Juan Carlos Lacal

1444 BACKGROUND: ES-285 is a novel marine-derived antitumor agent isolated from the clam Mactromerys polynyma. It has structural analogy with the sphingoid bases sphinganine and sphingosine. It has shown antitumor activity against a variety of human tumor cells including liver, prostate, breast, kidney and melanoma. MATERIALS & METHODS: Four breast cancer-derived cell lines (MDA-MB-231, T47D, SKBR3 and MCF-7) were used in comparison with the corresponding normal breast epithelial cells (HMEC). The antiproliferative effects of ES-285 were determined using cressyl violet staining and flow cytometry. Western blot were used to determine caspase activation after ES-285 exposure. ES-285 (14C-ES-285) metabolism was evaluated by lipid extraction and subsequent analysis by thin layer chromatography using. High-performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) were used to identify the resulting metabolites. RESULTS: ES-285 shows a potent and selective antitumor ac...

Colorectal cancer (CRC) is the third major cause of cancer related deaths worldwide. 5-fluorouracil (5-FU) is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Despite the achieved improvements over the past years in cancer treatment, the design of novel targeted and combined therapies is still necessary. Choline kinase alpha (ChoKα), a phospholipid-related enzyme that plays a role in cell proliferation and transformation, has been found overexpressed in many different tumors, including colorectal tumors. ChoKα specific inhibitors, MN58b and TCD717, have potent antitumoral activity both in vitro and in vivo against several tumor-derived cell lines including CRC-derived tumour cells. TCD717 has recently entered clinical trials (http://clinicaltrials.gov/ct2/show/NCT01215864). We have evaluated the effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors. The sequential combination of ChoKα inhibitors with 5-FU 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. Key enzymes in the metabolic pathway of 5-FU such as Thymidylate synthase (TS) and Thymidine kinase (TK1) have been related to drug resistance, one of the major problems for the lack of success of 5-FU chemotherapy. TS is a key enzyme in the synthesis of DNA and the target enzyme of 5-FU. Several studies have demonstrated that the expression of TS mRNA or protein, predicts overall survival for colon cancer and correlates with resistance to 5-FU. Acquired resistance to 5-FU can also be caused by overproduction of TS that results from gene amplification. Regarding to 5-FU developed resistance, a salvaged pathway has been reported in which TK1 plays an important role. Thus, both TS and TK1 are directly involved in resistance to 5-FU therapy. To elucidate the mechanism of the observed synergistic effect, we examined the modulation of ChoKα inhibitors on the expression levels of TS and TK1. CRC cell lines treated with the sequential combination of ChoKα inhibitors and 5-FU show both mechanisms of TS inactivation, a formation of ternary complex due to 5-FU mechanism and a significant down-regulation of TS active due to ChoKα inhibitors, resulting in a total depletion of TS protein. We also observed that ChoKα inhibitors affects the levels of TK1, and provide the basis to support a new therapy focused on the combination of ChoKα inhibitors and 5-FU as a new approach for colorectal cancer patients. In addition, we suggest a promising role for ChoKα inhibitors as a new treatment for patients who had failed 5-FU chemotherapy or display high expression levels of TS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3514. doi:10.1158/1538-7445.AM2011-3514

BACKGROUND: Tumor resistance is one of the major problems for cancer treatment and new approaches to overcome resistance are required. TNF-Related Apoptosis Inducing Ligand (TRAIL) has recently emerged as a promising therapy due to its selectivity towards tumor cells and is currently being evaluated in phase I and phase II clinical trials. Choline kinase alpha (ChoKα) inhibition has also recently proven to be an efficient strategy to specifically induce tumor cell death without affecting normal cells. The aim of this study is to investigate the potential use of combinatorial treatment of TRAIL and MN58b, a specific ChoKα inhibitor, to potentiate tumor cell death. EXPERIMENTAL PROCEDURES: Two different colon cancer-derived cell lines were used: DLD-1 and SW620. The antiproliferative effect of MN58b, TRAIL, or both was determined using MTT method. Western blots were carried out to measure PARP degradation and caspase-3 activation, and Annexin V- PI staining was used to verify cell dea...

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 c...

Choline kinase alpha (ChoKalpha) is a metabolic enzyme involved in the synthesis of phosphatidylcholine, recently implicated in cancer onset since it is overexpressed in a variety of human cancers such as mammary, lung, colorectal and prostate adenocarcinomas. Furthermore, overexpression of ChoKalpha in human HEK293T cells confers them oncogenic properties with the induction of tumors after subcutaneous injection in nude mice. ChoKalpha levels in tumor samples have been analyzed using polyclonal antibodies and Western blotting. These techniques have considerable limitations and do not allow for a precise and efficient evaluation of the real significance of ChoK overexpression in human carcinogenesis. We developed a set of monoclonal antibodies with high specificity and sensitivity against ChoKalpha, and characterized their properties. We provide evidence that the newly generated MoAbs against ChoKalpha have potential use in cancer diagnosis by conventional immunohistochemistry techniques.

The compound 6a is a novel bisoxazol derivative with high cytotoxic properties in vitro against different human tumor-derived cell lines and with similar efficiency against epithelial, haematopoietic and mesenchymal tumor cells. Although the molecular mechanism is not yet fully defined, cell cycle analysis revealed that 6a induces efficiently G0/G1 phase arrest in colon adenocarcinoma HT-29 cells in a dose- and time-dependent manner. Induction of cell death is observed, a possible explanation for the antiproliferative profile of the molecule. The compound was well tolerated at doses that allowed to examine its antitumor activity against human xenografts of the HT-29 cell line implanted s.c. in nude mice. Treatment of mice with 4 mg/kg of the compound resulted in a 60% inhibition of tumor growth. These observations support the use of 6a for the generation of more potent derivatives that could be used as new anticancer agents.

An Ala-to-Thr substitution at position 59 activates the transforming properties of the p21ras protein without impairment of GTPase activity, a biochemical alteration associated with other activating mutations. To investigate the basis for the transforming properties of the Thr-59 mutant, we characterized guanine nucleotide release. This reaction exhibited a slow rate and stringent temperature requirements. To further dissect the release reaction, we used monoclonal antibodies directed against different epitopes of the p21 molecule. One monoclonal specifically interfered with nucleotide release, while others which recognized different regions of the molecule blocked nucleotide binding. Mutants with the Thr-59 substitution exhibited a three- to ninefold-higher rate of GDP and GTP release than normal p21 or mutants with other activating lesions. This alteration in the Thr-59 mutant would have the effect of increasing its rate of nucleotide exchange. In an intracellular environment with...

43 Fecha de publicación de la solicitud: 01.07.2007 ... 45 Fecha de anuncio de la concesión: 01.04.2008 ... 45 Fecha de publicación del folleto de la patente: 01.04.2008 ... 73 Titular/es: Consejo Superior de Investigaciones Científicas Serrano, 117 28006 Madrid, ...

Microinjection of macromolecules into living cells has proved to be a powerful technique for the functional analysis of gene products in living somatic cells. This approach is useful for the introduction of functional proteins, genes, inhibitors of enzyme activities and antibodies into living cells (Ansorge, 1982). Types of cells into which these molecules can be introduced include primary and established cell lines, fibroblasts and cells of various tissue types, including contractile cardiac cells. Some of the advantages of microinjection over other methods of introducing biological materials into cells (Celis, 1984; Graessmann et al., 1980; Pepperkok et al., 1988) are as follows: (1) Economy of the material; microinjection delivers small volumes (10−13-10−15 1) directly into specific cells which means that the amount of material needed is much less than would be required for other approaches (e.g., scrape loading). (2) Damage to the cell appears to be much less than with other methods as evidence by stress responses, etc. (3) There is much less limitation on the nature of the material injected; for example, nucleic acids, proteins, large and small molecules can all be introduced with little alteration of the technique. (4) Temporal experiments can be performed where the response at a given time to a particular reagent may be monitored. Also, two or more different proteins may be introduced consequently in a defined order into the cell by re-injecting the same cell more than once. It is not easy to do this by other approaches such as transfection. (5) Molecules which had been altered in vitro may be injected in order to determine the effect of post-translational modifications. (6) Direct comparison of the effect of the injected material may be made by comparing injected and uninjected cells or cells injected with control material on the same dish.

We have previously reported that rho genes, members of the ras superfamily, are tumorigenic when overexpressed in NIH 3T3 cells. As other known oncogenes, they also induce apoptosis after serum deprivation but not in the presence of growth factors. In the present study, we provide evidence that overexpression of the Aplysia Rho protein in NIH 3T3 cells induces the generation of phosphatidylcholine (PC)-derived second messengers as a result of activation of a PC-specific phospholipase D (PC-PLD) as previously reported for ras-transformed cells. In contrast, removal of serum in the Rho transfectants, but not in normal NIH 3T3 cells or cells transformed by the ras oncogene, induced the production of ceramides as a result of activation of an sphingomyelinase (SMase). Furthermore, the rho-expressing cells underwent apoptosis in the presence of serum when exogenous ceramides were added, and this process was accelerated if cells were treated with exogenous SMase. Thus, Rho proteins act as an initiation signal that is necessary but not sufficient for the induction of apoptosis in NIH 3T3 cells. We propose here that induction of apoptosis in NIH 3T3 cells requires two complementary signals: an initiation signal generated even in the presence of serum which 'primes' the cells, making them sensitive to a progression signal, triggered by serum removal, which we have identified as generation of ceramides.

rho genes have been found in both lower and higher eucaryotes. They code for proteins of 21 kDa, highly conserved in evolution, which belong to the superfamily of ras GTPases. Among the members of this superfamily there are proteins with a regulatory function, such as ras, and proteins involved in vesicular trafficking, such as the family of rab proteins. We have investigated the putative role of rho proteins from Aplysia californica as transforming GTPases utilizing the wild-type and a Val-14 mutant, equivalent to the oncogenic Val-12 mutation of ras genes found in animal and human tumors. Over-expression of either rho gene was sufficient to confer anchorage- and serum-independent growth. Moreover, when introduced into nude mice, selected clones generated from either gene were able to induce tumors, although those carrying the mutated version were more efficient. Pathological analysis indicated that generated tumors corresponded to well-differentiated fibrosarcomas with distinct an...

1<p>Data are represented in milliMolar.</p>2<p>Referenced to the lowest Km.</p><p>Km of the different isoforms of ChoK for each substrate is indicated in each case. The results were obtained from four independent experiments using the logarithmic Michaelis-Menten formula as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007819#s4" target="_blank">Material and Methods</a>.</p

The high prevalence of ras oncogenes in human tumors has given increasing impetus to efforts aimed at elucidating the structure and function of their p21 products. To identify functionally important domains of the p21 protein, antibodies were generated against synthetic peptides corresponding to various regions of the protein. Antibodies directed against a synthetic peptide fragment corresponding to amino acid residues 161 to 176 in the carboxy-terminal region of the H-ras-encoded p21 molecule specifically recognized H-ras-encoded p21 proteins. This antibody was also shown to strikingly and specifically inhibit the guanine nucleotide-binding function of the p21 protein. The inability of p21 protein to bind guanine nucleotides was associated with a lack of autophosphorylation or GTPase activities. These studies suggest that a region toward its carboxy terminus is directly or indirectly involved in the guanine nucleotide-binding function of the p21 molecule.

Lung cancer is one of the main causes of death in developed countries, and non-small cell lung cancer (NSCLC) is the most frequent type (80% of patients). In advanced NSCLC, platinum-based chemotherapy is the frontline palliative treatment, but less than 5% of patients achieve prolonged survival. Immunotherapy has recently been proposed as the standard of care (SoC) as either monotherapy or in combination with chemotherapy for advanced NSCLC. The levels of expression of PD-L1 are the only predictive biomarkers for patient assessment. Although around 30% of patients receiving immunotherapy achieve 5-year survival, a significant number does not benefit from this novel therapeutic approach. Therefore, there is a need for novel strategies to improve clinical outcomes. The expression level of choline kinase α (ChoKα) is increased in a large number of human tumors, including NSCLC tumors, and constitutes an independent prognostic factor for early-stage NSCLC patients. Thus, ChoKα has been...

We have generated deletion mutants of the H-ras p21 protein which lack residues 58 to 63 or 64 to 68 and contain either the normal glycine or an activating mutation, arginine, at position 12. None of the deleted proteins were recognized by monoclonal antibody Y13-259, and those mutants with activating mutations showed at least a 100-fold reduction in their transforming activities compared with the activities of their nondeleted counterparts. Alterations observed in the in vitro GTPase or GTP interchange properties of the deletion mutants were not consistent with the decrease in their transforming activities. Moreover, each mutant showed normal membrane localization, which is essential for its biological activity. Recently, a newly identified protein, designated GTPase-activating protein (GAP), was found to markedly increase GTPase activity of the normal ras p21 but not of p21 mutants bearing activating lesions (H. Adari, D. R. Lowy, B. M. Willumsen, C. J. Der, and F. McCormick, Scie...

The H-ras gene of the BALB murine sarcoma virus (BALB-MSV) was placed under the transcriptional control of the tightly regulated PL promoter of bacteriophage lambda in the expression vectors pEV-vrf-1 and pRC23. Upon derepression of the PL promoter, large amounts (10-20% of total cellular protein) of the H-ras gene product p21 are synthesized in Escherichia coli. We constructed three H-ras gene expression vectors, designated pJCL-H5, pJCL-E30, and pJCL-33. pJCL-H5 directs the synthesis of p21, a fusion protein whose four amino-terminal residues are replaced by eight amino acids coded for by plasmid sequences. The 13 5' coding nucleotides of the BALB-MSV H-ras gene missing in pJCL-H5 were regenerated in pJCL-E30 by inserting a pair of complementary synthetic oligodeoxynucleotides. As a result, pJCL-E30 encodes a p21 protein, p21T, of sequence identical to that of the transforming p21 protein of BALB-MSV. pJCL-33 is a derivative of pJCL-E30 in which the 12th codon, AAA, a lysine c...

Signal transduction induced by generations of second messengers from membrane phospholipids is a major regulatory mechanism in the control of cell proliferation. Indeed, oncogenic p21ras alters the intracellular levels of phospholipid metabolites in both mammalian cells and Xenopus oocytes. However, it is still controversial whether this alteration it is biologically significant. We have analyzed the ras-induced signal transduction pathway in Xenopus oocytes and have correlated its mechanism of activation with that of the three most relevant phospholipases (PLs). After microinjection, ras-p21 induces a rapid PLD activation followed by a late PLA2 activation. By contrast, phosphatidylcholine-specific PLC was not activated under similar conditions. When each of these PLs was studied for its ability to activate intracellular signalling kinases, all of them were found to activate maturation-promoting factor efficiently. However, only PLD was able to activate MAP kinase and S6 kinase II,...

Background: The Kennedy pathway generates phosphocoline and phosphoethanolamine through its two branches. Choline Kinase (ChoK) is the first enzyme of the Kennedy branch of synthesis of phosphocholine, the major component of the plasma membrane. ChoK family of proteins is composed by ChoKa and ChoKb isoforms, the first one with two different variants of splicing. Recently ChoKa has been implicated in the carcinogenic process, since it is over-expressed in a variety of human cancers. However, no evidence for a role of ChoKb in carcinogenesis has been reported. Methodology/Principal Findings: Here we compare the in vitro and in vivo properties of ChoKa1 and ChoKb in lipid metabolism, and their potential role in carcinogenesis. Both ChoKa1 and ChoKb showed choline and ethanolamine kinase activities when assayed in cell extracts, though with different affinity for their substrates. However, they behave differentially when overexpressed in whole cells. Whereas ChoKb display an ethanolami...

Choline kinase (ChoK) is a cytosolic enzyme that catalyzes the phosphorylation of choline to form phosphorylcholine (PCho) in the presence of ATP and magnesium. ChoK is required for the synthesis of key membrane phospholipids and is involved in malignant transformation in a large variety of human tumours. Active compounds against ChoK have been identified and proposed as antitumor agents. The ChoK inhibitory and antiproliferative activities of symmetrical bispyridinium and bisquinolinium compounds have been defined using quantitative structure–activity relationships (QSARs) and structural parameters. The design strategy followed in the development of the most active molecules is presented. The selective anticancer activity of these structures is also described. One promising anticancer compound has even entered clinical trials. Recently, ChoKα inhibitors have also been proposed as a novel therapeutic approach against parasites, rheumatoid arthritis, inflammatory processes, and patho...

Signal transduction induced by generation of second messengers from membrane phospholipids is considered a major regulatory mechanism in control of cell proliferation. We report here that in the Xenopus laevis oocytes model, microinjection of the three most relevant types of phospholipases acting on membrane phospholipids (A2, C, and D) are capable of inducing oocyte maturation with similar efficiencies. This effect is mediated by the generation of known second messengers such as lyso-phospholipids, arachidonic acid, diacylglycerol, and phosphatidic acid. Specific inhibitors of protein kinase C made it possible to identify alternative independent signalling pathways for induction of oocyte maturation. Our results indicate that while phospholipase C seems to be dependent on protein kinase C (PKC), phospholipase A2, and phospholipase D are completely independent of protein kinase C function. Thus, the oocyte system is a powerful tool for the analysis of the potential mitogenic activity of lipid metabolites. It is also an excellent tool for unravelling the different routes involved in the regulation of cell growth.

Mouse skin papillomas and squamous cell carcinomas induced by initiation with 7,12-dimethylbenz[a]anthracene and promotion with phorbol esters, such as 12-O-tetradecanoyl phorbol-13-acetate, frequently contain an activated Harvey ras gene. Six murine epidermal cells lines established from pooled skin papillomas previously tested negative in the NIH-3T3 assay, but have an altered differentiation program by a variety of criteria. The Harvey ras gene and its p21 protein product from these cell lines have been analyzed for alterations responsible for their altered growth and differentiation properties that were undetectable by 3T3 transfection assays. In comparison with primary papillomas and carcinomas, shown to have a point mutation in codon 61 of the Harvey ras gene, resulting in a p21 product with the diagnostic alteration in SDS-PAGE, the papilloma cell lines exhibited neither the codon 61 mutation, nor p21 product with altered migration in SDS-PAGE. These findings suggest that these papilloma cell lines contain a genetic lesion(s), other than Harvey ras activation, that may be responsible for their altered epithelial differentiation patterns and thus may serve as a useful model for identifying lesions involved in malignant conversion.

Rho GTPases are overexpressed in a variety of human tumors contributing to both tumor proliferation and metastasis. Recently, several studies demonstrate an essential role of transcriptional regulation in Rho GTPases-induced oncogenesis. Herein, we demonstrate that RhoA, Rac1, and Cdc42 promote the expression of cyclooxygenase-2 (COX-2) at the transcriptional level by a mechanism that is dependent on the transcription factor nuclear factor-κB (NF-κB), but not Stat3, a transcription factor required for RhoA-induced tumorigenesis. With respect to RhoA, this effect is dependent on ROCK, but not PKN. Treatment of RhoA-, Rac1-, and Cdc42-transformed epithelial cells with Sulindac and NS-398, two well-characterized nonsteroid antiinflammatory drugs (NSAIDs), results in growth inhibition as determined by cell proliferation assays. Accordingly, tumor growth of RhoA-expressing epithelial cells in syngeneic mice is strongly inhibited by NS-398 treatment. The effect of NSAIDs over RhoA-induced tumor growth is not exclusively dependent on COX-2 because DNA-binding of NF-κB is also abolished upon NSAIDs treatment, resulting in complete loss of COX-2 expression. Finally, treatment of RhoA-transformed cells with Bay11-7083, a specific NF-κB inhibitor, leads to inhibition of cell proliferation. We suggest that treatment of human tumors that overexpress Rho GTPases with NSAIDs and drugs that target NF-κB could constitute a valid antitumoral strategy.

Phospholipase D (PLD) is activated by a variety of stimuli, including mitogenic stimulation by growth factors and oncogene transformation. Activation of PLD by growth factors requires protein kinase C (PKC) since depletion of the enzyme by down-regulation or direct inhibition by specific drugs completely abrogates this effect. Transformation by the ras and src oncogenes is also associated with an increase in basal PLD activity. However, this effect is not dependent on PKC, suggesting that growth factors and oncogenes may activate PLD by two independent mechanisms. Here we demonstrate that activation of PLD by phorbol esters is greatly enhanced in ras-transformed cells, suggesting synergistic activation of PLD by ras oncogenes and PKC. Also, ras-transformed cells showed a dramatic attenuation of the PLD activation induced by growth factors, although receptor function was still detectable. This attenuation paralleled the specific uncoupling of the phosphatidylinositol-specific phospho...

Signal transduction is the major mechanism by which cells communicate among themselves through extracellular stimuli. Among the different structural components involved in signal transduction, protein kinases are one of the key elements in the process. Protein kinase C is a multimember family of kinases which has been involved in the regulation of diverse cellular functions. Regulation of cell growth in fibroblasts has been reported to be one of such functions. In particular the PKC zeta isoenzyme has been postulated to be transforming to NIH3T3 cells (Berra et al., 1993) and to serve as an effector for Ras proteins through the regulation of the NF kappa B transcription factor (Dominguez et al., 1993) and direct interaction (Díaz-Meco et al., 1994). We have investigated the effects of overexpressing the mouse wild-type PKC zeta in NIH3T3 cells. When compared to the parental NIH3T3 cells, we have found (1) no significant effect on cell morphology; (2) no difference in growth properti...

Management of locally advanced rectal cancer (RC) consists of neoadjuvant chemoradiotherapy (CRT) with fluoropyrimidines, followed by total mesorectal excision. We sought to evaluate the expression of selected genes, some of which were derived from a previous undirected SAGE (serial analysis of gene expression)-based approach, before and after CRT, to identify mechanisms of resistance. This retrospective cohort study included 129 consecutive patients. Quantitative polymerase chain reaction of 53 candidate genes was performed on the biopsy specimen before treatment and on the surgical specimen after CRT. A paired-samples t test was performed to determine genes that were significantly changed after CRT. The result was correlated with…

... Semin Oncol 29: 47-50 Marcucci G, Byrd JC, Dai G, Klisovic MI, Young DC, Cataland SR, Fisher DB, Lucas D, Chan KK, Porcu P, Lin ... Semin Oncol 29: 29-37 Pietras RJ, Poen JC, Gallardo D, Wongvipat N, Lee HJ, Slamon DJ (1999) Monoclonal antibody to HER-2/neuroreceptor ...