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Guma M.,University of California at San Diego | Sanchez-Lopez E.,University of California at San Diego | Lodi A.,University of Texas at Austin | Garcia-Carbonell R.,University of California at San Diego | And 4 more authors.
Annals of the Rheumatic Diseases | Year: 2015

Objectives: Little is known about targeting the metabolome in non-cancer conditions. Choline kinase (ChoKα), an essential enzyme for phosphatidylcholine biosynthesis, is required for cell proliferation and has been implicated in cancer invasiveness. Aggressive behaviour of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) led us to evaluate whether this metabolic pathway could play a role in RA FLS function and joint damage. Methods: Choline metabolic profile of FLS cells was determined by 1H magnetic resonance spectroscopy (1 HMRS) under conditions of ChoKα inhibition. FLS function was evaluated using the ChoKα inhibitor MN58b (IC50=4.2 μM). For arthritis experiments, mice were injected with K/BxN sera. MN58b (3 mg/kg) was injected daily intraperitoneal beginning on day 0 or day 4 after serum administration. Results: The enzyme is expressed in synovial tissue and in cultured RA FLS. Tumour necrosis factor (TNF) and platelet-derived growth factor (PDGF) stimulation increased ChoKα expression and levels of phosphocholine in FLS measured by Western Blot (WB) and metabolomic studies of choline-containing compounds in cultured RA FLS extracts respectively, suggesting activation of this pathway in RA synovial environment. A ChoKα inhibitor also suppressed the behaviour of cultured FLS, including cell migration and resistance to apoptosis, which might contribute to cartilage destruction in RA. In a passive K/BxN arthritis model, pharmacologic ChoKα inhibition significantly decreased arthritis in pretreatment protocols as well as in established disease. Conclusions: These data suggest that ChoKα inhibition could be an effective strategy in inflammatory arthritis. It also suggests that targeting the metabolome can be a new treatment strategy in non-cancer conditions. © 2015, BMJ Publishing Group. All rights reserved.

Sanchez-Lopez E.,University of California at San Diego | Zimmerman T.,CSIC - Biological Research Center | Gomez Del Pulgar T.,University Hospital Fundacion Jimenez Diaz | Moyer M.P.,INCELL Corporation | And 2 more authors.
Cell Death and Disease | Year: 2013

Endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that regulates protein synthesis and maturation. Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress has been involved in apoptosis induced by several cytotoxic agents. Choline kinase α (ChoKα), the first enzyme in the Kennedy pathway, is responsible for the generation of phosphorylcholine (PCho) that ultimately renders phosphatidylcholine. ChoKα overexpression and high PCho levels have been detected in several cancer types. Inhibition of ChoKα has demonstrated antiproliferative and antitumor properties; however, the mechanisms underlying these activities remain poorly understood. Here, we demonstrate that ChoKα inhibitors (ChoKIs), MN58b and RSM932A, induce cell death in cancer cells (T47D, MCF7, MDA-MB231, SW620 and H460), through the prolonged activation of ER stress response. Evidence of ChoKIs-induced ER stress includes enhanced production of glucose-regulated protein, 78 kDa (GRP78), protein disulfide isomerase, IRE1α, CHOP, CCAAT/enhancer-binding protein beta (C/EBPβ) and TRB3. Although partial reduction of ChoKα levels by small interfering RNA was not sufficient to increase the production of ER stress proteins, silencing of ChoKα levels also show a decrease in CHOP overproduction induced by ChoKIs, which suggests that ER stress induction is due to a change in ChoKα protein folding after binding to ChoKIs. Silencing of CHOP expression leads to a reduction in C/EBPβ, ATF3 and GRP78 protein levels and abrogates apoptosis in tumor cells after treatment with ChoKIs, suggesting that CHOP maintains ER stress responses and triggers the pro-apoptotic signal. Consistent with the differential effect of ChoKIs in cancer and primary cells previously described, ChoKIs only promoted a transient and moderated ER stress response in the non-tumorogenic cells MCF10A. In conclusion, pharmacological inhibition of ChoKα induces cancer cell death through a mechanism that involves the activation of exaggerated and persistent ER stress supported by CHOP overproduction. © 2013 Macmillan Publishers Limited All rights reserved.

Zimmerman T.,CSIC - Biological Research Center | Moneriz C.,University of Cartagena | Diez A.,Institute Investigacion Hospital 12 Of Octubre | Bautista J.M.,Institute Investigacion Hospital 12 Of Octubre | And 3 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2013

We have investigated the mechanism of action of inhibition of the choline kinase of P. falciparum (p.f.-ChoK) by two inhibitors of the human ChoKα, MN58b and RSM-932A, which have previously been shown to be potent antitumoral agents. The efficacy of these inhibitors against p.f.-ChoK is investigated using enzymatic and in vitro assays. While MN58b may enter the choline/phosphocholine binding site, RSM-932A appears to have an altogether novel mechanism of inhibition and is synergistic with respect to both choline and ATP. A model of inhibition for RSM-932A in which this inhibitor traps p.f.-ChoK in a phosphorylated intermediate state blocking phosphate transfer to choline is presented. Importantly, MN58b and RSM-932A have in vitro inhibitory activity in the low nanomolar range and are equally effective against chloroquine-sensitive and chloroquine-resistant strains. RSM-932A and MN58b significantly reduced parasitemia and induced the accumulation of trophozoites and schizonts, blocking intraerythrocytic development and interfering with parasite egress or invasion, suggesting a delay of the parasite maturation stage. The present data provide two new potent structures for the development of antimalarial compounds and validate p.f.-ChoK as an accessible drug target against the parasite. Copyright © 2013, American Society for Microbiology. All Rights Reserved.

Cristobal I.,University Hospital Fundacion Jimenez Diaz | Manso R.,IIS Fundacion Jimenez Diaz | Rincon R.,IIS Fundacion Jimenez Diaz | Carames C.,University Hospital Fundacion Jimenez Diaz | And 9 more authors.
Molecular Cancer Therapeutics | Year: 2014

Protein phosphatase 2A (PP2A) is a tumor suppressor that regulates many signaling pathways crucial for cell transformation. In fact, decreased activity of PP2A has been reported as a recurrent alteration in many types of cancer. Here, we show that PP2A is frequently inactivated in patients with colorectal cancer, indicating that PP2A represents a potential therapeutic target for this disease.We identified overexpression of the endogenous PP2A inhibitors SET and CIP2A, and downregulation of regulatory PP2A such as PPP2R2A and PPP2R5E, as contributing mechanisms to PP2A inhibition in colorectal cancer.Moreover, we observed that its restoration using FTY720 impairs proliferation and clonogenic potential of colorectal cancer cells, induces caspase-dependent apoptosis, and affects AKT and extracellular signal-regulated kinase-1/2 activation status. Interestingly, treatment with FTY720 showed an additive effect with 5- fluorouracil, SN-38, and oxaliplatin, drugs used in standard chemotherapy in patients with colorectal cancer. These results suggest that PP2A activity is commonly decreased in colorectal cancer cells, and that the use of PP2A activators, such as FTY720, might represent a potential novel therapeutic strategy in colorectal cancer. Mol Cancer Ther; 13(4); 938-47. © 2014 AACR.

Garcia-Olmo D.,Hospital Fundacion Jimenez Diaz | Garcia-Olmo D.,University Hospital Fundacion JimenezDiaz | Guadalajara H.,Hospital Fundacion Jimenez Diaz | Rubio-Perez I.,Hospital Universitario La Paz | And 5 more authors.
World Journal of Gastroenterology | Year: 2015

AIM: To study the results of stem-cell therapy under a Compassionate-use Program for patients with recurrent anal fistulae. METHODS: Under controlled circumstances, and approved by European and Spanish laws, a Compassionate-use Program allows the use of stem-cell therapy for patients with very complex anal fistulae. Candidates had previously undergone multiple surgical interventions that had failed to resolve the fistulae, and presented symptomatic recurrence. The intervention consisted of limited surgery (with closure of the internal opening), followed by local implant of stem cells in the fistulatract wall. Autologous expanded adipose-derived stem cells were the main cell type selected for implant. The first evaluation was performed on the 8th postoperative week; outcome was classified as response or partial response. Evaluation one year after the intervention confirmed if complete healing of the fistula was achieved. RESULTS: Ten patients (8 male) with highly recurrent and complex fistulae were treated (mean age: 49 years, range: 28-76 years). Seven cases were non-Crohn's fistulae, and three were Crohn's-associated fistulae. Previous surgical attempts ranged from 3 to 12. Two patients presented with preoperative incontinence (Wexner scores of 12 and 13 points). After the intervention, six patients showed clinical response on the 8th postoperative week, with a complete cessation of suppuration from the fistula. Three patients presented a partial response, with an evident decrease in suppuration. A year later, six patients (60%) remained healed, with complete reepithelization of the external opening. Postoperative Wexner Scores were 0 in six cases. The two patients with previous incontinence improved their scores from 12 to 8 points and from 13 to 5 points. No adverse reactions or complications related to stem-cell therapy were reported during the study period. CONCLUSION: Stem cells are safe and useful for treating anal fistulae. Healing can be achieved in severe cases, sparing fecal incontinence risk, and improving previous scoring. © 2015 Baishideng Publishing Group Inc. All rights reserved.

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