Potential applications for sigma receptor ligands in cancer diagnosis and therapy

This neurochemical study reviews the potential applications for sigma receptor ligands, which may include endogenous DMT, in cancer diagnosis and therapy.

Abstract

“Sigma receptors (sigma-1 and sigma-2) represent two independent classes of proteins. Their endogenous ligands may include the hallucinogen N,N-dimethyltryptamine (DMT) and sphingolipid-derived amines which interact with sigma-1 receptors, besides steroid hormones (e.g., progesterone) which bind to both sigma receptor subpopulations. The sigma-1 receptor 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 only in tumor tissue. Sigma ligands may therefore be used to selectively eradicate tumors. 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 radioligands for positron emission tomography (PET) imaging. Right now, the mostpromising 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 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. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.”

Authors: Aren van Waarde, Anna A. Rybczynska, Nisha K. Ramakrishnan, Kiichi Ishiwata, Philip H. Elsinga & Rudi A. J. O. Dierckx

Summary

  1. Introduction

Sigma receptors are unique proteins integrated in several organs including liver, kidney and brain. They may be ligated by steroid hormones, sphingolipid-derived amines and N,N-dimethyltryptamine.

Recent evidence suggests that DMT, a well-known hallucinogen, may be an endogenous sigma-1 agonist. DMT is a substrate for the serotonin transporter and the vesicular monoamine transporter 2, which may allow its accumulation in neurons to the micromolar levels needed for sigma-1 receptor activation. DMT is stored in vesicles and inhibits the enzyme indole-N-methyl transferase (INMT), which is localized to postsynaptic sites of C-terminals of mouse motoneurons. INMT may be a candidate gene in the prevention of cancer progression.

Molecular biology techniques have indicated that sigma-1 receptors play critical roles in the mammalian nervous system. Sigma-1 knockout mice are viable and fertile, but display depressive behavior under certain forms of stress and increased anxiety in the open-field, passive avoidance and elevated plus-maze tests.

By knockdown of the sigma-1 receptor, many cellular functions are impaired, including steroid biogenesis, protein ubiquitination, organization of the actin cytoskeleton and Nrf2-mediated responses to oxidative stress.

A study published one year before this one identified the sigma-2 receptor as the progesterone receptor membrane component 1 (PGRMC1). Knockdown of PGRMC1 reduced the binding of a radioiodinated sigma-2 receptor ligand to HeLa cells and reduced the ability of sigma-2 agonists to induce caspase-3 activation in HeLa cells. Treatment of lung cancer cells with PGRMC1 or sigma-2 receptor ligands induced similar, dose-dependent upregulations of the PGRMC1 protein. The PGRMC1 and sigma-2 receptor proteins had the same intracellular localization.

A study by Xu et al. showed 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.

Although the close identity of the sigma-2 receptor and PGRMC1 appears to have been established, some questions remain unanswered. These include the molecular masses of both proteins, the fact that anti-cancer drugs require the agonist action at sigma-2 receptors but an antagonist action at PGRMC1, and the fact that PGRMC1 stimulates P450 activity.

Sigma-1 and sigma-2 receptors are usually treated as members of a common sigma receptor “family”, but their structures are unrelated, and the sigma-1 receptor is actually a chaperone protein.

  1. Receptor overexpression in tumors and tumor cell lines

Sigma-2 receptors are overexpressed in rapidly proliferating normal cells and cancer cells from animal and human origins. However, sigma-2 receptor ligands induce very little apoptosis in proliferating human stem cells, suggesting that they may be used to selectively eliminate tumor cells.

RT-4, a human urinary bladder tumor cell line, expresses sigma-2 receptors at a high density and sigma-1 receptors at a lower density. It can be used for a sigma-2 receptor binding assay.

Sigma-2 receptors were found to be over-expressed in 12 out of 15 samples of human small cell lung carcinoma and 6 out of 15 samples of human adenocarcinomas. Sigma-2 receptor levels were also significantly elevated in the plasma of lung cancer patients.

A subsequent study from the same group found that the sigma-2 receptor increases the expression of neutrophil gelatinase-associated lipocalin/lipocalin-2 (NGAL) and decreases the activity of matrix metalloproteinase-9 (MMP9) in samples of the culture medium, suggesting that the sigma-2 receptor may be an important target for inhibiting tumor invasion and cancer progression.

RPMI 8226, a cell line from human multiple myeloma, expresses sigma-1 receptors at high density, and may be a good alternative to guinea pig brain homogenates for assessing sigma-1 affinity of novel drugs.

Sigma-1 receptor expression was detected in three different human esophageal squamous cell carcinoma cell lines and in patients’ tumor samples. The expression of sigma-1 receptor may be a factor predicting ESCC classification and ESCC development.

In an animal study, sigma-1 receptors were found to be overexpressed in spontaneous pituitary tumors. A radioligand for sigma-1 receptors, like 11C-SA4503, was used to detect pituitary adenomas and microadenomas.

  1. Sigma ligands for diagnostic imaging

The development of radiolabeled sigma receptor ligands for diagnostic imaging using positron emission tomography (PET) or single photon emission computed tomography (SPECT) was reviewed in our previous review article.

3.1. Novel sigma-1 receptor probes

A spirocyclic, radiofluorinated sigma-1 receptor ligand was prepared by German investigators and showed higher affinity to sigma-1 receptors, a longer physical half life, and negligible affinity towards emopamil binding protein. The novel compound fluspidine showed high brain uptake, a regional distribution of radioactivity consistent with binding to sigma-1 receptors, and moderate in vivo metabolism. It was found to have selectivities for the sigma-1 subtype of the dopamine D2/sigma receptor ligand haloperidol and emopamil binding protein inhibitor tamoxifen.

A fluoromethyl derivative of 18F-fluspidine was prepared, but failed as a PET tracer because of rapid metabolism, entry of two radiometabolites into the brain, and a continuous washout from the central nervous system.

Investigators from Stanford University prepared a benzothiazolone sigma-1 receptor ligand, [18F]FTC-146, and found that it reduced the brain uptake of 18F-FTC-146 by 80 to 82% at 60 min after injection. In a later publication from the same group, [18F]FTC-146 was evaluated in rats and squirrel monkeys. The tracer showed specific in vivo binding in the monkey brain and was rapidly metabolized, with 15% intact parent compound remaining in monkey plasma at 50 to 110 min after injection.

Chinese investigators prepared 18F-labeled 1-(1,3-benzodioxol-5-ylmethyl)-4-(4-(2-fluoroethoxy)benzyl)piperazine as a potential sigma-1 receptor ligand for PET imaging. This compound showed favorable biodistribution data in mice and rats and a low nanomolar affinity to sigma-1 receptors.

A team at Kanazawa University in Japan produced a radiobrominelabeled vesamicol analog as a sigma receptor imaging agent for PET and SPECT, and evaluated the compound in vitro and in vivo. The authors concluded that the compound has considerable potential as a sigma receptor ligand for tumor imaging.

Analogs of prezamicol and trozamicol have been prepared. The most promising compound, 11C-labeled 1-[2-(3,4-dimethoxyphenyl)ethyl)]-4-(3-phenylpropyl)-piperazine, shows sigma-1 receptor selectivity in the living brain, but also significant affinity to emopamil binding protein.

3.2. Novel sigma-2 receptor probes

Abate et al. prepared benzamides with PB28 and RHM-1 moieties, hoping to create a tracer for sigma-2 receptors that would combine good brain uptake with good target-to-nontarget ratios and in vivo selectivity for the sigma-2 subtype. Unfortunately, the compounds were substrates for the drug efflux pump P-glycoprotein.

Analogs of PB28 with reduced lipophilicity were prepared, but displayed little sigma-2 subtype selectivity and were also a substrate for P-glycoprotein.

An Australian research team synthesized two novel phthalimido ligands for sigma-2 receptors, which showed high affinity and good selectivity for the sigma-2 subtype in binding assays involving membrane fragments isolated from rat brain. However, the tracers failed to demonstrate specific binding to tumor cells in nude mice bearing A375 tumors.

A sigma-2 receptor ligand, [18F]ISO-1, was evaluated in two different rodent models of breast cancer and showed promising results in indicating both the proliferative status of a tumor and its growth rate. This suggests that sigma-2 receptor imaging can be used in the selection and design of an appropriate treatment strategy.

A structurally similar ligand, RHM-4, showed promising preliminary results in mice bearing mammary tumors. It showed excellent tumor-to-muscle ratios of 8 to 10 at 120 min after injection.

A potential SPECT tracer for imaging of sigma-2 receptors in tumors has been proposed as well. The tracer showed moderate affinity to sigma-2 receptors and moderate sigma-2 subtype selectivity, and was well-uptake into brain and in tumors in C6 rat glioma cells and male ICR mice.

  1. Sigma ligands for targeted drug delivery

Sigma receptors are overexpressed in many kinds of tumors and have been considered as an attractive target for anticancer drug delivery.

Sigma-ligand-based drug targeting has been explored in two different ways: first, by conjugating sigma ligands with various nanoparticles, and second, by conjugating antisense oligonucleotides or antitumor peptides with sigma ligands.

Initial targeting attempts made use of anisamide and haloperidol, but more recent attempts have used compounds with high selectivity for the sigma-2 receptor subtype.

Conjugation of a sigma ligand to a nanoparticle may cause loss of affinity of the ligand to its target receptor. However, the loss of affinity appeared to be negligible.

  1. Cytotoxic effects of sigma ligands

Sigma-1 and sigma-2 receptors are overexpressed in a large variety of tumors and can activate apoptotic pathways. Sigma ligands are potentially useful as anticancer drugs.

Abate and co-workers described a series of 1-cyclohexyl-4-(4-arylcyclohexyl)piperazines that inhibit sigma-1 and sigma-2 receptors, human sterol isomerase and P-glycoprotein, resulting in significant growth inhibition in P-gp overexpressing tumor cells.

A later study from the same group developed tetrahydroisoquinoloines that had high selectivity and affinity for the sigma-2 subtype and were capable of reversing P-gp-mediated drug resistance. These compounds could be evaluated for the treatment of multidrug-resistant tumors.

Researchers conjugated haloperidol with cationic lipids to enhance the apoptosis-inducing ability of haloperidol without impairing its targeting ability. The most promising compound, HP-C8, showed very strong cytotoxicity towards breast cancer cells.

Haloperidol conjugates with other lipid chain lengths were tried, but the toxicity of the C8 conjugate was much higher than that 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.

Other modifications of haloperidol were tried aswell, including the phenylbutyrate ester of metabolite II of haloperidol, which combines inhibition of histone deacetylase with sigma receptorbinding. This molecule has a much lower IC50 value than 4-phenylbutyric acid or haloperidol metabolite II.

A series of adamantane phenylalkylamines were 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 and displayed significant antiproliferative activity to human colon, breast, ovarian, brain, pancreatic and liver cancer cells.

Selective sigma-1 or sigma-2 receptor ligands may also be employed for therapeutic purposes. Fenpropimorph-derived sigma-1 receptor ligands, conformationally restricted sigma-1 receptor ligands, and hydroxyethyl substituted piperazines have shown considerable cytotoxicity in various tumor cell lines.

A study of our own group found that rimcazole (26 mg/kg body weight) treatment resulted in a greater than 4-fold reduction of tumor weight in nude mice bearing A375M human melanoma xenografts after 2 weeks. The treatment was not associated with any significant adverse side effect.

Sigma ligands can enhance the pharmacological action of other anticancer drugs, such as gemcitabine and doxorubicin, by killing tumor cells and inhibiting tumor growth.

  1. Mechanisms underlying inhibition of tumor growth

Although small-molecule ligands targeting sigma-1 and/or sigma-2 receptors have been known to inhibit cancer cell proliferation, induce tumor cell death and suppress tumor growth, the mechanisms involved in cell death have not been well-defined.

Sigma-1 chaperone protein regulates the expression of several ion channels and G-protein coupled membrane receptors, including voltage-gated potassium channels, small conductance Ca2+ activated K+ channels, voltage-gated sodium channels, acid-sensing ion channels, volume-regulated chloride channels, and dopamine D1 receptors.

Investigators noticed that treatment of tumor cell lines with certain sigma-1 ligands visibly diminished cell size. They performed a study in various cell lines and found that sigma-1 ligands reduce cellular protein synthesis by repressing the cap-dependent initiation of translation and phosphorylation of the translational regulator proteins p70S6K, S6 and 4E-BP1.

Interleukin-24 (IL-24) induces ER stress, production of reactive oxygen species and calcium mobilization resulting in the apoptosis of cancer cells. The sigma-1 receptor is a key mediator of IL-24-induced cancer-specific apoptosis.

The sigma ligand [3H]PB28 accumulates in nuclear fractions of neuroblastoma and breast cancer cells, probably due to its high affinity for histone H2A/H2B dimers.

Sigma-2 induced apoptosis can be caspase-independent and may therefore also occur in cells with caspase dysfunction. Siramesine, a sigma-2 receptor agonist, caused apoptosis and secretory granule permeabilization in mast cells in a caspase-independent manner. However, the effect of siramesine appears to be cell-type dependent, and may be due to the fact that siramesine interacts with multiple molecular targets inside cells.

  1. Conclusions

Novel, potent and subtype-selective radioligands for PET imaging of sigma receptors have become available, and may be used for tumor detection, tumor staging, evaluation of therapeutic strategies and anti-tumor drug development.

Study details

Compounds studied
DMT

Topics studied
Chemistry Neuroscience

Study characteristics
Literature Review