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.

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.

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.

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.

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.