Teresa Gómez del Pulgar

Prevention of Alzheimer's Disease Pathology by Cannabinoids: Neuroprotection Mediated by Blockade of Microglial Activation

Alzheimers disease (AD) is characterized by enhanced β-amyloid peptide (βA) deposition along with glial activation in senile plaques, selective neuronal loss, and cognitive deficits. Cannabinoids are neuroprotective agents against excitotoxicity in vitro and acute brain damage in vivo. This background prompted us to study the localization, expression, and function of cannabinoid receptors in AD and the possible protective role of cannabinoids after βA treatment, both in vivo and in vitro. Here, we show that senile plaques in AD patients express cannabinoid receptors CB1 and CB2, together with markers of microglial activation, and that CB1-positive neurons, present in high numbers in control cases, are greatly reduced in areas of microglial activation. In pharmacological experiments, we found that G-protein coupling and CB1 receptor protein expression are markedly decreased in AD brains. Additionally, in AD brains, protein nitration is increased, and, more specifically, CB1 and CB2 proteins show enhanced nitration. Intracerebroventricular administration of the synthetic cannabinoid WIN55,212-2 to rats prevent βA-induced microglial activation, cognitive impairment, and loss of neuronal markers. Cannabinoids (HU-210, WIN55,212-2, and JWH-133) block βA-induced activation of cultured microglial cells, as judged by mitochondrial activity, cell morphology, and tumor necrosis factor-α release; these effects are independent of the antioxidant action of cannabinoid compounds and are also exerted by a CB2-selective agonist. Moreover, cannabinoids abrogate microglia-mediated neurotoxicity after βA addition to rat cortical cocultures. Our results indicate that cannabinoid receptors are important in the pathology of AD and that cannabinoids succeed in preventing the neurodegenerative process occurring in the disease.

Inhibition of tumor angiogenesis by cannabinoids

Cannabinoids, the active components of marijuana and their derivatives, induce tumor regression in rodents (8). However, the mechanism of cannabinoid antitumoral action in vivo is as yet unknown. Here we show that local administration of a nonpsychoactive cannabinoid to mice inhibits angiogenesis of malignant gliomas as determined by immunohistochemical analyses and vascular permeability assays. In vitro and in vivo experiments show that at least two mechanisms may be involved in this cannabinoid action: the direct inhibition of vascular endothelial cell migration and survival as well as the decrease of the expression of proangiogenic factors (vascular endothelial growth factor and angiopoietin‐2) and matrix metalloproteinase‐2 in the tumors. Inhibition of tumor angiogenesis may allow new strategies for the design of cannabinoid‐based antitumoral therapies.

Choline kinase as a link connecting phospholipid metabolism and cell cycle regulation: Implications in cancer therapy

Choline kinase alpha (ChoKalpha) is an enzyme involved in the metabolism of phospholipids recently found to play a relevant role in the regulation of cell proliferation, oncogenic transformation and human carcinogenesis. In addition, this novel oncogene has been recently defined as a prognostic factor in human cancer, and as a promising target for therapy since its specific inhibitors display efficient antitumoral activity in vivo. However, the mechanism by which this enzyme is involved in the regulation of these processes is not yet understood. Using differential microarray analysis, we identify target genes that provide the basis for the understanding of the molecular mechanism for the regulation of cell proliferation and transformation mediated by over-expression of the human ChoKalpha. These results fully support a critical role of this enzyme in the regulation of the G1-->S transition at different levels, and its relevant role in human carcinogenesis. The molecular basis to understand the connection between phospholipids metabolism and cell cycle regulation through choline kinase is reported.

Involvement of human choline kinase alpha and beta in carcinogenesis: A different role in lipid metabolism and biological functions

We have summarized here the importance of ChoKα1 in human carcinogenesis. ChoKα1 displays its oncogenic activity through activation of specific signaling pathways that influence on cell proliferation and survival. It is overexpressed in a large number of human tumors with an incidence of 40-60% of all tumors investigated. Currently, there is an active effort in the development of strategies to knockdown the activity of ChoKα through specific siRNA or small molecules inhibitors. Results from genetic silencing or from treatment with MN58b, a well characterized ChoKα inhibitor showing antiproliferative and antitumoral effect in mice xenografts, provide strong support to this concept, indicating that the design of new antitumoral drugs must be selective against this isoform. However, affecting the other two known isoforms of ChoK may have also therapeutic consequences since the physiologically active form of ChoK may be constituted by homo or heterodimers. Furthermore, alteration of the ChoKβ activity might lead to a change in the lipid content of the cells of particular tissues such as skeletal muscle as described in the ChoKβ null mice (Sher et al., 2006). Finally, the identification of the ChoKα1 isoform as an excellent novel tool for the diagnosis and prognosis of cancer patients may have clinical consequences of immediate usefulness. On one hand, the use of specific monoclonal antibodies against ChoKα1 as a tool for diagnosis in paraffin embedded samples from patient biopsies, through standard immunohistochemistry techniques, can now be achieved (Gallego-Ortega et al., 2006). On the other hand, it has been recently described the prognostic value of determination of ChoKα1 expression levels in non-small cell lung cancer using real time quantitative PCR technology (Ramírez de Molina et al., 2007). Therefore, further research should be supported on the utility of ChoK isoforms as a promising area to improve cancer diagnosis and treatment.

Combined 5-FU and ChoKα Inhibitors as a New Alternative Therapy of Colorectal Cancer: Evidence in Human Tumor-Derived Cell Lines and Mouse Xenografts

Background Colorectal cancer (CRC) is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU) is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα), an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy. Methodology/Principal Findings ChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS) and thymidine kinase (TK1) levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action. Conclusion/Significance Our data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors.

Phospholipid profiling identifies acyl chain elongation as a ubiquitous trait and potential target for the treatment of lung squamous cell carcinoma

Lung cancer is the leading cause of cancer death. Beyond first line treatment, few therapeutic options are available, particularly for squamous cell carcinoma (SCC). Here, we have explored the phospholipidomes of 30 human SCCs and found that they almost invariably (in 96.7% of cases) contain phospholipids with longer acyl chains compared to matched normal tissues. This trait was confirmed using in situ 2D-imaging MS on tissue sections and by phospholipidomics of tumor and normal lung tissue of the L-IkkαKA/KA mouse model of lung SCC. In both human and mouse, the increase in acyl chain length in cancer tissue was accompanied by significant changes in the expression of acyl chain elongases (ELOVLs). Functional screening of differentially expressed ELOVLs by selective gene knockdown in SCC cell lines followed by phospholipidomics revealed ELOVL6 as the main elongation enzyme responsible for acyl chain elongation in cancer cells. Interestingly, inhibition of ELOVL6 drastically reduced colony formation of multiple SCC cell lines in vitro and significantly attenuated their growth as xenografts in vivo in mouse models. These findings identify acyl chain elongation as one of the most common traits of lung SCC discovered so far and pinpoint ELOVL6 as a novel potential target for cancer intervention.

Human urine proteomics: Building a list of human urine cancer biomarkers

In the last decade, several reports have focused on the identification and characterization of proteins present in urine. In an effort to build a list of proteins of interest as biomarkers, we reviewed the largest urine proteomes and built two updated lists of proteins of interest (available as supplementary tables). The first table includes a consensus list of 443 proteins found in urine by independent laboratories and reported on the top three largest urine proteomes currently published. This consensus list of proteins could serve as biomarkers to diagnose, monitor and manage a number of diseases. Here, we focus on a reduced list of 35 proteins with potential interest as cancer biomarkers in urine following two criteria: first, proteins previously detected in urine using bottom-up proteomic experiments, and second, those suggested as cancer protein biomarkers in human plasma. In an effort to standardize the information presented and its use in future studies, here we include the updated International Protein Index (v. 3.80) and primary Swiss-Prot accession numbers, official gene symbols and recommended full names. The main variables that influence urine proteomic experiments are also discussed.

Antiplasmodial Activity and Mechanism of Action of RSM-932A, a Promising Synergistic Inhibitor of Plasmodium falciparum Choline Kinase

ABSTRACT 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.

Decreased PLK1 expression denotes therapy resistance and unfavourable disease-free survival in rectal cancer patients receiving neoadjuvant chemoradiotherapy

AIM Polo-like kinase 1 (Plk1) plays a key role in mitotic cell division and DNA damage repair. It has been observed that either up-regulated or down-regulated Plk1 could induce mitotic defects that results in aneuploidy and tumorigenesis, probably depending on the context. Few previous reports have associated Plk1 expression with prognosis and response to radiotherapy in rectal carcinomas. The aim of this study is to investigate the prognostic impact of Plk1 expression and its role in predicting response to neoadjuvant cheomoradiotherapy in rectal cancer. METHODS AND RESULTS Immunohistochemical analysis of Plk1 expression was performed in the pre-treatment tumour specimens from 75 rectal cancer patients. We analysed the assocation between Plk1 expression and clinicopathological parameters, pathologic response and outcome. Opposed to previous reports on this issue, low expression of Plk1 was significantly associated with a high grade of differentiation (P=0.0007) and higher rate of distant metastasis (P=0.014). More importantly, decreased levels of Plk1 were associated with absence of response after neoadjuvant therapy (P=0.049). Moreover, low Plk1 expression emerged as an unfavourable prognostic factor for disease-free survival in the non-responder group of patients (P=0.037). CONCLUSIONS Decreased Plk1 expression was associated with poor pathologic response and worse disease-free survival in rectal cancer patients receiving neoadjuvant chemoradiotherapy, suggesting Plk1 as a clinically relevant marker to predict chemoradiotherapy response and outcome.

UNR/CDSE1 expression as prognosis biomarker in resectable pancreatic ductal adenocarcinoma patients: A proof-of-concept

Pancreatic ductal adenocarcinoma is an aggressive form of pancreatic cancer and the fourth leading cause of cancer-related death. When possible, curative approaches are based on surgical resection, though not every patient is a candidate for surgery. There are clinical guidelines for the management of these patients that offer different treatment options depending on the clinical and pathologic characteristics. However, the survival rates seen in this kind of patients are still low. The CDSE1 gene is located upstream of NRAS and encodes an RNA-binding protein termed UNR. The aim of this study was to analyze UNR expression and its correlation with outcome in patients with resectable pancreatic ductal adenocarcinoma (PDAC). For this, samples from resectable PDAC patients who underwent duodenopancreatectomy were used to evaluate UNR protein expression by immunohistochemistry using a tissue microarray. Here, we observed that low UNR expression was significantly associated with shorter progression-free survival after surgery (P = 0.010). Moreover, this prognostic marker remained significant after Cox proportional hazards model (P = 0.036). We further studied the role of CDSE1 expression in patient’s prognosis using data from public repositories (GEO and TGCA), confirming our results. Interestingly, CDSE1 expression correlated with that of genes characteristic of an immunogenic molecular subtype of pancreatic cancer. Based on these findings, UNR may be considered a potential prognostic biomarker for resectable PDAC and may serve to guide subsequent adjuvant treatment decisions.