Dissecting the Role of Autophagy-Related Proteins in Cancer Metabolism and Plasticity.

Modulation of autophagy as an anticancer strategy has been widely studied and evaluated in several cell models. However, little attention has been paid to the metabolic changes that occur in a cancer cell when autophagy is inhibited or induced. In this review, we describe how the expression and regulation of various autophagy-related (ATGs) genes and proteins are associated with cancer progression and cancer plasticity. We present a comprehensive review of how deregulation of ATGs affects cancer cell metabolism, where inhibition of autophagy is mainly reflected in the enhancement of the Warburg effect. The importance of metabolic changes, which largely depend on the cancer type and form part of a cancer cell's escape strategy after autophagy modulation, is emphasized. Consequently, pharmacological strategies based on a dual inhibition of metabolic and autophagy pathways emerged and are reviewed critically here.

Reduction of Ca2+ Entry by a Specific Block of KCa3.1 Channels Optimizes Cytotoxic Activity of NK Cells against T-ALL Jurkat Cells.

Degranulation mediated killing mechanism by NK cells is dependent on store-operated Ca2+ entry (SOCE) and has optimum at moderate intracellular Ca2+ elevations so that partial block of SOCE optimizes the killing process. In this study, we tested the effect of the selective blocker of KCa3.1 channel NS6180 on SOCE and the killing efficiency of NK cells from healthy donors and NK-92 cells against T-ALL cell line Jurkat. Patch-clamp analysis showed that only one-quarter of resting NK cells functionally express KCa3.1 current, which increases 3-fold after activation by interleukins 15 and 2. Nevertheless, blockage of KCa3.1 significantly reduced SOCE and intracellular Ca2+ rise induced by IL-15 or target cell recognition. NS6180 (1 µM) decreased NK degranulation at zero time of coculture with Jurkat cells but already after 1 h, the degranulation reached the same level as in the control. Monitoring of target cell death by flow cytometry and confocal microscopy demonstrated that NS6180 significantly improved the killing ability of NK cells after 1 h in coculture with Jurkat cells and increased the Jurkat cell fraction with apoptotic and necrotic markers. Our data evidence a strong dependence of SOCE on KCa3.1 activity in NK cells and that KCa3.1 specific block can improve NK cytotoxicity.

In vitro simulation of the acute lymphoblastic leukemia niche: a critical view on the optimal approximation for drug testing.

Acute lymphoblastic leukemia with the worst prognosis is related to minimal residual disease. Minimal residual disease not only depends on the individual peculiarities of leukemic clones but also reflects the protective role of the acute lymphoblastic leukemia microenvironment. In this review, we discuss in detail cell-to-cell interactions in the 2 leukemic niches, more explored bone marrow and less studied extramedullary adipose tissue. A special emphasis is given to multiple ways of interactions of acute lymphoblastic leukemia cells with the bone marrow or extramedullary adipose tissue microenvironment, indicating observed differences in B- and T-cell-derived acute lymphoblastic leukemia behavior. This analysis argued for the usage of coculture systems for drug testing. Starting with a review of available sources and characteristics of acute lymphoblastic leukemia cells, mesenchymal stromal cells, endothelial cells, and adipocytes, we have then made an update of the available 2-dimensional and 3-dimensional systems, which bring together cellular elements, components of the extracellular matrix, or its imitation. We discussed the most complex available 3-dimensional systems like "leukemia-on-a-chip," which include either a prefabricated microfluidics platform or, alternatively, the microarchitecture, designed by using the 3-dimensional bioprinting technologies. From our analysis, it follows that for preclinical antileukemic drug testing, in most cases, intermediately complex in vitro cell systems are optimal, such as a "2.5-dimensional" coculture of acute lymphoblastic leukemia cells with niche cells (mesenchymal stromal cells, endothelial cells) plus matrix components or scaffold-free mesenchymal stromal cell organoids, populated by acute lymphoblastic leukemia cells. Due to emerging evidence for the correlation of obesity and poor prognosis, a coculture of adipocytes with acute lymphoblastic leukemia cells as a drug testing system is gaining shape.

Dexamethasone-Induced Fatty Acid Oxidation and Autophagy/Mitophagy Are Essential for T-ALL Glucocorticoid Resistance.

ALL is a highly aggressive subtype of leukemia that affects children and adults. Glucocorticoids (GCs) are a critical component of the chemotherapeutic strategy against T-ALL. Cases of resistance to GC therapy and recurrent disease require novel strategies to overcome them. The present study analyzed the effects of Dex, one of the main GCs used in ALL treatment, on two T-ALL cell lines: resistant Jurkat and unselected CCRF-CEM, representing a mixture of sensitive and resistant clones. In addition to nuclear targeting, we observed a massive accumulation of Dex in mitochondria. Dex-treated leukemic cells suffered metabolic reprogramming from glycolysis and glutaminolysis towards lipolysis and increased FAO, along with increased membrane polarization and ROS production. Dex provoked mitochondrial fragmentation and induced autophagy/mitophagy. Mitophagy preceded cell death in susceptible populations of CCRF-CEM cells while serving as a pro-survival mechanism in resistant Jurkat. Accordingly, preventing FAO or autophagy greatly increased the Dex cytotoxicity and overcame GC resistance. Dex acted synergistically with mitochondria-targeted drugs, curcumin, and cannabidiol. Collectively, our data suggest that GCs treatment should not be neglected even in apparently GC-resistant clinical cases. Co-administration of drugs targeting mitochondria, FAO, or autophagy can help to overcome GC resistance.

The G-Protein-Coupled Estrogen Receptor Agonist G-1 Inhibits Proliferation and Causes Apoptosis in Leukemia Cell Lines of T Lineage.

The G-protein-coupled estrogen receptor (GPER) mediates non-genomic action of estrogen. Due to its differential expression in some tumors as compared to the original healthy tissues, the GPER has been proposed as a therapeutic target. Accordingly, the non-steroidal GPER agonist G-1, which has often demonstrated marked cytotoxicity in experimental models, has been suggested as a novel anticancer agent for several sensitive tumors. We recently revealed that cell lines derived from acute T-cell (query) lymphoblastic leukemia (T-ALL) express the GPER. Here, we address the question whether G-1 is cytotoxic to T-ALL. We have shown that G-1 causes an early rise of intracellular Ca2+, arrests the cell cycle in G2/M, reduces viability, and provokes apoptosis in T-ALL cell lines. Importantly, G-1 caused destabilization and depolymerization of microtubules. We assume that it is a disturbance of the cytoskeleton that causes G-1 cytotoxic and cytostatic effects in our model. The observed cytotoxic effects, apparently, were not triggered by the interaction of G-1 with the GPER as pre-incubation with the highly selective GPER antagonist G-36 was ineffective in preventing the cytotoxicity of G-1. However, G-36 prevented the intracellular Ca2+ rise provoked by G-1. Finally, G-1 showed only a moderate negative effect on the activation of non-leukemic CD4+ lymphocytes. We suggest G-1 as a potential antileukemic drug.

Cannabidiol on the Path from the Lab to the Cancer Patient: Opportunities and Challenges.

Cannabidiol (CBD), a major non-psychotropic component of cannabis, is receiving growing attention as a potential anticancer agent. CBD suppresses the development of cancer in both in vitro (cancer cell culture) and in vivo (xenografts in immunodeficient mice) models. For critical evaluation of the advances of CBD on its path from laboratory research to practical application, in this review, we wish to call the attention of scientists and clinicians to the following issues: (a) the biological effects of CBD in cancer and healthy cells; (b) the anticancer effects of CBD in animal models and clinical case reports; (c) CBD's interaction with conventional anticancer drugs; (d) CBD's potential in palliative care for cancer patients; (e) CBD's tolerability and reported side effects; (f) CBD delivery for anticancer treatment.

Major vacuolar TPC1 channel in stress signaling: what matters, K+, Ca2+ conductance or an ion-flux independent mechanism?

Two-pore cation channel, TPC1, is ubiquitous in the vacuolar membrane of terrestrial plants and mediates the long distance signaling upon biotic and abiotic stresses. It possesses a wide pore, which transports small mono- and divalent cations. K+ is transported more than 10-fold faster than Ca2+, which binds with a higher affinity within the pore. Key pore residues, responsible for Ca2+ binding, have been recently identified. There is also a substantial progress in the mechanistic and structural understanding of the plant TPC1 gating by membrane voltage and cytosolic and luminal Ca2+. Collectively, these gating factors at resting conditions strongly reduce the potentially lethal Ca2+ leak from the vacuole. Such tight control is impressive, bearing in mind high unitary conductance of the TPC1 and its abundance, with thousands of active channel copies per vacuole. But it remains a mystery how this high threshold is overcome upon signaling, and what type of signal is emitted by TPC1, whether it is Ca2+ or electrical one, or a transduction via protein conformational change, independent on ion conductance. Here we discuss non-exclusive scenarios for the TPC1 integration into Ca2+, ROS and electrical signaling.

Tamoxifen Sensitizes Acute Lymphoblastic Leukemia Cells to Cannabidiol by Targeting Cyclophilin-D and Altering Mitochondrial Ca2+ Homeostasis.

Cytotoxic effects of cannabidiol (CBD) and tamoxifen (TAM) have been observed in several cancer types. We have recently shown that CBD primarily targets mitochondria, inducing a stable mitochondrial permeability transition pore (mPTP) and, consequently, the death of acute lymphoblastic leukemia (T-ALL) cells. Mitochondria have also been documented among cellular targets for the TAM action. In the present study we have demonstrated a synergistic cytotoxic effect of TAM and CBD against T-ALL cells. By measuring the mitochondrial membrane potential (ΔΨm), mitochondrial calcium ([Ca2+]m) and protein-ligand docking analysis we determined that TAM targets cyclophilin D (CypD) to inhibit mPTP formation. This results in a sustained [Ca2+]m overload upon the consequent CBD administration. Thus, TAM acting on CypD sensitizes T-ALL to mitocans such as CBD by altering the mitochondrial Ca2+ homeostasis.

Overcoming Glucocorticoid Resistance in Acute Lymphoblastic Leukemia: Repurposed Drugs Can Improve the Protocol.

Glucocorticoids (GCs) are a central component of multi-drug treatment protocols against T and B acute lymphoblastic leukemia (ALL), which are used intensively during the remission induction to rapidly eliminate the leukemic blasts. The primary response to GCs predicts the overall response to treatment and clinical outcome. In this review, we have critically analyzed the available data on the effects of GCs on sensitive and resistant leukemic cells, in order to reveal the mechanisms of GC resistance and how these mechanisms may determine a poor outcome in ALL. Apart of the GC resistance, associated with a decreased expression of receptors to GCs, there are several additional mechanisms, triggered by alterations of different signaling pathways, which cause the metabolic reprogramming, with an enhanced level of glycolysis and oxidative phosphorylation, apoptosis resistance, and multidrug resistance. Due to all this, the GC-resistant ALL show a poor sensitivity to conventional chemotherapeutic protocols. We propose pharmacological strategies that can trigger alternative intracellular pathways to revert or overcome GC resistance. Specifically, we focused our search on drugs, which are already approved for treatment of other diseases and demonstrated anti-ALL effects in experimental pre-clinical models. Among them are some "truly" re-purposed drugs, which have different targets in ALL as compared to other diseases: cannabidiol, which targets mitochondria and causes the mitochondrial permeability transition-driven necrosis, tamoxifen, which induces autophagy and cell death, and reverts GC resistance through the mechanisms independent of nuclear estrogen receptors ("off-target effects"), antibiotic tigecycline, which inhibits mitochondrial respiration, causing energy crisis and cell death, and some anthelmintic drugs. Additionally, we have listed compounds that show a classical mechanism of action in ALL but are not used still in treatment protocols: the BH3 mimetic venetoclax, which inhibits the anti-apoptotic protein Bcl-2, the hypomethylating agent 5-azacytidine, which restores the expression of the pro-apoptotic BIM, and compounds targeting the PI3K-Akt-mTOR axis. Accordingly, these drugs may be considered for the inclusion into chemotherapeutic protocols for GC-resistant ALL treatments.

Acute Lymphoblastic Leukemia Cell Lines in Immunology Research.

A considerable portion of our knowledge on T and B cell biology is acquired from research using acute lymphoblastic leukemia (ALL) cell lines, which are invaluable tools used in many immunology and leukemia studies. Here, we discuss the advantages and limitations of ALL cell lines and provide guidelines on their proper usage.

Phenolic Compounds Cannabidiol, Curcumin and Quercetin Cause Mitochondrial Dysfunction and Suppress Acute Lymphoblastic Leukemia Cells.

Anticancer activity of different phenols is documented, but underlying mechanisms remain elusive. Recently, we have shown that cannabidiol kills the cells of acute lymphoblastic leukemia (ALL) by a direct interaction with mitochondria, with their consequent dysfunction. In the present study, cytotoxic effects of several phenolic compounds against human the T-ALL cell line Jurkat were tested by means of resazurin-based metabolic assay. To unravel underlying mechanisms, mitochondrial membrane potential (∆Ψm) and [Ca2+]m measurements were undertaken, and reactive oxygen species generation and cell death were evaluated by flow cytometry. Three out of eight tested phenolics, cannabidiol, curcumin and quercetin, which displayed a significant cytotoxic effect, also dissipated the ∆Ψm and induced a significant [Ca2+]m increase, whereas inefficient phenols did not. Dissipation of the ∆Ψm by cannabidiol was prevented by cyclosporine A and reverted by Ru360, inhibitors of the permeation transition pore and mitochondrial Ca2+ uniporter, respectively. Ru360 prevented the phenol-induced [Ca2+]m rise, but neither cyclosporine A nor Ru360 affected the curcumin- and quercetin-induced ∆Ψm depolarization. Ru360 impeded the curcumin- and cannabidiol-induced cell death. Thus, all three phenols exert their antileukemic activity via mitochondrial Ca2+ overload, whereas curcumin and quercetin suppress the metabolism of leukemic cells by direct mitochondrial uncoupling.

Kv1.3 Current Voltage Dependence in Lymphocytes is Modulated by Co-Culture with Bone Marrow-Derived Stromal Cells: B and T Cells Respond Differentially.

BACKGROUND/AIMS: Kv1.3 channel is the only voltage-dependent potassium channel in plasma membrane of human lymphocytes. Bearing in mind a rather steep voltage-dependence of Kv1.3 activation and inactivation, its modulation by B and T cells activation and by co-culture with stromal bone-marrow cells was addressed. METHODS: Patch-clamp technique in the whole cell mode was applied to human resting and activated human B and T cells, in monoculture and co-culture with stromal OP9 cells. RESULTS: Polyclonal activation of B and T cells in monoculture caused Kv1.3 current in B cells to activate at more negative and in T cells at more positive potentials, whereas the inactivation of Kv1.3 current in resting T cells occurred at more negative voltages. Co-culture with OP9 cells abolished the shift of voltage dependence upon the polyclonal activation but fixed the substantial difference between B and T cells, resting or activated, with both activation and inactivation negatively shifted by 15 mV for T lymphocytes. However, activated B cells displayed an incomplete inactivation, which was augmented by the co-culture. Neither activation nor co-culture caused substantial changes in the Kv1.3 current density. CONCLUSION: The combination of activation and inactivation processes yields the fraction of steady-state Kv1.3 current (window current), which was higher in activated B cells, partly due to an incomplete inactivation. A relatively smaller window current in resting B cells and resting T cells in co-culture correlated with a more depolarized resting membrane potential. Rather than insignificant changes in the Kv1.3 channels functional expression, the modulation of their voltage dependence by activation and co-culture with bone-marrow stromal cells was essential for the control of membrane potential.

Modulation of Ion Transport Across Plant Membranes by Polyamines: Understanding Specific Modes of Action Under Stress.

This work critically discusses the direct and indirect effects of natural polyamines and their catabolites such as reactive oxygen species and γ-aminobutyric acid on the activity of key plant ion-transporting proteins such as plasma membrane H+ and Ca2+ ATPases and K+-selective and cation channels in the plasma membrane and tonoplast, in the context of their involvement in stress responses. Docking analysis predicts a distinct binding for putrescine and longer polyamines within the pore of the vacuolar TPC1/SV channel, one of the key determinants of the cell ionic homeostasis and signaling under stress conditions, and an additional site for spermine, which overlaps with the cytosolic regulatory Ca2+-binding site. Several unresolved problems are summarized, including the correct estimates of the subcellular levels of polyamines and their catabolites, their unexplored effects on nucleotide-gated and glutamate receptor channels of cell membranes and Ca2+-permeable and K+-selective channels in the membranes of plant mitochondria and chloroplasts, and pleiotropic mechanisms of polyamines' action on H+ and Ca2+ pumps.

Cannabidiol directly targets mitochondria and disturbs calcium homeostasis in acute lymphoblastic leukemia.

Anticancer properties of non-psychoactive cannabinoid cannabidiol (CBD) have been demonstrated on tumors of different histogenesis. Different molecular targets for CBD were proposed, including cannabinoid receptors and some plasma membrane ion channels. Here we have shown that cell lines derived from acute lymphoblastic leukemia of T lineage (T-ALL), but not resting healthy T cells, are highly sensitive to CBD treatment. CBD effect does not depend on cannabinoid receptors or plasma membrane Ca2+-permeable channels. Instead, CBD directly targets mitochondria and alters their capacity to handle Ca2+. At lethal concentrations, CBD causes mitochondrial Ca2+ overload, stable mitochondrial transition pore formation and cell death. Our results suggest that CBD is an attractive candidate to be included into chemotherapeutic protocols for T-ALL treatment.

Mitochondria as emerging targets for therapies against T cell acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) comprises a heterogeneous group of hematologic malignancies, arising from diverse genetic alterations in the early lymphocyte development. T-cell subtype of ALL (T-ALL) accounts for about 15% and 25% of ALL in children and adults, respectively. Being less frequent among ALL subtypes, T-ALL represents a high-risk factor for poor prognosis due to its aggressiveness and resistance to common antileukemic drugs. Mitochondria were widely explored recently as a target for anticancer treatment because they are involved in a metabolic reprogramming of a cancer cell and play key roles in reactive oxygen species generation, Ca2+ signaling, and cell death induction. Accordingly, a new class of anticancer compounds named mitocans has been developed, which target mitochondria at distinct crucial points to promote their dysfunction and subsequent cell death. The present review analyses the role of mitochondria in malignant reprogramming and emerging therapeutic strategies targeting mitochondria as an "Achilles' heel" in T-ALL, with an emphasis on BH3 mimetics, sequestering pro-survival BCL proteins and voltage-dependent anion channel (VDAC)1-directed drugs, which promote the suppression of aerobic glycolysis, VDAC1 closure, mitochondrial Ca2+ overload, stoppage of the oxidative phosphorylation, oxidative stress, and release of proapoptotic factors.

Tamoxifen induces toxicity, causes autophagy, and partially reverses dexamethasone resistance in Jurkat T cells.

Estrogens demonstrate biological activity in numerous organ systems, including the immune system, and exert their effects through estrogen receptors (ER) of two types: intracellular ERα and ERß that activate transcriptional factors and membrane G protein-coupled ER GPER. The latter is capable to mediate fast activation of cytosolic signaling pathways, influencing transcriptional events in response to estrogens. Tamoxifen (TAM), widely used in chemotherapy of ERα-positive breast cancer, is considered as an ERα antagonist and GPER agonist. TAM was shown to possess "off-target" cytotoxicity, not related to ER in various tumor types. The present work was designed to study biological effects of TAM on the glucocorticoid (GC)-resistant cell line Jurkat, derived from acute lymphoblastic leukemia of T lineage (T-ALL). We have shown that T-ALL cell lines, in contrast to healthy T cells, express only GPER, but not ERα or ERß. TAM compromised mitochondrial function and reduced the viability and proliferation of Jurkat cells. Additionally, TAM induced autophagy in a GPER-dependent manner. Gene expression profiling revealed the up-regulation of autophagy-related gene ATG5. Interestingly, TAM sensitized Jurkat cells to dexamethasone (DEX) treatment, which may be related to its capacity to cause autophagy. We suggest that TAM-based adjuvant therapy may represent a novel strategy in T-ALL patients handling.

Differential Activity of Voltage- and Ca2+-Dependent Potassium Channels in Leukemic T Cell Lines: Jurkat Cells Represent an Exceptional Case.

Activation of resting T cells relies on sustained Ca2+ influx across the plasma membrane, which in turn depends on the functional expression of potassium channels, whose activity repolarizes the membrane potential. Depending on the T-cells subset, upon activation the expression of Ca2+- or voltage-activated K+ channels, KCa or Kv, is up-regulated. In this study, by means of patch-clamp technique in the whole cell mode, we have studied in detail the characteristics of Kv and KCa currents in resting and activated human T cells, the only well explored human T-leukemic cell line Jurkat, and two additional human leukemic T cell lines, CEM and MOLT-3. Voltage dependence of activation and inactivation of Kv1.3 current were shifted up to by 15 mV to more negative potentials upon a prolonged incubation in the whole cell mode and displayed little difference at a stable state in all cell lines but CEM, where the activation curve was biphasic, with a high and low potential components. In Jurkat, KCa currents were dominated by apamine-sensitive KCa2.2 channels, whereas only KCa3.1 current was detected in healthy T and leukemic CEM and MOLT-3 cells. Despite a high proliferation potential of Jurkat cells, Kv and KCa currents were unexpectedly small, more than 10-fold lesser as compared to activated healthy human T cells, CEM and MOLT-3, which displayed characteristic Kv1.3high:KCa3.1high phenotype. Our results suggest that Jurkat cells represent perhaps a singular case and call for more extensive studies on primary leukemic T cell lines as well as a verification of the therapeutic potential of specific KCa3.1 blockers to combat acute lymphoblastic T leukemias.

Two-pore cation (TPC) channel: not a shorthanded one.

Two-pore cation (TPC) channels form functional dimers in membranes, delineating acidic intracellular compartments such as vacuoles in plants and lysosomes in animals. TPC1 is ubiquitously expressed in thousands of copies per vacuole in terrestrial plants, where it is known as slow vacuolar (SV) channel. An SV channel possesses high permeability for Na+, K+, Mg2+, and Ca2+, but requires high (tens of µM) cytosolic Ca2+ and non-physiological positive voltages for its full activation. Its voltage dependent activation is negatively modulated by physiological concentrations of vacuolar Ca2+, Mg2+and H+. Double control of the SV channel activity from cytosolic and vacuolar sides keeps its open probability at a minimum and precludes a potentially harmful global Ca2+ release. But this raises the question of what such' inactive' channel could be good for? One possibility is that it is involved in ultra-local Ca2+ signalling by generating 'hotspots' - microdomains of extremely high cytosolic Ca2+. Unexpectedly, recent studies have demonstrated the essential role of the TPC1 in the systemic Ca2+ signalling, and the crystal structure of plant TPC1, which became available this year, unravels molecular mechanisms underlying voltage and Ca2+ gating. This review emphasises the significance of these ice-breaking findings and sets a new perspective for the TPC1-based Ca2+ signalling.

Antiprotozoal drug nitazoxanide enhances parasitemia, tissue lesions and mortality caused by Trypanosoma cruzi in murine model.

Chagas' disease is caused by unicellular parasite Trypanosoma cruzi (T. cruzi). It is endemic throughout Latin America, but nowadays has become a global challenge due to tourism and migration. Non-treated infection may result in health-threatening complications and lead to death. Current medications for this infection are nifurtimox (NFT) and benznidazol. Both drugs may cause side effects and are ineffective in the chronic phase. Therefore, new antichagasic compounds are urgently required. Nitazoxanide (NTZ) is a broad spectrum antiparasitic drug, proposed recently as a potential candidate to be added to the list of essential medicines for integrated neglected tropical disease control and elimination. Although the effect of NTZ against T. cruzi epimastigotes in vitro was reported, the corresponding experiments in animal models of T. cruzi infection have never been undertaken. The present work was designed to fill this gap and evaluate the effect of NTZ on experimental murine trypanosomiasis, in comparison with classical antichagasic agent NFT. Highly sensitive to T. cruzi BALB/c mice were infected using Albarrada T. cruzi strain, recently isolated in Mexico. Experimental groups were either left untreated, or otherwise treated with NFT, NTZ (100 and 1000 mg/kg), or with both drugs simultaneously. The severity of the infection was estimated based on criteria such as parasitemia, lesions in target tissues (heart, muscles and lungs) and mortality. Despite the expected protective effect, NTZ drastically aggravates the course of T. cruzi infection. Namely, parasitemia, tissue lesions and mortality caused by T. cruzi infection were significantly higher in NTZ-treated mice groups, even in comparison with untreated infected animals. NTZ by itself no produced mortality o tissue damage, and NFT showed an expected protective effect. Our results indicate that NTZ cannot be considered for Chagas' disease treatment. Moreover, NTZ should be used with caution in patients positive for T. cruzi infection.

Cholinergic Machinery as Relevant Target in Acute Lymphoblastic T Leukemia.

Various types of non-neuronal cells, including tumors, are able to produce acetylcholine (ACh), which acts as an autocrine/paracrine growth factor. T lymphocytes represent a key component of the non-neuronal cholinergic system. T cells-derived ACh is involved in a stimulation of their activation and proliferation, and acts as a regulator of immune response. The aim of the present work was to summarize the data about components of cholinergic machinery in T lymphocytes, with an emphasis on the comparison of healthy and leukemic T cells. Cell lines derived from acute lymphoblastic leukemias of T lineage (T-ALL) were found to produce a considerably higher amount of ACh than healthy T lymphocytes. Additionally, ACh produced by T-ALL is not efficiently hydrolyzed, because acetylcholinesterase (AChE) activity is drastically decreased in these cells. Up-regulation of muscarinic ACh receptors was also demonstrated at expression and functional level, whereas nicotinic ACh receptors seem to play a less important role and not form functional channels in cells derived from T-ALL. We hypothesized that ACh over-produced in T-ALL may act as an autocrine growth factor and play an important role in leukemic clonal expansion through shaping of intracellular Ca(2+) signals. We suggest that cholinergic machinery may be attractive targets for new drugs against T-ALL. Specifically, testing of high affinity antagonists of muscarinic ACh receptors as well as antagomiRs, which interfere with miRNAs involved in the suppression of AChE expression, may be the first choice options.