Skeletal muscle contraction kinetics and AMPK responses are modulated by the adenine nucleotide degrading enzyme AMPD1.

AMP deaminase 1 (AMPD1; AMP → IMP + NH3) deficiency in skeletal muscle results in an inordinate accumulation of AMP during strenuous exercise, with some but not all studies reporting premature fatigue and reduced work capacity. To further explore these inconsistencies, we investigated the extent to which AMPD1 deficiency impacts skeletal muscle contractile function of different muscles and the [AMP]/AMPK responses to different intensities of fatiguing contractions. To reduce AMPD1 protein, we electroporated either an inhibitory AMPD1-specific miRNA encoding plasmid or a control plasmid, into contralateral EDL and SOL muscles of C57BL/6J mice (n = 48 males, 24 females). After 10 days, isolated muscles were assessed for isometric twitch, tetanic, and repeated fatiguing contraction characteristics using one of four (None, LOW, MOD, and HIGH) duty cycles. AMPD1 knockdown (∼35%) had no effect on twitch force or twitch contraction/relaxation kinetics. However, during maximal tetanic contractions, AMPD1 knockdown impaired both time-to-peak tension (TPT) and half-relaxation time (½ RT) in EDL, but not SOL muscle. In addition, AMPD1 knockdown in EDL exaggerated the AMP response to contractions at LOW (+100%) and MOD (+54%) duty cycles, but not at HIGH duty cycle. This accumulation of AMP was accompanied by increased AMPK phosphorylation (Thr-172; LOW +25%, MOD +34%) and downstream substrate phosphorylation (LOW +15%, MOD +17%). These responses to AMPD1 knockdown were not different between males and females. Our findings demonstrate that AMPD1 plays a role in maintaining skeletal muscle contractile function and regulating the energetic responses associated with repeated contractions in a muscle- but not sex-specific manner.NEW & NOTEWORTHY AMP deaminase 1 (AMPD1) deficiency has been associated with premature muscle fatigue and reduced work capacity, but this finding has been inconsistent. Herein, we report that although AMPD1 knockdown in mouse skeletal muscle does not change maximal isometric force, it negatively impacts muscle function by slowing contraction and relaxation kinetics in EDL muscle but not SOL muscle. Furthermore, AMPD1 knockdown differentially affects the [AMP]/AMPK responses to fatiguing contractions in an intensity-dependent manner in EDL muscle.

5'-Phosphonate modified oligoadenylates as potent activators of human RNase L.

The oligoadenylate synthetase-ribonuclease L pathway is a major player in the interferon-induced antiviral defense mechanism of cells. Upon sensing viral dsRNA, 5'-phosphorylated 2',5'-oligoadenylates are synthesized, and subsequently activate latent RNase L. To determine the influence of 5'-phosphate end on the activation of human RNase L, four sets of 5'-phosphonate modified oligoadenylates were prepared on solid-phase. The ability of these 5'-modified oligoadenylates bearing shortened, isosteric and prolonged phosphonate linkages to activate RNase L was explored. We found that isosteric linkages and linkages prolonged by one atom were in general well tolerated by the enzyme with the EC50 values comparable to that of the natural activator. In contrast, linkages shortened by one atom or prolonged by two atoms exhibited decrease in the activity.

3-methyadenine inhibits lipopolysaccharides-induced pulmonary inflammation at the early stage of silicosis via blocking NF-κB signaling pathway.

Occupational exposure to silica dust is related to pulmonary inflammation and silicosis. Lipopolysaccharides (LPSs) could aggravate apoptosis in alveolar macrophages (AMs) of human silicosis through autophagy, yet how the reduction of autophagy attenuated LPS-induced lung injury and the related mechanisms need to be investigated. In the study, we aim to understand the role of 3-methyladenine (3-MA), an inhibitor of autophagy, in LPS-mediated inflammatory responses and fibrosis. We collected AMs from observers/silicosis patients. The results showed that LPS induced NF-κB-related pulmonary inflammation in observers and silicosis patients, as confirmed by an increase in the expression of IL-1ß, IL-6, TNF-α, and p65, which could be inhibited by 3-MA treatment. In mice models, at the early stage (7d) of silicosis, but not the late (28d) stage, blocking autophagy reversed the increased levels of IL-1ß, IL-6, TNF-α, and p65 caused by LPS. Mechanism study revealed that LPS triggered the expression of LC3 II, p62, and cleaved caspase-3 at the early stage exposed to silica, which could be restored by 3-MA, while there was no difference in the expression of LAMP1 either at the early or late stage of silicosis in different groups. Similarly, 3-MA treatment did not prevent fibrosis characterized by destroyed alveoli, collagen deposition, and increased expression of α-SMA and Col-1 induced by LPS at the late stage of silicosis. The results suggested that 3-MA has a role in the protection of lung injury at the early stage of silicosis and provided an experimental basis for preventive strategies of pulmonary inflammation and silicosis.

Structure and mechanism of a Type III CRISPR defence DNA nuclease activated by cyclic oligoadenylate.

The CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes. On binding invading RNA species, Type III CRISPR systems generate cyclic oligoadenylate (cOA) signalling molecules, potentiating a powerful immune response by activating downstream effector proteins, leading to viral clearance, cell dormancy or death. Here we describe the structure and mechanism of a cOA-activated CRISPR defence DNA endonuclease, CRISPR ancillary nuclease 1 (Can1). Can1 has a unique monomeric structure with two CRISPR associated Rossman fold (CARF) domains and two DNA nuclease-like domains. The crystal structure of the enzyme has been captured in the activated state, with a cyclic tetra-adenylate (cA4) molecule bound at the core of the protein. cA4 binding reorganises the structure to license a metal-dependent DNA nuclease activity specific for nicking of supercoiled DNA. DNA nicking by Can1 is predicted to slow down viral replication kinetics by leading to the collapse of DNA replication forks.

Antithrombotic P2Y12 receptor antagonists: recent developments in drug discovery.

The P2Y12 receptor is one of eight known P2Y receptor subtypes, and belongs to the G-protein-coupled receptor (GPCR) family. The P2Y12 receptor is highly expressed on blood platelets and in the brain. Potent, selective, peripherally acting antagonists for the P2Y12 receptor are used clinically as antithrombotic drugs. Several different scaffolds have been identified as P2Y12 receptor antagonists, including irreversibly acting thienotetrahydropyridines (prodrugs), and reversible competitive antagonists, including adenine nucleotide analogs, piperazinyl-glutamate-quinolines, -pyridines, and -pyrimidines, and anthraquinone derivatives. Here, we provide an overview of the different scaffolds that have been developed as P2Y12 receptor antagonists, some of which have become important therapeutics.

The effects of adenine nucleotide perfusion on interstitial adenosine production in rat skeletal muscle.

The purpose of the present study was to utilize the microdialysis technique in rat skeletal muscle to perfuse varying concentrations of AMP, ADP, and ATP into the interstitium to examine the effects that these adenine nucleotides have on the production of adenosine in the interstitial space. Interstitial adenosine production appears to be related to the type (ATP, ADP, or AMP) and concentration (2-60 µmol/L) of the adenine nucleotide perfused. Interstitial adenosine levels increased (P < 0.05) from baseline (0.18 ± 0.02 and 0.22 ± 0.02 µmol/L) to 0.23 ± 0.02 and 0.41 ± 0.05 µmol/L following 5 and 30 µmol/L AMP perfusion, respectively. Similarly, perfusion with 30 µmol/L ADP and 30, 40, and 60 µmol/L ATP resulted in an increase (P < 0.05) in interstitial adenosine concentration from baseline (0.25 ± 0.02, 0.26 ± 0.02, 0.19 ± 0.03, and 0.14 ± 0.02 µmol/L) to 0.30 ± 0.02, 0.32 ± 0.02, 0.36 ± 0.04, and 0.33 ± 0.04 µmol/L, respectively. Interestingly, the most prominent increase in interstitial adenosine production occurred during the perfusion of 60 µmol/L ATP (126% increase from baseline). These data strongly suggest that interstitial ATP may play a more potent role in stimulating interstitial adenosine production as compared with ADP or AMP. In addition, interstitial adenosine production can occur independent of muscle contraction (voluntary or involuntary) or hypoxia when adequate concentrations of adenine nucleotides are available.

Inhibition of breast cancer cell growth by the combination of clofarabine and sulforaphane involves epigenetically mediated CDKN2A upregulation.

Many antineoplastic nucleoside analogue-based combinatorial strategies focused on remodelling aberrant DNA methylation patterns have been developed. The number of studies demonstrate high efficacy of bioactive phytochemicals in support of conventional chemotherapy. Our recent discoveries of the epigenetic effects of clofarabine (2'-deoxyadenosine analogue, antileukaemic drug) and clofarabine-based combinations with dietary bioactive compounds in breast cancer cells led us to look for more DNA methylation targets of these cancer-preventive agents. In the present study, using methylation-sensitive restriction analysis (MSRA) and qPCR, we showed that clofarabine in combination with sulforaphane, a phytochemical from cruciferous vegetables, significantly reactivates DNA methylation-silenced CDKN2A tumour suppressor and inhibits cancer cell growth at a non-invasive breast cancer stage.

Inhibition of mitochondrial UCP1 and UCP3 by purine nucleotides and phosphate.

Mitochondrial membrane uncoupling protein 3 (UCP3) is not only expressed in skeletal muscle and heart, but also in brown adipose tissue (BAT) alongside UCP1, which facilitates a proton leak to support non-shivering thermogenesis. In contrast to UCP1, the transport function and molecular mechanism of UCP3 regulation are poorly investigated, although it is generally agreed upon that UCP3, analogous to UCP1, transports protons, is activated by free fatty acids (FFAs) and is inhibited by purine nucleotides (PNs). Because the presence of two similar uncoupling proteins in BAT is surprising, we hypothesized that UCP1 and UCP3 are differently regulated, which may lead to differences in their functions. By combining atomic force microscopy and electrophysiological measurements of recombinant proteins reconstituted in planar bilayer membranes, we compared the level of protein activity with the bond lifetimes between UCPs and PNs. Our data revealed that, in contrast to UCP1, UCP3 can be fully inhibited by all PNs and IC50 increases with a decrease in PN-phosphorylation. Experiments with mutant proteins demonstrated that the conserved arginines in the PN-binding pocket are involved in the inhibition of UCP1 and UCP3 to different extents. Fatty acids compete with all PNs bound to UCP1, but only with ATP bound to UCP3. We identified phosphate as a novel inhibitor of UCP3 and UCP1, which acts independently of PNs. The differences in molecular mechanisms of the inhibition between the highly homologous transporters UCP1 and UCP3 indicate that UCP3 has adapted to fulfill a different role and possibly another transport function in BAT.

New insights on the regulation of the adenine nucleotide pool of erythrocytes in mouse models.

The observation that induced torpor in non-hibernating mammals could result from an increased AMP concentration in circulation led our investigation to reveal that the added AMP altered oxygen transport of erythrocytes. To further study the effect of AMP in regulation of erythrocyte function and systemic metabolism, we generated mouse models deficient in key erythrocyte enzymes in AMP metabolism. We have previously reported altered erythrocyte adenine nucleotide levels corresponding to altered oxygen saturation in mice deficient in both CD73 and AMPD3. Here we further investigate how these Ampd3-/-/Cd73-/- mice respond to the administered dose of AMP in comparison with the control models of single enzyme deficiency and wild type. We found that Ampd3-/-/Cd73-/- mice are more sensitive to AMP-induced hypometabolism than mice with a single enzyme deficiency, which are more sensitive than wild type. A dose-dependent rightward shift of erythrocyte p50 values in response to increasing amounts of extracellular AMP was observed. We provide further evidence for the direct uptake of AMP by erythrocytes that is insensitive to dipyridamole, a blocker for ENT1. The uptake of AMP by the erythrocytes remained linear at the highest concentration tested, 10mM. We also observed competitive inhibition of AMP uptake by ATP and ADP but not by the other nucleotides and metabolites tested. Importantly, our studies suggest that AMP uptake is associated with an erythrocyte ATP release that is partially sensitive to inhibition by TRO19622 and Ca++ ion. Taken together, our study suggests a novel mechanism by which erythrocytes recycle and maintain their adenine nucleotide pool through AMP uptake and ATP release.

Phase I Study of Clofarabine and 2-Gy Total Body Irradiation as a Nonmyeloablative Preparative Regimen for Hematopoietic Stem Cell Transplantation in Pediatric Patients with Hematologic Malignancies: A Therapeutic Advances in Childhood Leukemia Consortium Study.

Clofarabine is a purine nucleoside analog with immunosuppressive and antileukemic activity and its inclusion in reduced-intensity regimens could potentially improve outcomes. We performed a prospective phase I study of clofarabine combined with 2 Gy total body irradiation (TBI) as a nonmyeloablative preparative regimen for allogeneic stem cell transplantation in pediatric patients who were considered at high risk of mortality from standard myeloablative regimens. The main goal of the study was to delineate the maximum feasible dose (MFD) of clofarabine in combination with 2 Gy TBI. Eighteen patients, 1 to 21 years of age and in complete remission, were enrolled in 2 strata (matched related donor and unrelated donor) and evaluated for day100 dose-limiting events (DLE) (nonengraftment, nonrelapse mortality [NRM], and severe renal insufficiency) after receiving clofarabine at the starting dose level of 40 mg/m2. All 6 patients (3 in each stratum) engrafted with no day 100 DLE seen in the first cohort. The dose was increased to 52 mg/m2 in the next and an expanded cohort (total of 12 patients) and no DLE were observed at day 100 and at the 1-year study endpoint. The regimen was well tolerated with transient transaminitis and gastrointestinal and skin reactions as the common reversible toxicities observed with clofarabine. The dose of 52 mg/m2 of clofarabine was deemed the MFD. Disease relapse led to mortality in 6 (33%) patients during follow-up with 1-year event-free survival and overall survival of 60% (95% confidence interval [CI], 34 to 79) and 71% (95% CI, 44 to 87), respectively. This regimen leads to successful engraftment using both related and unrelated donors with exceptionally low rates of NRM.

SAMHD1 enhances nucleoside-analogue efficacy against HIV-1 in myeloid cells.

SAMHD1 is an intracellular enzyme that specifically degrades deoxynucleoside triphosphates into component nucleoside and inorganic triphosphate. In myeloid-derived dendritic cells and macrophages as well as resting T-cells, SAMHD1 blocks HIV-1 infection through this dNTP triphosphohydrolase activity by reducing the cellular dNTP pool to a level that cannot support productive reverse transcription. We now show that, in addition to this direct effect on virus replication, manipulating cellular SAMHD1 activity can significantly enhance or decrease the anti-HIV-1 efficacy of nucleotide analogue reverse transcription inhibitors presumably as a result of modulating dNTP pools that compete for recruitment by viral polymerases. Further, a variety of other nucleotide-based analogues, not normally considered antiretrovirals, such as the anti-herpes drugs Aciclovir and Ganciclovir and the anti-cancer drug Clofarabine are now revealed as potent anti-HIV-1 agents, under conditions of low dNTPs. This in turn suggests novel uses for nucleotide analogues to inhibit HIV-1 in differentiated cells low in dNTPs.

Prexasertib, a Chk1/Chk2 inhibitor, increases the effectiveness of conventional therapy in B-/T- cell progenitor acute lymphoblastic leukemia.

During the last few years many Checkpoint kinase 1/2 (Chk1/Chk2) inhibitors have been developed for the treatment of different type of cancers. In this study we evaluated the efficacy of the Chk 1/2 inhibitor prexasertib mesylate monohydrate in B-/T- cell progenitor acute lymphoblastic leukemia (ALL) as single agent and in combination with other drugs. The prexasertib reduced the cell viability in a dose and time dependent manner in all the treated cell lines. The cytotoxic activity was confirmed by the increment of apoptotic cells (Annexin V/Propidium Iodide staining), by the increase of γH2A.X protein expression and by the activation of different apoptotic markers (Parp-1 and pro-Caspase3 cleavage). Furthermore, the inhibition of Chk1 changed the cell cycle profile. In order to evaluate the chemo-sensitizer activity of the compound, different cell lines were treated for 24 and 48 hours with prexasertib in combination with other drugs (imatinib, dasatinib and clofarabine). The results from cell line models were strengthened in primary leukemic blasts isolated from peripheral blood of adult acute lymphoblastic leukemia patients. In this study we highlighted the mechanism of action and the effectiveness of prexasertib as single agent or in combination with other conventional drugs like imatinib, dasatinib and clofarabine in the treatment of B-/T-ALL.

Pharmacological hypothermia: a potential for future stroke therapy?

Mild physical hypothermia after stroke has been associated with positive outcomes. Despite the well-studied beneficial effects of hypothermia in the treatment of stroke, lack of precise temperature control, intolerance for the patient, and immunosuppression are some of the reasons which limit its clinical translation. Pharmacologically induced hypothermia has been explored as a possible treatment option following stroke in animal models. Currently, there are eight classes of pharmacological agents/agonists with hypothermic effects affecting a multitude of systems including cannabinoid, opioid, transient receptor potential vanilloid 1 (TRPV1), neurotensin, thyroxine derivatives, dopamine, gas, and adenosine derivatives. Interestingly, drugs in the TRPV1, neurotensin, and thyroxine families have been shown to have effects in thermoregulatory control in decreasing the compensatory hypothermic response during cooling. This review will briefly present drugs in the eight classes by summarizing their proposed mechanisms of action as well as side effects. Reported thermoregulatory effects of the drugs will also be presented. This review offers the opinion that these agents may be useful in combination therapies with physical hypothermia to achieve faster and more stable temperature control in hypothermia.

The shifting landscape of KATP channelopathies and the need for 'sharper' therapeutics.

ATP-sensitive potassium (KATP) channels play fundamental roles in the regulation of endocrine, neural and cardiovascular function. Small-molecule inhibitors (e.g., sulfonylurea drugs) or activators (e.g., diazoxide) acting on SUR1 or SUR2 have been used clinically for decades to manage the inappropriate secretion of insulin in patients with Type 2 diabetes, hyperinsulinism and intractable hypertension. More recently, the discovery of rare disease-causing mutations in KATP channel-encoding genes has highlighted the need for new therapeutics for the treatment of certain forms of neonatal diabetes mellitus, congenital hyperinsulinism and Cantu syndrome. Here, we provide a high-level overview of the pathophysiology of these diseases and discuss the development of a flexible high-throughput screening platform to enable the development of new classes of KATP channel modulators.

Adenine nucleotide-dependent and redox-independent control of mitochondrial malate dehydrogenase activity in Arabidopsis thaliana.

Mitochondrial metabolism is important for sustaining cellular growth and maintenance; however, the regulatory mechanisms underlying individual processes in plant mitochondria remain largely uncharacterized. Previous redox-proteomics studies have suggested that mitochondrial malate dehydrogenase (mMDH), a key enzyme in the tricarboxylic acid (TCA) cycle and redox shuttling, is under thiol-based redox regulation as a target candidate of thioredoxin (Trx). In addition, the adenine nucleotide status may be another factor controlling mitochondrial metabolism, as respiratory ATP production in mitochondria is believed to be influenced by several environmental stimuli. Using biochemical and reverse-genetic approaches, we addressed the redox- and adenine nucleotide-dependent regulation of mMDH in Arabidopsis thaliana. Recombinant mMDH protein formed intramolecular disulfide bonds under oxidative conditions, but these bonds did not have a considerable effect on mMDH activity. Mitochondria-localized o-type Trx (Trx-o) did not facilitate re-reduction of oxidized mMDH. Determination of the in vivo redox state revealed that mMDH was stably present in the reduced form even in Trx-o-deficient plants. Accordingly, we concluded that mMDH is not in the class of redox-regulated enzymes. By contrast, mMDH activity was lowered by adenine nucleotides (AMP, ADP, and ATP). Each adenine nucleotide suppressed mMDH activity with different potencies and ATP exerted the largest inhibitory effect with a significantly lower K(I). Correspondingly, mMDH activity was inhibited by the increase in ATP/ADP ratio within the physiological range. These results suggest that mMDH activity is finely controlled in response to variations in mitochondrial adenine nucleotide balance.

Dual anti-HIV mechanism of clofarabine.

BACKGROUND: HIV-1 replication kinetics inherently depends on the availability of cellular dNTPs for viral DNA synthesis. In activated CD4(+) T cells and other rapidly dividing cells, the concentrations of dNTPs are high and HIV-1 reverse transcription occurs in an efficient manner. In contrast, nondividing cells such as macrophages have lower dNTP pools, which restricts efficient reverse transcription. Clofarabine is an FDA approved ribonucleotide reductase inhibitor, which has shown potent antiretroviral activity in transformed cell lines. Here, we explore the potency, toxicity and mechanism of action of clofarabine in the human primary HIV-1 target cells: activated CD4(+) T cells and macrophages. RESULTS: Clofarabine is a potent HIV-1 inhibitor in both activated CD4(+) T cells and macrophages. Due to its minimal toxicity in macrophages, clofarabine displays a selectivity index over 300 in this nondividing cell type. The anti-HIV-1 activity of clofarabine correlated with a significant decrease in both cellular dNTP levels and viral DNA synthesis. Additionally, we observed that clofarabine triphosphate was directly incorporated into DNA by HIV-1 reverse transcriptase and blocked processive DNA synthesis, particularly at the low dNTP levels found in macrophages. CONCLUSIONS: Taken together, these data provide strong mechanistic evidence that clofarabine is a dual action inhibitor of HIV-1 replication that both limits dNTP substrates for viral DNA synthesis and directly inhibits the DNA polymerase activity of HIV-1 reverse transcriptase.

The combination effects of bendamustine with antimetabolites against childhood acute lymphoblastic leukemia cells.

Bendamustine combined with other drugs is clinically efficacious for some adult lymphoid malignancies, but to date there are no reports of the use of such combinatorial approaches in pediatric patients. We investigated the in vitro activity of bendamustine combined with other antimetabolite drugs on B cell precursor acute lymphoblastic leukemia (BCP-ALL) cell lines established from pediatric patients with refractory or relapsed ALL. We also developed a mathematically drown improved isobologram method to assess the data objectively. Three BCP-ALL cell lines; YCUB-2, YCUB-5, and YCUB-6, were simultaneously exposed to various concentrations of bendamustine and cladribine, cytarabine, fludarabine, or clofarabine. Cell growth inhibition was determined using the WST-8 assay. Combinatorial effects were estimated using our improved isobologram method with IC80 (drug concentration corresponding to 80 % of maximum inhibition). Bendamustine alone inhibited ALL cell growth with mean IC80 values of 11.30-18.90 µg/ml. Combinations of bendamustine with other drugs produced the following effects: (1) cladribine; synergistic-to-additive on all cell lines; (2) cytarabine; synergistic-to-additive on YCUB-5 and YCUB-6, and synergistic-to-antagonistic on YCUB-2; (3) fludarabine; additive-to-antagonistic on YCUB-5, and synergistic-to-antagonistic on YCUB-2 and YCUB-6; (4) clofarabine; additive-to-antagonistic on all cell lines. Flow cytometric analysis also showed the combination effects of bendamustine and cladribine. Bendamustine/cladribine or bendamustine/cytarabine may thus represent a promising combination for salvage treatment in childhood ALL.

Purinergic signaling during macrophage differentiation results in M2 alternative activated macrophages.

Macrophages represent a highly heterogenic cell population of the innate immune system, with important roles in the initiation and resolution of the inflammatory response. Purinergic signaling regulates both M1 and M2 macrophage function at different levels by controlling the secretion of cytokines, phagocytosis, and the production of reactive oxygen species. We found that extracellular nucleotides arrest macrophage differentiation from bone marrow precursors via adenosine and P2 receptors. This results in a mature macrophage with increased expression of M2, but not M1, genes. Similar to adenosine and ATP, macrophage growth arrested with LPS treatment resulted in an increase of the M2-related marker Ym1. Recombinant Ym1 was able to affect macrophage proliferation and could, potentially, be involved in the arrest of macrophage growth during hematopoiesis.

Sulforaphane Alone and in Combination with Clofarabine Epigenetically Regulates the Expression of DNA Methylation-Silenced Tumour Suppressor Genes in Human Breast Cancer Cells.

BACKGROUND/AIM: Sporadic breast cancer is frequently associated with aberrant DNA methylation patterns that are reversible and responsive to environmental factors, including diet. In the present study, we investigated the effects of sulforaphane (SFN), a phytochemical from cruciferous vegetables, on the methylation and expression of PTEN and RARbeta2 tumour suppressor genes as well as on the expression of regulators of DNA methylation reaction, DNMT1 , p53 , and p21 , in MCF-7 and MDA-MB-231 human breast cancer cells with different invasive potential. We also evaluate the role of SFN epigenetic effects in support of therapy with clofarabine (ClF) that was recently shown to modulate the epigenome as well. METHODS: Promoter methylation and gene expression were estimated using methylation-sensitive restriction analysis and real-time PCR, respectively. RESULTS: In both MCF-7 and MDA-MB-231 cells, SFN at IC 50 (22 and 46 µ M , respectively) and a physiologically relevant 10 µ M concentration lead to hypomethylation of PTEN and RARbeta2 promoters with concomitant gene upregulation. The combination of SFN and ClF enhances these effects, resulting in an increase in cell growth arrest and apoptosis at a non-invasive breast cancer stage. CONCLUSIONS: Our findings provide evidence that SFN activates DNA methylation-silenced tumour suppressor genes in breast cancer cells and may contribute to SFN-mediated support of therapy with an anti-cancer drug, ClF, increasing its applications in solid tumours.