Basic and Translational Science
TRPV2 Activation Induces
Apoptotic Cell Death in Human
T24 Bladder Cancer Cells: A Potential
Therapeutic Target for Bladder Cancer
Takahiro Yamada, Takashi Ueda, Yasuhiro Shibata, Yosuke Ikegami, Masaki Saito,
Yusuke Ishida, Shinya Ugawa, Kenjiro Kohri, and Shoichi Shimada
OBJECTIVES To investigate the functional expression of the transient receptor potential vanilloid 2 (TRPV2)
channel protein in human urothelial carcinoma (UC) cells and to determine whether calcium
inﬂux into UC cells through TRPV2 is involved in apoptotic cell death.
METHODS The expression of TRPV2 mRNA in bladder cancer cell lines (T24, a poorly differentiated UC
cell line and RT4, a well-differentiated UC cell line) was analyzed using reverse transcriptase-
polymerase chain reaction. The calcium permeability of TRPV2 channels in T24 cells was
investigated using a calcium imaging assay that used cannabidiol (CBD), a relatively selective
TRPV2 agonist, and ruthenium red (RuR), a nonselective TRPV channel antagonist. The death
of T24 or RT4 cells in the presence of CBD was evaluated using a cellular viability assay.
Apoptosis of T24 cells caused by CBD was conﬁrmed using an annexin-V assay and small
interfering RNA (siRNA) silencing of TRPV2.
RESULTS TRPV2 mRNA was abundantly expressed in T24 cells. The expression level in UC cells was
correlated with high-grade disease. The administration of CBD increased intracellular calcium
concentrations in T24 cells. In addition, the viability of T24 cells progressively decreased with
increasing concentrations of CBD, whereas RT4 cells were mostly unaffected. Cell death
occurred via apoptosis caused by continuous inﬂux of calcium through TRPV2.
CONCLUSIONS TRPV2 channels in UC cells are calcium-permeable and the regulation of calcium inﬂux
through these channels leads directly to the death of UC cells. TRPV2 channels in UC cells may
be a potential new therapeutic target, especially in higher-grade UC cells. UROLOGY 76:
509.e1–509.e7, 2010. © 2010 Elsevier Inc.
Bladder carcinoma is the second most common
malignancy of the urinary tract and nearly 90% of
all primary tumors of the bladder are urothelial
Currently, intravesical instillation of
bacillus Calmette-Guérin (BCG) is the most effective
and widely used agent for the treatment of superﬁcial
In addition, other agents, including mitomycin C,
have been used to prevent recurrence.
rence after intravesical instillation remains frequent. As a
result, the development of new drugs that target only UC
cells is desirable.
Transient receptor potential (TRP) channels are
-permeable channels that contribute to intracellular
homeostasis. Recently, there has been increasing
evidence for the association of TRP channels with can-
cer. The expression levels of members of the TRP Ca-
nonical (TRPC), Melastatine (TRPM), and vanilloid
(TRPV) families are correlated with the emergence
and/or progression of certain epithelial cancers.
TRPV1 expression has been shown to progressively de-
crease as tumor stage increases and cell differentiation
In contrast, the expression of TRPV2 mRNA
and proteins was enhanced in higher-grade UC speci-
mens and UC cell lines.
Therefore, modulators of the
TRP channels expressed in high-grade UC cells may be
attractive targets for the medical treatment of malignant
Here, we examined whether TRPV2 channels are
functional in 2 UC cell lines: T24, a poorly differentiated
line of UC cells, and RT4, which is well-differentiated.
The ﬁrst two authors contributed equally to this work.
From the Department of Neurobiology and Anatomy, Graduate School of Medical
Sciences, Nagoya City University, Nagoya, Japan; Department of Nephro-urology,
Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan; and
Department of Neuroscience and Cell Biology, Osaka University, Graduate School of
Medicine, Osaka, Japan
Reprint requests: T. Ueda, Department of Neurobiology and Anatomy, Graduate
School of Medical Sciences, Nagoya City University, Kawasumi, Mizuho-cho, Mizuho-
ku, Nagoya, Aichi 467– 8601, Japan. E-mail: firstname.lastname@example.org
©2010ElsevierInc. 0090-4295/10/$34.00 509.e1
All Rights Reserved doi:10.1016/j.urology.2010.03.029
We used both physiological and pharmacologic ap-
proaches. Although there is a lack of selective pharma-
cologic tools speciﬁc to TRPV2, it has been recently
reported that cannabidiol (CBD) may be a relatively
selective TRPV2 agonist.
We therefore used CBD as a
selective TRPV2 agonist in the present study. In addi-
tion, we investigated the association between TRPV2
and UC cell death. The most interesting ﬁnding was that
continuous exposure to CBD-mediated apoptotic cell
death via TRPV2 in T24 cells. This investigation of the
functional properties of UC cells is an important step in
the development of novel strategies for antitumor ther-
MATERIAL AND METHODS
The Center for Experimental Animal Sciences at Nagoya City
University approved the following experiments.
Cell Lines and Culture Conditions
Bladder cancer cell lines (T24 and RT4) were obtained from
the American Type Culture Collection (ATCC, Rockville,
MD). The T24 line is a poorly differentiated bladder UC cell
line, whereas the RT4 line is a well-differentiated bladder UC
cell line. Both cell lines were cultured in McCoy’s 5 A modiﬁed
medium (Life Technologies, Carlsbad, CA), containing 5%
fetal calf serum, at 37 °C in a 5% CO
with water vapor.
Reverse Transcription Polymerase Chain Reaction (RT-
PCR). First, 3
subjected to random-primed reverse transcription using Super-
Script II (Life Technologies). Next, 2.5% of the samples were
ampliﬁed through 35 cycles of PCR with the following primers. For
human TRPV1 (product length "372 bp; GenBank accession
No. NM080706): 5=-CTGCGGACCCACTCCAAAAGGA-3=
(sense) and 5=-AGAGCAGCAGGCTCTCCAGATC-3=(anti-
sense); human TRPV2 (327 bp; NM016113): 5=-CTGCA-
CATCGCCATTGAGAAGA-3=(sense) and 5=-TTGGAG-
GAGCCCATCATACATG-3=(antisense); human TRPV3 (288
bp; NM145068): 5=-GCTGAAGAAGCGCATCTTTGCA-3=
(sense) and 5=-TCATAGGCCTCCTCTGTGTACT-3=(anti-
sense); human TRPV4 (379 bp; NM021625): 5=-TACCTGT-
GTGCCATGGTCATCT-3=(sense) and 5=-TGCTATAG-
GTCCCCGTCAGCTT-3=(antisense); human TRPM8 (621 bp;
(sense) and 5=-TCCTCTGAGGTGTCGTTGGCTTT-3=(anti-
sense); human TRPA1 (541 bp; NM007332): 5=-GACCA-
CAATGGCTGGACAGCT-3=(sense) and 5=-GTACCATT-
-actin (298 bp;
(sense) and 5=-GCGGATGTCCACGTCACACTTCA-3=(an-
tisense). Beta-actin was used for quantiﬁcation of the samples. The
PCR products obtained were separated by electrophoresis in a 1%
agarose gel. We checked the molecular identity and homogeneity
of the resulting PCR products using DNA sequencing and con-
ﬁrmed through the BLAST database that the primers were speciﬁc
for each TRP channel.
Measurement of Intracellular
Calcium Concentrations ([Ca
Experiments were performed 48-72 hours after subculture. T24
cells were incubated with a ﬂuorescent Ca
acetoxymethyl ester, 10
M, Life Technologies) in assay buffer
for 45 minutes at room temperature. The loading solution was
washed thoroughly for 10 minutes and the cells were stimulated
with CBD (3 and 30
-phorbol 12,13-didecanoate (4
M), capsaicin (1
M), menthol (300
M), or ruthenium red (RuR, 25-50
M) using a bath
perfusion system at a ﬂow rate of 2-3 mL/min. We recorded
changes using an Olympus IX-70 microscope equipped
with the Argus/HiSCA system (Hamamatsu Photonics, Hama-
matsu, Japan). Acquisition and analysis of the ﬂuorescence
images were performed with Argus/HiSCA, version 1.65 soft-
The assay buffer solution was prepared according to a
previously described method.
For further analysis, we selected
cells showing similar internal calcium mobilization after 4
PDD was applied (10-15 cells).
Transfection of Small Interfering RNA (siRNA)
T24 cells were seeded onto 60-mm dishes and incubated for 24
hours at 37 °C. The cells were then washed with McCoy’s 5 A
modiﬁed medium and transfected with a siRNA for TRPV2
(Silencer predesigned siRNA, ID s28081 or s28082) (Life Tech-
nologies) or scrambled siRNA as a negative control (Silencer
negative control # 1 siRNA; AM4611) (Life Technologies)
using lipofectamine 2000 (Life Technologies). At 48 hours after
transfection, transfected cells were replated, further incubated
for 48 hours, and used in RT-PCR, calcium imaging analysis,
and an annexin V assay.
Cellular Viability Assay
The viabilities of T24 and RT4 cells were evaluated using the
Cell Counting Kit-8 (Dojin, Kumamoto, Japan), based on the
mide (MTT) assay, according to the protocol described by
Yamamura et al. (2008).
These cells were subcultured in
96-well plates and incubated at 37°C in a 5% CO
saturated with water vapor for 24-48 hour (approximately 5000
cells/well); they were then incubated for 24 hour in medium
containing various concentrations of CBD with or without RuR
M). Cellular viabilities in the absence and presence of
CBD were quantitated colorimetrically by examining absor-
bance at 450 nm (A
). Control cells were treated in exactly
the same way, but with a solution lacking CBD (0.1%-2.0%
methanol). We conﬁrmed that methanol concentrations up to
2.0% had no effect on cellular responses. The relative viabilities
were determined using the following equation: Relative viabil-
ity (%) "(A
of CBD-treated cells/A
of control cells) #
Apoptosis in T24 cells was evaluated using an annexin-V assay
(FITC annexin V apoptosis Detection Kit I, 556 547) (BD
Biosciences, San Jose, CA), which was performed according to
the manufacturer’s protocol. Brieﬂy, after incubation in a me-
dium containing 30
M CBD for 15 hour, a single-cell suspen-
sion (100 000 cells in 100
L) was stained with annexin-V
FITC and Propidium Iodide (PI) for 15 minutes at room tem-
perature in the dark. After this incubation, two-color (FL1 and
FL2) ﬂow cytometry analysis was conducted using a FacsCalibur
509.e2 UROLOGY 76 (2), 2010
machine (BD Biosciences). Ten thousand cells per sample were
Pooled data are shown as mean $SE. Statistical signiﬁcance
was determined using analysis of variance with Bonferroni’s
comparison test. Signiﬁcant differences are indicated in the
ﬁgures as P%.01 or P%.001.
Expression of TRP Channel mRNA in T24 Cells
To investigate the expression of TRPV2 and other TRP
channels in poorly differentiated bladder UC cells (T24),
we isolated total RNA from T24 cells and performed
RT-PCR with primer sets for various TRP channels. As
shown in Fig. 1A, products with the predicted sizes and
nucleotide sequences for TRPV2 and TRPV4 mRNA
were ampliﬁed from the cells (lanes 2 and 4, respec-
tively). In contrast, PCR products corresponding to
TRPV1, TRPV3, TRPM8, or TRPA1 were not detected
in the T24 cells (lanes 1, 3, 5, and 6, respectively).
Previous reports have shown that the expression levels of
TRPV2 mRNA in UC cells increase with cancer grade.
To conﬁrm this ﬁnding, we compared the expression
levels of TRPV2 mRNA in T24 cells to those in RT4
cells. As shown in Fig. 1B, TRPV2 transcripts were more
highly expressed in T24 cells than in RT4 cells.
Functional Analysis of TRPV2 in T24 Cells
To conﬁrm the presence of functional TRPV2 channels
in T24 cells, we physiologically investigated the effects of
an agonist and an antagonist of TRPV2 on RT4 and T24
cells. In RT4 cells, although 4
-PDD (a TRPV4-selec-
tive agonist) caused [Ca
to increase signiﬁcantly
"0.62 $0.06%, 36/42, 85.7% of the cells),
CBD displayed no apparent effect on any of the cells
"0.03 $0.005, 0/42) (Fig. 2A). In
contrast, T24 cells responded to 4
-PDD (60/69, 87.0%)
(Fig. 2B). Moreover, almost all the cells exhibited a
dose-dependent increase in [Ca
in response to CBD
"0.20 $0.02%, 157/165, 95.2% and
"0.40 $0.06%, 47/49, 95.9%) (Fig.
2C, 2D). These responses were fully or partially sup-
pressed by 50
M RuR (Fig. 2C, 2D), suggesting the
presence of functional TRPV2 channels in the T24 cells.
We also conﬁrmed that T24 cells failed to respond to
capsaicin (a TRPV1-selective agonist), menthol (a TRPM8
agonist), or carvacrol (an agonist of TRPV3 or TRPA1)
(data not shown). These ﬁndings, together with the RT-
PCR data, imply that TRPV2 is abundantly expressed in
T24 cells (high-grade UC cells), but not in RT4 cells
(low-grade UC cells).
Silencing of TRPV2 in T24 Cells
To determine the involvement of TRPV2 in CBD-in-
duced activation of T24 cells, we performed siRNA si-
lencing and evaluated the effect of TRPV2 knockdown
using RT-PCR and a calcium imaging assay. T24 cells
were transfected with 2 different siRNA sequences. As
shown in Fig. 3(A), TRPV2 mRNA expression was sup-
pressed by both siRNA1 and siRNA2, whereas neither
the control nor the negative control siRNA had any
effect on TRPV2 mRNA expression. The invariable ex-
pression of TRPV4 mRNA indicates that these siRNA
sequences selectively suppressed TRPV2 gene expression.
Moreover, in a calcium imaging assay, siRNA1 and
siRNA2 markedly suppressed CBD-induced [Ca
creases (Fig. 3B, 3C), whereas neither siRNA had any
effect on 4
mobilization (Fig. 3B–
3D). These results indicate that CBD induces an increase
via TRPV2 channels in T24 cells. It should be
noted that the siRNA fully inhibited the increase in
that was induced by 30
M CBD as well as 3
CBD (Fig. 3C), whereas RuR was less effective in sup-
pressing it (Fig. 2D). Thus, RuR treatment might be
insufﬁcient to suppress robust TRPV2 activation.
Viability of T24 Cells in the
Presence of a TRPV2 Agonist
It has been shown that continuous Ca
-permeable channels and high [Ca
cells via apoptosis and necrosis.
We examined the effects
inﬂux through TRPV2 channels on the survival
of T24 cells using a colorimetric quantitative kit based on
the MTT assay. T24 and RT4 cells were incubated for 24
hours in media containing various concentrations of
CBD, as shown in Fig. 4A. The viability of T24 cells was
found to be dependent on the concentration of CBD
Figure 1. RT-PCR analysis of the expression of mRNAs for transient receptor potential vanilloid 2 (TRPV2) and other TRP
channels in T24 and RT4 cells. (A) TRP mRNA expression in T24 cells. The sizes of the DNA standards are indicated in the
left margin. (B) TRPV2 and TRPV4 mRNA expression in T24 cells and RT4 cells. The controls,
-actin fragments, are shown
in the lower panel. RT (') indicates templates without reverse transcriptase.
UROLOGY 76 (2), 2010 509.e3
Figure 2. Representative [Ca
response in RT4 (A) and T24 cells (B-D).(A) RT4 cells exhibited an increase in [Ca
in response to 4
M), but not to cannabidiol (CBD, 3
M). The 4
-PDD–induced response was completely
inhibited by extracellular ruthenium red (RuR, 25
M). (B) 4
M) also induced an RuR-sensitive increase in [Ca
in T24 cells. (C) Moreover, in most T24 cells (approximately 95% of the cells examined), 3
M CBD evoked a marked
increase in [Ca
, which was fully suppressed by extracellular RuR (50
M). (D) In contrast, the responses induced by 30
M CBD was reduced by approximately 50% with the addition of extracellular RuR (50
M) in T24 cells.
Figure 3. siRNA-mediated knockdown of TRPV2 in T24 cells. The expression of TRPV2 and TRPV4 was analyzed at the mRNA
level by RT-PCR (A) and at the functional level by a calcium imaging assay (B-D).(B) The effect of 3
M CBD and 4
on siRNA1-transfected T24 cells. (C) The effect of 30
M CBD and 4
-PDD on siRNA1-transfected T24 cells. Ionomycin (3
M), a calcium ionophore, was used to prove that cells were viable. (B, C, and D) The siRNAs did not have any effects on
responses. Statistically signiﬁcant differences are expressed as **P%.001 vs. scrambled siRNA.
Experimental data were obtained from 10 cells.
509.e4 UROLOGY 76 (2), 2010
(ranging from 1.5-60
M), with higher concentrations
resulting in more cell death. When comparing the sur-
vival of T24 cells to the survival of RT4 cells, however,
M CBD was most effective in inducing T24-speciﬁc
cell death. CBD did not affect RT4 cells at a concentra-
tion of 30
M, whereas it signiﬁcantly reduced T24 cell
viability, by 40%; 50
M RuR signiﬁcantly suppressed
the effect of CBD (Fig. 4B).
Induction of Apoptosis Via TRPV2 in T24 Cells
To investigate whether CBD induces apoptosis in T24
cells and whether TRPV2 is involved in this process, we
combined an annexin-V assay and the siRNA strategy to
detect apoptosis. In the early stages of apoptosis, phos-
phatidylserine (PS) is translocated from the inner side of
the plasma membrane to the outer layer, causing PS to
become exposed on the external surface of the cells.
Annexin-V is a Ca
protein with a high afﬁnity for PS.
As shown in Fig. 4C,
treatment with 30
M CBD for 15-hour induced apo-
ptosis in T24 cells transfected with scrambled siRNA
(annexin-V-FITC!/PI'"11.4%, n "3), similar to
what occurred in control cells (data not shown). This
CBD-induced apoptosis decreased signiﬁcantly in the
cells transfected with siRNA1 (annexin-V-FITC!/PI',
1.31%, n "3) (Fig. 4D). Similar results were also ob-
tained in the cells transfected with siRNA2 (data not
shown). Moreover, the silencing was effective against
necrotic cell death (upper right quadrant; annexin-V-
FITC!/PI!), indicating that CBD-induced apoptosis
and necrosis in T24 cells are the result of continuous
inﬂux through TRPV2 channels.
In the present study, we focused on a Ca
TRP channels expressed abundantly in human bladder
cancer cells, and we examined, using Ca
Figure 4. (A) Viability of T24 cells in the presence of a TRPV2 agonist. Cell death from exposure to different concentrations of
M) was observed in T24 cells after 24 hours of culture. Experimental data were obtained from 8-12 wells. (B)
Exposure to 30
partially, but not fully, suppressed the effect of CBD on T24 cells. Statistically signiﬁcant differences are expressed as *P%.01,
**P%.001 vs. RT4 cells or T24 cells treated with RuR. Experimental data were obtained from 8-12 wells. (C and D) CBD-induced
apoptosis of T24 cells as determined by the Annexin-V assay and effect of TRPV2 siRNA after exposure to 30
hours. Cells in the lower left quadrant (Annexin-V-FITC'/PI')areviable,thoseinthelowerrightquadrant(Annexin-V-FITC!/PI')
are early apoptotic, and those in the upper right quadrants (Annexin-V-FITC!/PI!)arelateapoptoticornecrotic.Transfectionof
T24 cells with scrambled siRNA apparently caused apoptosis and necrosis in response to CBD (C),whereastransfectionwith
TRPV2 siRNA1 did not (D).Datawereobtainedfrom3separateexperimentsandrepresentativeresultsareshown.
UROLOGY 76 (2), 2010 509.e5
ysis and biochemical approaches, whether the regulation
of channel activity could lead to an inhibitory effect on
the viability of UC cells. Our RT-PCR gene expression
analysis clearly showed that, among the TRP channels
examined, TRPV2 was abundantly expressed in the T24
line, a line of poorly differentiated bladder UC cells;
however, it was not expressed in the RT4 line, which is
a differentiated UC cell line. Because TRPM8 and
TRPV1 transcripts, which are undetectable in T24 cells,
have been reported to be upregulated in prostate can-
TRP channels may be differentially expressed in
a variety of cancers.
We also demonstrated that CBD induces the inﬂux of
into human T24 UC cells, which express TRPV2
endogenously, but not in RT4 cells, which lack TRPV2
channel activity. Among the newly identiﬁed TRPV2 ago-
nists, CBD is particularly interesting because it was the
most potent and selective and its in vivo mechanism of
action and molecular targets remains unknown.
was previously shown to activate human TRPV1 recep-
CBD acts as an allosteric modulator of
It is an antagonist of CB1 and CB2
In addition, the effect of CBD
was also observed in human MDA-MB-231 breast carci-
noma, in which it acts through the direct and indirect
activation of the cannabinoid receptors CB2 and TRPV1
and the cannabinoid/vanilloid receptor-independent el-
evation of [Ca
Because T24 cells lack capsaicin-
sensitive TRPV1 channel responses (our unpublished
data) and silencing with TRPV2 siRNA almost abolished
the stimulatory effect of CBD on [Ca
in T24 cells
(Fig. 3B, 3C), we concluded that CBD predominantly
stimulates TRPV2 channels and increases [Ca
A growing number of studies have demonstrated that
increases in [Ca
regulate various signaling mecha-
nisms that control a variety of cellular processes such as
proliferation, metabolism, and gene transcription; yet,
under certain conditions, increases in [Ca
Indeed, the activation of Ca
channels, such as TRPV1 and TRPM8 induced apoptosis
in human U373 glioma cells
and suppressed the viabil-
ity of human G-361 melanoma cells,
suggests that TRP channels could be potential targets in
cancer treatment. Therefore, we examined whether con-
tinuous activation of TRPV2 by CBD can suppress cell
viability and trigger cell death in T24 UC cells. Interest-
ingly, the survival of T24 cells decreased dramatically in
the presence of CBD, with a concentration-dependent
effect. This CBD-induced growth inhibition in T24 cells
implied that Ca
permeability via TRPV2 channels
partly contributes to the regulation of cellular viability.
Although no morphologic changes were observed in
TRPV2-transfected RT4 cells and TRPV2-knockdown
T24 cells (our unpublished data), details of the patho-
physiological signiﬁcance of TRPV2 in UC cells should
be addressed in further research.
We further conﬁrmed, using an annexin-V assay in
combination with siRNA technology, that continuous
CBD treatment induces apoptotic cell death, and that
this effect is mediated by TRPV2 activation (Fig. 4C,
4D). A previous study reported that, in human breast car-
cinoma, CBD induces apoptosis via the direct and indirect
activation of CB2 and TRPV1 receptors, cannabinoid/va-
nilloid receptor-independent elevation of [Ca
active oxygen species.
In T24 cells, however, silencing of
TRPV2 mostly blocked CBD-induced apoptosis by 89%
(1.31% vs 11.4% of control), suggesting that TRPV2 chan-
nels mainly contribute to CBD-induced apoptotic cell
death in high-grade UC cells. CBD has been shown to
exert both central and peripheral actions with a broad
spectrum of therapeutic effects on pain, neuroprotection,
anxiety, nausea, cerebral ischemia, type 1 diabetes, rheu-
matoid arthritis, multiple sclerosis, and cancer.
has a wider distribution pattern: it is found in the brain,
skin, spleen, lung, stomach, intestines, prostate, and pe-
TRPV2 may not only constitute a
viable new drug target for high-grade bladder carcinoma,
but may also comprise a mechanism by which CBD exerts
its clinically beneﬁcial effects in vivo.
Our results clariﬁed the differential TRPV2 expression in
UC cells by demonstrating that they contribute to
changes in the levels of [Ca
in high-grade UC cells,
but not in low-grade UC cells. Moreover, continuous
activation of TRPV2 triggered apoptosis in high-grade
UC cells. This study provides new insight into the de-
velopment of novel strategies for antitumor therapeutics.
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