From mixed sigma-2 receptor/P-glycoprotein targeting agents to selective P-glycoprotein modulators: Small structural changes address the mechanism of interaction at the efflux pump

Article abstract of DOI:10.1016/j.bbamem.2014.08.022

Generations of modulators of the efflux pump P-glycoprotein (P-gp) have been produced as tools to counteract the Multidrug Resistance (MDR) phenomenon in tumor therapy, but clinical trials were not successful so far. With the aim of contributing to the development of novel P-gp modulators, we started from recently studied high-affinity sigma-2 (σ₂) receptor ligands that showed also potent interaction with P-gp. For σ₂ receptors high-affinity binding, a basic N-atom is a strict requirement. Therefore, we reduced the basic character of the N-atom present in these ligands, and we obtained potent P-gp modulators with poor or null σ₂ receptor affinity. We also evaluated whether modulation of P-gp by these novel compounds involved consumption of ATP (as P-gp substrates do), as a source of energy to support the efflux. Surprisingly, even small structural changes resulted in opposite behavior, with amide 13 depleting ATP, in contrast to its isomer 18. Two compounds, 15 and 25, emerged for their potent activity at P-gp, and deserve further investigations as tools for P-gp modulation.

Full text of DOI:10.1016/j.bbamem.2014.08.022

Potential Applications for Sigma Receptor Ligands in Cancer Diagnosis and
Therapy
Aren van Waarde, Anna A. Rybczynska, Nisha K. Ramakrishnan, Ki-
ichi Ishiwata, Philip H. Elsinga, Rudi A. Dierckx
PII:
DOI:
Reference:
S0005-2736(14)00304-6
doi: 10.1016/j.bbamem.2014.08.022
BBAMEM 81671
To appear in:
BBA - Biomembranes
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3 June 2014
4 August 2014
19 August 2014
Please cite this article as: Aren van Waarde, Anna A. Rybczynska, Nisha K. Ramakr-
ishnan, Kiichi Ishiwata, Philip H. Elsinga, Rudi A. Dierckx, Potential Applications for
Sigma Receptor Ligands in Cancer Diagnosis and Therapy, BBA - Biomembranes (2014),
doi: 10.1016/j.bbamem.2014.08.022
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ACCEPTED MANUSCRIPT
Potential Applications for Sigma Receptor Ligands in Cancer Diagnosis and Therapy
Aren van Waardea, Anna A Rybczynskaa, Nisha K Ramakrishnana, Kiichi Ishiwatab, Philip H
Elsingaa, Rudi A Dierckxa,c
a University of Groningen, University Medical Center Groningen, Department of Nuclear
Medicine and Molecular Imaging, Hanzeplein 1, 9713 GZ Groningen, The Netherlands
b Tokyo Metropolitan Institute of Gerontology, Research Team for Neuroimaging, 35-2
Sakae-Cho, Itabashi-Ku, Tokyo 173-0015, Japan
c University of Ghent, University Hospital, De Pintelaan 185, 9000 Ghent, Belgium
Corresponding author:
Dr. Aren van Waarde, University of Groningen, University Medical Center Groningen,
Department of Nuclear Medicine and Molecular Imaging, Hanzeplein 1, 9713 GZ Groningen,
The Netherlands, Telephone +31-50-3613215, Telefax +31-50-3611687, E-mail:
a.van.waarde@umcg.nl
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Abstract
Sigma receptors (sigma-1 and sigma-2) represent two independent classes of proteins with a
unique pharmacological profile. Their endogenous ligands may include the hallucinogen N,N-
dimethyltryptamine (DMT) and sphingolipid-derived amines which interact with sigma-1
receptors, besides steroid hormones such as progesterone which bind to both sigma receptor
subpopulations. The sigma-1 receptor gene has been cloned. Its product is a ligand-regulated
molecular chaperone with various ion channels and G-protein-coupled membrane receptors as
clients. The sigma-2 receptor was identified as the progesterone receptor membrane
component 1 (PGRMC1). Although sigma receptors are over-expressed in tumors and up-
regulated in rapidly dividing normal tissue, their ligands induce significant cell death and
apoptosis only in tumor tissue and hardly in proliferating normal cells such as stem cells.
Sigma ligands may therefore be used to selectively eradicate tumors, either as single agents or
adjuvants in chemotherapy. Multiple mechanisms appear to underlie cell killing after
administration of sigma ligands, and the signaling pathways are dependent both on the type of
ligand and the type of tumor cell. Recent evidence suggests that the sigma-2 receptor is a
potential tumor and serum biomarker for human lung cancer and an important target for
inhibiting tumor invasion and cancer progression. Current radiochemical efforts are focused
on the development of subtype-selective sigma receptor ligands for positron emission
tomography (PET) imaging. Right now, the most promising tracers are [18F]fluspidine and
[18F]FTC-146 for sigma-1 receptors and [11C]RHM-1 and [18F]ISO-1 for the sigma-2 subtype.
Nanoparticles coupled to sigma-2 ligands (or non-subtype-selective sigma ligands) have
shown considerable potential for targeted delivery of antitumor drugs in animal models of
cancer, but clinical studies exploring this strategy in cancer patients have not yet been
reported.
Keywords: antitumor agents; chemotherapy; drug targeting; positron emission tomography;
sigma-1 receptor; sigma-2 receptor.
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1. Introduction
Sigma receptors were originally described as a subtype of the opioid receptor family, but later
shown to be unique proteins integrated in plasma, mitochondrial and endoplasmatic reticulum
membranes of several organs including liver, kidney and brain. Two subtypes of sigma
receptors have been identified, termed sigma-1 and sigma-2 [1]. As discussed previously [2],
the endogenous ligands for these receptors have not been identified with certainty but may
include steroid hormones (particularly progesterone), sphingolipid-derived amines and N,N-
dimethyltryptamine (DMT).
Recent evidence has been presented in support of the hypothesis that DMT, a well-known
hallucinogen, may in fact be an endogenous sigma-1 agonist. DMT is a substrate for the
serotonin transporter (with even higher affinity than serotonin itself) and is also a substrate for
the vesicular monoamine transporter 2. These transporter proteins may allow accumulation of
DMT (and other tryptamines) in neurons to the micromolar levels needed for sigma-1
receptor activation [3]. Consistent with this hypothesis, radiolabeled DMT enters the brain of
living rabbits very rapidly (10 s) and is there retained in intact form for at least 7 days,
whereas the compound is cleared from the rest of the body via the renal route [4]. Although
the authors labeled DMT with 131I and thus modified its structure, the in vivo behavior of the
2-iodo derivative is expected to be similar to that of the parent indolealkylamine. Persistence
of DMT in the mammalian brain can be explained by the fact that DMT is stored in vesicles
and thereby protected from degradation by monoamine oxidase. Using immunocytochemical
techniques, indole-N-methyl transferase (INMT), the enzyme that converts tryptamine to
DMT, was shown to be localized to postsynaptic sites of C-terminals of mouse motoneurons
in close proximity to sigma-1 receptors which are enriched at these sites [5]. Moreover, DMT
inhibits INMT non-competetively by binding to an allosteric site on the enzyme molecule.
DMT formation may therefore be regulated via a negative feedback loop [6]. Interestingly,
downregulation of INMT has been associated with tumor recurrence (e.g., of malignant
prostate and lung cancers) and inmt was identified as a candidate gene in prevention of cancer
progression [7].
Molecular biology techniques have indicated that sigma-1 receptors play critical roles in the
mammalian nervous system. As discussed previously [2], sigma-1 knockout (KO) mice are
viable and fertile and do not display any overt phenotype, although the response of such
animals to painful stimuli is strongly suppressed and KO mice display depressive behavior
under certain forms of stress. A more recent study has reported gender-related alterations in
KO mice. Male knockouts show signs of increased anxiety in the open-field, passive
avoidance and elevated plus-maze tests. They also show increased depressive-like behavior in
the forced swimming test, but no memory changes. Female knockouts show deficits in
spontaneous alternation or water maze learning, and avoidance escape latency. These
symptoms of impaired memory appear to be related to changes in steroid tonus, since female
KO mice have decreased plasma levels of 17ß-estradiol compared to wild-type mice and
treatment with 17ß-estradiol reverses their memory deficits [8].
By transduction of cultured rat hippocampal neurons with siRNA for the sigma-1 receptor and
gene expression analysis using a rat genome cDNA array, knockdown of the sigma-1 receptor
was shown to impair many cellular functions, including steroid biogenesis, protein
ubiquination, organization of the actin cytoskeleton and Nrf2-mediated responses to oxidative
stress [9]. Several studies have shown that sigma-1 receptors play an important role in the
protection of retinal cells against various forms of damage. KO mice suffer from late onset
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inner retinal dysfunction [10]. They show accelerated retinal ganglion cell death after optical
nerve crush [11] and a more rapid loss of retinal function in diabetes [12]
In an article which preceded the current overview [2], we wrote that the identity of the sigma-
2 receptor was unknown and the existence of this subtype had only been proven
pharmacologically. However, within one year a study was published [13] in which sigma-2
receptors were irreversibly labeled using WC-21, a ligand containing an azide moiety for
photoaffinity tagging and a fluorescein isothiocyanate (FITC) group for visualization of the
sigma-2 protein. By matrix-assisted laser desorption/ionization-mass spectrometry analysis,
the membrane-bound protein which was labeled by WC-21 in rat liver was identified as
progesterone receptor membrane component 1 (PGRMC1). Knockdown of the PGRMC1
protein with specific sRNA reduced the binding of a radioiodinated sigma-2 receptor ligand in
HeLa cells and of a fluorescent sigma-2 receptor ligand in human embryonic kidney 293T
cells. Moreover, knockdown of PGRMC1 reduced the ability of sigma-2 agonists to induce
caspase-3 activation in HeLa cells. Overexpression of PGRMC1 by transfection of HeLa cells
with PGRMC1 cDNA was associated with a striking (60%) increase of the cellular binding of
the radioiodinated sigma-2 ligand. Treatment of A549 lung cancer cells with a PGRMC1
ligand (AG-205) or a sigma-2 receptor ligand (WC-26) induced similar, dose-dependent
upregulations of the PGRMC1 protein. Both the PGRMC1 ligand AG-205 and various sigma-
2 receptor ligands (DTG, siramesine, SV119, WC-26) displaced bound radioiodinated sigma-
2 receptor ligand in tumour cell membrane homogenates in a concentration-dependent
manner. Thus, these ligands appeared to bind to the same site. Confocal microscopy indicated
that PGRMC1 and the sigma-2 receptor protein had the same intracellular localization, viz. in
mitochondria and endoplasmatic reticulum [13]. Based on this combined evidence, the
PGRMC1 protein complex was identified as the putative sigma-2 receptor binding site.
It had already been known for a long time that sigma-2 ligands inhibit high-affinity
progesterone binding to a microsomal fraction of porcine liver, suggesting that high-affinity
progesterone binding sites are part of a complex including sigma receptors, if not themselves
sigma receptors [14]. But the important study of Xu et al. [13] provided actual proof for
identity of the sigma-2 receptor and PGRMC1. Since PGRMC1 is a known protein, the cDNA
sequence of the sigma-2 receptor gene may in fact already have been determined, both in
porcine [15] and human [16] tissues.
Although the close identity of the sigma-2 receptor and PGRMC1 appears to have been
established, some questions remain unanswered. First, the molecular masses of both proteins
appear to be different. Values of 22 to 28 kDa have been reported for PGRMC1 [17-19]
whereas a value of only 21.5 kDa has been determined for the sigma-2 receptor protein [20].
These differences may be related to post-translational processing or splice variants of a single
protein. Second, inhibition of tumor cell proliferation has been reported to require agonist
action of anticancer drugs at sigma-2 receptors [21,22] but antagonist action at PGRMC1
[23,24]. This contradiction may be more apparent than real, since agonist or antagonist
actions at sigma receptors have not been well-defined. Compounds which were originally
classified as sigma agonists may in fact be antagonists, and vice versa [25-27]. Caspase-3
activation by sigma-2 receptor ligands has been proposed to serve as a functional assay for
differentiating sigma-2 agonists, partial agonists and antagonists [27]. Finally, PGRMC1 is
known to bind to P450 resulting in stimulation of its activity and increased cholesterol
synthesis [28], but for the sigma-2 receptor such binding has not been reported. Future
research in this exciting field may resolve these discrepancies and may result in further
confirmation of the identity of PGRMC1 and the sigma-2 receptor protein.
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Although sigma-1 and sigma-2 receptors are usually treated as members of a common sigma
receptor “family” and share affinity for certain artificial ligands and steroids like
progesterone, the structures of the sigma-1 and sigma-2 receptor proteins are in fact unrelated.
While the sigma-2 receptor seems to belong to a progesterone receptor complex, the sigma-1
receptor has been characterized as a chaperone protein [29,30]. The function and intracellular
location of this protein are altered by ligands but the protein can be active even in the absence
of ligands. Thus, the notion of “agonists” and “antagonists”, which is commonly applied to
receptor ligands, may not be applicable to compounds which interact with sigma-1 receptors.
2. Receptor overexpression in tumors and tumor cell lines
Both subtypes of sigma receptors are overexpressed in rapidly proliferating normal cells and
cancer cells from animal and human origin. Particularly the sigma-2 receptor is an interesting
tumor imaging target since it is expressed about 10-fold more in proliferating tumor cells
compared with quiescent tumor cells and because ligand binding to this receptor can result in
tumor cell death both via apoptotic and non-apoptotic mechanisms (reviewed in [2]). A
comprehensive overview of sigma receptor expression in various cell lines, bovine tumors
and human tumors was presented in [2]. Here, only some recent findings will be reported.
Using a fluorescently labeled ligand (SW120), investigators from the National Institutes of
Health showed that sigma-2 receptors are highly expressed in proliferating human stem cells
(bone marrow stromal, neural progenitor, amniotic fluid, hematopoietic and embryonic)
compared to differentiated lineage-restricted cells. These results provided additional support
for the hypothesis that sigma-2 receptor expression is a biomarker of the proliferative status of
cells. However, in contrast to tumor cells where sigma-2 ligands induce significant apoptosis,
very little apoptosis was observed in ligand-treated stem cells [31]. Thus, sigma-2 receptor
ligands may be used to selectively eliminate tumor cells.
RT-4, a human urinary bladder tumor cell line, expresses sigma-2 receptors at very high and
sigma-1 receptors at lower density (2108 and 279 fmol/mg protein, respectively). Membrane
homogenates of this cell line can be employed for a sigma-2 receptor binding assay, using the
non-subtype-selective sigma ligand [3H]ditolylguanidine (DTG) in the presence of 2 µM non-
radioactive (+)-pentazocine to block sigma-1 receptors. Ki-values of sigma-2 ligands
determined in that assay are in good accordance with Ki-values estimated in rat liver
preparations. RT-4-derived membrane fragments are therefore an animal-friendly alternative
to rat liver homogenates and can be used to determine the affinity of ligands to human sigma-
2 receptors [32].
Sigma-2 receptors were found to be significantly over-expressed in 12 out of 15 samples of
human small cell lung carcinoma and were particularly elevated in poorly differentiated
tumors. In vitro treatment of small cell lung carcinoma cells (A549, NCI-H226) with the
PGRMC1/sigma-2 receptor ligand AG-205 or with short interfering RNA for the sigma-2
receptor resulted in growth inhibition and in the case of the NCI-H226 line also in loss of
viability through a caspase-3- and caspase-8-independent pathway. Sigma-2 receptors were
significantly over-expressed in 6 out of 15 samples of human adenocarcinomas, and sigma-2
receptor expression was inversely correlated with patient survival. Sigma-2 receptors
appeared to be associated with secretory vesicles in lung cancer cells and to be secreted by
these cells. Sigma-2 receptor levels in the plasma of lung cancer patients were therefore
significantly elevated compared to plasma of cancer-free individuals where these levels were
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very low. The authors concluded that the sigma-2 receptor is a potential tumor and serum
biomarker as well as a therapeutic target for lung cancer [33].
In a subsequent study from the same group, knockdown of the PGRMC1/sigma-2 receptor in
A549 cells was found to block the expression of neutrophil gelatinase-associated
lipocalin/lipocalin-2 (NGAL) and to decrease the activity of matrix metalloproteinase-9
(MMP9) in samples of the culture medium. NGAL is a secreted glycoprotein that binds to
MMP9 and protects it from degradation. MMP9 activity is essential for breakdown of the
extracellular matrix and is involved in tumor invasion and survival. Transfection of cells in
which the sigma-2-receptor had been knocked down with a plasmid encoding sigma-2
receptor/PGRMC1 resulted in re-expression of sigma-2 receptors and restoration of the
NGAL levels. NGAL proved to be essential for the formation and growth of A549 in vivo
tumors, since A549 cells in which NGAL had been knocked down showed strongly impaired
xenograft formation in athymic nude mice. By incubating normal cells and cells in which the
sigma-2 receptor had been knocked down with inhibitors of the epidermal growth factor
receptor (EGFR), Akt and ERK, evidence was obtained for the hypothesis that the sigma-2
receptor increases NGAL levels by activating the transcription factor NFkappaB via EGFR
[34] (see Figure 1). Since NGAL expression is dependent on the sigma-2 receptor, the latter
protein may be an important target for inhibiting tumor invasion and cancer progression.
RPMI 8226, the hematopoietic cell line of human multiple myeloma, expresses sigma-1
receptors at high density (Bmax 477 fmol/mg protein, 122,000 binding sites per cell). Fourteen
different sigma-1 ligands showed similar competition with [3H]-(+)-pentazocine for binding
to sigma-1 receptors in membrane fragments isolated from this cell line and the estimated Ki
values were in good accordance with Ki-values assessed in guinea pig brain preparations.
Thus, RPMI 8226-derived membrane fragments may be an animal-friendly alternative to
guinea pig brain homogenates for assessment of the sigma-1 affinity of novel drugs [35].
Flow cytometry analysis and Western blotting indicated strong expression of the sigma-1
receptor in three different human esophageal squamous cell carcinoma (ESCC) cell lines
(KYSE150, KYSE180 and, particularly, EC109). In these cell lines, further
immunocytochemistry showed that the receptor protein is located mainly in the cytoplasm and
the nucleus. Immunohistochemical analysis of patient samples indicated that the sigma-1
receptor is overexpressed in ESCC compared to normal epithelium. Levels of total sigma-1
receptor protein were significantly correlated with pathologic Tumor, Node, Metastasis
(TNM) classification of the tumors, and levels of nuclear sigma-1 receptor protein were
correlated both with TNM classification and the presence of lymph node metastases. Thus,
sigma-1 receptor expression may be a factor predicting ESCC classification and ESCC
development [36].
In an animal study from our own laboratory, sigma-1 receptors were found to be
overexpressed (> 2-fold) in spontaneous pituitary tumors as compared to the normal pituitary.
This overexpression was detected as an increase in uptake (> 3-fold) and binding potential
11
(BPND, > 2-fold) of the sigma-1 receptor ligand C-SA4503. In microPET scans of aged rats
using this tracer, even very small tumors (e.g., a specimen of 17 mg) were clearly visualized.
Thus, PET with a radioligand for sigma-1 receptors (like 11C-SA4503) may have promise for
the detection of pituitary adenomas and microadenomas. It is not yet clear whether such scans
can discriminate between symptomatic, hormone-secretive and nonsymptomatic, nonsecretive
neoplasms [37].
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3. Sigma ligands for diagnostic imaging
The overexpression of sigma receptors in tumor cells motivated the development of
radiolabeled sigma ligands for diagnostic imaging using positron emission tomography (PET,
see Figure 2) or single photon emission computed tomography (SPECT). An overview of the
development of sigma receptor ligands for imaging purposes was provided in our previous
review article [2]. At the time of writing, the most commonly applied sigma-1 receptor
ligands were radioiodinated benzamides, the piperidine 18F-FPS and the piperazines 11C-
SA4503 and 18F-FESA5845. In recent years, radiochemical efforts have focused on the
development of selective radioligands for each sigma receptor subtype. A comprehensive
review concerning the development of sigma-2 ligands for PET, SPECT, optical imaging,
photoaffinity labeling and binding assays was published in 2013 [38].
3.1 Novel sigma-1 receptor probes
A spirocyclic, radiofluorinated sigma-1 receptor ligand, 1’-benzyl-3-(2-[18F]fluoroethyl)-3H-
spiro[[2]benzofuran-1,4’-piperidine] or [18F]fluspidine, was prepared by German investigators
(see Figure 3). Compared to 11C-SA4503, this compound could offer the advantages of a very
high affinity to sigma-1 receptors (Ki 0.59 nM vs 17 nM), a longer physical half life (109.8
min vs 20.4 min) and negligible affinity towards emopamil binding protein, a vertebrate sterol
isomerase which is located in endoplasmatic reticulum membranes and is involved in the
biosynthesis of cholesterol. Biodistribution studies in female CD-1 mice and ex vivo phosphor
storage imaging of brain slices indicated that the novel compound is a promising probe for
molecular imaging of sigma-1 receptors. Tracer uptake in brain, heart, lung and spleen was
significantly inhibited after pretreatment of animals with the dopamine D2/sigma receptor
ligand haloperidol (1 mg/kg), but not reduced after treatment of mice with the emopamil
binding protein inhibitor tamoxifen (1 mg/kg). Specific binding appeared to be at least 75% of
total cerebral uptake of radioactivity at 60 min after injection. [18F]Fluspidine showed a high
brain uptake (3.9 to 4.7 % ID/g), a regional distribution of radioactivity consistent with
binding to sigma-1 receptors, and moderate in vivo metabolism. At 60 min after injection,
67% of total plasma radioactivity still represented parent compound. Blood-brain barrier
passage of radioactive metabolites was not observed [39]. In a subsequent publication from
the same group, the (R)- and (S)-enantiomers of fluspidine were prepared. Ki-values of these
enantiomers at sigma-1 receptors were 0.57 nM and 2.3 nM, respectively, and selectivities for
the sigma-1 subtype were 2895- and 390-fold. Although incubation of the (R)- and (S)-
enantiomers with rat liver microsomes led to the identification of seven and eight metabolites,
respectively, the (S)-enantiomer was metabolically more stable than the eutomer [40].
A subsequent publication from the same group highlighted that small modifications of the
chemical structure of a radioligand can have serious consequences for its pharmacokinetics.
18
An analog of F-fluspidine was prepared in which the fluoroethyl moiety was replaced by a
fluoromethyl group. Although this compound had almost equally high sigma-1 receptor
affinity as 18F-fluspidine (Ki 0.74 nM vs 0.59 nM) and a comparable selectivity for the sigma-
1 subtype (743-fold vs. 1334-fold), the fluoromethyl derivative failed as a PET tracer because
of rapid metabolism, entry of two radiometabolites into the brain, low target-to-nontarget
ratios in ex vivo autoradiography and a continuous washout from the central nervous system
[41].
Investigators from Stanford University prepared a benzothiazolone sigma-1 receptor ligand,
6-(3-fluoropropyl)-3-(2-(azepan-1-yl)ethyl)benzo[d]thiazol-2(3H)-one, or [18F]FTC-146
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[42,43] (see Figure 3). Pretreatment of normal BalbC mice with equal doses of three different
nonradioactive sigma-1 ligands (haloperidol, FTC-146 or BD1047) reduced the brain uptake
of [18F]FTC-146 by 80 to 82% at 60 min after injection. The fraction of plasma radioactivity
representing intact parent compound was 60% at 30 min and 50% at 60 min. Radioactive
metabolites did not enter the brain [42]. In a later publication from the same group, [18F]FTC-
146 was evaluated in rats and squirrel monkeys. Biodistribution studies in control and
haloperidol- or BD1047-pretreated rats indicated a large specific binding fraction in sigma-1
receptor-expression organs (brain, pancreas, spleen and liver, 85%, > 80%, > 65% and > 70
%, respectively). The regional distribution of radioactivity in the rat brain as observed by ex
vivo autoradiography was consistent with the known distribution of sigma-1 receptors. Tracer
metabolism in rats appeared to be more rapid than in mice, about 20% of total plasma
radioactivity representing intact parent compound at 60 min after injection. However, blood-
brain barrier passage of radioactive metabolites did not occur. [18F]FTC-146 showed specific
in vivo binding also in the monkey brain, and cerebral uptake of the tracer was reduced by
68% to 77% after pretreatment of monkeys with haloperidol. Metabolism in squirrel monkeys
appeared to be about equally rapid as in rats, 15% intact parent compound remaining in
monkey plasma between 50 and 110 min [43]. [18F]FTC-146 has therefore favorable
characteristics for in vivo studies of sigma-1 receptors in the brain.
Chinese investigators prepared 18F-labeled 1-(1,3-benzodioxol-5-ylmethyl)-4-(4-(2-
fluoroethoxy)benzyl)piperazine (nicknamed [18F]XW-6, see Figure 3) as a potential sigma-1
receptor ligand for PET imaging. This compound shows a low nanomolar affinity to sigma-1
receptors (Ki 1.85 nM), a rather high selectivity for the sigma-1 subtype (157-fold) and an
optimal logD (at pH 7.4) for penetration in tumors and the brain (+2.57). Favorable
biodistribution data were acquired in mice and rats with > 70% specific binding in the mouse
brain at 60 min after injection, and sigma-1 receptor-mediated uptake in several target organs
(brain, lungs, kidneys, heart and spleen) as shown by the impact of pretreatment of animals
with haloperidol. Low uptake of radioactivity in bone suggested the absence of defluorination.
Thus, this piperazine ligand appears to be a suitable radiotracer for in vivo imaging [44].
A team at Kanazawa University in Japan produced a radiobromine-labeled vesamicol analog
as a sigma receptor imaging agent for PET. This compound, called (+)-[77Br]pBrV, showed a
nanomolar affinity for sigma-1 receptors (Ki 2.5 nM), but only 9-fold selectivity for the
sigma-1 subtype (see Figure 3). In vitro binding of the compound to the DU-145 prostate
cancer cell line was examined and biodistribution experiments in DU-145 tumor-bearing mice
were performed. Specific binding of the probe to sigma receptors was observed both in vitro
and in vivo. Judged by tumor-to-blood ratios of radioactivity, > 80% of the tumor uptake was
blocked after pretreatment of animals with the non-radioactive sigma ligands, haloperidol and
SA4503. The authors concluded that (+)-[76Br]pBrV has considerable potential as a sigma
receptor ligand for tumor imaging [45]. For SPECT imaging, a radioiodinated vesamicol, (+)-
[125I]-IV-OH (see Figure 3), was also prepared and evaluated in a similar way. Tumor-to-
blood ratios of that compound were 3- to 5-fold reduced after pretreatment of mice with non-
radioactive haloperidol, SA4503, or (+)-pIV, and brain-to-blood ratios were reduced up to
2.5-fold, suggesting specific in vivo binding of (+)-[125I]-IV-OH to sigma receptors [46].
Analogs of prezamicol and trozamicol have also been prepared. Four compounds which were
most promising showed nanomolar (1.4 to 4.0 nM) or subnanomolar (0.48 nM) affinities to
sigma-1 receptors, sigma-1 subtype selectivities ranging from 1111-fold to 3627-fold, logP
values between +2.6 and +2.8, and low affinity for the vesicular acetylcholine transporter
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18
11
[47]. All four can be labeled with F or C, but in vivo data for such radioligands have not
yet been presented.
The well-known sigma-1 receptor ligand 11C-labeled 1-2-(3,4-dimethoxyphenyl)ethyl]-4-(3-
phenylpropyl)piperazine ([11C]SA4503) can not only bind to sigma-1 receptors but shows
also significant in vitro affinity to emopamil binding protein (Ki 1.7 nM; [39,48]. For this
reason, Toyohara et al. re-evaluated the in vivo selectivity of [11C]SA4503 to sigma-1
receptors in the brain. Mice were injected with [11C]SA4503 after pretreatment with various
doses of the sigma-1 receptor ligands haloperidol, ifenprodil and trifluperidol, the emopamil
binding protein blockers tamoxifen and trifluoperazine, or saline. The brain uptake of
[11C]SA4503 was dose-dependently decreased by sigma-1 receptor ligands, but not by
inhibitors of the emopamil binding protein. Thus, [11C]SA4503 shows sigma-1 selective
binding in the living brain [49].
3.2 Novel sigma-2 receptor probes
In an attempt to develop novel probes for sigma-2 receptors, Abate et al. prepared benzamides
which combined moieties of 1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-
yl)piperazine (PB28) and 2-methoxy-5-methyl-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-
isoquinolin-2-yl)butyl)benzamide (RHM-1) in a single molecule. PB28 is a sigma ligand with
sub-nM sigma-2 affinity (Ki 0.68 nM), but no subtype selectivity and a high degree of
nonspecific binding, making the 11C-labeled compound unsuitable for in vivo imaging. RHM-
1 (see Figure 4) is a sigma-2 ligand with lower affinity (Ki 8.68 nM) but strong sigma-2
subtype selectivity (248-fold); moreover, [11C]-RHM-1 is capable of visualizing tumor lesions
in an animal model. By preparing hybrid molecules, the authors hoped to create a tracer
which would combine high brain uptake with good target-to-nontarget ratios and in vivo
selectivity for the sigma-2 subtype. Their hybrid molecules showed considerable subtype
selectivity and appropriate lipophilicity, but unfortunately turned out to be substrates for the
drug efflux pump P-glycoprotein which precludes their entry into the brain [50]. The most
promising compound from this series was later labeled with 18F and used for preclinical
imaging studies. Although it had good affinity for sigma-2 receptors (Ki 9.2 nM), a high
selectivity for the sigma-2 subtype (330-fold) and was lipophilic (clogP 3.5), it failed to enter
the rat brain, probably because of P-glycoprotein-mediated efflux (EC50 5.0 µM) [51].
In another study from the same group, analogues of PB28 with reduced lipophilicity were
prepared in an attempt to reduce their nonspecific binding. The propylene linker and the
tetralin C4 position of PB28 were modified for this purpose. However, the analogues
displayed little sigma-2 subtype selectivity (maximally 2-fold) and the most promising
candidate with appropriate logD7.4 (+2.38) turned out to be also a substrate for P-glycoprotein
[52].
An Australian research team synthesized two novel phthalimido ligands for sigma-2
receptors: 2-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-5-fluoroisoindoline-
1,3-dione (18F-SIG343) and 2-(5-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-pentyl-5-
fluoroisoindoline-1,3-dione (18F-SIG353). The compounds demonstrated high affinity (Ki
values 8 and 2.4 nM) and good selectivity for the sigma-2 subtype (200- and 110-fold,
respectively) in binding assays involving membrane fragments isolated from rat brain. In vitro
blocking studies with subtype-selective nonradioactive ligands in A375 (human amelanotic
18
melanoma) cells demonstrated that the cellular binding of F-SIG343 was sigma-2 receptor-
18
mediated, in contrast to F-SIG353 which did not display any specific binding. Both tracers
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were evaluated in nude mice bearing A375 tumors. Particularly for 18F-SIG343, specific
binding was observed in sigma-receptor containing organs (brain, liver and lung) as tracer
uptake was significantly reduced after pretreatment with haloperidol or non-radioactive
SIG343. However, tracer uptake in the tumors was low (about 1% ID/g at 2 h post injection)
and although adequate tumor-to-muscle ratios were reached (4 or 7 at 2 h post injection,
respectively) and metabolic stability of the tracers was also adequate, no specific signal from
sigma-2 receptors could be detected in tumor cells. Thus, in this particular animal model,
[18F]SIG343 and [18F]SIG353 failed as probes for tumor imaging [53].
In contrast to these negative findings, very promising results were obtained with the sigma-2
receptor ligand 2-(2-[18F]fluoroethoxy)-N-(4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)-
yl)butyl)-5-methyl-benzamide ([18F]ISO-1, see Figure 4). This tracer was evaluated in two
different rodent models of breast cancer: female nude mice bearing tumors of murine
mammary tumor 66 cells, and Sprague-Dawley rats with N-methyl-N-nitrosourea-induced
tumors. Rats were subsequently treated either with bexarotene (220 mg/kg in the diet) or
vorozole (1.25 mg/kg body weight by gavage). In the first model, the tumor-to-background
ratio of [18F]ISO-1 corresponded closely and linearly to the ratio of proliferative over
quiescent cells in each tumor as determined by flow cytometry. Changes of [18F]ISO-1 uptake
during chemotherapy in the second model were significantly correlated with changes in tumor
volume determined by repeated magnetic resonance imaging (MRI) scans. These data suggest
that PET studies with a sigma-2 ligand can indicate both the proliferative status of a tumor
and its growth rate. Thus, sigma-2 receptor imaging can be used in the selection and design of
an appropriate treatment strategy [54]. A first human study with [18F]ISO-1 in thirty cancer
patients confirmed these positive expectations. In this heterogeneous group (13 patients with
primary breast cancer, 10 with head and neck cancer and 7 with lymphoma), tumor-to-muscle
uptake ratios and the maximum standardized uptake value of [18F-ISO-1 were significantly
correlated with expression of the proliferation marker Ki-67 [55].
Promising preliminary results were also obtained for a structurally similar ligand, N-(4-(6,7-
dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-fluoroethoxy)-5-iodo-3-methoxyben-
zamide (nicknamed RHM-4, see Figure 4). Because of its chemical structure, this compound
can be labeled either with 18F (for PET), or wih radioiodine (for SPECT). Both labeled
molecules showed similar in vivo pharmacokinetics in mice bearing mammary 66 tumors.
Excellent tumor-to-muscle ratios of 8 to 10 were observed at 120 min after injection [56].
A potential SPECT tracer for imaging of sigma-2 receptors in tumors has been proposed as
well. Chinese investigators prepared 99mTc 4-(4-cyclohexylpiperazine-1-yl)-butan-1-one-1-
cyclopentadienyltricarbonyl technetium for this purpose. The corresponding rhenium complex
was also prepared and was used to determine in vitro binding parameters of this novel ligand.
It showed moderate affinity to sigma-2 receptors (Ki 64.4 nM) and moderate sigma-2 subtype
selectivity (12.5-fold). The lipophilicity of the 99mTc-labeled ligand was about optimal for
uptake into brain and in tumors (logD +2.52). In vitro binding experiments and biodistribution
studies were performed, using C6 rat glioma cells and male ICR mice, respectively. A high
tracer uptake was observed in (sigma-2 receptor-expressing) C6 cells which was reduced
substantially (up to > 90%) after co-incubation with haloperidol. In living mice, specific
binding of the tracer was observed in receptor-expressing organs (brain, heart, lung, spleen),
but the reduction of tracer uptake after haloperidol pretreatment was not greater than 51% at 2
h post injection. Very promising results with the same tracer were obtained in C6 glioma
brain tumor-bearing Sprague Dawley rats. Cerebral binding of the tracer at 2 h post injection
was for 67% specific and the tumors were well-visualized (tumor-to-brain ratio > 2 ). Thus, it
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may prove possible to develop 99mTc-labeled SPECT tracers for sigma-2 receptor imaging
[57].
4. Sigma Ligands for Targeted Drug Delivery
Since sigma receptors (particularly the sigma-2 subtype) are over-expressed in many kinds of
tumors, they have been considered as an attractive target not only for cancer diagnosis but
also for anticancer drug delivery. An overview of sigma ligand-based drug targeting
experiments is presented in Table 1. Chemical structures of the sigma ligands which have
been employed in these experiments are depicted in Figure 5.
Two different approaches towards sigma-ligand-based drug targeting have been explored (see
Table 1). First, sigma ligands were conjugated with various nanoparticles, either via a
polyethylene glycol (PEG) spacer or via direct covalent linking at the end of a long alkyl
chain of the sigma ligand. Before injection into experimental animals, the nanoparticles were
filled with various cytostatic or cytotoxic agents. Second, antisense oligonucleotides or
antitumor peptides were conjugated with sigma ligands by direct covalent linking, and the
resulting hybrid molecules were systemically administered.
Initial targeting attempts made use of anisamide and haloperidol – ligands which bind to both
subtypes of sigma receptors and may in addition bind to neurotransmitter binding sites (e.g,
dopamine D2 and D3 receptors). More recent attempts have employed compounds with high
selectivity for the sigma-2 receptor subtype (SV119, SW43). The latter approach may result
in lower brain uptake and greater uptake in the target tumors.
Conjugation of a sigma ligand to a nanoparticle (either directly or via a PEG spacer) may be
associated with loss of affinity of the ligand to its target receptor. For this reason, most
authors checked whether the tumor uptake of the targeted nanoparticles, oligonucleotides or
peptides was sigma-receptor specific by comparing uptake in the presence and absence of a
pharmacological dose of a sigma ligand (ditolylguanidine or haloperidol). Since uptake was
strongly reduced after pretreatment of animals with sigma ligands, the conjugates retained
sufficient affinity for the intended receptor and the loss of affinity induced by conjugation
appeared to be negligible (see Table 1).
Both targeted nanoparticles and antitumor peptides coupled to sigma ligands have shown
considerable potential in preclinical in vitro and in vivo models of anti-tumor therapy (Table
1), but attempts to apply such strategies to treatment of cancer patients have not yet been
reported.
5. Cytotoxic effects of sigma ligands
Since both subtypes of sigma receptors are overexpressed in a large variety of tumors and can
activate apoptotic pathways, sigma ligands are potentially useful as anticancer drugs either for
single agent or adjuvant chemotherapy. A few comprehensive reviews on the perspectives of
sigma-1 and sigma-2 receptor ligands in cancer therapy were published in recent years
[74,75]. Not only drugs selective for sigma-1 and/or sigma-2 receptors have been prepared,
but also mixed action compounds combining sigma receptor interaction with binding to other
targets.
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Abate and co-workers described a series of 1-cyclohexyl-4-(4-arylcyclohexyl)piperazines
which combine high affinity to sigma-1 and sigma-2 receptors with inhibition of human ∆8-∆7
sterol isomerase and inhibition of P-glycoprotein (P-gp), an efflux pump involved in tumor
multidrug resistance [76]. The most promising compound of the series, cis-11, showed Ki
values of 0.26, 7.92 and 7.6 nM at the first three targets. In transfected canine kidney
epithelial cells over-expressing P-gp, 0.1 µM doxorubicin alone did not have any
antiproliferative effect. However, when doxorubicin was combined with 30 µM cis-11, 70%
cell death was observed within 24 h and a combination with 50 µM cis-11 resulted even in
90% cell death. Administration of 50 µM cis-11 alone caused 50% cell death in the same
assay. Thus, this class of compounds is capable of inducing significant growth inhibiton in P-
gp overexpressing tumor cells [76].
A later study from the same group concerned the development of tetrahydroisoquinoloines
which combine selectivity and high affinity for the sigma-2 subtype with capability of
reversing P-gp-mediated drug resistance. The two most promising compounds from the series
showed nanomolar (Ki 5.34 nM) or picomolar (Ki 0.04 nM) affinity at rodent sigma-2
receptors, and high or extremely high selectivity for the sigma-2 subtype (260-fold and >
50,000-fold, respectively). They were capable of reversing P-gp-mediated resistance and re-
establishing the antitumor effect of doxorubicin in P-gp-overexpressing MCF7adr human
breast cancer cells. The compound with picomolar affinity showed even stronger
antiproliferative action in doxorubicin-resistant cells than in parent cells, a phenomenon that
has been called “collateral sensitivity” and may be based on a P-gp-catalyzed ATP-degrading
futile cycle [77]. Thus, these tetrahydroisoquinolines could be evaluated for the treatment of
multidrug-resistant tumors, and be administered either alone or in combination with classic
chemotherapeutic agents.
In an attempt to enhance the apoptosis-inducing ability of haloperidol without impairing its
targeting ability, medicinal chemists conjugated the tertiary OH group of haloperidol with
cationic lipids of varying chain lengths. Such conjugation might not affect the affinity of
haloperidol to sigma-1 or sigma-2 receptors, but could increase cellular entry of the drug
because of the presence of the cationic lipid chain. The most promising compound of the
series, HP-C8, contained a lipid chain with 8 carbon atoms. It showed very strong cytotoxicity
towards MCF-7 and MDA-MB-231 (breast cancer) cells at doses at least 100-fold lower than
parent haloperidol, and at least 10-fold lower than haloperidol conjugates with other lipid
chain lengths that were tried (4, 12 and 16 carbon atoms). Toxicity of the C8 conjugate was
also much higher than of haloperidol and the C8 lipid administered in combination.
Moreover, HP-C8 was 2- to 3-fold more toxic to tumor cells than normal cells (COS-1, HEK-
293), and the compound induced apoptosis in cancer cells but not in normal cells.
Downregulation of sigma-1 receptors in MCF-7 cells resulted in increased viability of cells
treated with HP-C8, thus, the cytotoxicity of HP-C8 appears to be sigma receptor-mediated.
Five intraperitoneal injections of 7.5 mg/kg HP-C8 at 2 to 3 day intervals in mice bearing
B16F10 tumors resulted in greater than 3-fold tumor growth reduction in this animal model of
melanoma. Experiments with HP-C8 in human umbilical vein endothelial (HUVEC) cells
suggested that the compound may also be capable of suppressing angiogenesis and the
formation of neovasculature in the tumor environment. Cationic lipid-modified haloperidol
derivatives are therefore an interesting novel class of anticancer drugs [78].
Other modifications of haloperidol were tried as well. An Italian publication concerned the
phenylbutyrate ester of metabolite II of haloperidol. This molecule, called (±)-MRJF4,
combines inhibition of histone deacetylase with sigma receptor binding. It has affinities to
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sigma receptors in the 10-7 M range (Ki 162 and 105 nM at sigma-1 and sigma-2,
respectively), but negligible affinity to dopamine D2 and D3 receptors (Ki > 5000 nM). IC50
values of (±)-MRJF4 for growth inhibition in prostate cancer cell lines (LNCaP and PC3) are
much lower (11 and 13 µM) than those of 4-phenylbutyric acid (2324 and 2273 µM),
haloperidol metabolite II (177 and 208 µM) or an equimolar mixture of 4-phenylbutyric acid
and haloperidol (190 and 165 µM). Blocking experiments with (+)-pentazocine and AC927
suggested that both subtypes of sigma receptors are involved in the cytotoxic effects of (±)-
MRJF4 with a prevalence of the sigma-2 subtype [79].
A series of adamantane phenylalkylamines was tested for sigma receptor affinity and
antiproliferative activity. The most interesting compound of the series bound to both sigma-1
and sigma-2 receptors with affinities in the 10-8 M range (IC50 values 48 and 85 nM,
respectively). It displayed significant antiproliferative activity to colon (HCT-116, HCT-15),
prostate (DU145, PC3), breast cancer (MCF7), ovarian (OVCAR-5), brain tumor (U251),
leukemia (HL-60), pancreatic (BX-PC3) and liver cancer (SKHep1) cells. Activation of
caspase-3 and apoptosis was observed in addition to cell cycle arrest at the sub-G1 level.
Three cycles of treatment with this adamantane (40 mg/kg doses administered on 3
consecutive days per week during a period of 3 weeks) resulted in more than 3-fold inhibition
of the growth of BX-PC3 xenografts in SCID mice. A different chemotherapy protocol (two
cycles of treatment, 55 mg/kg doses administered on 5 consecutive days per week during a
period of 3 weeks) resulted in significant but smaller growth reductions of PC3, DU145 and
OVCAR5 tumors [80]. These adamantane phenylalkylamines may therefore be considered as
potential antitumor agents, although rather high drug doses were required for inhibition of
tumor growth.
Selective sigma-1 or sigma-2 receptor ligands may also be employed for therapeutic purposes.
High affinity sigma-1 receptor ligands derived from spipethiane with sigma-1 subtype
selectivities of 2630- and 29512-fold showed significant cytostatic effects in the human breast
cancer cell line MCF-7/ADR [81]. A series of indole-based sigma receptor ligands was
recently prepared, and cytotoxicity of these compounds was tested in liver (HUH7), breast
(MCF7) and colon (HCT-116) cancer cell lines. The most promising ligand bound to both
sigma-1 and sigma-2 receptors with affinities in the submicromolar range (Ki values 126 and
130 nM, respectively). It showed similar (MCF7) or significantly (2- to 8-fold) greater
cytotoxicity than 5-fluorouracil in the tested cell lines. Thus, indole-based sigma ligands may
be useful for cancer therapy [82]. High-affinity fenpropimorph-derived sigma-1 receptor
ligands [83], conformationally restricted sigma-1 receptor ligands with 7,9-
diazabicyclo[4.2.2]decane scaffold [84] and hydroxyethyl substituted piperazines [85] have
also shown considerable cytotoxicity in various tumor cell lines.
A publication of our own group examined the impact of daily intraperitoneal injections of
rimcazole (26 mg/kg body weight) in nude mice bearing A375M human melanoma xenografts
[86]. This treatment regimen resulted in a greater than 4-fold reduction of tumor weight in
comparison to controls after 2 weeks. A transient increase of the tumor uptake of the glucose
analog [18F]FDG was observed after the first week of treatment. Rimcazole treatment was not
associated with any significant adverse side effect [86]. In murine models of pancreatic
cancer, sigma-2 ligands are accumulated preferentially in the tumor cells where they are
internalized by both receptor-mediated and caveolin-mediated endocytosis. In preclinical
studies, such ligands are capable of inducing apoptosis in the tumors with limited toxicity at
non-target sites. The animal and in vitro data raise hope that sigma-2 ligands may be used to
induce apoptosis in pancreatic tumors when resistance to traditional chemotherapies has
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developed [87]. At least half of the samples of human pancreatic adenocarcinoma appear to
show overexpression of sigma-2 receptors.
Sigma ligands can not only kill tumor cells and inhibit tumor growth, but they can also
significantly enhance the pharmacological action of other anticancer drugs. At concentrations
of the individual agents having hardly any cytotoxic effects, combined administration can
result in potent killing of tumor cells. In a preclinical model of pancreatic cancer (Panc02
tumors in C57BL/6 mice), combination treatment of the animals with gemcitabine and the
novel sigma-2 receptor ligand SW43 was superior to single agent treatment and resulted in
stabilization of tumor volume during the treatment period with no significant adverse effects
[88]. In an in vitro model of melanoma, the sigma ligand rimcazole synergistically enhanced
the inhibition of A375M tumor cell growth induced by anti-MCSP:TRAIL [89]. During in
vitro studies of colon and breast cancer cells, the P-glycoprotein inhibitor and sigma-1
receptor ligand MC70 strongly enhanced the effectiveness of doxorubicin [90].
6. Mechanisms underlying inhibition of tumor growth
Although it had been known for a long time that small-molecule ligands targeting sigma-1
and/or sigma-2 receptors can inhibit cancer cell proliferation, induce tumor cell death and
suppress tumor growth in pre-clinical models, the cellular pathways activated by sigma
ligands and the mechanisms involved in cell death had not been well-defined. Early studies in
this area have been reviewed previously [2], thus only more recent and novel findings will be
discussed here. Recently published reviews have focused on the interaction of sigma-1
receptors with ion channels [91,92] and sigma-2 receptor-mediated tumor cell death [74].
Ion channels and G-protein coupled membrane receptors have been identified as the main
clients for the sigma-1 chaperone protein. Protein-protein interactions are known to occur
between sigma-1 receptors and voltage-gated potassium channels (Kv1.2, Kv1.4 and Kv1.5)
[93-97], small conductance Ca2+ activated K+ current (SK) channels [98], voltage-gated
sodium channels (Nav1.5) [99-101], voltage-gated L-type calcium channels [102], acid-
sensing ion channels (ASIC1a) [103,104], volume-regulated chloride channels (VRCC) [105],
the GluN1 subunit of the NMDA receptor [106], dopamine D1 receptors [107,108], dopamine
D2 receptors [109], and histamine H3 receptors [107]. Moreover, the sigma-1 receptor
regulates the expression of the human ether-a-gogo (HERG) channel by stimulating channel
subunit biosynthesis [110]. This channel promotes the progression of many primary human
cancers through modulation of extracellular matrix adhesive interactions [110]. The
mentioned protein-protein interactions and effects of sigma-1 receptors on channel subunit
biosynthesis may be involved in the antitumor effects of sigma-1 ligands, since ion channels
are important actors in the control of cancer growth and invasiveness [91,95,105,110-112].
American investigators noticed that treatment of tumor cell lines with certain sigma-1 ligands
visibly diminished cell size. Based on this observation, they performed a study in various cell
lines (T47D, MDA468 and MCF7 human breast carcinoma, PC3 and LNCaP prostate
adenocarcinoma) which were treated with putative sigma-1 agonists (PRE084, (+)SKF-
10047) and antagonists (IPAG, haloperidol). Antagonists but not agonists were found to
reduce cellular protein synthesis by repressing the cap-dependent initiation of translation and
phosphorylation of the translational regulator proteins p70S6K, S6 and 4E-BP1. Knockdown
of sigma-1 receptors in the cells with specific siRNA had the same effect as treatment of cells
with sigma-1 antagonists, confirming that the observed translational repression was indeed
related to diminished sigma-1 receptor function. Thus, sigma-1 receptors appear to play a role
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in protein synthesis and sigma-1 ligands may be used to reversibly modulate the cellular
synthetic machinery [113]. In a subsequent publication from the same group, MDA-MB-468
and T47D (breast adenocarcinoma) cells were treated with 10 µM concentrations of the
previously mentioned sigma-1 agonists and antagonists. Evidence was now obtained
suggesting that autophagy is involved in sigma ligand-induced decreases in cell size.
Antagonists (but not agonists) induced ER stress and activated the unfolded protein response
in a dose-dependent, time-dependent and reversible manner, extended treatment resulting in
autophagy and finally in apoptosis. Knockdown of sigma-1 receptors suppressed these effects
of sigma-1 antagonists, suggesting that these responses are indeed sigma-1 receptor-mediated
[114].
A recent paper has indicated that the sigma-1 receptor is a key mediator of interleukin-24 (IL-
24)-induced cancer-specific apoptosis. IL-24 generated from an adenovirus expressing this
cytokine induces endoplasmatic reticulum (ER) stress, production of reactive oxygen species
and calcium mobilization resulting in apoptosis of cancer cells. Treatment of cancer cells with
the sigma-1 agonist (+)-SK-10047 blocks these responses. A direct interaction between IL-24
and sigma-1 receptors is suggested by the fact that these two molecules co-localize in
immunocytochemical studies and co-immunoprecipitate after the application of either a
sigma-1 receptor or an IL-24 antibody. Thus, the ER stress response, ROS production and
calcium mobilization appear to be triggered by IL-24 via a sigma-1 receptor-dependent
pathway and IL-24 appears to induce apoptosis through a sigma-1 receptor antagonistic
mechanism [115].
The sigma ligand [3H]PB28 was found to accumulate up to fivefold in nuclear compared to
cytosolic fractions of neuroblastoma (SK-N-SH) and breast cancer (MCF-7) cells, probably
due to the fact that it binds with high affinity (0.5 nM) to histone H2A/H2B dimers [116].
Apparently, some sigma ligands can directly interact with nuclear material and may thus exert
antiproliferative and cytotoxic effects.
Sigma-2 induced apoptosis can be caspase-independent and may therefore also occur in cells
with caspase dysfunction [117]. SW43, a sigma-2 subtype-selecitive ligand, was shown to
induce apoptosis in Bxpc3 and Aspc1 pancreatic cancer cells by permeabilization of
lysosomal membranes and increasing cellular oxidative stress, but independent of caspase-3
activation, since the caspase-3 inhibitor DEVD-FMK did not counteract these effects and did
not protect cells against the antitumor drug [118]. Siramesine, which has been classified as a
sigma-2 receptor agonist, caused apoptosis and secretory granule permeabilization in mast
cells also in a caspase-independent manner [119]. However, the effect of siramesine appears
to be cell-type dependent. In human keratinocytes (HaCaT), cell death induced by siramesine
was accompanied by caspase activation and triggered by destabilization of mitochondria
rather than lysosomes. The divergent effects of siramesine in different cell lines may be due to
the fact that siramesine is not a selective sigma-2 receptor ligand, but interacts with multiple
molecular targets inside cells [120]. The cytotoxic and DNA-fragmenting effects of sigma-2
subtype-selective ligands other than SW43 or siramesine (WC26, SV119 and RHM-138) were
dependent on caspase-3 since they were counteracted by the caspase-3 inhibitor ZVAD-FMK.
These ligands induced autophagy and decreased the activity of the mTOR (mammalian target
of rapamycin) pathway [121]. The sigma-2 receptor/PGRMC1 has been shown to bind to key
components of the autophagy machinery (microtubule-associated protein 1 light chain 3, UV
radiation resistance associated/UV radiation associated gene) and to be required for the
degradative activity of autophagy. Inhibition of PGRMC1 by RNAi or small molecule
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antagonists results in accumulation of autophagy substrates and of aberrant mitochondria
[122].
In summary: multiple mechanisms appear to underlie inhibition of tumor growth and cell
death after administration of sigma ligands, and the signaling pathways are dependent both on
the type of ligand and the type of tumor cell, resulting in conflicting reports in the oncological
literature.
7. Conclusions
Novel, potent and subtype-selective radioligands for PET imaging of sigma receptors have
become available. In vivo studies with these ligands may provide important information about
the biology of sigma receptors and may lead to application of PET imaging for tumor
detection, tumor staging, evaluation of therapeutic strategies and anti-tumor drug
development. Treatment of preclinical in vitro and in vivo models of cancer with target- and
subtype-selective, nonradioactive sigma ligands may result in greater understanding of the
mechanisms underlying sigma ligand-induced cell death and may lead to clinical applications.
8. Acknowledgements
During their PhD research on sigma receptors, AAR and NKR were supported by University
funding (Ubbo Emmius Bursary Position).
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Figure 1. Proposed signaling pathway which links the sigma-2 receptor to NGAL expression
and tumor invasion (redrawn after [34])
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Figure 2. PET images of the head-and-neck region of a single rat made with the glucose
18
11
analog F-FDG (left) and the sigma-1 receptor ligand C-SA4503 (right). The animal had a
C6 glioma tumor implanted in its right shoulder. Visible are the tumor (T), brain (B),
Harderian gland and nasal epithelium. Note that the tumor is well-visualized by both tracers,
but the areas with strong 18F-FDG uptake and those with elevated binding of the sigma-1
ligand do not match.
29