Targeting citrate as a novel therapeutic strategy in cancer treatment.

An important feature shared by many cancer cells is drastically altered metabolism that is critical for rapid growth and proliferation. The distinctly reprogrammed metabolism in cancer cells makes it possible to manipulate the levels of metabolites for cancer treatment. Citrate is a key metabolite that bridges many important metabolic pathways. Recent studies indicate that manipulating the level of citrate can impact the behaviors of both cancer and immune cells, resulting in induction of cancer cell apoptosis, boosting immune responses, and enhanced cancer immunotherapy. In this review, we discuss the recent developments in this emerging area of targeting citrate in cancer treatment. Specifically, we summarize the molecular basis of altered citrate metabolism in both tumors and immune cells, explore the seemingly conflicted growth promoting and growth inhibiting roles of citrate in various tumors, discuss the use of citrate in the clinic as a novel biomarker for cancer progression and outcomes, and highlight the new development of combining citrate with other therapeutic strategies in cancer therapy. An improved understanding of complex roles of citrate in the suppressive tumor microenvironment should open new avenues for cancer therapy.

Impact of specific inhibitors on metallo-ß-carbapenemases detected in Escherichia coli and Klebsiella pneumoniae isolates.

Carbapenems are widely regarded as the drugs of choice for the treatment of severe infections caused by extended-spectrum beta lactamases producing Enterobacteriaceae. The emergence of carbapenem-resistant organisms is worrisome due to the limited treatment options. Detection of carbapenemase-producing bacteria is critical for the choice of appropriate therapy. However, Inhibition of carbapenemases is an alternative approach to combat resistance to carbapenms. In this study, Escherichia coli and Klebsiella pneumoniae carbapenem resistant isolates were recovered from 300 clinical isolates. They were subjected phenotypically for detection of class B metallo-carbapenemase (MBL) producers (by carbapenem disks with or without EDTA), and were subjected for confirmation genotypically by PCR. In addition, the synergistic activities of MBL-inhibitors in combination with carbapenems were elucidated. Two E. coli and 15 K. pneumoniae isolates were carbapenem resistant. The genes encoding blaNDM-1 carbapenemase were detected in 16/17 isolates solely, or collaboratively with either blaVIM, or blaIMP or both in all carbapenem resistant isolates, by PCR method. The VIM-carbapenemase was encoded by one isolate. In pre-clinical trials for development of MBL-specific inhibitors, Sub-inhibitory concentrations of citric acid, malic acid, ascorbic acid and ciprofloxacin in combination with imipenem or meropenem exerted synergistic activities against metallo-carbapenemases. Their activities are probably attributed to the chelation of zinc ions in the active site of carbapenemase. Conclusively, these promising combined therapies might represent a new strategy for combating such serious infections caused by metallo-B-carbapenemase producers of K. pneumoniae and E. coli isolates.

Effect of the association between citric acid and EDTA on root surface etching.

OBJECTIVE: This study aims to compare the clot stabilization on root surfaces conditioned with citric acid and ethylenediamine-tetraacetic acid (EDTA). MATERIALS AND METHODS: Scaled root samples (n = 100) were set in fve groups: group I-control group (saline solution); group II (24% EDTA); group III (25% citric acid); group IV (EDTA + citric acid); group V (citric acid + EDTA). Fifty samples were assessed using the root surface modifcation index (RSMI). The other 50 received a blood drop after conditioning. Clot formation was assessed using blood elements adhesion index (BEAI). A blind examiner evaluated photomicrographs. Statistical analysis considered p < 0.05. RESULTS: Groups-III and G-V attained the best results for RSMI and BEAI in comparison to control. The worst results for clot stabilization were seen in group-II. EDTA employment before citric acid (group-IV) reduced clot formation in comparison to citric acid use alone (group-III). CONCLUSION: Root conditioning with citric acid alone and before EDTA had the best results for smear layer removal and clot stabilization. EDTA inhibited clot stabilization on root surface and must have a residual activity once it has diminished clot adhesion to root even after citric acid conditioning. Thus, EDTA can be used to neutralize citric acid effects on periodontal cells without affecting clot stabilization. Clinical signifcance: To demonstrate that citric acid use on root surfaces previously affected by periodontal disease may favor clot stabilization and may have a benefcial effect on surgical outcomes. Also, EDTA can be used to neutralize citric acid effects on periodontal cells.

Pathogenic consequences in semen quality of an autoimmune response against the prostate gland: from animal models to human disease.

We have recently proposed an autoimmune etiology in approximately 35% of chronic nonbacterial prostatitis patients, the most frequent form of prostatitis observed, because they exhibit IFN-gamma-secreting lymphocytes specific to prostate Ags. Interestingly, this particular group of patients, but not the rest of chronic nonbacterial prostatitis patients, also presented striking abnormalities in their semen quality. In this work, we use an experimental animal model of autoimmune prostatitis on Wistar rats developed in our laboratory to investigate when, where, and how sperm cells from autoimmune prostatitis individuals are being damaged. As in patients, a marked reduction in sperm concentration, almost null sperm motility and viability, and an increased percentage of apoptotic spermatozoa were detected in samples from animals with the disease. Prostate-specific autoantibodies as well as elevated levels of NO, TNF-alpha, and IFN-gamma were also detected in their seminal plasma. In contrast, epididymal spermatozoa remain intact, indicating that sperm damage occurs at the moment of joining of prostate secretion to sperm cells during ejaculation. These results were further supported by experiments in which mixture of normal sperm cells with autoimmune seminal plasma were performed. We hypothesize that sperm damage in experimental autoimmune prostatitis can be the consequence of an inflammatory milieu, originally produced by an autoimmune response in the prostate; a diminished prostate functionality, evidenced by reduced levels of citric acid in semen or by both mechanisms simultaneously. Once more, we suggest that autoimmunity to prostate may have consequences on fertility.

Killing of Enterococcus faecalis by MTAD and chlorhexidine digluconate with or without cetrimide in the presence or absence of dentine powder or BSA.

The antibacterial efficacy of irrigating solutions and local disinfectants used in endodontics appears poorer in vivo than in vitro. One explanation may be inactivation by compounds present in the root canal. MTAD (a mixture of tetracycline isomer, acid, and detergent) is a new root canal irrigation solution with antibacterial activity. The aim of this study was to investigate the antibacterial activity of MTAD and chlorhexidine towards two strains of Enterococcus faecalis and the inhibitory effects of dentine and bovine serum albumin on the antibacterial activity. Survival of bacteria exposed to the medicaments in the presence or absence of inhibitors was monitored in an in vitro model. Full concentration (100%) MTAD and 0.2% chlorhexidine rapidly killed both strains. Combining chlorhexidine with cetrimide further reduced the time required for killing. The presence of dentine or BSA caused a marked delay in killing by both medicaments. The two E. faecalis strains tested showed minor differences in their susceptibility to the disinfectants.

Agaric acid induces mitochondrial permeability transition through its interaction with the adenine nucleotide translocase. Its dependence on membrane fluidity.

The effect of agaric acid as inducer of mitochondrial permeability transition was studied. It was found that: (i) agaric acid (AA) promoted efflux of accumulated Ca2+, collapse of transmembrane potential, and mitochondrial swelling; (ii) these effects depend on membrane fluidity; (iii) ADP inhibited the effect of AA on Ca2+ efflux, and (iv) AA blocked binding of the sulfhydryl reagent, eosin-5-maleimide, to the adenine nucleotide translocase. It is proposed that AA induces pore opening through binding of the citrate moiety to the ADP/ATP carrier; this interaction must be stabilized by insertion of the alkyl chain in the lipid milieu of the membrane.

Effect of a novel NK1 receptor selective antagonist (NKP608) on citric acid induced cough and airway obstruction.

The effects of an orally administered novel and selective NK1 antagonist, NKP608, on cough and airway obstruction, induced by citric acid in guinea pigs, were investigated. Guinea pigs were pre-treated with 0.03, 0.3 and 1 mg kg(-1) of NKP608, the NK2 antagonist, SR48968 or both 2 h prior to challenge with citric acid (0.6 M) for a 10 min period. Guinea pigs pre-treated with 0.03, 0.3 and 1mgkg(-1) of NKP608 exhibited a significant reduction of 77, 74 and 79%, respectively, in the numbers of cough compared to vehicle pre-treated animals (P<0.05). SR48968, 10 mg kg(-1), alone did not significantly affect the citric acid-induced cough but when co-administered with 1 mg kg(-1) of NKP608, there was a significant 90% reduction in cough. NKP608 did not significantly reduce the citric acid-induced increase in Penh at any of the doses used. SR48968 significantly reduced the citric acid induced airway obstruction by about 50%. However, when SR48968 was co-administered with NKP608, there was a greater (73%) decrease in the airway obstruction compared with SR48968 alone. These data show that NKP608, a selective NK1 receptor antagonist, is a potent inhibitor of citric acid induced cough in guinea pigs and may therefore have value in the therapy of clinical cough.

Effect of mineral supplements to citric acid on enamel erosion.

The aim of this study was to evaluate the effect of mineral supplements to citric acid (1%; pH 2.21) on enamel erosion under controlled conditions in an artificial mouth. From each of 156 bovine incisors one polished enamel sample was prepared. The samples were divided among 13 experimental groups (n=12). In group 1 citric acid only was used (control). In groups 2-10 either calcium, phosphate or fluoride in various low concentrations was admixed to the citric acid. In groups 11-13 the citric acid was supplemented with a mixture of calcium, phosphate and fluoride. For demineralisation the specimens were rinsed with the respective solution for 1 min, immediately followed by a remineralisation period with artificial saliva (1 min). The specimens were cycled through this alternating procedure five times followed by rinsing for 8 h with artificial saliva. The de- and remineralisation cycle was repeated three times for each specimen interrupted by the 8 h-remineralisation periods. Before and after the experiments, the specimens were examined using microhardness testing (Knoop hardness) and laser profilometry. Hardness loss and enamel dissolution was significantly higher for the controls as compared to the remaining groups. Significantly lowest hardness loss for all groups was recorded for group 12 with admixture of calcium, phosphate and fluoride to citric acid. The significantly highest enamel loss was recorded for the controls compared to all other samples. Groups 3 and 4 revealed significantly lower and higher tissue loss compared to the remaining groups (2-13), respectively. The other groups did not differ significantly from each other. Modification of citric acid with calcium, phosphate and fluoride exerts a significant protective potential with respect to dental erosion. However, with the low concentrations applied enamel dissolution could not be completely prevented.

Accessibility of cysteine residues in a cytoplasmic loop of CitS of Klebsiella pneumoniae is controlled by the catalytic state of the transporter.

The citrate transporter CitS of Klebsiella pneumoniae is a secondary transporter that transports citrate in symport with two sodium ions and one proton. Treatment of CitS with the alkylating agent N-ethylmaleimide resulted in a complete loss of transport activity. Treatment of mutant proteins in which the five endogenous cysteine residues were mutated into serines in different combinations revealed that two cysteine residues located in the C-terminal cytoplasmic loop, Cys-398 and Cys-414, were responsible for the inactivation. Labeling with the membrane impermeable methanethiosulfonate derivatives MTSET and MTSES in right-side-out membrane vesicles showed that the cytoplasmic loop was accessible from the periplasmic side of the membrane. The membrane impermeable but more bulky maleimide AmdiS did not inactivate the transporter in right-side-out membrane vesicles. Inactivation by N-ethylmaleimide, MTSES, and MTSET was prevented by the presence of the co-ion Na(+). Protection was obtained upon binding 2 Na(+), which equals the transport stoichiometry. In the absence of Na(+), the substrate citrate had no effect on the inactivation by permeable or impermeable thiol reagents. In contrast, when subsaturating concentrations of Na(+) were present, citrate significantly reduced inactivation suggesting ordered binding of the substrate and co-ion; citrate is bound after Na(+). In the presence of the proton motive force, the reactivity of the Cys residues was increased significantly for the membrane permeable N-ethylmaleimide, while no difference was observed for the membrane impermeable thiol reagents. The results are discussed in the context of a model for the opening and closing of the translocation pore during turnover of the transporter.

The effect of Phyllanthus niruri on urinary inhibitors of calcium oxalate crystallization and other factors associated with renal stone formation.

OBJECTIVE: To evaluate the effect of an aqueous extract of Phyllanthus niruri (Pn), a plant used in folk medicine to treat lithiasis, on the urinary excretion of endogenous inhibitors of lithogenesis, citrate, magnesium and glycosaminoglycans (GAGs). MATERIALS AND METHODS: The effect of chronic (42 days) administration of Pn (1.25 mg/mL/day, orally) was evaluated in a rat model of urolithiasis induced by the introduction of a calcium oxalate (CaOx) seed into the bladder of adult male Wistar rats. The animals were divided into four groups: a sham control (16 rats); a control+Pn (six); CaOx+water instead of Pn (14); and CaOx+Pn (22). Plasma and urine were collected after 42 days of treatment for biochemical analysis and the determination of urinary excretion of citrate, magnesium and GAGs. The animals were then killed and the calculi analysed. RESULTS: The creatinine clearance or urinary and plasma concentrations of Na+, K+, Ca2+, oxalate, phosphate and uric acid were unaffected by Pn or the induction of lithiasis. Treatment with Pn strongly inhibited the growth of the matrix calculus and reduced the number of stone satellites compared with the group receiving water. The calculi were eliminated or dissolved in some treated animals (three of 22). The urinary excretion of citrate and magnesium was unaffected by Pn treatment. However, the mean (sd) urinary concentration of GAGs was significantly lower in rats treated with CaOx+Pn, at 5.64 (0.86) mg/g creatinine, than when treated with CaOx + water, at 11.78 (2.21) mg/g creatinine. In contrast, the content of GAGs in the calculi was higher in the CaOx + Pn rats, at 48.0 (10.4) g/g calculus, than in the CaOx + water group, at 16.6 (9.6) g/g calculus. CONCLUSION: These results show that Pn has an inhibitory effect on crystal growth, which is independent of changes in the urinary excretion of citrate and Mg, but might be related to the higher incorporation of GAGs into the calculi.

Citrate and succinate transport in proximal tubule cells.

Urinary citrate, which inhibits calcium nephrolithiasis, is determined by proximal reabsorption via an apical dicarboxylate transporter. Citrate is predominantly trivalent at physiological pH, but citrate(-2) is transported at the apical membrane. We now demonstrate that low-Ca solutions induce transport of citrate(-2) and succinate in opossum kidney cells. With 1.2 mM extracellular Ca, citrate uptake was pH insensitive and not competed by succinate(-2). In contrast, with low extracellular Ca, citrate uptake increased twofold, was inhibited by succinate (and other dicarboxylates), was stimulated by lowering extracellular pH (consistent with citrate(-2) transport), and increased further by lowering extracellular Mg. The effect of Ca was incrementally concentration dependent, between 0 and 1.2 mM. The effect of Ca was not simply complexation with citrate because succinate (which is complexed significantly less) was affected by Ca similarly. Incubation of cells for 48 h in a low-pH media increased citrate transport (studied at control pH) more than twofold, suggesting induction of transporters.

Sour transduction involves activation of NPPB-sensitive conductance in mouse taste cells.

We examined the sour taste transduction mechanism in the mouse by applying whole cell patch-clamp technique to nondissociated taste cells from the fungiform papillae. Localized stimulation with 0.5 M NaCl and 25 mM citric acid (pH 3.0) of the apical membrane enabled us to obtain responses from single taste cells under a quasi-natural condition. Of 28 taste cells examined, 11 cells (39%) responded to 0. 5 M NaCl alone and 2 cells (7%) responded to 25 mM citric acid alone, indicating the presence of salty- and sour-specific taste cells. Ten cells (36%) responded to both NaCl and citric acid and 5 cells (18%) responded to neither salt nor citric acid. Amiloride reversibly suppressed NaCl-induced responses in mouse taste cells but not citric acid-induced responses. On the other hand, a Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), reversibly suppressed all the citric-acid-induced responses. Most of the NaCl-induced current responses displayed an inwardly rectifying property, whereas all the citric-acid-induced responses displayed an outwardly rectifying property. The reversal potential for NPPB-sensitive component in citric-acid-induced current responses was -2 +/- 7 mV (mean +/- SE, n = 4), which was close to the equilibrium potential of Cl- (ECl), whereas the reversal potential for NPPB-insensitive component was 34 +/- 8 mV (n = 4). The reversal potential of citric-acid-induced current responses (19 +/- 8 mV, n = 4) was mostly present at the middle point between reversal potentials of NPPB-sensitive and -insensitive current components. In some taste cells, an inorganic cation channel blocker, Cd2+, suppressed citric-acid-induced responses, but an inorganic stretch-activated cation channel blocker, Gd3+, did not affect these responses. These results suggest that salt- and acid-induced responses were mediated by differential transduction mechanisms in mouse taste cells and that NPPB-sensitive Cl- channels play a more important role to sour taste transduction rather than amiloride-sensitive Na+ channels. However, the fact that the reversal potentials of citric-acid-induced responses had more positive than ECl suggests that Ca2+ or H+ permeable and poorly selective cation channels, which should be amiloride insensitive, may be activated by citric acid.

In vitro modulation of proliferation and melanization of melanoma cells by citrate.

B16/F10 murine melanoma cells were grown for 24 and 36 h in Dulbecco's modified Eagle medium in presence of 10-20 mM trisodium citrate. The intracellular melanin concentration and the melanin secreted in the extracellular medium was estimated. It is observed that 20 mM citrate stimulates extracellular melanin secretion in B16/F10 melanoma cells by 200% at 36 h treatment. The intracellular melanin content increased by 90%. This stimulatory effect of citrate was totally abolished when these cells were grown in presence of 1 mM phenyl thiourea, a specific inhibitor of tyrosinase activity. Citrate (0.1-5 mM) had no effect on dopa oxidase activity either at pH 5.0 or at pH 6.8. There was no increase in the tyrosinase specific activity in presence of citrate. The increased melanin synthesis was shown to be due to stimulation of cellular tyrosine hydroxylase activity by citrate. It has been suggested that enhanced melanin synthesis results in an increased production of metabolites that are toxic to the growth of melanoma cells. We have studied the effect of citrate on cellular proliferation. Following 24 and 36 h treatment with citrate, the cells exhibited a dose-dependent decrease in proliferation. In presence of 20 mM citrate the cell number was only up to 50% of the control cultures after 36 h of incubation. The growth retardation was not due to cytotoxicity. Citrate, a natural metabolite, is a unique molecule which may be involved in the regulation of melanin biosynthetic pathway, since it enhances melanogenesis by increasing the hydroxylase activity of tyrosinase which is the regulatory enzyme of this pathway. These observations add further support to the critical role of intramelanosomal pH in regulation of melanogenesis.

An excess of topical calcium and magnesium reverses the therapeutic effect of citrate on the development of corneal ulcers after alkali injury.

Our purpose was to determine whether chelation of Ca2+ and Mg2+ is the mechanism by which sodium citrate inhibits corneal ulceration in the alkali-injured rabbit eye. The right eyes of 60 albino rabbits (2-2.5 kg) were alkali-injured by filling a 12-mm-diameter plastic well placed on the corneal surface with 0.4 ml of 1 N NaOH. After 35 s the alkali was aspirated, and the well was rinsed with physiological saline. Animals were randomly distributed to three treatment groups of equal size. Two drops of the following topical medications were administered on the hour (14 times per day) for 35 days: physiological saline, 10% citrate in saline, and 346 mM Ca2+, 346 mM Mg2+, and 10% citrate in saline. During the experiment, significantly fewer ulcerations occurred in the citrate-treated eyes (five of 20, 25%) than in the saline-treated eyes (13 of 20, 65%) or in the calcium-magnesium-citrate-treated eyes (15 of 20, 75%). When ulcerations did develop in the citrate group, they occurred significantly later and were less severe than those in the saline and calcium-magnesium-citrate groups. There was a significant increase in the number of eyes with signs of band keratopathy and translucent areas in the calcium-magnesium-citrate group when compared with the other two groups. As in previous studies, sodium citrate significantly inhibited the development of corneal ulcers after alkali injury. The annullment of the favorable effect of citrate on ulceration in the alkali-injured eye by the addition of calcium and magnesium shows that the mechanism of action of citrate is the chelation of these divalent cations.