The Interaction of the Endocannabinoid Anandamide and Paracannabinoid Lysophosphatidylinositol during Cell Death Induction in Human Breast Cancer Cells.

Endocannabinoid anandamide (AEA) and paracannabinoid lysophosphatidylinositol (LPI) play a significant role in cancer cell proliferation regulation. While anandamide inhibits the proliferation of cancer cells, LPI is known as a cancer stimulant. Despite the known endocannabinoid receptor crosstalk and simultaneous presence in the cancer microenvironment of both molecules, their combined activity has never been studied. We evaluated the effect of LPI on the AEA activity in six human breast cancer cell lines of different carcinogenicity (MCF-10A, MCF-7, BT-474, BT-20, SK-BR-3, MDA-MB-231) using resazurin and LDH tests after a 72 h incubation. AEA exerted both anti-proliferative and cytotoxic activity with EC50 in the range from 31 to 80 µM. LPI did not significantly affect the cell viability. Depending on the cell line, the response to the LPI-AEA combination varied from a decrease in AEA cytotoxicity to an increase in it. Based on the inhibitor analysis of the endocannabinoid receptor panel, we showed that for the former effect, an active GPR18 receptor was required and for the latter, an active CB2 receptor. The data obtained for the first time are important for the understanding the manner by which endocannabinoid receptor ligands acting simultaneously can modulate cancer growth at different stages.

The Mechanisms of GPR55 Receptor Functional Selectivity during Apoptosis and Proliferation Regulation in Cancer Cells.

GPR55 is a non-canonical cannabinoid receptor, important for cancer proliferation. Depending on the ligand, it induces either cell proliferation or death. The objective of the study was to establish the mechanisms of this multidirectional signaling. Using the CRISPR-Cas9 system, the GPR55, CB1, CB2, and GPR18 receptor knockouts of the MDA-MB-231 line were obtained. After the CB2 receptor knockout, the pro-apoptotic activity of the pro-apoptotic ligand docosahexaenoyl dopamine (DHA-DA) slightly increased, while the pro-proliferative activity of the most active synthetic ligand of the GPR55 receptor (ML-184) completely disappeared. On the original cell line, the stimulatory effect of ML-184 was removed by the CB2 receptor blocker and by GPR55 receptor knockout. Thus, it can be confidently assumed that when proliferation is stimulated with the participation of the GPR55 receptor, a signal is transmitted from the CB2 receptor to the GPR55 receptor due to the formation of a heterodimer. GPR18 was additionally involved in the implementation of the pro-apoptotic effect of DHA-DA, while the CB1 receptor is not involved. In the implementation of the pro-apoptotic action of DHA-DA, the elimination of Gα13 led to a decrease in cytotoxicity. The obtained data provide novel details to the mechanism of the pro-proliferative action of GPR55.

N-Acyl Dopamines Induce Apoptosis in Endometrial Stromal Cells from Patients with Endometriosis.

Endometriosis is characterized by the formation and development of endometrial tissues outside the uterus, based on an imbalance between proliferation and cell death, leading to the uncontrolled growth of ectopic foci. The potential target for the regulation of these processes is the endocannabinoid system, which was found to be involved in the migration, proliferation, and survival of tumor cells. In this paper, we investigated the effect of endocannabinoid-like compounds from the N-acyl dopamine (NADA) family on the viability of stromal cells from ectopic and eutopic endometrium of patients with ovarian endometriosis. N-arachidonoyldopamine, N-docosahexaenoyldopamine, and N-oleoyldopamine have been shown to have a five-times-more-selective cytotoxic effect on endometrioid stromal cells. To study the mechanisms of the toxic effect, inhibitory analysis, measurements of caspase-3/9 activity, reactive oxygen species, and the mitochondrial membrane potential were performed. It was found that NADA induced apoptosis via an intrinsic pathway through the CB1 receptor and downstream serine palmitoyltransferase, NO synthase activation, increased ROS production, and mitochondrial dysfunction. The higher selectivity of NADA for endometriotic stromal cells and the current lack of effective drug treatment can be considered positive factors for further research of these compounds as possible therapeutic agents against endometriosis.

ACTH(6-9)PGP Peptide Protects SH-SY5Y Cells from H2O2, tert-Butyl Hydroperoxide, and Cyanide Cytotoxicity via Stimulation of Proliferation and Induction of Prosurvival-Related Genes.

Stabilized melanocortin analog peptide ACTH(6-9)PGP (HFRWPGP) possesses a wide range of neuroprotective activities. However, its mechanism of action remains poorly understood. In this paper, we present a study of the proproliferative and cytoprotective activity of the adrenocorticotropic hormone fragment 6-9 (HFRW) linked with the peptide prolyine-glycyl-proline on the SH-SY5Y cells in the model of oxidative stress-related toxicity. The peptide dose-dependently protected cells from H2O2, tert-butyl hydroperoxide, and KCN and demonstrated proproliferative activity. The mechanism of its action was the modulation of proliferation-related NF-κB genes and stimulation of prosurvival NRF2-gene-related pathway, as well as a decrease in apoptosis.

GPR55 Receptor Activation by the N-Acyl Dopamine Family Lipids Induces Apoptosis in Cancer Cells via the Nitric Oxide Synthase (nNOS) Over-Stimulation.

GPR55 is a GPCR of the non-CB1/CB2 cannabinoid receptor family, which is activated by lysophosphatidylinositol (LPI) and stimulates the proliferation of cancer cells. Anandamide, a bioactive lipid endocannabinoid, acts as a biased agonist of GPR55 and induces cancer cell death, but is unstable and psychoactive. We hypothesized that other endocannabinoids and structurally similar compounds, which are more hydrolytically stable, could also induce cancer cell death via GPR55 activation. We chemically synthesized and tested a set of fatty acid amides and esters for cell death induction via GPR55 activation. The most active compounds appeared to be N-acyl dopamines, especially N-docosahexaenoyl dopamine (DHA-DA). Using a panel of cancer cell lines and a set of receptor and intracellular signal transduction machinery inhibitors together with cell viability, Ca2+, NO, ROS (reactive oxygen species) and gene expression measurement, we showed for the first time that for these compounds, the mechanism of cell death induction differed from that published for anandamide and included neuronal nitric oxide synthase (nNOS) overstimulation with concomitant oxidative stress induction. The combination of DHA-DA with LPI, which normally stimulates cancer proliferation and is increased in cancer setting, had an increased cytotoxicity for the cancer cells indicating a therapeutic potential.

N-arachidonoyl dopamine inhibits epithelial-mesenchymal transition of breast cancer cells through ERK signaling and decreasing the cellular cholesterol.

N-acyl dopamines (NADAs) are bioactive lipids of the endovanilloid family with known cytotoxicity for the cancer cells; however, the available data on the participation of the endovanilloids in epithelial-mesenchymal transition (EMT) and cancer stemness are controversial. This study unveils the inhibitory role of N-arachidonoyl dopamine (AA-DA), a typical representative of the NADA family, in breast cancer cell migration, EMT, and stemness. AA-DA treatment also led to a decrease in cholesterol biosynthesis gene expressions, and addition of exogenous cholesterol reverted these AA-DA-mediated inhibitory effects. Notably, AA-DA treatment inhibited the key regulatory gene of the cholesterol biosynthesis pathway, sterol regulatory element-binding protein 1 (SREBP1), with concurrent repression of the endoplasmic reticulum kinase 1/2 (ERK1/2) pathway. Furthermore, U0126, an ERK inhibitor, inhibited SREBP1 and decreased cellular cholesterol level, unwinding the molecular mechanism behind AA-DA-mediated anticancer activity. Thus, we, for the first time, revealed that AA-DA counteracts breast cancer EMT via inhibition of ERK signaling and cholesterol content.

In Vivo Targeted Metabolomic Profiling of Prostanit, a Novel Anti-PAD NO-Donating Alprostadil-Based Drug.

Prostanit is a novel drug developed for the treatment of peripheral arterial diseases. It consists of a prostaglandin E1 (PGE1) moiety with two nitric oxide (NO) donor fragments, which provide a combined vasodilation effect on smooth muscles and vascular spastic reaction. Prostanit pharmacokinetics, however, remains poorly investigated. Thus, the object of this study was to investigate the pharmacokinetics of Prostanit-related and -affected metabolites in rabbit plasma using the liquid chromatography-mass spectrometry (LC-MS) approach. Besides, NO generation from Prostanit in isolated rat aorta and human smooth muscle cells was studied using the Griess method. In plasma, Prostanit was rapidly metabolized to 1,3-dinitroglycerol (1,3-DNG), PGE1, and 13,14-dihydro-15-keto-PGE1. Simultaneously, the constant growth of amino acid (proline, 4-hydroxyproline, alanine, phenylalanine, etc.), steroid (androsterone and corticosterone), and purine (adenosine, adenosine-5 monophosphate, and guanosine) levels was observed. Glycine, aspartate, cortisol, and testosterone levels were decreased. Ex vivo Prostanit induced both NO synthase-dependent and -independent NO generation. The observed pharmacokinetic properties suggested some novel beneficial activities (i.e., effect prolongation and anti-inflammation). These properties may provide a basis for future research of the effectiveness and safety of Prostanit, as well as for its characterization from a clinical perspective.

Novel bexarotene derivatives: Synthesis and cytotoxicity evaluation for glioma cells in 2D and 3D in vitro models.

Glioblastoma (GBM) is an aggressive and lethal form of brain cancer with a high invasion capacity and a lack of effective chemotherapeutics. Retinoid bexarotene (BXR) inhibits the neurospheroidal colony formation and migration of primary glioblastoma cells but has side effects. To enhance the BXR glioblastoma selectivity and cytotoxicity, we chemically modified it at the carboxyl group with either nitroethanolamine (NEA) bearing a NO-donating group (a well-known bioactivity enhancer; BXR-NEA) or with a dopamine (DA) moiety (to represent the highly toxic for various tumor cells N-acyldopamine family; BXR-DA). These two novel compounds were tested in the 2D (monolayer culture) and 3D (multicellular tumor spheroids) in vitro models. Both BXR-DA and BXR-NEA were found to be more toxic for rat C6 and human U-87MG glioma cells than the initial BXR. After 24 h incubation of the cells (monolayer culture) with the drugs, the IC50 values were in the range of 28-42, and 122-152 µM for BXR derivatives and BXR, respectively. The cell death occurred via apoptosis according to the annexin staining and caspase activation. The tumor spheroids demonstrated higher resistance to the treatment compared to that one of the monolayer cultures. BXR-DA and BXR-NEA were more specific against tumor cells than the parental drug, in particular the selectivity index was 1.8-2.7 vs. 1.3-1.5, respectively. Moreover, they inhibited cell migration more effectively than parental BXR according to a scratch assay. Cell spreading from the tumor spheroids was also inhibited. Thus, the obtained BXR derivatives could be promising for glioblastoma treatment.

5-alkylvinyl-1,2,4-triazole nucleosides: Synthesis and biological evaluation.

Some 5-substituted ribavirin analogues have a high antiviral and anticancer activity, but their mechanisms of action are obviously not the same as their parent compound. The SAR studies performed on 3 (5)-substituted 1,2,4-triazole nucleosides have shown a high dependency between the structure of the 3 (5)-substituent and the level of antiviral/anticancer activity. The most active substances of the row contain coplanar with the 1,2,4-triazole ring aromatic substituent which is connected by a rigid ethynyl bond. However, the compounds with the trans-vinyl linker also had antiviral activity. We decided to study the antitumor activity of ribavirin analogues with alkyl/aryl vinyl substituents in the 5th position of the 1,2,4-triazole ring. Protected nucleoside analogues with various 5-alkylvinyl substituents were obtained by Horner-Wadsworth-Emmons reaction from the common precursor and converted to the nucleosides. Arylvinyl nucleosides were synthesised according the reported procedures. All compounds did not show significant antiproliferative activity on several tumour cell lines. Coplanar aromatic motif in the 5-substituent for the anticancer activity manifestation was confirmed.

Arachidonoylcholine and Other Unsaturated Long-Chain Acylcholines Are Endogenous Modulators of the Acetylcholine Signaling System.

Cholines acylated with unsaturated fatty acids are a recently discovered family of endogenous lipids. However, the data on the biological activity of acylcholines remain very limited. We hypothesized that acylcholines containing residues of arachidonic (AA-CHOL), oleic (Ol-CHOL), linoleic (Ln-CHOL), and docosahexaenoic (DHA-CHOL) acids act as modulators of the acetylcholine signaling system. In the radioligand binding assay, acylcholines showed inhibition in the micromolar range of both α7 neuronal nAChR overexpressed in GH4C1 cells and muscle type nAChR from Torpedo californica, as well as Lymnaea stagnalis acetylcholine binding protein. Functional response was checked in two cell lines endogenously expressing α7 nAChR. In SH-SY5Y cells, these compounds did not induce Ca2+ rise, but inhibited the acetylcholine-evoked Ca2+ rise with IC50 9 to 12 µM. In the A549 lung cancer cells, where α7 nAChR activation stimulates proliferation, Ol-CHOL, Ln-CHOL, and AA-CHOL dose-dependently decreased cell viability by up to 45%. AA-CHOL inhibited human erythrocyte acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BChE) by a mixed type mechanism with Ki = 16.7 ± 1.5 µM and αKi = 51.4 ± 4.1 µM for AChE and Ki = 70.5 ± 6.3 µM and αKi = 214 ± 17 µM for BChE, being a weak substrate of the last enzyme only, agrees with molecular docking results. Thus, long-chain unsaturated acylcholines could be viewed as endogenous modulators of the acetylcholine signaling system.

Cytotoxicity of Endogenous Lipids N-acyl Dopamines and their Possible Metabolic Derivatives for Human Cancer Cell Lines of Different Histological Origin.

BACKGROUND/AIM: Dopamine amides of long chain fatty acids are a family of endogenous mammalian lipids with an unknown function; they are anti-proliferative for the C6 glioblastoma cell line. To assess their possible anti-cancer activity we evaluated their cytotoxicity for a set of cancer cell lines. MATERIALS AND METHODS: Anti-proliferative and cytotoxic actions of these substances were evaluated in HOS, IMR-32, MCF-7, Namalwa, K-562 and HEK 293 cell lines (18 h incubation time) using MTT and lactate dehydrogenase (LDH) tests, accordingly. RESULTS: All N-acyl dopamines (NADA) induced cell death in all cell lines tested with a 50% lethal dose (LD50) in the range of 0.5-80 µM, except for HEK-293. For HEK-293 only N-arachidonoyl epinephrine demonstrated an LD50 below 100 µM. CONCLUSION: According to the structure-activity relationship, N-acyl dopamines with an intact catechol group and a non-modified hydrophobic fatty acid residue are cytotoxic to cancer cell lines of various histological origins.