Overcoming Resistance of Caco-2 Cells to 5-Fluorouracil through Diruthenium Complex Encapsulation in PMMA Nanoparticles.

Drug resistance, one of the main drawbacks in cancer chemotherapy, can be tackled by employing a combination of drugs that target different biological processes in the cell, enhancing the therapeutic efficacy. Herein, we report the synthesis and characterization of a new paddlewheel diruthenium complex that includes 5-fluorouracil (5-FU), a commonly used anticancer drug. This drug was functionalized with a carboxylate group to take advantage of the previously demonstrated release capacity of carboxylate ligands from the diruthenium core. The resulting hydrophobic complex, [Ru2Cl(DPhF)3(5-FUA)] (Ru-5-FUA) (DPhF = N,N'-diphenylformamidinate; 5-FUA = 5-fluorouracil-1-acetate) was subsequently entrapped in poly(methyl methacrylate) (PMMA) nanoparticles (PMMA@Ru-5-FUA) via a reprecipitation method to be transported in biological media. The optimized encapsulation procedure yielded particles with an average size of 81.2 nm, a PDI of 0.11, and a zeta potential of 29.2 mV. The cytotoxicity of the particles was tested in vitro using the human colon carcinoma cell line Caco-2. The IC50 (half maximal inhibitory concentration) of PMMA@Ru-5-FUA (6.08 µM) was just slightly lower than that found for the drug 5-FU (7.64 µM). Most importantly, while cells seemed to have developed drug resistance against 5-FU, PMMA@Ru-5-FUA showed an almost complete lethality at ∼30 µM. Conversely, an analogous diruthenium complex devoid of the 5-FU moiety, [Ru2Cl(DPhF)3(O2CCH3)] (PMMA@RuA), displayed a reduced cytotoxicity at equivalent concentrations. These findings highlight the effect of combining the anticancer properties of 5-FU with those of diruthenium species. This suggests that the distinct modes of action of the two chemical species are crucial for overcoming drug resistance.

Molecular dissection of the membrane aggregation mechanisms induced by monomeric annexin A2.

Annexins are a multigene family of proteins involved in aggregation and fusion processes of biological membranes. One of its best-known members is annexin A2 (or p36), capable of binding to acidic phospholipids in a calcium-dependent manner, as occurs with other members of the same family. In its heterotetrameric form, especially with protein S100A10 (p11), annexin A2 has been involved as a determinant factor in innumerable biological processes like tumor development or anticoagulation. However, the subcellular coexistence of different pools of the protein, in which the monomeric form of annexin A2 is growing in functional relevance, is to date poorly described. In this work we present an exhaustive structural and functional characterization of monomeric human annexin A2 by using different recombinant mutants. The important role of the amphipathic N-terminal α-helix in membrane binding and aggregation has been analyzed. We have also studied the potential implication of lateral "antiparallel" protein dimers in membrane aggregation. In contrast to what was previously suggested, formation of these dimers negatively regulate aggregation. We have also confirmed the essential role of three lysine residues located in the convex surface of the molecule in calcium-free and calcium-dependent membrane binding and aggregation. Finally, we propose models for annexin A2-mediated vesicle aggregation mechanisms.

Structural and lipid-binding characterization of human annexin A13a reveals strong differences with its long A13b isoform.

Annexin A13 is the founder member of the vertebrate family of annexins, which are comprised of a tetrad of unique conserved domains responsible for calcium-dependent binding to membranes. Its expression is restricted to epithelial intestinal and kidney cells. Alternative splicing in the N-terminal region generates two isoforms, A13a and A13b, differing in a deletion of 41 residues in the former. We have confirmed the expression of both isoforms in human colon adenocarcinoma cells at the mRNA and protein levels. We have cloned, expressed, and purified human annexin A13a for the first time to analyze its structural characteristics. Its secondary structure and thermal stability differs greatly from the A13b isoform. The only tryptophan residue (Trp186) is buried in the protein core in the absence of calcium but is exposed to the solvent after calcium binding even though circular dichroism spectra are quite similar. Non-myristoylated annexin A13a binds in a calcium-dependent manner to acidic phospholipids but not to neutral or raft-like liposomes. Calcium requirements for binding to phosphatidylserine are around 6-fold lower than those required by the A13b isoform. This fact could account for the different subcellular localization of both annexins as binding to basolateral membranes seems to be calcium-dependent and myristoylation-independent.

4F2hc-silencing impairs tumorigenicity of HeLa cells via modulation of galectin-3 and ß-catenin signaling, and MMP-2 expression.

4F2hc is a type-II glycoprotein whose covalent-bound association with one of several described light chains yields a heterodimer mainly involved in large neutral amino acid transport. Likewise, it is well known that the heavy chain interacts with ß-integrins mediating integrin-dependent events such as survival, proliferation, migration and even transformation. 4F2hc is a ubiquitous protein whose overexpression has been related to tumor development and progression. Stable silencing of 4F2hc in HeLa cells using an artificial miRNA impairs in vivo tumorigenicity and leads to an ineffective proliferation response to mitogens. 4F2hc colocalizes with ß1-integrins and CD147, but this interaction does not occur in lipid rafts in HeLa cells. Moreover, silenced cells present defects in integrin- (FAK, Akt and ERK1/2) and hypoxia-dependent signaling, and reduced expression/activity of MMP-2. These alterations seem to be dependent on the inappropriate formation of CD147/4F2hc/ß1-integrin heterocomplexes on the cell surface, arising when CD147 cannot interact with 4F2hc. Although extracellular galectin-3 accumulates due to the decrease in MMP-2 activity, galectin-3 signaling events are blocked due to an impaired interaction with 4F2hc, inducing an increased degradation of ß-catenin. Furthermore, cell motility is compromised after protein silencing, suggesting that 4F2hc is related to tumor invasion by facilitating cell motility. Therefore, here we propose a molecular mechanism by which 4F2hc participates in tumor progression, favoring first steps of epithelial-mesenchymal transition by inhibition of ß-catenin proteasomal degradation through Akt/GSK-3ß signaling and enabling cell motility.

Long-chain fatty acids block allergic reaction against lipid transfer protein Sola l 7 from tomato seeds.

Due to the benefits of tomato as an antioxidant and vitamin source, allergy to this vegetable food is a clinically concerning problem. Sola l 7, a class I lipid transfer protein found in tomato seeds, has been identified as an allergen linked to severe anaphylaxis. However, the role of lipid binding in Sola l 7-induced allergy remains unclear. Here, the three-dimensional structure of recombinant Sola l 7 (rSola l 7) has been elucidated using nuclear magnetic resonance spectroscopy (NMR). Its interaction with free fatty acids has been deeply studied; fluorescence emission spectroscopy revealed that different long-chain fatty acids interact with the protein, affecting the only tyrosine residue present in Sola l 7. On the contrary, no changes in the overall secondary structure were observed after the analysis of the circular dichroism spectra in the presence of fatty acids. Unsaturated oleic and linoleic fatty acids presented higher affinity and promoted more significant changes than saturated or short-chain fatty acids. 1H-15N HSQC NMR spectra allowed to determine the regions of the protein that were modified when rSola l 7 interacts with the fatty acids, suggesting epitope modification after the interaction. For corroboration, IgG and IgE binding to rSola l 7 were assessed in the presence of free fatty acids, revealing that both IgE and IgG binding were significantly lower than in their absence, suggesting a potential protective role of unsaturated fatty acids in tomato allergy.

Walnut Jug r 1 is Responsible for Primary Sensitization among Patients Suffering Walnut-Hazelnut 2S Albumin Cross-Reactivity.

Walnut and hazelnut coallergy is a frequent manifestation in clinical practice whose molecular basis remains unclear. For this purpose, walnut-hazelnut cross-reactivity was evaluated in 20 patients allergic to one or both tree nuts and sensitized to their 2S albumins. Immunoblotting assays showed that 85% of patients recognized Jug r 1, walnut 2S albumin, which was associated with the development of severe symptoms; 50% of them corecognized hazelnut 2S albumin, Cor a 14. Both allergens were isolated using chromatographic techniques. Inhibition ELISAs revealed that Jug r 1 strongly inhibited the binding of Cor a 14-specific IgE, but Cor a 14 only partially inhibited Jug r 1-specific IgE binding. Our results showed that patients sensitized to walnut/hazelnut 2S albumins were not a homogeneous population. There were patients sensitized to specific epitopes of walnut 2S albumins and patients sensitized to cross-reactive epitopes between walnut and hazelnut, with Jug r 1 being the primary sensitizer.

Identification of New Allergens in Macadamia Nut and Cross-Reactivity with Other Tree Nuts in a Spanish Cohort.

The consumption of macadamia nuts has increased due to their cardioprotective and antioxidant properties. However, this rise is consistent with an increase in the cases of macadamia nut allergy, leading to severe reactions. Although two Macadamia integrifolia allergens (Mac i 1 and Mac i 2) have been identified in Australian and Japanese patients, the allergenic sensitization patterns in Western European populations, particularly in Spain, remain unclear. For this purpose, seven patients with macadamia nut allergy were recruited in Spain. Macadamia nut protein extracts were prepared and, together with hazelnut and walnut extracts, were used in Western blot and inhibition assays. IgE-reactive proteins were identified using MALDI-TOF/TOF mass spectrometry (MS). Immunoblotting assays revealed various IgE-binding proteins in macadamia nut extracts. Mass spectrometry identified three new allergens: an oleosin, a pectin acetylesterase, and an aspartyl protease. Cross-reactivity studies showed that hazelnut extract but not walnut extract inhibited macadamia nut oleosin-specific IgE binding. This suggests that oleosin could be used as marker for macadamia-hazelnut cross-reactivity. The results show an allergenic profile in the Spanish cohort different from that previously detected in Australian and Japanese populations. The distinct sensitization profiles observed highlight the potential influence of dietary habits and environmental factors exposure on allergenicity.

Histone deacetylase inhibitors upregulate MMP11 gene expression through Sp1/Smad complexes in human colon adenocarcinoma cells.

MMP-11 (stromelysin-3) is a matrix metalloproteinase associated with tumor progression and poor prognosis. Its expression was initially described exclusively in stromal cells surrounding tumors, but more recently it has also been detected in macrophages and hepatocarcinoma cells. Here we show MMP-11 expression in human epithelial colon adenocarcinoma cell lines (Caco-2, HT-29 and BCS-TC2). Treatment of BCS-TC2 cells with butyrate and trichostatin A (TSA) (histone deacetylase inhibitors) increases MMP11 promoter activity and protein expression. Using electrophoretic mobility shift assay (EMSA) and supershift assays, we demonstrate for the first time that Sp1 is able to bind to the GC-boxes within the MMP11 proximal promoter region; this binding has been confirmed by chromatin immunoprecipitation. Sp1 is involved in MMP11 basal expression and it is essential for the upregulation of transcription by histone deacetylase inhibitors as deduced from mutant constructs lacking the Sp1 sites and by inhibition of its binding to the promoter with mithramycin. This regulation requires the formation of Sp1/Smad2 heterocomplexes, which is stimulated by an increase in the acetylation status of Smad after butyrate or TSA treatments. We have also found that ERK1/2-mitogen-activated protein kinase (MAPK), but not p38-MAPK or JNK, is involved in the upregulation of MMP11 by HDAC inhibitors.

Resistance to butyrate impairs bile acid-induced apoptosis in human colon adenocarcinoma cells via up-regulation of Bcl-2 and inactivation of Bax.

A critical risk factor in colorectal carcinogenesis and tumor therapy is the resistance to the apoptotic effects of different compounds from the intestinal lumen, among them butyrate (main regulator of colonic epithelium homeostasis). Insensitivity to butyrate-induced apoptosis yields resistance to other agents, as bile acids or chemotherapy drugs, allowing the selective growth of malignant cell subpopulations. Here we analyze bile acid-induced apoptosis in a butyrate-resistant human colon adenocarcinoma cell line (BCS-TC2.BR2) to determine the mechanisms that underlay the resistance to these agents in comparison with their parental butyrate-sensitive BCS-TC2 cells. This study demonstrates that DCA and CDCA still induce apoptosis in butyrate-resistant cells through increased ROS production by activation of membrane-associated enzymes and subsequent triggering of the intrinsic mitochondrial apoptotic pathway. Although this mechanism is similar to that described in butyrate-sensitive cells, cell viability is significantly higher in resistant cells. Moreover, butyrate-resistant cells show higher Bcl-2 levels that confer resistance to bile acid-induced apoptosis sequestering Bax and avoiding Bax-dependent pore formation in the mitochondria. We have confirmed that this resistance is reverted using the Bcl-2 inhibitor ABT-263, thus demonstrating that the lower sensitivity of butyrate-resistant cells to the apoptotic effects of bile acids is mainly due to increased Bcl-2 levels.

Annexin-phospholipid interactions. Functional implications.

Annexins constitute an evolutionary conserved multigene protein superfamily characterized by their ability to interact with biological membranes in a calcium dependent manner. They are expressed by all living organisms with the exception of certain unicellular organisms. The vertebrate annexin core is composed of four (eight in annexin A6) homologous domains of around 70 amino acids, with the overall shape of a slightly bent ring surrounding a central hydrophilic pore. Calcium- and phospholipid-binding sites are located on the convex side while the N-terminus links domains I and IV on the concave side. The N-terminus region shows great variability in length and amino acid sequence and it greatly influences protein stability and specific functions of annexins. These proteins interact mainly with acidic phospholipids, such as phosphatidylserine, but differences are found regarding their affinity for lipids and calcium requirements for the interaction. Annexins are involved in a wide range of intra- and extracellular biological processes in vitro, most of them directly related with the conserved ability to bind to phospholipid bilayers: membrane trafficking, membrane-cytoskeleton anchorage, ion channel activity and regulation, as well as antiinflammatory and anticoagulant activities. However, the in vivo physiological functions of annexins are just beginning to be established.

Beyond allergic progression: From molecules to microbes as barrier modulators in the gut-lung axis functionality.

The "epithelial barrier hypothesis" states that a barrier dysfunction can result in allergy development due to tolerance breakdown. This barrier alteration may come from the direct contact of epithelial and immune cells with the allergens, and indirectly, through deleterious effects caused by environmental changes triggered by industrialization, pollution, and changes in the lifestyle. Apart from their protective role, epithelial cells can respond to external factors secreting IL-25 IL-33, and TSLP, provoking the activation of ILC2 cells and a Th2-biased response. Several environmental agents that influence epithelial barrier function, such as allergenic proteases, food additives or certain xenobiotics are reviewed in this paper. In addition, dietary factors that influence the allergenic response in a positive or negative way will be also described here. Finally, we discuss how the gut microbiota, its composition, and microbe-derived metabolites, such as short-chain fatty acids, alter not only the gut but also the integrity of distant epithelial barriers, focusing this review on the gut-lung axis.

Structural characterization and unfolding mechanism of human 4F2hc ectodomain.

4F2hc (CD98hc) is a multifunctional type II membrane glycoprotein involved in several functions as amino acid transport, cell fusion, ß1-integrin-signaling and transformation. 4F2hc ectodomain has been crystallized and its three-dimensional structure determined. We have carried out a spectroscopical/structural characterization of the recombinant ectodomain in order to obtain information on its dynamic structure in solution and on its ability to form homodimers by itself in the absence of the transmembrane helix and of the potential interactions with the plasma membrane. Analytical ultracentrifugation and crosslinking experiments showed that the ectodomain is monomeric in solution. The secondary structure determined by far-UV circular dichroism (CD) spectroscopy (around 30% α-helix and 20% ß-sheets, 12% antiparallel and 8% parallel) reveals a compact and thermally stable structure with a high melting temperature (57-59°C). Tryptophan residues are mainly buried and immobilized in the hydrophobic core of the protein as suggested by near-UV CD spectrum, the position of the Trp maximum fluorescence emission (323nm) and from the acrylamide quenching constant (2.6M(-1)). Urea unfolding equilibrium has been studied by far-UV CD and fluorescence spectroscopy to gain information on the folding/unfolding process of the ectodomain. The analyses suggest the existence of two intermediate states as reported for other TIM barrel-containing proteins rather than an independent unfolding of each domain [A, (ßα)(8) barrel; C, antiparallel ß(8) sandwich]. Folding seems to be directed by the initial formation of hydrophobic clusters within the first strands of the ß-barrel of domain A followed by additional hydrophobic interactions in domain C.

Deoxycholic and chenodeoxycholic bile acids induce apoptosis via oxidative stress in human colon adenocarcinoma cells.

The continuous exposure of the colonic epithelium to high concentrations of bile acids may exert cytotoxic effects and has been related to pathogenesis of colon cancer. A better knowledge of the mechanisms by which bile acids induce toxicity is still required and may be useful for the development of new therapeutic strategies. We have studied the effect of deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) treatments in BCS-TC2 human colon adenocarcinoma cells. Both bile acids promote cell death, being this effect higher for CDCA. Apoptosis is detected after 30 min-2 h of treatment, as observed by cell detachment, loss of membrane asymmetry, internucleosomal DNA degradation, appearance of mitochondrial transition permeability (MPT), and caspase and Bax activation. At longer treatment times, apoptosis is followed in vitro by secondary necrosis due to impaired mitochondrial activity and ATP depletion. Bile acid-induced apoptosis is a result of oxidative stress with increased ROS generation mainly by activation of plasma membrane enzymes, such as NAD(P)H oxidases and, to a lower extent, PLA2. These effects lead to a loss of mitochondrial potential and release of pro-apoptotic factors to the cytosol, which is confirmed by activation of caspase-9 and -3, but not caspase-8. This initial apoptotic steps promote cleavage of Bcl-2, allowing Bax activation and formation of additional pores in the mitochondrial membrane that amplify the apoptotic signal.

Inhibition of phosphatidylserine recognition heightens the immunogenicity of irradiated lymphoma cells in vivo.

Strategies to enhance the immunogenicity of tumors are urgently needed. Although vaccination with irradiated dying lymphoma cells recruits a tumor-specific immune response, its efficiency as immunogen is poor. Annexin V (AxV) binds with high affinity to phosphatidylserine on the surface of apoptotic and necrotic cells and thereby impairs their uptake by macrophages. Here, we report that AxV preferentially targets irradiated lymphoma cells to CD8+ dendritic cells for in vivo clearance, elicits the release of proinflammatory cytokines and dramatically enhances the protection elicited against the tumor. The response was endowed with both memory, because protected animals rejected living lymphoma cells after 72 d, and specificity, because vaccinated animals failed to reject unrelated neoplasms. Finally, AxV-coupled irradiated cells induced the regression of growing tumors. These data indicate that endogenous adjuvants that bind to dying tumor cells can be exploited to target tumors for immune rejection.

Biocompatibility and calcification of bovine pericardium employed for the construction of cardiac bioprostheses treated with different chemical crosslink methods.

The use of biological materials in the construction of bioprostheses requires the application of different chemical procedures to improve the durability of the material without producing any undesirable effects. A number of crosslinking methods have been tested in biological tissues composed mainly of collagen. The aim of this study was to evaluate the in vitro biocompatibility, the mechanical properties, and in vivo calcification of chemically modified bovine pericardium using glutaraldehyde acetals (GAAs) in comparison with glutaraldehyde (GA) treatment. Homsy's tests showed that the most cytotoxic treatment is GA whereas GAA treatments showed lower cytotoxicity. Regarding the mechanical properties of the modified materials, no significant differences in stress at rupture were detected among the different treatments. Zeta-Potential showed higher negative values for GA treatment (-4.9 +/- 0.6 mV) compared with GAA-0.625% (-2.2 +/- 0.5 mV) and GAA-1% (-2.2 +/- 0.4 mV), which presented values similar to native tissue. Similar results were obtained for calcium permeability coefficients which showed the highest values for GA treatment (0.12 +/- 0.02 mm(2)/min), being significantly lower for GAA treatments or non-crosslinked pericardium. These results confirmed the higher propensity of the GA-treated tissues for attraction of calcium cations and were in good agreement with the calcification degree obtained after 60 days implantation into young rats, which was significantly higher for the GA group (22.70 +/- 20.80 mg/g dry tissue) compared with GAA-0.625% and GAA-1% groups (0.49 +/- 0.28 mg/g dry tissue and 3.51 +/- 3.27 mg/g dry tissue, respectively; P < 0.001). In conclusion, GAA treatments can be considered a promising alternative to GA treatment.

Kinetic analysis of butyrate transport in human colon adenocarcinoma cells reveals two different carrier-mediated mechanisms.

Butyrate has antitumorigenic effects on colon cancer cells, inhibits cell growth and promotes differentiation and apoptosis. These effects depend on its intracellular concentration, which is regulated by its transport. We have analysed butyrate uptake kinetics in human colon adenocarcinoma cells sensitive to the apoptotic effects of butyrate (BCS-TC2, Caco-2 and HT-29), in butyrate-resistant cells (BCS-TC2.BR2) and in normal colonic cells (FHC). The properties of transport were analysed with structural analogues, specific inhibitors and different bicarbonate and sodium concentrations. Two carrier-mediated mechanisms were detected: a low-affinity/high-capacity (K(m)=109+/-16 mM in BCS-TC2 cells) anion exchanger and a high-affinity/low-capacity (K(m)=17.9+/-4.0 microM in BCS-TC2 cells) proton-monocarboxylate co-transporter that was energy-dependent and activated via PKCdelta (protein kinase Cdelta). All adenocarcinoma cells analysed express MCT (monocarboxylate transporter) 1, MCT4, ancillary protein CD147 and AE2 (anion exchanger 2). Silencing experiments show that MCT1, whose expression increases with butyrate treatment in butyrate-sensitive cells, plays a key role in high-affinity transport. Low-affinity uptake was mediated by a butyrate/bicarbonate antiporter along with a possible contribution of AE2 and MCT4. Butyrate treatment increased uptake in a time- and dose-dependent manner in butyrate-sensitive but not in butyrate-resistant cells. The two butyrate-uptake activities in human colon adenocarcinoma cells enable butyrate transport at different physiological conditions to maintain cell functionality. The high-affinity/low-capacity transport functions under low butyrate concentrations and may be relevant for the survival of carcinoma cells in tumour regions with low glucose and butyrate availability as well as for the normal physiology of colonocytes.

The tetraspanin CD9 inhibits the proliferation and tumorigenicity of human colon carcinoma cells.

The implication of the tetraspanin CD9 in cancer has received much recent attention and an inverse correlation between CD9 expression and the metastatic potential and cancer survival rate has been established for different tumor types. In contrast to the well-established role of CD9 in metastasis, very little is known about the involvement of this tetraspanin in the process of development of primary tumors. In the present study, we present evidence on the implication of CD9 in colon carcinoma tumorigenesis. We report here that ectopic expression of CD9 in colon carcinoma cells results in enhanced integrin-dependent adhesion and inhibition of cell growth. Consistently with these effects, treatment of these cells with anti-CD9-specific antibodies resulted in (i) increased beta1 integrin-mediated cell adhesion through a mechanism involving clustering of integrin molecules rather than altered affinity; (ii) induction of morphological changes characterized by the acquisition of an elongated cell phenotype; (iii) inhibition of cell proliferation with no significant effect on cell survival; (iv) increased expression of membrane TNF-alpha, and finally (v) inhibition of the in vivo tumorigenic capacity in nude mice. In addition, through the use of selective blockers of TNF-alpha, we have demonstrated that this cytokine partly mediates the antiproliferative effects of CD9. These results clearly establish for the first time a role for CD9 in the tumorigenic process.

Colorectal Cancer: From the Genetic Model to Posttranscriptional Regulation by Noncoding RNAs.

Colorectal cancer is the third most common form of cancer in developed countries and, despite the improvements achieved in its treatment options, remains as one of the main causes of cancer-related death. In this review, we first focus on colorectal carcinogenesis and on the genetic and epigenetic alterations involved. In addition, noncoding RNAs have been shown to be important regulators of gene expression. We present a general overview of what is known about these molecules and their role and dysregulation in cancer, with a special focus on the biogenesis, characteristics, and function of microRNAs. These molecules are important regulators of carcinogenesis, progression, invasion, angiogenesis, and metastases in cancer, including colorectal cancer. For this reason, miRNAs can be used as potential biomarkers for diagnosis, prognosis, and efficacy of chemotherapeutic treatments, or even as therapeutic agents, or as targets by themselves. Thus, this review highlights the importance of miRNAs in the development, progression, diagnosis, and therapy of colorectal cancer and summarizes current therapeutic approaches for the treatment of colorectal cancer.

Structure-function relationship in annexin A13, the founder member of the vertebrate family of annexins.

Annexin A13 is considered the original progenitor of the 11 other members of vertebrate annexins, a superfamily of calcium/phospholipid-binding proteins. It is highly tissue-specific, being expressed only in intestinal and kidney epithelial cells. Alternative splicing generates two isoforms, both of which bind to rafts. In view of the lack of structural information supporting the physiological role of this annexin subfamily, we have cloned, expressed and purified human annexin A13b to investigate its structural and functional properties. The N-terminus of annexin A13b: (i) destabilizes the conserved protein core, as deduced from the low melting temperature in the absence (44 degrees C) or presence of calcium (55 degrees C), and (ii) impairs calcium-dependent binding to acidic phospholipids, requiring calcium concentrations >400 microM. Truncation of the N-terminus restores thermal stability and decreases the calcium requirement for phospholipid binding, confirming its essential role in the structure-function relationship of this annexin. Non-myristoylated annexin A13b only binds to acidic phospholipids at high calcium concentrations. We show for the first time that myristoylation of annexin A13b enables the direct binding to phosphatidylcholine, raft-like liposomes and acidic phospholipids in a calcium-independent manner. The conformational switch induced by calcium binding, from a 'closed' to an 'open' conformation with exposure of Trp227, can be mimicked by a decrease in pH, a process that may be relevant for membrane interactions. Our studies confirm that the common structural and functional characteristics that are dependent on the protein core of vertebrate annexins are likely to be common conserved features, whereas their variable N-termini confer distinct functional properties on annexins, as we report for myristoylation of annexin A13b.

Acquisition of resistance to butyrate enhances survival after stress and induces malignancy of human colon carcinoma cells.

Acquired resistance to apoptosis by tumor cells remains a major obstacle for cancer treatment, and hence the analysis of resistance to apoptosis constitutes a major goal in the development of antitumoral drugs. We have established a butyrate-resistant human colon adenocarcinoma cell line (BCS-TC2.BR2) from nontumorigenic BCS-TC2 cells to analyze whether the acquisition of such phenotype confers resistance to apoptosis and stress. Although BCS-TC2.BR2 cells exhibited a more differentiated phenotype than the parental BCS-TC2 cells, higher butyrate concentrations remained capable of additionally enhancing their differentiation without inducing apoptosis. Survival rates of BCS-TC2.BR2 cells after glucose deprivation and heat shock were higher than those of parental cells, revealing a stress-resistant phenotype. These findings were accompanied by key differences between parental and butyrate-resistant cells in gene expression profiles and the acquisition of in vivo tumorigenicity. In conclusion, cells gaining resistance to an endogenous physiological modulator of growth, differentiation, and apoptosis concurrently acquired resistance to other agents that influence cell survival.