Elsevier

Bioorganic & Medicinal Chemistry

Volume 16, Issue 22, 15 November 2008, Pages 9644-9651
Bioorganic & Medicinal Chemistry

Acylamido analogs of endocannabinoids selectively inhibit cancer cell proliferation

https://doi.org/10.1016/j.bmc.2008.10.015Get rights and content

Abstract

A series of amide derivatives of long-chain fatty acids has been studied for their effects on the proliferation of cancer cells in vitro. Fatty acids ranged from palmitic to higher polyunsaturated types containing 22 carbon atoms. The amino portions of the molecules included ammonia, ethanolamine, various amino acids and dopamine. Several cell lines were used as models and these included HTB-125 (normal human breast cells), HTB-126 (human breast cancer cells), HeLa (cervical cancer cells), WI-38 (human embryonic lung cells), RAW264.7 (mouse macrophage tumor cells) and RBL-2H3 (rat basophilic leukemia cells). The HTB lines were obtained from the same donor, so, could be considered a matched pair, that is, normal control versus cancer cells and thus, provide a model for testing specificity of action for the acylamido analogs. While many compounds were efficacious in inhibiting the proliferation of various cell lines, only two analogs showed a high degree of specificity in the matched HTB cell lines. N-palmitoyl dopamine and N-palmitoyl tyrosine each demonstrated complete specificity of action at a concentration of 10 μM and were highly efficacious in both cases. No clear structure–activity pattern could be derived from these studies since the intensity of the inhibitory action seemed to depend on three factors, namely, the fatty acid, the amine group and the cell type.

Graphical abstract

A series of amide derivatives of long-chain fatty acids has been studied for their effects on the proliferation of cancer cells in vitro. Two analogs showed a high degree of specificity in matched breast cancer (tumor vs normal) cell lines obtained from the same donor. N-palmitoyl dopamine and N-palmitoyl tyrosine each demonstrated complete specificity of action at a concentration of 10 μM and were highly efficacious in both cases.

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Introduction

The discovery of an endogenous cannabinoid system (ECS) has fueled an intense interest in identifying the roles that this complex may have in the regulation of cell function and the possible significance for health care strategies.1 Various studies have addressed this question in both physiological and pathological processes at central as well as peripheral sites. Thus far, the endocannabinoid system has been shown to be involved in the immune system, energy balance, stress recovery, food intake, metabolic homeostasis, cardiovascular depressive effects, endocrine axes, fertility, pain modulation and neuroprotection. The ECS may also have a role in cancer biology because of its effects on cell proliferation.2

Many of the substances, and related analogs, that modulate this system are lipid in nature and generally are comprised of a long-chain fatty acid coupled to a more polar group such as ethanolamine, glycerol, dopamine or one of a number of amino acids (Fig. 1). The fatty acid component of the structure can range from a fully saturated acid such as palmitic to a higher polyunsaturated type such as arachidonic. To date, fairly extensive groups of these ligands or mediators have been identified and to some degree characterized.3 Thus, it is probably safe to say that the extent of this family of compounds is far greater than what has thus far been discovered.

Our own interest is focused primarily on the amino acid conjugates that we have named elmiric acids (EMA).4 These analogs do not produce typical cannabimimetic actions in animal models, however, they do show both analgesic5, 6, 7 and anti inflammatory8 properties in vivo. In addition, they exhibit a number of in vitro properties such as inhibition of fatty acid amide hydrolase (FAAH),9 effects on prostaglandin profiles,4 calcium mobilization and firing rates in dorsal root ganglia,10, 11 and cell proliferation.4

The plant derived cannabinoids, or phytocannabinoids, have been studied as possible anti cancer agents for some time.12 Various mechanisms have been suggested to help explain their anti proliferative actions and these may have relevance for the effects of the endocannabinoids as well.13, 14, 15, 16 Tetrahydrocannabinol (THC), the principle psychotropic ingredient in Cannabis, has long been used in the form of the drug dronabinol, to reduce the side effects of chemotherapy, but, its impact on the course of the disease in humans is not clear. Synthetic analogs of THC have been prepared and studied for a possible use in cancer therapy. Such a compound, ajulemic acid (AJA) is a peripheralized,17 non psychoactive analog with moderate anti cancer actions in preclinical models.18 Nabilone, another THC analog, is used clinically to control nausea and emesis in patients receiving chemotherapy, however, its possible effects on tumor progression has not been reported.19

We previously carried out a limited study on the inhibition of proliferation by several EMAs in a mouse macrophage tumor-derived cell line, RAW264.7.8 Full dose–response data were not obtained in that study; however, a complete inhibition of proliferation was seen between 1 and 10 μM. Only the effects of glycine and alanine conjugates were reported and their polyunsaturated derivatives appeared to be more active than the saturated analogs. The limited nature of the study did not allow any detailed conclusions regarding structure–activity relationships (SAR) to be made.

In subsequent experiments reported here, we sought to extend the previous work to a series of new analogs and to expand the number and variety of cell types studied with these compounds. In addition, more complete relationships between drug concentration and cell proliferation have been obtained. Of particular interest, is an observed selectivity of inhibition when the effect on tumor cells is compared with a matched normal control, which was observed for certain compounds in a model of human breast cancer.

Section snippets

Chemistry

Only one of the compounds used in this study, N-arachidonoyl-l-tyrosine; L-EMA-10 (16:0) (see Ref. 8 for nomenclature), was synthesized in the university laboratories using the procedures described below in Section 6.1.2. Briefly this involved condensation of the mixed anhydride palmitoyl-i-butylchloroformate with l-tyrosine methyl ester in the presence of triethylamine followed by saponification with lithium hydroxide to yield the EMA. The rest of the compounds came from a commercially

Time course

In Figure 2 the time course for the anti proliferative effect of one of the EMAs, N-palmitoyl tyrosine (10 μM) was compared to vehicle (DMSO) treated cells and AJA (10 μM), a cannabinoid analog with anti tumor activity.18 Equal numbers (2000 cells/well) of human tumor-derived breast cancer cells (HTB-126) were plated at time zero. All three treatments were applied at 24 h and cell numbers measured two hours later. Subsequent measurements, on parallel plates, were made at 48, 72 and 96 h post plating

Discussion

Almost a century has passed since Paul Ehrlich used the term “silver bullet” to describe salvarsan, his drug for the treatment of syphilis. Unfortunately, salvarsan did not exhibit the specificity of action he sought, but this has not discouraged generations of scientists since then from continuing the search for agents that will only target disease sites leaving healthy tissue unaffected. This has been particularly true in the area of cancer chemotherapy where specificity of action remains a

Conclusions

Future studies with the types of fatty acid amido compounds reported on here should be aimed at three important questions. First, the mechanism(s) responsible for the specificity of the anti proliferative actions should be addressed. Second, a more detailed SAR should be obtained that would allow the design of more potent selective analogs. And, finally, the in vivo potential of the more promising compounds should be studied with a view toward their becoming drug candidates. The data presented

Materials

All of the acylamido analogs studied were obtained from BIOMOL International, L.P., Plymouth Meeting, PA 19462 (Screen-WellTM Endocannabinoid Library). The only exception was N-palmitoyl tyrosine that was synthesized using a published procedure.8 All other chemicals were purchased from Sigma-Aldrich, St. Louis, MO 63178. All cell lines were obtained from American Type Culture Collection (ATCC), Manassas, VA 20108. Cell proliferation assay reagents were purchased from Promega Corporation,

Acknowledgments

This publication was made possible by grant DA17969 from National Institute on Drug Abuse, National Institutes of Health, Bethesda, MD. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institute on Drug Abuse. We thank Dr. Robert E. Zurier, Dr. Ethan S. Burstein and Ronald Rossetti for helpful discussions throughout the course of this study and during the preparation of the manuscript and we are also grateful to Dr.

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