cells to do-
cetaxel. TUBB3 over-expression, however, did not appear to
play a role in the acquired docetaxel resistance in the PC3-
D12 cell line.
It was recently reported that hypoxia-dependent TUBB3
expression is abolished in taxane-resistant cells through
methylation of the 3=enhancer [17].TUBB3 methylation
may represent a potential pretreatment molecular marker for
preferential response to docetaxel in patients presenting
Fig. 7. TUBB3 hypermethylation at the HIF1A binding site. Hypermethylation of the internal CpG dinucleotide in the putative HIF1A binding site
(5=-acgtg-3=) in the 3=UTR of the TUBB3 gene in benign prostate cell lines (PWR1E and RWPE1) and in prostate cancer cell lines 22RV1, DU145, LNCaP,
PC3, and RC58, and in 8 tumor specimens.
7J.C. Forde et al. / Urologic Oncology: Seminars and Original Investigations xx (2010) xxx

with prostate tumors. CpG methylation within the HIF1A
response element blocked transcription factor binding and
resulted in down-regulation of expression of target gene
TUBB3 [20]. Intriguingly, the response element is not
present in the 5=CpG island, but is located 168 bp into the
3=UTR of the TUBB3 gene, within a downstream enhancer
region. Hypermethylation of this site was evident in a panel
of prostate cell lines and 8 tumor specimens. This is the first
report of TUBB3 hypermethylation in prostate cancer.
These results highlight differential hypermethylation levels
between normal (50%) and malignant (80%) cell lines, with
particularly low amounts of 5-methylcytosine detected in
LNCaP cells (30%). Hypermethylation levels were also
variable in tumor specimens. High levels of hypermethyl-
ation at this site did not correlate with the reduced TUBB3
expression levels in DU145 compared with 22Rv1 cells,
however, the gene was down-regulated in hypoxia in both
cell types. These findings would support further evaluation
of TUBB3 methylation in a larger cohort of patients.
4. Conclusion
Our data suggest that the combination of the hypermeth-
ylation of target genes such as TUBB3 at their HIF1A
binding site with the HIF1A targeting property of docetaxel
may represent a possible expansion in the administration of
docetaxel in the management of prostate cancer.
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