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

  • 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.
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).
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.
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
-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.
In UC,
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:
©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-
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
atmosphere saturated
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
(sense) and 5=-AGAGCAGCAGGCTCTCCAGATC-3=(anti-
sense); human TRPV2 (327 bp; NM016113): 5=-CTGCA-
GAGCCCATCATACATG-3=(antisense); human TRPV3 (288
(sense) and 5=-TCATAGGCCTCCTCTGTGTACT-3=(anti-
sense); human TRPV4 (379 bp; NM021625): 5=-TACCTGT-
GTCCCCGTCAGCTT-3=(antisense); human TRPM8 (621 bp;
(sense) and 5=-TCCTCTGAGGTGTCGTTGGCTTT-3=(anti-
sense); human TRPA1 (541 bp; NM007332): 5=-GACCA-
GCGTTGAGGGCTGT-3=(antisense); human
-actin (298 bp;
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
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
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
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).
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) #
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
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.
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
examined (&[Ca
"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
M, &[Ca
"0.20 $0.02%, 157/165, 95.2% and
M, &[Ca
"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
-PDD-induced [Ca
mobilization (Fig. 3B–
3D). These results indicate that CBD induces an increase
in [Ca
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
influx through
-permeable channels and high [Ca
kill cancer
cells via apoptosis and necrosis.
We examined the effects
of Ca
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
-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
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
-PDD–induced [Ca
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
-dependent phospholipid-binding
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
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
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
- and
-opioid receptors.
It is an antagonist of CB1 and CB2
cannabinoid receptors.
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
in UC
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
are cyto-
Indeed, the activation of Ca
-permeable TRP
channels, such as TRPV1 and TRPM8 induced apoptosis
in human U373 glioma cells
and suppressed the viabil-
ity of human G-361 melanoma cells,
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
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-
ripheral blood.
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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UROLOGY 76 (2), 2010 509.e7
... Three studies indicated that cannabis appears to be protective against bladder cancer, and this was further strengthened by the results of 3 in vitro studies showing that cannabis had pro-apoptotic effects on bladder cancer cell lines [8,[31][32][33][34][35] (Table 3). There has been no relationship suggested between cannabis consumption and penile cancer [36] (Table 4). ...
... Yamada et al. showed that villinoid receptors are expressed more in high grade UC. They also demonstrated a dose-dependent relationship between cannabis administration and apoptosis mediated by calcium influx, via villinoid receptors [35]. This was supported by another study examining the activation of CB2 in primary UC of the bladder, where it was found to lead to cell death [33]. ...
Background Cannabis is the most commonly used illicit drug worldwide. An increasing number of jurisdictions are legalising cannabis for both medicinal and recreational use. The changing cannabis market has resulted in both an increase in the number of people consuming these compounds, and an increase in the frequency and quantity of cannabis being used. Endogenous and exogenous cannabinoids act on receptors across the entire body including the genitourinary system; however, there is a paucity of understanding of how cannabinoids affect genitourinary malignancy.Objective To present a narrative review of the available literature detailing the relationship between cannabis and the incidence, diagnosis, and management of genitourinary malignancy.MethodsA comprehensive search was undertaken using the Ovid MEDLINE, Ovid Embase, and Cochrane Central Register of Controlled Trials (CENTRAL) up to July 2021. Studies included case reports, case series, case-control studies, and in vitro studies.ResultsThe search identified 40 studies in total: 8 described the relationship between cannabis and testicular carcinoma, 20 related to prostate cancer, 5 to bladder cancer, 5 to renal cancer, 1 to penile cancer, and 1 study examined testicular carcinoma, renal cell carcinoma, bladder cancer, and prostate cancer.Conclusions Cannabis use has been linked to an increased risk of developing testicular tumours, whilst the evidence for bladder cancer is mixed. There is no apparent increase in risk for prostate cancer, penile cancer, or renal cell carcinoma; however, this evidence was based on a very small number of patients. There remains a lack of understanding of the relationship between cannabis and genitourinary malignancy. With an expected increase in cannabis use, monitoring for testicular tumour plus efforts to further understand its effects upon the genitourinary tract will aid diagnosis and management.
... The immunerelated analysis of TRPV2 showed that high expression of TRPV2 was correlated with better therapeutic effect and prognosis from multi-angle such as immunotyping, ESTIMATE score, and immune checkpoint co-expression, showed that TRPV was important to clinical application. Takahiro Yamada et al. confirmed that active TRPV2 channel could lead to apoptosis of T24 cells [29]. Besides, TRPV4 also had certain significance for pan-cancer, and played an obvious role in observing the level of immune infiltration and guiding immunotherapy. ...
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Background: Transient receptor potential cation channel subfamily V (TRPV) play an essential in cancer initiation, progression, and treatment. TRPV expression alteration are shown relate to multiple cancers prognosis and treatment of cancers but are less-studied in pan-cancer. In this study, we characterize the clinical prediction value of TRPV at pan-cancer level. Methods: Several databases were used to examine the transcript expression difference in tumor vs. normal tissue, copy-number variant (CNV) and single nucleotide polymorphisms (SNP) mutation of each TRPV members in pan-cancer, including The Cancer Genome Atlas (TCGA) and cBioPortal. We performed K-M survival curve and univariate Cox regression analyses to identify survival and prognosis value of TRPV. CellMiner were selected to explore drug sensitivity. We also analyzed association between tumor mutation burden (TMB), microsatellite instability (MSI), tumor immune microenvironment and TRPV family genes expression. Moreover, we investigated the relationship between TRPVs expression and effectiveness of immunotherapy in multiple cohorts, including one melanoma (GSE78220), one renal cell carcinoma (GSE67501), and three bladder cancer cohorts (GSE111636, IMvigor210, GSE176307 and our own sequencing dataset (TRUCE-01)), and further analyzed the changes of TRPVs expression before and after treatment (tislelizumab combined with nab-paclitaxel) of bladder cancer. Next, we made a special effort to investigate and study biological functions of TRPV in bladder cancer using gene set enrichment analysis (GSEA), and conducted immune infiltration analysis with TRPVs family genes expression, copy number or somatic mutations of bladder cancer by TIMER 2.0. Finally, real-time PCR and protein expression validation of TRPVs within 10 paired cancer and para-carcinoma tissue samples, were also performed in bladder cancer. Results: Only TRPV2 expression was lower in most cancer types among TRPV family genes. All TRPVs were correlated with survival changes. Amplification was the significant gene alternation in all TRPVs. Next, analysis between TRPVs and clinical traits showed that TRPVs were related to pathologic stage, TNM stage and first course treatment outcome. Moreover, TRPV expression was highly correlated with MSI and TMB. Immunotherapy is a research hotspot at present, our result showed the significant association between TRPVs expression and immune infiltration indicated that TRPV expression alternation could be used to guide prognosis. In addition, we also discovered that the expression level of TRPV1/2/3/4/6 was positively or negatively correlated with objective responses to anti-PD-1/PD-L1 across multiple immunotherapy cohort. Further analysis of drug sensitivity showed the value to treatment. Based on the above analysis, we next focused on TRPV family in bladder cancer. The result demonstrated TRPV also played an important role in bladder cancer. Finally, qPCR assay verified our analysis in bladder cancer. Conclusion: Our study firstly revealed expression and genome alternation of TRPV in pan-cancer. TRPV could be used to predict prognosis or instructing treatment of human cancers, especially bladder cancer.
... In macrophages, TRPV2 channel expression is essential for phagocytosis (Link et al., 2010;Lévêque et al., 2018), in pancreatic β-cells it influences insulin secretion (Hisanaga et al., 2009), in red blood cells it contributes to their response to osmotic challenges (Belkacemi et al., 2021), and its expression levels are significantly altered in multiple types of cancers (Monet et al., 2010;Yamada et al., 2010;Kudou et al., 2019;Siveen et al., 2020;Guéguinou et al., 2021). ...
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The cation-permeable TRPV2 channel is important for cardiac and immune cell function. Cannabidiol (CBD), a non-psychoactive cannabinoid of clinical relevance, is one of the few molecules known to activate TRPV2. Using the patch-clamp technique, we discover that CBD can sensitize current responses of the rat TRPV2 channel to the synthetic agonist 2-aminoethoxydiphenyl borate (2-APB) by over two orders of magnitude, without sensitizing channels to activation by moderate (40°C) heat. Using cryo-EM, we uncover a new small-molecule binding site in the pore domain of rTRPV2 in addition to a nearby CBD site that had already been reported. The TRPV1 and TRPV3 channels are also activated by 2-APB and CBD and share multiple conserved features with TRPV2, but we find that strong sensitization by CBD is only observed in TRPV3, while sensitization for TRPV1 is much weaker. Mutations at non-conserved positions between rTRPV2 and rTRPV1 in either the pore domain or the CBD sites failed to confer strong sensitization by CBD in mutant rTRPV1 channels. Together, our results indicate that CBD-dependent sensitization of rTRPV2 channels engages multiple channel regions, and that the difference in sensitization strength between rTRPV2 and rTRPV1 channels does not originate from amino acid sequence differences at the CBD binding site or the pore domain. The remarkably robust effect of CBD on TRPV2 and TRPV3 channels offers a promising new tool to both understand and overcome one of the major roadblocks in the study of these channels - their resilience to activation.
... Little is known about TRPV3's role in cancers. However, as it regulates the growth and survival of skin cells, its overexpression could be associated with the proliferation of lung cancer cells [151,152]. ...
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Mechanical forces are an inherent element in the world around us. The effects of their action can be observed both on the macro and molecular levels. They can also play a prominent role in the tissues and cells of animals due to the presence of mechanosensitive ion channels (MIChs) such as the Piezo and TRP families. They are essential in many physiological processes in the human body. However, their role in pathology has also been observed. Recent discoveries have highlighted the relationship between these channels and the development of malignant tumors. Multiple studies have shown that MIChs mediate the proliferation, migration, and invasion of various cancer cells via various mechanisms. This could show MIChs as new potential biomarkers in cancer detection and prognosis and interesting therapeutic targets in modern oncology. Our paper is a review of the latest literature on the role of the Piezo1 and TRP families in the molecular mechanisms of carcinogenesis in different types of cancer.
... Adult mice in which TRPV2 channel expression in cardiomyocytes was ablated died within two weeks and exhibited severe structural abnormalities of the heart, and TRPV2-deficient cardiomyocytes from neonatal mice were also highly dysfunctional after growth in culture (Katanosaka et al., 2014), indicating that TRPV2 channel expression in cardiomyocytes is required for normal development and function of the heart (Katanosaka et al., 2014;Iwata and Matsumura, 2019). In macrophages, TRPV2 channel expression is essential for phagocytosis (Link et al., 2010;Lévêque et al., 2018), in pancreatic β-cells it influences insulin secretion (Hisanaga et al., 2009), in red blood cells it contributes to their response to osmotic challenges (Belkacemi et al., 2021), and its expression levels are significantly altered in multiple types of cancers (Monet et al., 2010;Yamada et al., 2010;Kudou et al., 2019;Siveen et al., 2020;Guéguinou et al., 2021). ...
Full-text available
The cation-permeable TRPV2 channel is essential for cardiac and immune cells. Cannabidiol (CBD), a non-psychoactive cannabinoid of clinical relevance, is one of the few molecules known to activate TRPV2. Using the patch-clamp technique we discover that CBD can sensitize current responses of the rat TRPV2 channel to the synthetic agonist 2-aminoethoxydiphenyl borate (2-APB) by over two orders of magnitude, without sensitizing channels to activation by moderate (40 °C) heat. Using cryo-EM we uncover a new small-molecule binding site in the pore domain of rTRPV2 that can be occupied by CBD in addition to a nearby CBD site that had already been reported. The TRPV1 and TRPV3 channels share >40% sequence identity with TRPV2 are also activated by 2-APB and CBD, but we only find a strong sensitizing effect of CBD on the response of mouse TRPV3 to 2-APB. Mutations at non-conserved positions between rTRPV2 and rTRPV1 in either the pore domain or the CBD sites failed to confer strong sensitization by CBD in mutant rTRPV1 channels. Together, our results indicate that CBD-dependent sensitization of TRPV2 channels engages multiple channel regions and possibly involves more than one CBD and 2-APB sites. The remarkably robust effect of CBD on TRPV2 and TRPV3 channels offers a promising new tool to both understand and overcome one of the major roadblocks in the study of these channels – their resilience to activation.
Abstract The immune system is capable of identifying and eliminating cancer, a complicated illness marked by unchecked cellular proliferation. The significance of ion channels in the complex interaction between the immune system and cancer has been clarified by recent studies. Ion channels, which are proteins that control ion flow across cell membranes, have variety of physiological purposes, such as regulating immune cell activity and tumor development. Immune cell surfaces contain ion channels, which have been identified to control immune cell activation, motility, and effector activities. The regulation of immune responses against cancer cells has been linked to a number of ion channels, including potassium, calcium, and chloride channels. As an example, potassium channels are essential for regulating T cell activation and proliferation, which are vital for anti-tumor immunity. Calcium channels play a crucial role when immune cells produce cytotoxic chemicals in order to eliminate cancer cells. Chloride channels also affect immune cell infiltration and invasion into malignancies. Additionally, tumor cells’ own expressed ion channels have an impact on their behavior and in the interaction with the immune system. The proliferation, resistance to apoptosis, and immune evasion of cancer cells may all be impacted by changes in ion channel expression and function. Ion channels may also affect the tumor microenvironment by controlling angiogenesis, inflammatory responses, and immune cell infiltration. Ion channel function in the interaction between the immune system and cancer has important implications for cancer treatment. A possible method to improve anti-tumor immune responses and stop tumor development is to target certain ion channels. Small compounds and antibodies are among the ion channel modulators under investigation as possible immunotherapeutics. The complex interaction between ion channels, the immune system, and cancer highlights the significance of these channels for tumor immunity. The development of novel therapeutic strategies for the treatment of cancer will be made possible by unraveling the processes by which ion channels control immune responses and tumor activity. Hence, the main driving idea of the present chapter is trying to understand the possible function of ion channels in the complex crosstalk between cancer and immunoresponse. To this aim, after giving a brief journey of ion channels throughout the history, a classification of the main ion channels involved in cancer disease will be discussed. Finally, the last paragraph will focus on more recently advancements in the use of biomaterials as therapeutic strategy for cancer treatment. The hope is that future research will take advantage of the promising combination of ion channels, immunomodulation and biomaterials filed to provide better solutions in the treatment of cancer disease. Keywords Ion channelsImmune systemCancerTumor immunityPotassium channelsCalcium channelsChloride channelsImmunotherapyBiomaterials as immunotherapeutics agents
Background: The potential of membrane transporters activated in cancer stem cells (CSCs) as new therapeutic targets for cancer is attracting increasing interest. Therefore, the present study examined the expression profiles of ion transport-related molecules in the CSCs of esophageal adenocarcinoma (EAC). Methods: Cells that highly expressed aldehyde dehydrogenase 1 family member A1 (ALDH1A1) were separated from OE33 cells, a human Barrett's EAC cell line, by fluorescence-activated cell sorting. CSCs were identified based on the formation of tumorspheres. Gene expression profiles in CSCs were examined by a microarray analysis. Results: Among OE33 cells, ALDH1A1 messenger RNA levels were higher in CSCs than in non-CSCs. Furthermore, CSCs exhibited resistance to cisplatin and had the capacity to redifferentiate. The results of the microarray analysis of CSCs showed the up-regulated expression of several genes related to ion channels/transporters, such as transient receptor potential vanilloid 2 (TRPV2) and solute carrier family 12 member 2 (SLC12A2). The cytotoxicities of the TRPV2 inhibitor tranilast and the SLC12A2 inhibitor furosemide were higher at lower concentrations in CSCs than in non-CSCs, and both markedly reduced the number of tumorspheres. The cell population among OE33 cells that highly expressed ALDH1A1 also was significantly decreased by these inhibitors. Conclusions: Based on the present results, TRPV2 and SLC12A2 are involved in the maintenance of CSCs, and their specific inhibitors, tranilast and furosemide, respectively, have potential as targeted therapeutic agents for EAC.
Background: Transient receptor potential vanilloid 2 (TRPV2) is a member of the TRP superfamily of non-specific cation channels with functionally diverse roles. We herein investigated the effects of TRPV2 on the expression of programmed cell death-ligand 1 (PD-L1) and its binding ability to programmed cell death-1 (PD-1) in gastric cancer (GC). Methods: Knockdown (KD) experiments were performed on human GC cell lines using TRPV2 small-interfering RNA. The surface expression of PD-L1 and its binding ability to PD-1 were analyzed by flow cytometry. Eighty primary tissue samples were assessed by immunohistochemistry (IHC), and the relationships between IHC results, clinicopathological factors, and patient prognosis were analyzed. The molecular mechanisms underlying the effects of TRPV2 on the intracellular ion environment were also investigated. Results: TRPV2-KD decreased the expression level of PD-L1 in NUGC4 and MKN7 cells, thereby inhibiting its binding to PD-1. A survival analysis revealed that 5-year overall survival rates were significantly lower in the TRPV2 high expression and PD-L1-positive groups. In IHC multivariate analysis of GC patients, high TRPV2 expression was identified as an independent prognostic factor. Furthermore, a positive correlation was observed between the expression of TRPV2 and PD-L1. An immunofluorescence analysis showed that TRPV2-KD decreased the intracellular concentration of calcium ([Ca2+]i). Treatment with ionomycin/PMA (phorbol 12-myristate 13-acetate), which increased [Ca2+]i, upregulated the protein expression of PD-L1 and promoted its binding to PD-1. Conclusions: The surface expression of PD-L1 and its binding ability to PD-1 in GC were regulated by TRPV2 through [Ca2+]i, indicating the potential of TRPV2 as a biomarker and target of immune checkpoint blockage for GC.
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The TRP superfamily of channels (nomenclature as agreed by NC-IUPHAR [176, 1072]), whose founder member is the Drosophila Trp channel, exists in mammals as six families; TRPC, TRPM, TRPV, TRPA, TRPP and TRPML based on amino acid homologies. TRP subunits contain six putative TM domains and assemble as homo- or hetero-tetramers to form cation selective channels with diverse modes of activation and varied permeation properties (reviewed by [730]). Established, or potential, physiological functions of the individual members of the TRP families are discussed in detail in the recommended reviews and in a number of books [401, 686, 1155, 256]. The established, or potential, involvement of TRP channels in disease [1126] is reviewed in [448, 685], [688] and [464], together with a special edition of Biochemica et Biophysica Acta on the subject [685]. Additional disease related reviews, for pain [633], stroke [1135], sensation and inflammation [988], itch [130], and airway disease [310, 1051], are available. The pharmacology of most TRP channels has been advanced in recent years. Broad spectrum agents are listed in the tables along with more selective, or recently recognised, ligands that are flagged by the inclusion of a primary reference. See Rubaiy (2019) for a review of pharmacological tools for TRPC1/C4/C5 channels [805]. Most TRP channels are regulated by phosphoinostides such as PtIns(4,5)P2 although the effects reported are often complex, occasionally contradictory, and likely to be dependent upon experimental conditions, such as intracellular ATP levels (reviewed by [1009, 689, 801]). Such regulation is generally not included in the tables.When thermosensitivity is mentioned, it refers specifically to a high Q10 of gating, often in the range of 10-30, but does not necessarily imply that the channel's function is to act as a 'hot' or 'cold' sensor. In general, the search for TRP activators has led to many claims for temperature sensing, mechanosensation, and lipid sensing. All proteins are of course sensitive to energies of binding, mechanical force, and temperature, but the issue is whether the proposed input is within a physiologically relevant range resulting in a response. TRPA (ankyrin) familyTRPA1 is the sole mammalian member of this group (reviewed by [293]). TRPA1 activation of sensory neurons contribute to nociception [414, 890, 602]. Pungent chemicals such as mustard oil (AITC), allicin, and cinnamaldehyde activate TRPA1 by modification of free thiol groups of cysteine side chains, especially those located in its amino terminus [575, 60, 365, 577]. Alkenals with α, β-unsaturated bonds, such as propenal (acrolein), butenal (crotylaldehyde), and 2-pentenal can react with free thiols via Michael addition and can activate TRPA1. However, potency appears to weaken as carbon chain length increases [26, 60]. Covalent modification leads to sustained activation of TRPA1. Chemicals including carvacrol, menthol, and local anesthetics reversibly activate TRPA1 by non-covalent binding [424, 511, 1081, 1080]. TRPA1 is not mechanosensitive under physiological conditions, but can be activated by cold temperatures [425, 212]. The electron cryo-EM structure of TRPA1 [740] indicates that it is a 6-TM homotetramer. Each subunit of the channel contains two short ‘pore helices’ pointing into the ion selectivity filter, which is big enough to allow permeation of partially hydrated Ca2+ ions. TRPC (canonical) familyMembers of the TRPC subfamily (reviewed by [284, 778, 18, 4, 94, 446, 739, 70]) fall into the subgroups outlined below. TRPC2 is a pseudogene in humans. It is generally accepted that all TRPC channels are activated downstream of Gq/11-coupled receptors, or receptor tyrosine kinases (reviewed by [765, 953, 1072]). A comprehensive listing of G-protein coupled receptors that activate TRPC channels is given in [4]. Hetero-oligomeric complexes of TRPC channels and their association with proteins to form signalling complexes are detailed in [18] and [447]. TRPC channels have frequently been proposed to act as store-operated channels (SOCs) (or compenents of mulimeric complexes that form SOCs), activated by depletion of intracellular calcium stores (reviewed by [741, 18, 770, 820, 1121, 157, 726, 64, 158]). However, the weight of the evidence is that they are not directly gated by conventional store-operated mechanisms, as established for Stim-gated Orai channels. TRPC channels are not mechanically gated in physiologically relevant ranges of force. All members of the TRPC family are blocked by 2-APB and SKF96365 [347, 346]. Activation of TRPC channels by lipids is discussed by [70]. Important progress has been recently made in TRPC pharmacology [805, 619, 436, 102, 851, 191, 291]. TRPC channels regulate a variety of physiological functions and are implicated in many human diseases [295, 71, 885, 1031, 1025, 154, 103, 561, 913, 409]. TRPC1/C4/C5 subgroup TRPC1 alone may not form a functional ion channel [229]. TRPC4/C5 may be distinguished from other TRP channels by their potentiation by micromolar concentrations of La3+. TRPC2 is a pseudogene in humans, but in other mammals appears to be an ion channel localized to microvilli of the vomeronasal organ. It is required for normal sexual behavior in response to pheromones in mice. It may also function in the main olfactory epithelia in mice [1114, 723, 724, 1115, 539, 1168, 1109].TRPC3/C6/C7 subgroup All members are activated by diacylglycerol independent of protein kinase C stimulation [347].TRPM (melastatin) familyMembers of the TRPM subfamily (reviewed by [275, 346, 741, 1151]) fall into the five subgroups outlined below. TRPM1/M3 subgroupIn darkness, glutamate released by the photoreceptors and ON-bipolar cells binds to the metabotropic glutamate receptor 6 , leading to activation of Go . This results in the closure of TRPM1. When the photoreceptors are stimulated by light, glutamate release is reduced, and TRPM1 channels are more active, resulting in cell membrane depolarization. Human TRPM1 mutations are associated with congenital stationary night blindness (CSNB), whose patients lack rod function. TRPM1 is also found melanocytes. Isoforms of TRPM1 may present in melanocytes, melanoma, brain, and retina. In melanoma cells, TRPM1 is prevalent in highly dynamic intracellular vesicular structures [398, 708]. TRPM3 (reviewed by [714]) exists as multiple splice variants which differ significantly in their biophysical properties. TRPM3 is expressed in somatosensory neurons and may be important in development of heat hyperalgesia during inflammation (see review [941]). TRPM3 is frequently coexpressed with TRPA1 and TRPV1 in these neurons. TRPM3 is expressed in pancreatic beta cells as well as brain, pituitary gland, eye, kidney, and adipose tissue [713, 940]. TRPM3 may contribute to the detection of noxious heat [1017]. TRPM2TRPM2 is activated under conditions of oxidative stress (respiratory burst of phagocytic cells). The direct activators are calcium, adenosine diphosphate ribose (ADPR) [970] and cyclic ADPR (cADPR) [1118]. As for many ion channels, PI(4,5)P2 must also be present [1109]. Numerous splice variants of TRPM2 exist which differ in their activation mechanisms [239]. Recent studies have reported structures of human (hs) TRPM2, which demonstrate two ADPR binding sites in hsTRPM2, one in the N-terminal MHR1/2 domain and the other in the C-terminal NUDT9-H domain. In addition, one Ca2+ binding site in the intracellular S2-S3 loop is revealed and proposed to mediate Ca2+ binding that induces conformational changes leading the ADPR-bound closed channel to open [387, 1027]. Meanwhile, a quadruple-residue motif (979FGQI982) was identified as the ion selectivity filter and a gate to control ion permeation in hsTRPM2 [1120]. TRPM2 is involved in warmth sensation [848], and contributes to several diseases [76]. TRPM2 interacts with extra synaptic NMDA receptors (NMDAR) and enhances NMDAR activity in ischemic stroke [1164]. Activation of TRPM2 in macrophages promotes atherosclerosis [1165, 1147]. Moreover, silica nanoparticles induce lung inflammation in mice via ROS/PARP/TRPM2 signaling-mediated lysosome impairment and autophagy dysfunction [1028]. Recent studies have designed various compounds for their potential to selectively inhibit the TRPM2 channel, including ACA derivatives A23, and 2,3-dihydroquinazolin-4(1H)-one derivatives [1137, 1139]. TRPM4/5 subgroupTRPM4 and TRPM5 have the distinction within all TRP channels of being impermeable to Ca2+ [1072]. A splice variant of TRPM4 (i.e.TRPM4b) and TRPM5 are molecular candidates for endogenous calcium-activated cation (CAN) channels [327]. TRPM4 is active in the late phase of repolarization of the cardiac ventricular action potential. TRPM4 deletion or knockout enhances beta adrenergic-mediated inotropy [593]. Mutations are associated with conduction defects [404, 593, 879]. TRPM4 has been shown to be an important regulator of Ca2+ entry in to mast cells [993] and dendritic cell migration [52]. TRPM5 in taste receptor cells of the tongue appears essential for the transduction of sweet, amino acid and bitter stimuli [537] TRPM5 contributes to the slow afterdepolarization of layer 5 neurons in mouse prefrontal cortex [513]. Both TRPM4 and TRPM5 are required transduction of taste stimuli [246]. TRPM6/7 subgroupTRPM6 and 7 combine channel and enzymatic activities (‘chanzymes’) [172]. These channels have the unusual property of permeation by divalent (Ca2+, Mg2+, Zn2+) and monovalent cations, high single channel conductances, but overall extremely small inward conductance when expressed to the plasma membrane. They are inhibited by internal Mg2+ at ~0.6 mM, around the free level of Mg2+ in cells. Whether they contribute to Mg2+ homeostasis is a contentious issue. PIP2 is required for TRPM6 and TRPM7 activation [810, 1077]. When either gene is deleted in mice, the result is embryonic lethality [413, 1065]. The C-terminal kinase region of TRPM6 and TRPM7 is cleaved under unknown stimuli, and the kinase phosphorylates nuclear histones [479, 480]. TRPM7 is responsible for oxidant- induced Zn2+ release from intracellular vesicles [3] and contributes to intestinal mineral absorption essential for postnatal survival [622]. The putative metal transporter proteins CNNM1-4 interact with TRPM7 and regulate TRPM7 channel activity [40, 467]. TRPM8Is a channel activated by cooling and pharmacological agents evoking a ‘cool’ sensation and participates in the thermosensation of cold temperatures [63, 178, 224] reviewed by [1011, 562, 457, 649]. Direct chemical agonists include menthol and icilin[1086]. Besides, linalool can promote ERK phosphorylation in human dermal microvascular endothelial cells, down-regulate intracellular ATP levels, and activate TRPM8 [68]. Recent studies have found that TRPM8 has typical S4-S5 connectomes with clear selective filters and exowell rings [512], and have identified cryo-electron microscopy structures of mouse TRPM8 in closed, intermediate, and open states along the ligand- and PIP2-dependent gated pathways [1111]. Moreover, the last 36 amino acids at the carboxyl terminal of TRPM8 are key protein sequences for TRPM8's temperature-sensitive function [194]. TRPM8 deficiency reduced the expression of S100A9 and increased the expression of HNF4α in the liver of mice, which reduced inflammation and fibrosis progression in mice with liver fibrosis, and helped to alleviate the symptoms of bile duct disease [556]. Channel deficiency also shortens the time of hypersensitivity reactions in migraine mouse models by promoting the recovery of normal sensitivity [12]. A cyclic peptide DeC‐1.2 was designed to inhibit ligand activation of TRPM8 but not cold activation, which can eliminate the side effects of cold dysalgesia in oxaliplatin-treated mice without changing body temperature [9]. Analysis of clinical data shows that TRPM8-specific blockers WS12 can reduce tumor growth in colorectal cancer xenografted mice by reducing transcription and activation of Wnt signaling regulators and β-catenin and its target oncogenes, such as C-Myc and Cyclin D1 [732]. TRPML (mucolipin) familyThe TRPML family [782, 1132, 775, 1084, 190] consists of three mammalian members (TRPML1-3). TRPML channels are probably restricted to intracellular vesicles and mutations in the gene (MCOLN1) encoding TRPML1 (mucolipin-1) cause the neurodegenerative disorder mucolipidosis type IV (MLIV) in man. TRPML1 is a cation selective ion channel that is important for sorting/transport of endosomes in the late endocytotic pathway and specifically, fission from late endosome-lysosome hybrid vesicles and lysosomal exocytosis [822]. TRPML2 and TRPML3 show increased channel activity in low luminal sodium and/or increased luminal pH, and are activated by similar small molecules [319, 147, 877]. A naturally occurring gain of function mutation in TRPML3 (i.e. A419P) results in the varitint waddler (Va) mouse phenotype (reviewed by [782, 690]). TRPP (polycystin) familyThe TRPP family (reviewed by [216, 214, 300, 1061, 374]) or PKD2 family is comprised of PKD2 (PC2), PKD2L1 (PC2L1), PKD2L2 (PC2L2), which have been renamed TRPP1, TRPP2 and TRPP3, respectively [1072]. It should also be noted that the nomenclature of PC2 was TRPP2 in old literature. However, PC2 has been uniformed to be called TRPP2 [345]. PKD2 family channels are clearly distinct from the PKD1 family, whose function is unknown. PKD1 and PKD2 form a hetero-oligomeric complex with a 1:3 ratio. [905]. Although still being sorted out, TRPP family members appear to be 6TM spanning nonselective cation channels. TRPV (vanilloid) familyMembers of the TRPV family (reviewed by [995]) can broadly be divided into the non-selective cation channels, TRPV1-4 and the more calcium selective channels TRPV5 and TRPV6. TRPV1-V4 subfamilyTRPV1 is involved in the development of thermal hyperalgesia following inflammation and may contribute to the detection of noxius heat (reviewed by [762, 882, 922]). Numerous splice variants of TRPV1 have been described, some of which modulate the activity of TRPV1, or act in a dominant negative manner when co-expressed with TRPV1 [844]. The pharmacology of TRPV1 channels is discussed in detail in [329] and [1015]. TRPV2 is probably not a thermosensor in man [736], but has recently been implicated in innate immunity [547]. Functional TRPV2 expression is described in placental trophoblast cells of mouse [204]. TRPV3 and TRPV4 are both thermosensitive. There are claims that TRPV4 is also mechanosensitive, but this has not been established to be within a physiological range in a native environment [127, 530]. TRPV5/V6 subfamily TRPV5 and TRPV6 are highly expressed in placenta, bone, and kidney. Under physiological conditions, TRPV5 and TRPV6 are calcium selective channels involved in the absorption and reabsorption of calcium across intestinal and kidney tubule epithelia (reviewed by [1057, 205, 651, 270]).TRPV6 is reported to play a key role in calcium transport in the mouse placenta [1056].
Intracellular calcium (Ca2+) is an essential second messenger in eukaryotic cells regulating numerous cellular functions such as contraction, secretion, immunity, growth, and metabolism. Ca2+ signaling is also a key signal transducer in the intrinsic apoptosis pathway. The store-operated Ca2+ entry pathway (SOCE) is ubiquitously expressed in eukaryotic cells, and is the primary Ca2+ influx pathway in non-excitable cells. SOCE is mediated by the endoplasmic reticulum Ca2+ sensing STIM proteins, and the plasma membrane Ca2+-selective Orai channels. A growing number of studies have implicated SOCE in regulating cell death primarily via the intrinsic apoptotic pathway in a variety of tissues and in response to physiological stressors such as traumatic brain injury, ischemia reperfusion injury, sepsis, and alcohol toxicity. Notably, the literature points to excessive cytosolic Ca2+ influx through SOCE in vulnerable cells as a key factor tipping the balance towards cellular apoptosis. While the literature primarily addresses the functions of STIM1 and Orai1, STIM2, Orai2 and Orai3 are also emerging as potential regulators of cell death. Here, we review the functions of STIM and Orai proteins in regulating cell death and the implications of this regulation to human pathologies.
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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.
(−)-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
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.
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.
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.
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.
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.
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
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.