Cell Calcium
Unexpected Ca2+-mobilization of oxaliplatin via H1 histamine receptors
Potenzieri A, Riva B, Genazzani AA
ABSTRACT
Oxaliplatin is a widely used chemotherapeutic drug and represents the cornerstone of colorectal cancer therapy,
in combination with 5-fluorouracil and folinic acid. As with many chemotherapeutic agents, its use is associated
with a number of side effects, ranging from hypersensitivity reactions to haematological dyscrasias. Oxaliplatin
also induces acute and chronic peripheral neuropathy.
While it is likely that the haematological side effects are associated with its anti-proliferative effects and with
the ability to form DNA adducts, the molecular mechanisms underlying peripheral neuropathy and hypersensitivity reactions are poorly understood, and therefore the choice of adequate supportive therapies is
largely empirical.
Here we show that an acute low dose oxaliplatin application on DRG neurons is able to induce an increase in
intracellular calcium that is dependent on the Histamine 1 receptor (H1). Oxaliplatin-induced intracellular
calcium rises are blocked by two selective H1 antagonist, as well as by U73122, a PLC inhibitor, and by 2-APB, a
non-specific IP3 receptor blocker. Moreover, expression of the H1 receptor on HEK293 t cells unmasks an oxaliplatin-induced Ca2+-rise. Last, activation of H1 via either histamine or oxaliplatin activates TRPV1 receptors,
a mechanism that has been associated with itch. These data, together with literature data that has shown that
anti-histamine agents reduce the incidence of oxaliplatin-induced hypersensitivity, may provide a molecular
mechanism of this side effect in oncological patients.
1. Introduction
Oxaliplatin (OHP) is a pillar chemotherapeutic agent in colorectal
cancer therapy, mainly in combination with 5-fluorouracil and folinic
acid [1]. Its primary mechanism of action can be re-conducted to the
formation of inter-strand and intrastrand adducts with DNA.
While the formation of DNA adducts underlies most likely the
haematological effects of OHP, e.g. anemia, thrombocytopenia and
neutropenia, it is less likely to be responsible for other side effects,
including peripheral neuropathy and hypersensitivity reactions [2,3].
OHP-induced peripheral neuropathy is thought to arise from sensitization of TRP channels in dorsal root ganglia (DRG) neurons [4]. Indeed, either pharmacological antagonism or genetic ablation of TRPA1
and TRPV1 channels renders rodents less susceptible to OHP-induced
cold allodynia [5,6].
Modification of the Ca2+-toolkit by OHP has been shown in DRG
neurons, in which prolonged exposure to OHP (24 h) leads to a sensitization of ATP-induced Ca2+-release [7]. We have recently hypothesized that these modifications are most likely a result of pH acidification
of the cytosol [8]. In other words, OHP affects indirectly calcium
signalling by modifying intracellular pH and therefore modulating pHsensitive channels, including TRPA1 and TRPV1, or by triggering
transcriptional rearrangements [9]. Indeed, a change in the qualitative
and quantitative components of the calcium toolkit has been shown to
affect Ca2+-signals in a number of conditions [10].
Yet, these mechanisms are unlikely to explain acute side effects,
including hypersensitivity reactions. In the present manuscript, we set
to investigate the acute effects of OHP on DRG neurons, as contrasting
effects have been reported. Shultze et al. reported that low micromolar
concentrations of OHP do not induce any Ca2+-signal in DRG cells or in
the SH-SY5Y neuroblastoma cells [7], while Kawashiri et al. showed
that OHP, at non-therapeutic concentrations induced an increase in
intracellular Ca2+ secondary to voltage-operated Ca2+-channel
opening [11]. Such contrasting reports may well be attributed to the
concentrations used, given that it is highly likely that the higher the
concentration, the more non-specific protein adducts will be formed.
We now report that acute treatment of DRG neurons with therapeutic concentrations of OHP (0.1 μg/ml) leads to Ca2+-release that is
dependent on type 1 histamine receptors (H1). How this occurs is at
present unknown, although the fact that the effect is antagonized by a
https://doi.org/10.1016/j.ceca.2019.102128
Received 12 August 2019; Received in revised form 20 November 2019; Accepted 20 November 2019
Abbreviations: OHP, Oxaliplatin; DRG, dorsal root ganglia ⁎ Corresponding author.
E-mail address: [email protected] (A. Genazzani).
Cell Calcium 86 (2020) 102128
Available online 03 December 2019
0143-4160/ © 2019 Elsevier Ltd. All rights reserved.
T
competitive antagonist of H1 would lead to speculate that OHP binds
close to the histamine site. Furthermore, activation of H1 receptors with
OHP, as it has been previously reported for histamine [12], leads to
opening and desensitization of TRPV1 receptors. The interaction between TRPV1 and H1 receptors in DRG neurons has been associated
with histamine-induced itch [12]. These data, together with the
knowledge that histamine H1 antagonists reduce the percentage of
patients hypersensitivity reactions and the itch induced by OHP [13],
might therefore provide a molecular mechanism for this side effect and
a rationale for a supportive therapy.
2. Materials and methods
2.1. Animals and husbandry
BALB/C male mice aged 4–8 weeks were purchased from Envigo
(San Pietro al Natisone, Italy). Care and husbandry of animals were in
conformity with the institutional guidelines in compliance with national and international laws and policies. Mice were housed in cages in
22 °C monitored rooms with 12 h light/dark cycles with ad libitum access to food and water and weaned at 23 days old by sex. The procedures were approved by the local animal-health and ethical Committee
(Università del Piemonte Orientale) and were authorized by the national authority (Istituto Superiore di Sanità; authorization number N.
22/2013). All mice were euthanized under deep CO2-induced anaesthesia.
2.2. Drugs
Oxaliplatin (OHP), trazodone, haloperidol, ketanserin, cetirizine,
loratadine, histamine (HIST), capsazepine, capsaicin and icilin were
purchased from Sigma-Aldrich Inc., Italy. Nigericin, BCECF (2′,7′-Bis-
(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester)
and Fura 2-AM were purchased from Life Technologies, Italy. These
drugs, with the exception of capsaicin (reconstituted in 100 % EtOH),
capsazepine (reconstituted in 100 % methanol) and histamine (reconstituted in milliQ (MilliPore) water), were dissolved in 100 % dimethyl
sulfoxide (DMSO) and stored at −20 °C, according to manufacturers’
specifications. Working concentrations of these drugs were freshly
prepared for each experiment by diluting DMSO, methanol or EtOH to
0.1 % in milliQ (MilliPore) water.
2.3. Isolation and primary cell culture of mouse DRG neurons
DRG obtained from adult BALB/C mice (4/8-wk-old) were excised
and collected in a dish containing cold F12 (Nutrient Mixture F12 Ham)
medium (Sigma Aldrich Inc.). Working under a dissecting microscope
and using fine forceps, the surrounding membranes were gently teased
away from each DRG; nerves and sheath were cut. All de-sheathed DRG
were then transferred into a sterile 35 mm dish containing collagenase
from Clostridium hystoliticum 0.125 % (Sigma Aldrich Inc., Italy) and
DNase (Sigma Aldrich Inc., Italy) in F12 (Nutrient Mixture F12 Ham)
medium and incubated at 37 °C for 1 h. After incubation, DRG were
triturated using a 1000 μl tip. Myelin and nerve debris were eliminated
by centrifugation through a bovine serum albumin (BSA) cushion. Cell
pellets were re-suspended in Bottenstein and Sato medium (BS) composed of 30 % F12 (Nutrient Mixture F12 Ham medium), 40 % DMEM
(Dulbecco’s Modified Eagle’s medium (Sigma Aldrich Inc., Italy), 30 %
Neurobasal A medium (Life Technologies, Italy), 100 X N2 supplement
(Life Technologies, Italy), penicillin 10 U/mL and streptomycin
100 mg/mL (Sigma Aldrich Inc., Italy), supplemented with
Recombinant Human β-NGF, Recombinant Murine GDNF and
Recombinant Human NT3 (Peprotech, USA) and plated onto 24 mm
glass coverslips pre-coated with laminin (Sigma Aldrich Inc., Italy).
2.4. Cell cultures
Human embryonic kidney (HEK293t) cells were obtained from
ATCC (Rockville, MD, USA) and were cultured in Dulbecco’s Modified
Eagle’s Medium (DMEM; Sigma-Aldrich Inc., Italy), supplemented with
10 % heat-inactivated FBS (Gibco, Italy), L-glutamine 50 mg/mL
(Sigma-Aldrich, Italy), 10 U/mL penicillin, and 100 mg/mL streptomycin (Sigma-Aldrich Inc., Italy) at 37 °C, under a 5 % CO2 humidified
atmosphere.
Human primary lung airway smooth muscle cells (ASM) were obtained from ATCC (Rockville, MD, USA) and were cultured in Vascular
Cell Basal Medium (VCBM; ATCC, Rockville, MD, USA) supplemented
with 5 % heat-inactivated fetal bovine serum (FBS), L-glutamine 50 mg/
mL (Sigma-Aldrich, Italy), 10 U/mL penicillin, and 100 mg/mL streptomycin, 0,5 % antibiotic-antimycotic (ThermoFisher, Italy), 5 ng/ml of
basic-fibroblasts growth factor and 5 ng/ml epidermal growth factor
(Immunotools), 50 μg/ml of ascorbic acid, 10 ng/ml of insulin (SigmaAldrich Inc., Italy).
For calcium experiments, cell lines were seeded on poly-L-lysine
(Sigma-Aldrich Inc., Italy) coated glass coverslips at concentrations
10 × 103 per mL (24 mm diameter coverslips in 6-well plates).
2.5. Generation of Hek Cells overexpressing H1 and TRPV1 receptors
HEK293 t cells overexpressing H1 receptors were maintained in a
37 °C, 5 % CO2 humidified incubator as described for HEK293 t cells.
The human H1 (pH1R-P2A-mCherry-N1)receptor was purchased from
Addgene. The Rat pcDNA3-TRPV1 was a kind gift from Prof. Asia
Fernandez Carvajal and was confirmed by sequencing. For cell transfection, 7 μg of plasmid DNA was transfected using lipid reagent lipofectamine (Lipofectamine 2000 Transfection Reagent, Life
Technologies). 24 h after transfection, PCR was performed to evaluate
the gene expression.
2.6. Gene expression evaluation by PCR
Total RNA was isolated from HEK, HEK-H1, HEK-H1-TRPV1 cells
using TRI-Reagent® and reverse transcribed according to the manufacturer’s instructions (SENSIFAST, Aurogene, Italy). cDNA was then
stored at −20 °C until further used. The PCR reaction was conducted
using 2x DreamTaq Hot Start Green PCR Master Mix (ThermoFisher,
Italy), forward and reverse specific primers (H1: FW: 5′- CTTGGTCAC
AGTAGGGCTCA-3′, REV: 5′-TGGGGAACCTGTACATCGTC-3′; TRPV1:
TRPV1, FW:5′-CCTGCATTGACACCTGTGAG-3′; REV: 5′-AGAAGATGC
GCTTGACAAATC-3′), and cDNA obtained as previously described. The
electrophoresis of the PCR products were performed in a 2 % agarose
gel (Euroclone spa, Italy).
2.7. Fura-2 Ca2+ measurements and image analysis
DRG cultures, HEK or HEK-H1 cells were loaded with 5 μM Fura-2
AM in presence of 0.02 % of Pluronic-127 (both from Life Technologies,
Italy) and 10 μM sulfinpyrazone (Sigma Adrich Inc., Italy) in
Krebs–Ringer buffer (KRB, 135 mM NaCl, 5 mM KCl, 0.4 mM KH2PO4,
1 mM MgSO4, 5.5 mM glucose, 20 mM HEPES, pH 7.4) containing 2 mM
CaCl2 (30 min, room temperature). Cells were washed and incubated
with KRB for 15 min to allow de-esterification of Fura-2. Coverslips
were mounted into acquisition chamber and places on the stage of a
Leica DMI6000 epifluorescent microscope equipped with S Fluor ×40/
1.3 objective. The probe was excited by alternate 340 and 380 nm using
a Polychrome IV monochromator (Till Photonics, Munich, Germany)
and the Fura-2 emission light was filtered through 520/20 bandpass
filter and collected by a cooled CCD camera (Hamamatsu, Japan). The
fluorescence signals were acquired and processed using MetaFluor
software (Molecular Device, Sunnyvale, CA, USA). To quantify the
differences in the amplitudes of Ca2+ transients the ratio values were
A. Potenzieri, et al. Cell Calcium 86 (2020) 102128
2
normalized using the formula ΔF/F0. Only responsive cells were used
for statistical analysis. Responsive cells were defined as having increase
that varied by at least an order of magnitude compared to the the basal
calcium.
2.8. Measurement of intracellular pH in DRG neurons by epifuorescent
microscopy with BCECF
DRG cells were plated onto 24 mm round cover-slips, incubated
with 1 μM BCECF (Life Technologies, Italy) in KRB containing 2 mM
CaCl2 (15 min, room temperature) and re-suspended in KRB (pH 7.4).
Coverslips were mounted into acquisition chamber and places on the
stage of a Leica DMI6000 epifluorescent microscope equipped with S
Fluor ×40/1.3 objective. Cells were alternatively excited at 490/
450 nm (monochromator Polychrome IV, Till Photonics, Germany) and
the fluorescent signals were collected and analysed every 10 s using
MetaFluor software (Molecular Devices, Sunny-vale, CA, USA).
Intracellular pH was calculated by comparing 525/610 nm emission
fluorescence ratios with calibration curves obtained by pH equilibration
using the proton ionophore nigericin (10 μM) and the Intracellular pH
Calibration Buffer Kit (pH 7.5-5.5, Life Technologies, Italy).
2.9. Statistical analysis
Data are presented as mean ± SEM or Median and IQR. The normality of data distributions was assessed using Shapiro–Wilk test.
Parametric (unpaired t-test and One-way analysis of variance (ANOVA)
followed by Tukey’s post-hoc) or non parametric (Mann-Whitney U test
and One-way Kruskal-Wallis H test followed by Dunn’s post-hoc) statistical analysis was used for comparisons of data. All statistical assessments were two-sided and a value of P < 0.05 was considered
statistically significant. Statistical analyses were performed using
GraphPad Prism software (GraphPad Software, Inc., USA). In in vitro
experiments, the n number was calculated on the number of cells, and
the number of independent experiments (defined as cultures performed
on different days) is given in the respective figure legends.
3. Results
3.1. Oxaliplatin increases intracellular calcium through activation of Gq
coupled receptors
Contrasting evidence exists in the literature on whether acute
treatment with OHP induces an acute Ca2+-response in cells [4]. To
analyse this aspect, we exposed DRG cultures to concentrations between 0.003 μg/mL and 0.3 μg μg/mL (75−750 nM), and found that
OHP dose-dependently increased intracellular Ca2+ (Fig. 1A and B)
with small differences between the different concentration used (responding cells were: OHP 0.03 μg/mL: 38 ± 11.1 %; OHP 0.1 μg/mL
53 ± 10 %; OHP 0.3 μg/mL: 60 ± 17 %). Therapeutically relevant
concentrations of OHP (0.1 μg/mL) induced a rapid rise in intracellular
calcium, which was evident also when cells were placed in a Ca2+-free
solution (Fig. 1C and E for quantification), suggesting that the source of
the ion was an intracellular store. When cells were analysed further, no
oscillations or other signals were observed over the course of 10 min
apart from the first spike (data not shown). To further investigate the
mechanism of the Ca2+-rise, cells were pre-incubated for 10 min with a
PLC inhibitor, U73122 (5 μM), or with a non-specific IP3 channel inhibitor, 2-APB (50 μM). Both agents abolished the response to OHP
(Fig. 1D and E), proving that OHP activates a receptor coupled to Gq
and its downstream PLC-IP3 receptor pathway.
3.2. Oxaliplatin is an agonist of the H1 histamine receptor
To pinpoint the receptor that might have been responsible for OHPinduced Ca2+-release, we used a number of multi-receptor antagonists
and compared the number of responding cells in the presence or absence of these antagonists (OHP 0.1 μg/mL responding cells were
53 ± 10 %; n = 122). In the presence of haloperidol (500 nM), that at
these concentrations blocks D2, D3, D4, H1, M1, α1, and 5HT2A-C
[14], or trazodone (1 μM), a H1, 5HT2A-C, 5HT1, α1 antagonist [14],
OHP elicited responses in 11 % (2/18) and 0 % (0/33) of cells, respectively. On the other hand, ketanserin, a specific 5HT2A-C antagonist was largely unable to affect responses, with 67 % of cells (30/45)
responding to OHP. This led us to hypothesize that H1 receptors might
have mediated the OHP response.
To verify the hypothesis generated with multi-receptor antagonists,
we then challenged DRG neurons pre-treated with H1 selective antagonists (cetirizine, 50 nM; loratidine, 100 nM). As it can be observed
in Fig. 2A and B, both antagonists fully blocked the response to OHP. To
further validate this hypothesis, we took advantage of the desensitization of H1 receptor after exposure with high histamine concentration
[15] and evaluated whether Ca2+-responses to OHP and histamine
cross-desensitized. As shown in Fig. 2C and D, when cells were challenged with high concentrations of histamine (300 μM) they were unable to respond to OHP (0.1 μg/mL) while they still responded to ATP
(100 μM), indicating that the effect was not due to a non specific desensitazion of its downstream cascade. The opposite experiment was
also true (Fig. 2E and F), as when cells were challenged with high
concentrations of OHP (3 μg/ml) they were unable to respond to a
challenge to histamine, but still responded to ATP. Homologous or
heterologous desensitization did not occur, instead, when lower concentrations of either OHP (0.1 μg/mL) or histamine (10 μM) were used
(supplementary Fig. S1). These data indicate that, surprisingly, OHP
behaves as an agonist of the H1 receptor. In our cultures, the % of
histamine-responsive neurons was 52.4 ± 6.3 % (n = 246) of the total
population, which was comparable to the OHP-sensitive population
(53 ± 0.1 %; see above). It should be noticed that previous reports
suggested that the number of histamine-sensitive neurons was lower
[16], and this discrepancy might be explained by culture methods,
strains of mice used or other factors.
The hypothesis that H1 is the receptor mediating OHP-induced
Ca2+-release is further substantiated by the fact that we observed a
response both to histamine or OHP in ASM (Fig. S2), a bronchial
smooth muscle cell line which is known to respond to histamine [17],
while we did not observe a response with either agent in HEK293 t cells
(Fig. 3), in which we could not detect a transcript for H1 (Fig. 3A,
inset). To conclusively confirm that OHP releases Ca2+ via H1 receptors, we therefore decided to transiently express H1 in HEK293 t
cells (Fig. 3A, inset). As shown in Fig. 3A and B, the presence of H1
receptors conferred sensitivity of cells to both histamine (10 μM) and
OHP (0.1 μg/ml).
3.3. H1 activation and cytosolic pH acidification are distinct phenomena
We have previously shown that OHP induces intracellular acidification in DRG neurons and that this sensitizes TRPA1 and blunts
responses to TRPV1 channels [8]. In the original report [8], we showed
that this acidification occurs after a 6 -h treatment although further
characterization has shown that acidification occurs as early as 30 min
[18]. We therefore decided to evaluate whether these two phenomena
were linked, i.e. whether Ca2+-responses are linked to cytosolic acidification, or, viceversa, whether it is the acidification that is responsible
for the Ca2+-release. We first determined whether we could detect a
significant pH change in the first five minutes of addition of OHP, but
this was not the case (pH CTRL 7.04 ± 0.02 n = 41; OHP 6.97 ± 0.03
n = 56). Furthermore, we evaluated whether OHP, in the presence of
H1 antagonists was still able to acidify the cytosol after 30 min. Indeed,
this was the case (pH CTRL 7.04 ± 0.02; n = 38; OHP 6.73 ± 0.049
n = 49; CTZ + OHP: 6,77 ± 0.026 n = 25). Last, we could detect a pH
change in HEK293 t cells after 30 min [18], despite the fact that these
cells do not possess H1 receptors transcripts and neither OHP nor
A. Potenzieri, et al. Cell Calcium 86 (2020) 102128
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histamine induced Ca2+-release. These data suggest that the effect of
OHP on H1 receptors and the effect on intracellular pH are mediated by
separate mechanisms.
3.4. OHP, like histamine, activates TRPV1 receptors
It has been previously reported that histamine is able to modulate
TRPV1 responses, partially contributing to histamine-induced itching
[12]. As shown in Fig. 4A, pre-treatment of DRG cells with histamine
led to a significant reduction in the subsequent Ca2+-response induced
Fig. 1. Oxaliplatin increases intracellular calcium through activation of Gq coupled receptors.
A Representatives Ca2+-traces from DRG neurons treated with OHP. Traces are representative of three independent cultures. The number of cells evaluated is
indicated in the figure. B Box and whisker plots show median and IQR of peak of calcium changes. Kruskal-Wallis H test followed by Dunn’s post-hoc. *P < 10−6 vs
OHP 0.03 μg/mL, + P < 10−6 vs OHP 0.1 μg/mL. C Representative Ca2+-traces from DRG neurons treated with 0.1 μg/mL of OHP in the presence or absence of EGTA
50 μM. Traces are representative of three independent cultures. The number of cells evaluated is indicated in the figure. D Representative Ca2+-traces from DRG
neurons treated with 0.1 μg/mL of OHP with or without pre-incubation with the PLC inhibitor U73122 (5 μM) or the non-specific IP3R blocker 2-APB (50 μM). Traces
are representative of three independent cultures. The number of cells evaluated is indicated in the figure. E Box and whisker plots show median and IQR of peak of
calcium changes. Kruskal-Wallis H test followed by Dunn’s post-hoc. *P < 10−6 vs OHP, + P < 10−6 vs OHP Ø Ca2+.
A. Potenzieri, et al. Cell Calcium 86 (2020) 102128
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Fig. 2. H1 antagonists prevent OHP-induced Ca2+-increases.
A Representatives Ca2+-traces from DRG neurons treated with 0.1 μg/mL of OHP in the presence or absence of cetirizine (50 nM) or loratadine (100 nM). Traces are
representative of four independent cultures. The number of cells evaluated is indicated in the figure. B Box and whisker plots show median and IQR of peak of Ca2+
changes. Kruskal-Wallis H test followed by Dunn’s post-hoc. *P < 10−6 vs OHP. C and E Representative Ca2+-traces from DRG neurons treated with 300 μM
histamine (C) or 3 μg/mL OHP (E) to desensitize the H1 receptor and then treated with OHP 0.1 μg/mL (C) or histamine 10 μM (E), respectively. ATP was used as a
control at the end of each experiment. Traces are representative of four independent cultures. The number of cells evaluated is indicated in the figure. D Box and
whisker plots show median and IQR of OHP peak of Ca2+ induced by OHP. Kruskal-Wallis H test followed by Dunn’s post-hoc. *P < 10−6 vs OHP DES. F Box and
whisker plots show median and IQR of the Ca2+-peak induced by histamine. Kruskal-Wallis H test followed by Dunn’s post-hoc. *P < 10−6 vs HIST DES.
A. Potenzieri, et al. Cell Calcium 86 (2020) 102128
5
by capsaicin both when compared to cells that did not respond to histamine (presumably due to the absence of the H1 receptor). An identical result was obtained when cells were pre-treated with OHP. More
importantly, the effect was totally reversed when experiments were
performed in the presence of cetirizine (Fig. 4B, C). Furthermore, the
number of capsaicin responsive cells decrease after treatment with
histamine or OHP, an effect that suggest a desensitization of TRPV1
receptors and that was abolished by pre-treatment with cetirizine
(Fig. 4D). It should be noticed that in our hands the number of capsaicin-sensitive cells is higher (90.5 ± 3.1 %) than what previously
reported by others [19,20], as is the case for histamine-sensitive neurons. The characterization of the diameters of our cell culture population has been recently published [8]. Last, we performed experiments in
the presence of the selective antagonist of TRPV1, capsazepine (10 μM).
As shown in Fig. 4E–G, this inhibitor reduced both the amplitude of the
Ca2+-peak and the area under the curve, providing further evidence for
the involvement of TRPV1 in the H1-induced signals.
To verify whether OHP had a direct effect on TRPV1 receptors, we
tested OHP on HEK cells expressing TRPV1. In these cells, no activity by
OHP or histamine was observed (Fig. S3). To further investigate whether the abolition of the capsaicin response observed in DRG neurons
pre-treated with OHP or histamine was the consequence of the desensitization of TRPV1 channels, we co-expressed H1 and TRPV1 receptors in HEK293 t cells (HEK H1-V1) and we compared the response
elicited by OHP or histamine with cells transfected exclusively with H1.
HEK-H1-V1 cells, as expected, responded to histamine, OHP and capsaicin. Yet, if cells were pre-treated with OHP or histamine, no response
to capsaicin was observed (Fig. 5A and B). Yet, when comparing the
area under the curve (Fig. 5D) of the responses to histamine or OHP,
this was significantly greater in HEK-H1-V1 cells compared to HEK-H1
cells. There was no difference, instead, in the peak Ca2+ elicited in the
two cell types (Fig. 5C), suggesting that Ca2+-entry through the TRPV1
channel contributed to the overall response to histamine/OHP and that
the lack of response to capsaicin observed in DRG neurons is due to
TRPV1 desensitization after activation via H1 receptors. Indeed, capsazepine was able to reduce the Ca2+-responses to histamine or OHP in
HEK-H1-V1 cells, although statistical significance was evident only for
the area under the curve (Fig. 5E–G).
4. Discussion
In the present manuscript we show, that OHP, at concentrations that
are therapeutically relevant in chemotherapy, triggers a Ca2+-response
that is mediated by H1 receptors. Furthermore, we show that such activation, similarly to histamine [12], is able to trigger the activation of
TRPV1 receptors in DRG neurons, a receptor implicated both in pruritus
and in neuropathic pain [21,22].
Our data are in contrast to previous reports that OHP, at similar
concentrations, was unable to elicit acute Ca2+-signals. A number of
factors may account for this discrepancy, including difference in species
and age of the donors for the culture (early postnatal rats vs adult mice).
Hypersensitivity reactions to OHP are rather frequent [3]. A large
trial with OHP (MOSAIC) has reported an incidence of 10.3 % of patients but observational studies suggest that in real practice the incidence could well be higher [23]. To minimize the possibility of hypersensitivity reactions, a pre-medication protocol with
immunosuppressants, mainly dexamethasone is usually employed [3].
Yet, antihistamine agents have also been empirically used [23]. When a
retrospective cohort analysis was undertaken in which patients treated
with dexamethasone alone were compared with patients treated with
dexamethasone and anti-histamine agents, hypersensitivity reactions
were reduced from 53 % to 11 % [13]. Unfortunately, to our knowledge
this retrospective finding was never used to design a prospective trial.
The serendipitous finding reported in the present manuscript was
obtained in DRG neurons, and it is possible that the pruritus and itch
observed in some OHP-treated patients is secondary to this system.
While we did not investigate the effect of OHP on extra-neuronal cellular systems (except for bronchial smooth muscle), which most likely
are the major contributors to the hypersensitivity reactions, the agonistic effect of OHP is likely to be similar, as shown also in heterologous
HEK293t cells.
In conclusion, therefore, therapeutically relevant concentration of
OHP, in vitro, both in DRG neurons, ASM cells and in transfected
HEK293t cells acts as a H1 agonist. We believe that these data provide a
strong rationale to envisage antihistamine agents as supportive therapy
for OHP-treated patients and for a trial to be conducted.
Author contribution
AP designed, performed and analysed all experiments and wrote the
manuscript. BR performed some experiments and data-analyses. AAG
supervised the project and wrote the manuscript.
Declaration of Competing Interest
The authors declares no conflict of interest.
Acknowledgements
This work was funded by Fondazione Cariplo to AAG (grant number
2013.0842).
Fig. 3. H1 receptors expression is required for OHP-induced intracellular calcium rises.
A Representative Ca2+-traces from HEK WT and HEK-H1 treated with 10 μM histamine or 0.1 μg/mL OHP. Traces are representative of three independent experiments. The number of cells evaluated is indicated in the figure. B Box and whisker plots show median and IQR of peak of calcium changes. Kruskal-Wallis H test
followed by Dunn’s post-hoc. *P < 10−6 vs HEK WT, +P < 10−6 vs HEK WT.
A. Potenzieri, et al. Cell Calcium 86 (2020) 102128
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Fig. 4. H1 receptors agonist activates TRPV1 channels in DRG neurons.
A Representative Ca2+-traces from DRG neurons treated with or without 0.1 μg/mL of OHP or 10 μM histamine and then exposed to capsaicin (200 nM). B
Representative Ca2+-traces from DRG neurons treated with 0.1 μg/mL of OHP in the presence or absence of CTZ and then exposed to capsaicin. Traces shown in A, B
are representative of four independent cultures. The number of cells evaluated is indicated in the figure. C Box and whisker plots show median and IQR of peak of
capsaicin induced calcium changes. Kruskal-Wallis H test followed by Dunn’s post-hoc. *P < 10−6 vs CTRL, + P < 10−6 vs CTZ + OHP. D Bar graph showing the
percentage of capsaicin responsive neurons. Histograms show the mean ± S.E.M of four separate experiments. One-way analysis of variance followed by Tukey’s
post-hoc.*P < 10−6 vs CTRL, + P < 10−6 vs CTZ + OHP. E Representative Ca2+-traces from DRG neurons treated with OHP or histamine in the presence or absence
of capsazepine (10 μM). Traces shown in A, B are representative of three independent cultures. The number of cells evaluated is indicated in the figure. F Box and
whisker plots show median and IQR of peak of calcium changes. Kruskal-Wallis H test followed by Dunn’s post-hoc. * P = 0.017, + P= 0.012 G Box and whisker plots
show median and IQR of AUC of calcium changes. Kruskal-Wallis H test followed by Dunn’s post-hoc.* P = 0.005, + P= 0.04.
A. Potenzieri, et al. Cell Calcium 86 (2020) 102128
7
Fig. 5. H1 receptors agonists activate and desensitize TRPV1 in HEK cell co-expressing H1-TRPV1 receptors.
A Representative Ca2+-traces from HEK H1-V1 cells treated with or without 10 μM histamine or 0.1 μg/mL OHP. Traces are representative of three independent
experiments. For each treatment about 100 cells were evaluated. B Box and whisker plots show median and IQR of peak of calcium changes. Kruskal-Wallis H test
followed by Dunn’s post-hoc. *P < 10−6 vs CTRL. C, D Comparison of max peak (C) and AUC (D) of HEK cells transfected with H1 and TRPV1 receptors or H1 alone.
*P = 0.0028 vs HEK-H1, +P = 0.0495 vs HEK-H1. E Representative Ca2+-traces from HEK H1-V1 cells treated with OHP or histamine in the presence or absence of
capsazepine (10 μM). Traces are representative of three independent experiments. The number of cells evaluated is indicated in the figure. F Box and whisker plots
show median and IQR of peak of calcium changes. Kruskal-Wallis H test followed by Dunn’s post-hoc. G Box and whisker plots show median and IQR of AUC of
calcium changes. Kruskal-Wallis H test followed by Dunn’s post-hoc. *P < 10−6 vs OHP, +P = 0.0008 vs HIST.
A. Potenzieri, et al. Cell Calcium 86 (2020) 102128
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