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TRPV2 Activation Induces Apoptotic Cell Death in Human T24 Bladder Cancer Cells: A Potential Therapeutic Target for Bladder Cancer

Authors:
  • Nagoya City University, Nagoya, Japan

Abstract and Figures

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. 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. 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. 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.
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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.
MATERIAL AND
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
carcinomas (UCs).
1
Currently, intravesical instillation of
bacillus Calmette-Guérin (BCG) is the most effective
and widely used agent for the treatment of superficial
UC.
2
In addition, other agents, including mitomycin C,
have been used to prevent recurrence.
3
However, recur-
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
Ca
2!
-permeable channels that contribute to intracellular
Ca
2!
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.
4,5
In UC,
TRPV1 expression has been shown to progressively de-
crease as tumor stage increases and cell differentiation
declines.
6
In contrast, the expression of TRPV2 mRNA
and proteins was enhanced in higher-grade UC speci-
mens and UC cell lines.
7
Therefore, modulators of the
TRP channels expressed in high-grade UC cells may be
attractive targets for the medical treatment of malignant
UC.
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: tueda@med.nagoya-cu.ac.jp
©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.
8
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-
apeutics.
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
2
atmosphere saturated
with water vapor.
Reverse Transcription Polymerase Chain Reaction (RT-
PCR). First, 3
!
goftotalRNAisolatedfromT24orRT4cellswas
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;
NM024080): 5=-CCTGTTCCTCTTTGCGGTGTGGAT-3=
(sense) and 5=-TCCTCTGAGGTGTCGTTGGCTTT-3=(anti-
sense); human TRPA1 (541 bp; NM007332): 5=-GACCA-
CAATGGCTGGACAGCT-3=(sense) and 5=-GTACCATT-
GCGTTGAGGGCTGT-3=(antisense); human
"
-actin (298 bp;
NM001101): 5=-GATCCTCACCGAGCGCGGCTACA-3=
(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
2!
]
i
)
Experiments were performed 48-72 hours after subculture. T24
cells were incubated with a fluorescent Ca
2!
indicator (fura-2
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
!
M), 4
#
-phorbol 12,13-didecanoate (4
#
-
PDD, 10
!
M), capsaicin (1
!
M), menthol (300
!
M), carvacrol
(500
!
M), or ruthenium red (RuR, 25-50
!
M) using a bath
perfusion system at a flow rate of 2-3 mL/min. We recorded
(Ca
2!
)
i
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-
ware.
9
The assay buffer solution was prepared according to a
previously described method.
10
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
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro-
mide (MTT) assay, according to the protocol described by
Yamamura et al. (2008).
11
These cells were subcultured in
96-well plates and incubated at 37°C in a 5% CO
2
atmosphere
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
(50
!
M). Cellular viabilities in the absence and presence of
CBD were quantitated colorimetrically by examining absor-
bance at 450 nm (A
450
). 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
450
of CBD-treated cells/A
450
of control cells) #
100.
Annexin-V Assay
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
analyzed.
Statistical Analysis
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.
RESULTS
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.
7
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
2!
]
i
to increase significantly
(&[Ca
2!
]
i
"0.62 $0.06%, 36/42, 85.7% of the cells),
CBD displayed no apparent effect on any of the cells
examined (&[Ca
2!
]
i
"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
2!
]
i
in response to CBD
(3
!
M, &[Ca
2!
]
i
"0.20 $0.02%, 157/165, 95.2% and
30
!
M, &[Ca
2!
]
i
"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
2!
]
i
in-
creases (Fig. 3B, 3C), whereas neither siRNA had any
effect on 4
#
-PDD-induced [Ca
2!
]
i
mobilization (Fig. 3B–
3D). These results indicate that CBD induces an increase
in [Ca
2!
]
i
via TRPV2 channels in T24 cells. It should be
noted that the siRNA fully inhibited the increase in
[Ca
2!
]
i
that was induced by 30
!
M CBD as well as 3
!
M
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
2!
influx through
Ca
2!
-permeable channels and high [Ca
2!
]
i
kill cancer
cells via apoptosis and necrosis.
4
We examined the effects
of Ca
2!
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
2!
]
i
response in RT4 (A) and T24 cells (B-D).(A) RT4 cells exhibited an increase in [Ca
2!
]
i
in response to 4
#
-PDD (10
!
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
#
-PDD (10
!
M) also induced an RuR-sensitive increase in [Ca
2!
]
i
in T24 cells. (C) Moreover, in most T24 cells (approximately 95% of the cells examined), 3
!
M CBD evoked a marked
increase in [Ca
2!
]
I
, 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
#
-PDD
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
4
#
-PDD–induced [Ca
2!
]
i
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,
30
!
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
2!
-dependent phospholipid-binding
protein with a high affinity for PS.
12
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
Ca
2!
influx through TRPV2 channels.
COMMENT
In the present study, we focused on a Ca
2!
-permeable
TRP channels expressed abundantly in human bladder
cancer cells, and we examined, using Ca
2!
imaging anal-
Figure 4. (A) Viability of T24 cells in the presence of a TRPV2 agonist. Cell death from exposure to different concentrations of
CBD (1.5-60
!
M) was observed in T24 cells after 24 hours of culture. Experimental data were obtained from 8-12 wells. (B)
Exposure to 30
!
MCBDfor24hoursdidnotaffectthesurvivalofRT4cells,incontrasttoT24cells.Moreover,50
!
MRuR
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
!
MCBDfor15
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-
cers,
13,14
TRP channels may be differentially expressed in
a variety of cancers.
We also demonstrated that CBD induces the influx of
Ca
2!
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.
8
CBD
was previously shown to activate human TRPV1 recep-
tors.
15
CBD acts as an allosteric modulator of
!
- and
$
-opioid receptors.
16
It is an antagonist of CB1 and CB2
cannabinoid receptors.
17
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
2!
]
i
.
18
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
2!
]
i
in T24 cells
(Fig. 3B, 3C), we concluded that CBD predominantly
stimulates TRPV2 channels and increases [Ca
2!
]
i
in UC
cells.
A growing number of studies have demonstrated that
increases in [Ca
2!
]
i
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
2!
]
i
are cyto-
toxic.
4,19
Indeed, the activation of Ca
2!
-permeable TRP
channels, such as TRPV1 and TRPM8 induced apoptosis
in human U373 glioma cells
20
and suppressed the viabil-
ity of human G-361 melanoma cells,
11
respectively. This
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
2!
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
2!
]
i
,andre-
active oxygen species.
18
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.
21
TRPV2
has a wider distribution pattern: it is found in the brain,
skin, spleen, lung, stomach, intestines, prostate, and pe-
ripheral blood.
22,23
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.
CONCLUSIONS
Our results clarified the differential TRPV2 expression in
UC cells by demonstrating that they contribute to
changes in the levels of [Ca
2!
]
i
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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UROLOGY 76 (2), 2010 509.e7
... TRPV3 is a Ca 2+ -permeable, non-selective cation channel and widely expressed throughout the tissues but especially abundant in various epithelial tissues, including the specialized epithelial cells, keratinocytes of the skin [84,85]. In the skin keratinocytes, TRPV3 can be activated at least by heat and its activation can regulate various downstream functions, including skin barrier formation, wound healing, sensing of temperature, itch and pain [20,71,[86][87][88][89][90]. In general, TRPV3 seems to be important for the health of the skin, and loss of TRPV3 function leads to skin inflammations, dermatitis, itchiness and hair loss. ...
... In the prostate carcinoma model, trafficking of TRPV2 to the plasma membrane correlated with enhanced cell migration through the phosphoinositide 3-kinase (PI3K) pathway [231]. In bladder cancer cells, TRPV2 also enhanced cell migration and invasion but did not affect cell proliferation in vitro [89,232,233]. In hepatoma and hepatocarcinoma models, TRPV2 may also increase drug resistance [234] and suppression of TRPV2 activity in nude mice xenografts reduced tumor growth and invasion [226]. ...
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Biophysical cues from the cellular microenvironment are detected by mechanosensitive machineries that translate physical signals into biochemical signaling cascades. At the crossroads of extracellular space and cell interior are located several ion channel families, including TRP family proteins, that are triggered by mechanical stimuli and drive intracellular signaling pathways through spatio-temporally controlled Ca2+-influx. Mechanosensitive Ca2+-channels, therefore, act as critical components in the rapid transmission of physical signals into biologically compatible information to impact crucial processes during development, morphogenesis and regeneration. Given the mechanosensitive nature of many of the TRP family channels, they must also respond to the biophysical changes along the development of several pathophysiological conditions and have also been linked to cancer progression. In this review, we will focus on the TRPV, vanilloid family of TRP proteins, and their connection to cancer progression through their mechanosensitive nature.
... For example, it has been reported that inhibition of TRPV2 expression reduces the proliferation and invasion of esophageal squamous cell carcinoma cells [25]. In urothelial carcinoma and endometrial carcinoma, TRPV2 is highly expressed in cancer tissues, and the TRPV2 agonist cannabidiol activates apoptosis [28,29]. Several studies in the past few years have also identified a role for TRPV2 in cardiac function, and have suggested that inhibition of TRPV2 activity may be a potential target for the treatment of cardiomyopathy and heart failure [30][31][32][33]. ...
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Cataracts are a serious complication of diabetes. In long-term hyperglycemia, intracellular Ca2+ concentration ([Ca2+]i) and reactive oxygen species (ROS) are increased. The apoptosis of lens epithelial cells plays a key role in the development of cataract. We investigated a potential role for transient receptor potential vanilloid 2 (TRPV2) in the development of diabetic cataracts. Immunohistochemical and Western blotting analyses showed that TRPV2 expression levels were significantly increased in the lens epithelial cells of patients with diabetic cataracts as compared with senile cataract, as well as in both a human lens epithelial cell line (HLEpiC) and primary rat lens epithelial cells (RLEpiCs) cultured under high-glucose conditions. The [Ca2+]i increase evoked by a TRPV2 channel agonist was significantly enhanced in both HLEpiCs and RLEpiCs cultured in high-glucose media. This enhancement was blocked by the TRPV2 nonspecific inhibitor ruthenium red and by TRPV2-specific small interfering (si)RNA transfection. Culturing HLEpiCs or RLEpiCs for seven days in high glucose significantly increased apoptosis, which was inhibited by TRPV2-specific siRNA transfection. In addition, ROS inhibitor significantly suppressed the ROS-induced increase of TRPV2-mediated Ca2+ signal and apoptosis under high-glucose conditions. These findings suggest a mechanism underlying high-glucose–induced apoptosis of lens epithelial cells, and offer a potential target for developing new therapeutic options for diabetes-related cataracts.
... In other studies, AEA has been shown to induce receptor-independent apoptosis of nonmelanoma cancer cells via ER stress [72] and to elicit reduced viability of cholangiocarcinoma cells via activation of GPR55, with the latter eliciting increased recruitment of the death receptor Fas in membrane lipid rafts [73]. Furthermore, activation of TRPV2 has been revealed to be involved in the proapoptotic effect of CBD on human bladder cancer cells [74]. Mostly, however, the proapoptotic effects of CBD have been associated with receptorindependent mechanisms, although corresponding inhibitor experiments with receptor antagonists have not always been performed. ...
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Drugs that target the endocannabinoid system are of interest as pharmacological options to combat cancer and to improve the life quality of cancer patients. From this perspective, cannabinoid compounds have been successfully tested as a systemic therapeutic option in a number of preclinical models over the past decades. As a result of these efforts, a large body of data suggests that the anticancer effects of cannabinoids are exerted at multiple levels of tumour progression via different signal transduction mechanisms. Accordingly, there is considerable evidence for cannabinoid-mediated inhibition of tumour cell proliferation, tumour invasion and metastasis, angiogenesis and chemoresistance, as well as induction of apoptosis and autophagy. Further studies showed that cannabinoids could be potential combination partners for established chemotherapeutic agents or other therapeutic interventions in cancer treatment. Research in recent years has yielded several compounds that exert promising effects on tumour cells and tissues in addition to the psychoactive Δ ⁹ -tetrahydrocannabinol, such as the non-psychoactive phytocannabinoid cannabidiol and inhibitors of endocannabinoid degradation. This review provides an up-to-date overview of the potential of cannabinoids as inhibitors of tumour growth and spread as demonstrated in preclinical studies.
... Moreover, the activity and expression of the TRPV2 receptor was induced by cannabidiol in glioblastoma cells that was accompanied by increases drug uptake, induced Ca 2+ influx, and induced apoptosis [52]. A functional study revealed that TRPV2 over-expressed in poorly differentiated urothelial carcinoma cells (T24 cells) and CBD administration induced intracellular calcium level and decreased cell viability and increased apoptosis in such cells [53]. In addition, activation of TRPV2 receptor by CBD sensitized Triple-negative breast cancer cells to doxorubicin uptake and induced apoptosis, cleaved caspase-3, and cleaved PARP. ...
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Cancer, as a mysterious and complex disease, has a multi-stage molecular process that uses the cellular molecular machine and multiple signaling pathways to its advantage. Cannabinoids, as terpenophenolic compounds and their derivatives, showed influences on immune system responses, inflammation, and cell growth that have sparked a growing interest in exploring their effects on cancer cell fate, as well. A large body of evidence in experimental models indicating the involvement of cannabinoids and their related receptors in cancer cell growth, development, and fate. In accordance, the present study provided insights regarding the strengths and limits of cannabinoids and their receptors in critical steps of tumorigenesis and its underlying molecular pathways such as; cancer cell proliferation, type of cell death pathway, angiogenesis, invasion, metastasis and, immune system response. Based on the results of the present study and due to the contribution of cannabinoids in various cancer cell growth control processes, these compounds cancer can be considered worthwhile in finding new alternatives for cancer therapy.
... TRPV2 expression is increased in high-grade and high-stage urothelial cancer tissues and cell lines [15]. TRPV2 activation increases the intracellular calcium levels, decreases cell viability and induces apoptotic death of BC cell lines [16]. Furthermore, Liu et al. have shown that TRPV2 mediated migration and invasion through metalloproteinase-2 activation in BC cancer cell lines [17]. ...
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The Transient Receptor Potential Vanilloid type 2 (TRPV2) channel is highly selective for Ca²⁺ and can be activated by lipids, such as LysoPhosphatidylCholine (LPC). LPC analogues, such as the synthetic alkyl-ether-lipid edelfosine or the endogenous alkyl-ether-lipid Platelet Activating Factor (PAF), modulates ion channels in cancer cells. This opens the way to develop alkyl-ether-lipids for the modulation of TRPV2 in cancer. Here, we investigated the role of 2-Acetamido-2-Deoxy-l-O-Hexadecyl-rac-Glycero-3-PhosphatidylCholine (AD-HGPC), a new alkyl-ether-lipid (LPC analogue), on TRPV2 trafficking and its impact on Ca²⁺ -dependent cell migration. The effect of AD-HGPC on the TRPV2 channel and tumour process was further investigated using calcium imaging and an in vivo mouse model. Using molecular and pharmacological approaches, we dissected the mechanism implicated in alkyl-ether-lipids sensitive TRPV2 trafficking. We found that TRPV2 promotes constitutive Ca²⁺ entry, leading to migration of highly metastatic breast cancer cell lines through the PI3K/Akt-Girdin axis. AD-HGPC addresses the functional TRPV2 channel in the plasma membrane through Golgi stimulation and PI3K/Akt/Rac-dependent cytoskeletal reorganization, leading to constitutive Ca²⁺ entry and breast cancer cell migration (without affecting the development of metastasis), in a mouse model. We describe, for the first time, the biological role of a new alkyl-ether-lipid on TRPV2 channel trafficking in breast cancer cells and highlight the potential modulation of TRPV2 by alkyl-ether-lipids as a novel avenue for research in the treatment of metastatic cancer.
... In contrast to THC, CBD does not produce psychoactive effects as it is neither a substrate of cannabinoid receptors CB1 nor CB2, and it has proven to be well tolerated in humans [141]. CBD at higher concentration (30 μM) induced cell death in human T24 bladder cancer cells through activating TRPV2 [142], and CBD decreased glioma stem-like cell viability with an IC50 of nearly 20 μM in a TRPV2-dependent manner [79]. CBD could also reduce lipopolysaccharide (LPS)-mediated BBB disruption [123] and protect BBB integrity in multiple sclerosis mice models [143]. ...
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The gliovascular unit (GVU) is composed of the brain microvascular endothelial cells forming blood-brain barrier and the neighboring surrounding "mural" cells (e.g., pericytes) and astrocytes. Modulation of the GVU/BBB features could be observed in a variety of vascular, immu-nologic, neuro-psychiatric diseases, and cancers, which can disrupt the brain homeostasis. Ca 2+ dynamics have been regarded as a major factor in determining BBB/GVU properties, and previous studies have demonstrated the role of transient receptor potential vanilloid (TRPV) channels in modulating Ca 2+ and BBB/GVU properties. The physiological role of thermosensitive TRPV channels in the BBB/GVU, as well as their possible therapeutic potential as targets in treating brain diseases via preserving the BBB are reviewed. TRPV2 and TRPV4 are the most abundant isoforms in the human BBB, and TRPV2 was evidenced to play a main role in regulating human BBB integrity. Interspecies differences in TRPV2 and TRPV4 BBB expression complicate further preclinical validation. More studies are still needed to better establish the physiopathological TRPV roles such as in astrocytes, vascular smooth muscle cells, and pericytes. The effect of the chronic TRPV modulation should also deserve further studies to evaluate their benefit and innocuity in vivo.
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Cannabidiol (CBD), as a major phytocannabinoid of Cannabis sativa, has emerged as a promising natural compound in the treatment of diseases. Its diverse pharmacological effects with limited side effects have promoted researchers to pursue new therapeutic applications. It has little affinity for classical cannabinoid receptors (CB1 and CB2). Considering this and its diverse pharmacological effects, it is logical to set up studies for finding its putative potential targets other than CB1 and CB2. A class of ion channels, namely transient potential channels (TRP), has been identified during two recent decades. More than 30 members of this family have been studied, so far. They mediate diverse physiological functions and are associated with various pathological conditions. Some have been recognized as key targets for natural compounds such as capsaicin, menthol, and CBD. Studies show that CBD has agonistic effects for TRPV1-4 and TRPA1 channels with antagonistic effects on the TRPM8 channel. In this article, we reviewed the recent findings considering the interaction of CBD with these channels. The review indicated that TRP channels mediate, at least in part, the effects of CBD on seizure, inflammation, cancer, pain, acne, and vasorelaxation. This highlights the role of TRP channels in CBD-mediated effects, and binding to these channels may justify part of its paradoxical effects in comparison to classical phytocannabinoids.
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Cell-cell interactions between cancer cells and neighboring adipose tissue-derived stromal cells (ATSCs) are known to regulate the aggressiveness of cancer cells. In addition, the radiation-induced bystander effect is an important modulator of cancer cell kinetics. Radiation therapy is often given for urinary cancer, but the biological effects of the irradiated cancer stroma, including adipose tissue, on urothelial carcinoma (UC) remain unclear. We investigated the bystander effect of irradiated ATSCs on UC using a collagen gel culture method to replicate irradiated ATSC-cancer cell interactions after a single 12-Gy dose of irradiation. Proliferative activity, invasive capacity, protein expression and nuclear translocation of p53 binding protein-1 (53BP1) were analyzed. Irradiated ATSCs significantly inhibited the growth and promoted the apoptosis of UC cells in comparison to non-irradiated controls. The invasiveness of UC cells was increased by irradiated ATSCs, but not irradiated fibroblasts. Nuclear translocation of 53BP1 protein due to the bystander effect was confirmed in the irradiated group. Irradiated ATSCs regulated the expressions of the insulin receptor, insulin-like growth factor-1 and extracellular signal-regulated kinase-1/2 in UC. In conclusion, the bystander effect of irradiated ATSCs is a critical regulator of UC, and the actions differed depending on the type of mesenchymal cell involved. Our alternative culture model is a promising tool for further investigations into radiation therapy for many types of cancer.
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The poor water-solubility of Cannabidiol (CBD) seriously hinders its pharmacological activities such as anti-anxiety, anti-epilepsy, anti-oxidation and anti-cancer, etc. In this paper, we successfully designed and synthesized two bridged cyclodextrins (CDs) dimers with different length of bridged chains (succinic acid (SACDD) and 3,3'-thiodipropionic acid (TPACDD)) to encapsulate CBD, a reported DMCD (2,6-Di-O-methyl-β-cyclodextrin) with single cavity was used as a control complex. Their characteristics and inclusion complexation behaviors were investigated via XRD, SEM, NMR and TGA, the obtained data suggests CBD is successfully encapsulated into two cavity of bridged CD dimers with stronger stability constants, compared with DMCD. Water solubility of CBD is significantly promoted by 6.76 × 10⁵ and 4.52 × 10⁵ folds after formation of inclusion complexes, as well as the antioxidant activity in vitro (5.26-fold enhanced). MTT assays shows they remained effective anti-tumor activity, while they barely show cytotoxicity to normal cell. Our work might provide a strategy for development and application of water-solubility CBD.
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It is known that nucleus pulposus cells (NPs) play an important role in intervertebral disc degeneration (IVDD), and a previous study indicated that the stiffness of NP tissue changes during the degeneration process. However, the mechanism underlying the cellular response to ECM stiffness is still unclear. To analyze the effects of extracellular matrix (ECM) with different degrees of stiffness on NPs, we prepared polyacrylamide (PA) gels with different elastic moduli, and cells grown under different stiffness conditions were obtained and analyzed. The results showed that the spreading morphology of NPs changed significantly under increased ECM elastic modulus conditions and that TRPV2 and the PI3K / AKT signaling pathway were activated by stiffer ECM. At the same time, mitochondria released cytochrome c (Cyt c) and activated caspase proteins to promote the apoptosis of NPs. After TRPV2 was specifically knocked out, the activation of the PI3K / AKT signaling pathway decreased, and the release of Cyt c and NP apoptosis were reduced. These results indicate that TRPV2 is closely linked to the detection of extracellular mechanical signals, and that conversion of mechanical and biological signals plays an important role in regulating the biological behavior of cells. This study offers a new perspective on the cellular and biochemical events underlying IVDD which could result in novel treatments.
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We studied the localization and physiological functions of the transient receptor potential (TRP) channels TRPV1 (TRP vanilloid 1) and TRPV4 (TRP vanilloid 4) in the mouse bladder, because both channels are thought to be mechanosensors for bladder distention. RT-PCR specifically amplified TRPV4 transcripts from the urothelial cells, whereas TRPV1 transcripts were barely detectable. ISH experiments showed that TRPV4 transcripts were abundantly expressed in the urothelium, whereas TRPV1 transcripts were not detectable in the urothelial cells. Immunoblotting and IHC studies showed that TRPV4 proteins were mainly localized at the basal plasma membrane domains of the basal urothelial cells. In contrast, TRPV1-immunoreactivities were found not in the urothelial cells but in the nerve fibers that innervate the urinary bladder. In Ca(2+)-imaging experiments, 4alpha-phorbol 12,13-didecanoate, a TRPV4 agonist, and hypotonic stimuli induced significant increases in intracellular calcium ion concentration ([Ca(2+)](i)) in isolated urothelial cells, whereas capsaicin, a TRPV1 agonist, showed no marked effect on the cells. These findings raise the possibility that, in mouse urothelial cells, TRPV4 may contribute to the detection of increases in intravesical pressure related to the micturition reflex.
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(−)-Cannabidiol (CBD) is a non-psychotropic component of Cannabis with possible therapeutic use as an anti-inflammatory drug. Little is known on the possible molecular targets of this compound. We investigated whether CBD and some of its derivatives interact with vanilloid receptor type 1 (VR1), the receptor for capsaicin, or with proteins that inactivate the endogenous cannabinoid, anandamide (AEA). CBD and its enantiomer, (+)-CBD, together with seven analogues, obtained by exchanging the C-7 methyl group of CBD with a hydroxy-methyl or a carboxyl function and/or the C-5′ pentyl group with a di-methyl-heptyl (DMH) group, were tested on: (a) VR1-mediated increase in cytosolic Ca2+ concentrations in cells over-expressing human VR1; (b) [14C]-AEA uptake by RBL-2H3 cells, which is facilitated by a selective membrane transporter; and (c) [14C]-AEA hydrolysis by rat brain membranes, which is catalysed by the fatty acid amide hydrolase. Both CBD and (+)-CBD, but not the other analogues, stimulated VR1 with EC50=3.2 – 3.5 μM, and with a maximal effect similar in efficacy to that of capsaicin, i.e. 67 – 70% of the effect obtained with ionomycin (4 μM). CBD (10 μM) desensitized VR1 to the action of capsaicin. The effects of maximal doses of the two compounds were not additive. (+)-5′-DMH-CBD and (+)-7-hydroxy-5′-DMH-CBD inhibited [14C]-AEA uptake (IC50=10.0 and 7.0 μM); the (−)-enantiomers were slightly less active (IC50=14.0 and 12.5 μM). CBD and (+)-CBD were also active (IC50=22.0 and 17.0 μM). CBD (IC50=27.5 μM), (+)-CBD (IC50=63.5 μM) and (−)-7-hydroxy-CBD (IC50=34 μM), but not the other analogues (IC50>100 μM), weakly inhibited [14C]-AEA hydrolysis. Only the (+)-isomers exhibited high affinity for CB1 and/or CB2 cannabinoid receptors. These findings suggest that VR1 receptors, or increased levels of endogenous AEA, might mediate some of the pharmacological effects of CBD and its analogues. In view of the facile high yield synthesis, and the weak affinity for CB1 and CB2 receptors, (−)-5′-DMH-CBD represents a valuable candidate for further investigation as inhibitor of AEA uptake and a possible new therapeutic agent. British Journal of Pharmacology (2001) 134, 845–852; doi:10.1038/sj.bjp.0704327
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Vanilloid receptor subtype-1 (TRPV1), the founding member of the vanilloid receptor-like transient receptor potential channel family, is a non-selective cation channel that responds to noxious stimuli such as low pH, painful heat and irritants.In the present study, we show, as means of reverse transcriptase-polymerase chain reaction and Western blot analysis, that the vanilloid TRPV1 receptor is expressed in the prostate epithelial cell lines PC-3 and LNCaP as well as in human prostate tissue. The kinetic parameters inferred from [125I]-resiniferatoxin binding were in concordance with data of TRPV1 receptors expressed in other tissues. The contribution of the endogenously expressed TRPV1 channel to intracellular calcium concentration increase in the prostate cells was studied by measuring changes in Fura-2 fluorescence by fluorescence microscopy. Addition of capsaicin, (R)-methanandamide and resiniferatoxin to prostate cells induced a dose-dependent increase in the intracellular calcium concentration that was reversed by the vanilloid TRPV1 receptor antagonist capsazepine. These results indicate that the vanilloid TRPV1 receptor is expressed and functionally active in human prostate cells.
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The transient receptor potential melastatin subfamily (TRPM), which is a mammalian homologue of cell death-regulated genes in Caenorhabditis elegans and Drosophila, has potential roles in the process of the cell cycle and regulation of Ca(2+) signaling. Among this subfamily, TRPM8 (also known as Trp-p8) is a Ca(2+)-permeable channel that was originally identified as a prostate-specific gene upregulated in tumors. Here we showed that the TRPM8 channel was expressed in human melanoma G-361 cells, and activation of the channel produced sustainable Ca(2+) influx. The application of menthol, an agonist for TRPM8 channel, elevated cytosolic Ca(2+) concentration in a concentration-dependent manner with an EC(50) value of 286 microM in melanoma cells. Menthol-induced responses were significantly abolished by the removal of external Ca(2+). Moreover, inward currents at a holding potential of -60 mV in melanoma cells were markedly potentiated by the addition of 300 microM menthol. The most striking finding was that the viability of melanoma cells was dose-dependently depressed in the presence of menthol. These results reveal that a functional TRPM8 protein is expressed in human melanoma cells to involve the mechanism underlying tumor progression via the Ca(2+) handling pathway, providing us with a novel target of drug development for malignant melanoma.
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In a prospective controlled study the influence of long-term mitomycin C instillation therapy on tumor recurrence, progression and patient survival after transurethral resection of superficial bladder tumors was evaluated. This report is an update of a randomized controlled study that was stopped 1.5 years ago. The results show that long-term mitomycin C instillation therapy improves recurrence rate, progression rate and survival in patients with superficial bladder cancer.
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In the early stages of apoptosis changes occur at the cell surface, which until now have remained difficult to recognize. One of these plasma membrane alterations is the translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer layer, by which PS becomes exposed at the external surface of the cell. Annexin V is a Ca2+ dependent phospholipid-binding protein with high affinity for PS. Hence this protein can be used as a sensitive probe for PS exposure upon the cell membrane. Translocation of PS to the external cell surface is not unique to apoptosis, but occurs also during cell necrosis. The difference between these two forms of cell death is that during the initial stages of apoptosis the cell membrane remains intact, while at the very moment that necrosis occurs the cell membrane looses its integrity and becomes leaky. Therefore the measurement of Annexin V binding to the cell surface as indicative for apoptosis has to be performed in conjunction with a dye exclusion test to establish integrity of the cell membrane.
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Pain-producing heat is detected by several classes of nociceptive sensory neuron that differ in their thermal response thresholds. The cloned capsaicin receptor, also known as the vanilloid receptor subtype 1 (VR1), is a heat-gated ion channel that has been proposed to mediate responses of small-diameter sensory neurons to moderate (43 degrees C) thermal stimuli. VR1 is also activated by protons, indicating that it may participate in the detection of noxious thermal and chemical stimuli in vivo. Here we identify a structurally related receptor, VRL-1, that does not respond to capsaicin, acid or moderate heat. Instead, VRL-1 is activated by high temperatures, with a threshold of approximately 52 degrees C. Within sensory ganglia, VRL-1 is most prominently expressed by a subset of medium- to large-diameter neurons, making it a candidate receptor for transducing high-threshold heat responses in this class of cells. VRL-1 transcripts are not restricted to the sensory nervous system, indicating that this channel may be activated by stimuli other than heat. We propose that responses to noxious heat involve these related, but distinct, ion-channel subtypes that together detect a range of stimulus intensities.
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1The situation is similar for most industrialised western nations, with incidence rates of 18 to 30 new cases per 10 0 000 men placing bladder cancer among the top five cancers in this sex. It is unclear why women are affected a third to a quarter less often than men. In 75% of patients the disease is diagnosed in its early superficial stage, usually as a result of gross or microscopic blood in the urine. However, with an overall 65% recurrence rate and 30% progression rate, even these patients need lifelong medical vigilance involving periodic internal inspections of their bladders. The cause of bladder cancer is unknown, but the disease has been strongly associated with exposure to certain aromatic chemicals, notably aniline dyes and benzidine compounds. Cigarette smoking with its release of -naphthalene and -napthalene into the urine is estimated to cause over one half of bladder cancers found in men and one third of those found in women. 2 Over 90% of bladder cancers found in such settings are of the transitional cell type, with squamous cell carcinoma usually occuring in patients with chronic infections such as bilhariasis. Among the transitional cell carcinomas, depth of penetration (stage) and degree of cellular anaplasia (grade) are the most important factors for prognosis. One particular mucosal high-grade lesion called carcinoma-in-situ is not surgically accessible because of its diffuse surface-spreading behaviour. Untreated carcinoma-in-situ will progress to muscleinvasive disease in about 80% of cases within 5 years. 3
Article
We have identified and cloned a novel gene, trp-p8, by screening a prostate-specific subtracted cDNA library. The 5694-bp cDNA has a 3312-bp open reading frame, which codes for a 1104 amino acid putative protein with seven transmembrane domains. The predicted protein revealed significant homology with the transient receptor potential (trp) family of Ca(2+) channel proteins. Northern blot analysis indicated that trp-p8 expression within normal human tissues is mostly restricted to prostate epithelial cells. In situ hybridization analysis showed that trp-p8 mRNA expression was at moderate levels in normal prostate tissue and appears to be elevated in prostate cancer. Notably, trp-p8 mRNA was also expressed in a number of nonprostatic primary tumors of breast, colon, lung, and skin origin, whereas transcripts encoding trp-p8 were hardly detected or not detected in the corresponding normal human tissues.