The Efficacy of Intratissue Percutaneous Electrolysis (EPI®) and Nutritional Factors for the Treatment of Induced Tendinopathy in Wistar Rats: Hepatic Intermediary Metabolism Effects.

Achilles tendinopathy (TP) is characterized as the third most common disease of the musculoskeletal system, and occurs in three phases. There is currently no evidence of effective treatment for this medical condition. In this study, the modulatory effects of the minimally invasive technique intratissue percutaneous electrolysis (EPI) and combinations of EPI with four nutritional factors included in the diet, hydroxytyrosol (HT), maslinic acid (MA), glycine, and aspartate (AA), on hepatic intermediary metabolism was examined in Wistar rats with induced tendinopathy at various stages of TP. Results obtained showed that induced tendinopathy produced alterations in the liver intermediary metabolisms of the rats. Regarding carbohydrate metabolism, a reduction in the activity of pro-inflammatory enzymes in the later stages of TP was observed following treatment with EPI alone. Among the combined treatments using nutritional factors with EPI, HT+EPI and AA+EPI had the greatest effect on reducing inflammation in the late stages of TP. In terms of lipid metabolism, the HT+EPI and AA+EPI groups showed a decrease in lipogenesis. In protein metabolism, the HT+EPI group more effectively reduced the inflammatory effects of induced TP. Treatment with EPI combined with nutritional factors might help regulate intermediary metabolism in TP disease and reduce the inflammation process.

Effect of Nutraceutical Factors on Hepatic Intermediary Metabolism in Wistar Rats with Induced Tendinopathy.

Tendinopathy (TP) is a complex clinical syndrome characterized by local inflammation, pain in the affected area, and loss of performance, preceded by tendon injury. The disease develops in three phases: Inflammatory phase, proliferative phase, and remodeling phase. There are currently no proven treatments for early reversal of this type of injury. However, the metabolic pathways of the transition metabolism, which are necessary for the proper functioning of the organism, are known. These metabolic pathways can be modified by a number of external factors, such as nutritional supplements. In this study, the modulatory effect of four dietary supplements, maslinic acid (MA), hydroxytyrosol (HT), glycine, and aspartate (AA), on hepatic intermediary metabolism was observed in Wistar rats with induced tendinopathy at different stages of the disease. Induced tendinopathy in rats produces alterations in the liver intermediary metabolism. Nutraceutical treatments modify the intermediary metabolism in the different phases of tendinopathy, so AA treatment produced a decrease in carbohydrate metabolism. In lipid metabolism, MA and AA caused a decrease in lipogenesis at the tendinopathy and increased fatty acid oxidation. In protein metabolism, MA treatment increased GDH and AST activity; HT decreased ALT activity; and the AA treatment does not cause any alteration. Use of nutritional supplements of diet could help to regulate the intermediary metabolism in the TP.

Involvement of the PI3K/AKT Intracellular Signaling Pathway in the AntiCancer Activity of Hydroxytyrosol, a Polyphenol from Olea europaea, in Hematological Cells and Implication of HSP60 Levels in Its Anti-Inflammatory Activity.

Hydroxytyrosol (HT), the main representative of polyphenols of olive oil, has been described as one of the most powerful natural antioxidants, also showing anti-inflammatory, antimicrobial, cardioprotective and anticancer activity in different type of cancers, but has been little studied in hematological neoplasms. The objective of this work was to evaluate the anticancer potential of HT in acute human leukemia T cells (Jurkat and HL60) and the anti-inflammatory potential in murine macrophages (Raw264.7). For this, cytotoxicity tests were performed for HT, showing IC50 values, at 24 h, for Jurkat, HL60 and Raw264.7 cells, of 27.3 µg·mL-1, 109.8 µg·mL-1 and 45.7 µg·mL-1, respectively. At the same time, HT caused cell arrest in G0/G1 phase in both Jurkat and HL60 cells by increasing G0/G1 phase and significantly decreasing S phase. Apoptosis and cell cycle assays revealed an antiproliferative effect of HT, decreasing the percentage of dividing cells and increasing apoptosis. Furthermore, HT inhibited the PI3K signaling pathway and, consequently, the MAPK pathway was activated. Inflammation tests revealed that HT acts as an anti-inflammatory agent, reducing NO levels in Raw264.7 cells previously stimulated by lipopolysaccharide (LPS). These processes were confirmed by the changes in the expression of the main markers of inflammation and cancer. In conclusion, HT has an anticancer and anti-inflammatory effect in the cell lines studied, which were Raw264.7, Jurkat, and HL60, and could be used as a natural drug in the treatment of liquid cancers, leukemias, myelomas and lymphomas.

Nutraceutical Role of Polyphenols and Triterpenes Present in the Extracts of Fruits and Leaves of Olea europaea as Antioxidants, Anti-Infectives and Anticancer Agents on Healthy Growth.

There is currently a worldwide consensus and recognition of the undoubted health benefits of the so-called Mediterranean diet, with its intake being associated with a lower risk of mortality. The most important characteristics of this type of diet are based on the consumption of significant amounts of fruit, vegetables, legumes, and nuts, which provide, in addition to some active ingredients, fiber and a proportion of vegetable protein, together with extra virgin olive oil (EVOO) as the main sources of vegetable fat. Fish and meat from poultry and other small farm animals are the main sources of protein. One of the main components, as already mentioned, is EVOO, which is rich in monounsaturated fatty acids and to a lesser extent in polyunsaturated fatty acids. The intake of this type of nutrient also provides an important set of phytochemicals whose health potential is widely spread and agreed upon. These phytochemicals include significant amounts of anthocyanins, stilbenes, flavonoids, phenolic acids, and terpenes of varying complexities. Therefore, the inclusion in the diet of this type of molecules, with a proven healthy effect, provides an unquestionable preventive and/or curative activity on an important group of pathologies related to cardiovascular, infectious, and cancerous diseases, as well as those related to the metabolic syndrome. The aim of this review is therefore to shed light on the nutraceutical role of two of the main phytochemicals present in Olea europaea fruit and leaf extracts, polyphenols, and triterpenes, on healthy animal growth. Their immunomodulatory, anti-infective, antioxidant, anti-aging, and anti-carcinogenic capabilities show them to be potential nutraceuticals, providing healthy growth.

Efficient In Vitro and In Vivo Anti-Inflammatory Activity of a Diamine-PEGylated Oleanolic Acid Derivative.

Recent evidence has shown that inflammation can contribute to all tumorigenic states. We have investigated the anti-inflammatory effects of a diamine-PEGylated derivative of oleanolic acid (OADP), in vitro and in vivo with inflammation models. In addition, we have determined the sub-cytotoxic concentrations for anti-inflammatory assays of OADP in RAW 264.7 cells. The inflammatory process began with incubation with lipopolysaccharide (LPS). Nitric oxide production levels were also determined, exceeding 75% inhibition of NO for a concentration of 1 µg/mL of OADP. Cell-cycle analysis showed a reversal of the arrest in the G0/G1 phase in LPS-stimulated RAW 264.7 cells. Furthermore, through Western blot analysis, we have determined the probable molecular mechanism activated by OADP; the inhibition of the expression of cytokines such as TNF-α, IL-1ß, iNOS, and COX-2; and the blocking of p-IκBα production in LPS-stimulated RAW 264.7 cells. Finally, we have analyzed the anti-inflammatory action of OADP in a mouse acute ear edema, in male BL/6J mice treated with OADP and tetradecanoyl phorbol acetate (TPA). Treatment with OADP induced greater suppression of edema and decreased the ear thickness 14% more than diclofenac. The development of new derivatives such as OADP with powerful anti-inflammatory effects could represent an effective therapeutic strategy against inflammation and tumorigenic processes.

Phytochemicals: "A Small Defensive Advantage for Plants and Fungi; a Great Remedy for the Health of Mankind".

In the chronology of Biochemistry, as a new science that emerged in the mid-nineteenth century after its separation from Organic Chemistry and Physiology, its beginnings were characterized by an intense search and subsequent isolation and characterization of different organic compounds that were part of the chemical composition of living organisms [...].

Are Ancestral Medical Practices the Future Solution to Today's Medical Problems?

Our cells and organs are threatened and, in most cases, constantly subjected to the aggression of numerous situations, both endogenous, characterized by unfavorable genetics, and exogenous, by deficient or inadequate nutrition, and even by a hostile environment; in most cases, they ultimately cause a cascade of degenerative and cardiovascular diseases, cancer, and infections, as well as those related to the metabolic syndrome, all of which eventually generate irreversible damage to the organism and, consequently, a significant deterioration in its survival [...].

Unveiling the Differential Antioxidant Activity of Maslinic Acid in Murine Melanoma Cells and in Rat Embryonic Healthy Cells Following Treatment with Hydrogen Peroxide.

Maslinic acid (MA) is a natural triterpene from Olea europaea L. with multiple biological properties. The aim of the present study was to examine MA's effect on cell viability (by the MTT assay), reactive oxygen species (ROS levels, by flow cytometry) and key antioxidant enzyme activities (by spectrophotometry) in murine skin melanoma (B16F10) cells compared to those on healthy cells (A10). MA induced cytotoxic effects in cancer cells (IC50 42 µM), whereas no effect was found in A10 cells treated with MA (up to 210 µM). In order to produce a stress situation in cells, 0.15 mM H2O2 was added. Under stressful conditions, MA protected both cell lines against oxidative damage, decreasing intracellular ROS, which were higher in B16F10 than in A10 cells. The treatment with H2O2 and without MA produced different responses in antioxidant enzyme activities depending on the cell line. In A10 cells, all the enzymes were up-regulated, but in B16F10 cells, only superoxide dismutase, glutathione S-transferase and glutathione peroxidase increased their activities. MA restored the enzyme activities to levels similar to those in the control group in both cell lines, highlighting that in A10 cells, the highest MA doses induced values lower than control. Overall, these findings demonstrate the great antioxidant capacity of MA.

Antiproliferative and Pro-Apoptotic Effect of Uvaol in Human Hepatocarcinoma HepG2 Cells by Affecting G0/G1 Cell Cycle Arrest, ROS Production and AKT/PI3K Signaling Pathway.

Natural products have a significant role in the development of new drugs, being relevant the pentacyclic triterpenes extracted from Olea europaea L. Anticancer effect of uvaol, a natural triterpene, has been scarcely studied. The aim of this study was to understand the anticancer mechanism of uvaol in the HepG2 cell line. Cytotoxicity results showed a selectivity effect of uvaol with higher influence in HepG2 than WRL68 cells used as control. Our results show that uvaol has a clear and selective anticancer activity in HepG2 cells supported by a significant anti-migratory capacity and a significant increase in the expression of HSP-60. Furthermore, the administration of this triterpene induces cell arrest in the G0/G1 phase, as well as an increase in the rate of cell apoptosis. These results are supported by a decrease in the expression of the anti-apoptotic protein Bcl2, an increase in the expression of the pro-apoptotic protein Bax, together with a down-regulation of the AKT/PI3K signaling pathway. A reduction in reactive oxygen species (ROS) levels in HepG2 cells was also observed. Altogether, results showed anti-proliferative and pro-apoptotic effect of uvaol on hepatocellular carcinoma, constituting an interesting challenge in the development of new treatments against this type of cancer.

Expression inactivation of SMARCA4 by microRNAs in lung tumors.

SMARCA4 is the catalytic subunit of the SWI/SNF chromatin-remodeling complex, which alters the interactions between DNA and histones and modifies the availability of the DNA for transcription. The latest deep sequencing of tumor genomes has reinforced the important and ubiquitous tumor suppressor role of the SWI/SNF complex in cancer. However, although SWI/SNF complex plays a key role in gene expression, the regulation of this complex itself is poorly understood. Significantly, an understanding of the regulation of SMARCA4 expression has gained in importance due to recent proposals incorporating it in therapeutic strategies that use synthetic lethal interactions between SMARCA4-MAX and SMARCA4-SMARCA2. In this report, we found that the loss of expression of SMARCA4 observed in some primary lung tumors, whose mechanism was largely unknown, can be explained, at least partially by the activity of microRNAs (miRNAs). We reveal that SMARCA4 expression is regulated by miR-101, miR-199 and especially miR-155 through their binding to two alternative 3'UTRs. Importantly, our experiments suggest that the oncogenic properties of miR-155 in lung cancer can be largely explained by its role inhibiting SMARCA4. This new discovered functional relationship could explain the poor prognosis displayed by patients that independently have high miR-155 and low SMARCA4 expression levels. In addition, these results could lead to application of incipient miRNA technology to the aforementioned synthetic lethal therapeutic strategies.

A New HPLC-MS Method for Measuring Maslinic Acid and Oleanolic Acid in HT29 and HepG2 Human Cancer Cells.

Maslinic acid (MA) and oleanolic acid (OA), the main triterpenic acids present in olive, have important properties for health and disease prevention. MA selectively inhibits cell proliferation of the HT29 human colon-cancer cell line by inducing selective apoptosis. For measuring the MA and OA concentration inside the cell and in the culture medium, a new HPLC-MS procedure has been developed. With this method, a determination of the amount of MA and OA incorporated into HT29 and HepG2 human cancer-cell lines incubated with different concentrations of MA corresponding to 50% growth inhibitory concentration (IC50), IC50/2, IC50/4, and IC50/8 has been made. The results demonstrate that this method is appropriate for determining the MA and OA concentration in different types of cultured cells and reveals the specific dynamics of entry of MA into HT29 and HepG2 cells.

The natural triterpene maslinic acid induces apoptosis in HT29 colon cancer cells by a JNK-p53-dependent mechanism.

BACKGROUND: Maslinic acid, a pentacyclic triterpene found in the protective wax-like coating of the leaves and fruit of Olea europaea L., is a promising agent for the prevention of colon cancer. We have shown elsewhere that maslinic acid inhibits cell proliferation to a significant extent and activates mitochondrial apoptosis in colon cancer cells. In our latest work we have investigated further this compound's apoptotic molecular mechanism. METHODS: We used HT29 adenocarcinoma cells. Changes genotoxicity were analyzed by single-cell gel electrophoresis (comet assay). The cell cycle was determined by flow cytometry. Finally, changes in protein expression were examined by western blotting. Student's t-test was used for statistical comparison. RESULTS: HT29 cells treated with maslinic acid showed significant increases in genotoxicity and cell-cycle arrest during the G0/G1 phase after 72 hours' treatment and an apoptotic sub-G0/G1 peak after 96 hours. Nevertheless, the molecular mechanism for this cytotoxic effect of maslinic acid has never been properly explored. We show here that the anti-tumoral activity of maslinic acid might proceed via p53-mediated apoptosis by acting upon the main signaling components that lead to an increase in p53 activity and the induction of the rest of the factors that participate in the apoptotic pathway. We found that in HT29 cells maslinic acid activated the expression of c-Jun NH2-terminal kinase (JNK), thus inducing p53. Treatment of tumor cells with maslinic acid also resulted in an increase in the expression of Bid and Bax, repression of Bcl-2, release of cytochrome-c and an increase in the expression of caspases -9, -3, and -7. Moreover, maslinic acid produced belated caspase-8 activity, thus amplifying the initial mitochondrial apoptotic signaling. CONCLUSION: All these results suggest that maslinic acid induces apoptosis in human HT29 colon-cancer cells through the JNK-Bid-mediated mitochondrial apoptotic pathway via the activation of p53. Thus we propose a plausible sequential molecular mechanism for the expression of the different proteins responsible for the intrinsic mitochondrial apoptotic pathway. Further studies with other cell lines will be needed to confirm the general nature of these findings.

Effects of Erythrodiol on the Antioxidant Response and Proteome of HepG2 Cells.

Erythrodiol (EO) is a pentacyclic triterpenic alcohol found in olive tree leaves and olive oil, and it has important effects on the health properties and quality of olive oil. In this study, we characterized the cytotoxic effects of EO on human hepatocarcinoma (HepG2) cells by studying changes in cell viability, reactive oxygen species (ROS) production, antioxidant defense systems, and the proteome. The results reveal that EO markedly decreased HepG2 cell viability without changing ROS levels. The concentrations of glutathione and NADPH were significantly reduced, with selective changes in the activity of several antioxidant enzymes: glutathione peroxidase, glutathione reductase, glucose 6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase. Proteomic data reveal that EO led to the complete elimination or decreased abundance of 41 and 3 proteins, respectively, and the abundance of 29 proteins increased. The results of functional enrichment analysis show that important metabolic processes and the nuclear transport of mature mRNA were impaired, whereas AMP biosynthesis and cell cycle G2/M phase transition were induced. The transcription factors and miRNAs involved in this response were also identified. These potent antiproliferative effects make EO a good candidate for the further analysis of its hepatic antitumor effects in in vivo studies.

A Diamine-PEGylated Oleanolic Acid Derivative Induced Efficient Apoptosis through a Death Receptor and Mitochondrial Apoptotic Pathway in HepG2 Human Hepatoma Cells.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Our recent studies have shown that the diamine-(PEG)ylated oleanolic acid (OADP) has strong anti-tumor effects in HCCs. In this study, we evaluated the anti-tumor mechanisms of OADP in the HepG2 HCC cell line. The cytotoxicity results showed that HepG2 cell viability was markedly reduced, with a very low 50% of cell growth inhibitory concentration (IC50, 0.14 µg/mL). We then investigated the anti-tumor mechanisms of OADP in HepG2 cells. The flow-cytometry analysis was used to evaluate cell apoptosis, indicating that 74-95% of cells were apoptotic. OADP caused cell cycle arrest in the G0/G1 phase and the loss of the mitochondrial membrane potential (MMP). Western blot analysis was performed to assess the expression levels of key proteins associated with the underlying molecular mechanism. The results showed the clear upregulation of caspase-8, caspase-9, caspase-3, Bak, p21, and p53, accompanied by the downregulation of Bcl-2. Similar results were obtained by the cotreatment with OADP and the c-Jun N-terminal kinase (JNK) inhibitor SP600125. Agents such as OADP, which are capable of activating extrinsic and intrinsic apoptotic pathways, may represent potential HCC cancer therapies.

NADPH production, a growth marker, is stimulated by maslinic acid in gilthead sea bream by increased NADP-IDH and ME expression.

NADPH plays a central role in reductive biosynthesis of membrane lipids, maintenance of cell integrity, protein synthesis and redox balance maintenance. Hence, NADPH is involved in the growth and proliferation processes. In addition, it has been shown that changes in nutritional conditions produced changes in NADPH levels and growth rate. Maslinic acid (MA), a pentacyclic triterpene of natural origin, is able to stimulate NADPH production, through regulation of the two oxidative phase dehydrogenases of the pentose phosphate pathway. Our main objective was to study the effects of MA on the kinetic behaviour and on the molecular expression of two NADPH-generating systems, NADP-dependent isocitrate dehydrogenase (NADP-IDH) and malic enzyme (ME), in the liver and white muscle of gilthead sea bream (Sparus aurata). Four groups of 12g of a mean body mass were fed for 210days in a fish farm, with diets containing 0 (control), and 0.1g of MA per kg of diet. Two groups were fed ad libitum (C-AL and MA-AL) and another's two, with restricted diet of 1% of fish weight (C-R and MA-R). Results showed that MA significantly increased the main kinetic parameter of the NADPH-forming enzymes (NADP-IDH and ME). In this sense, specific activity, maximum velocity, catalytic efficiency and activity ratio values were higher in MA conditions than control groups. Moreover, these changes were observed in both feeding regimen, AL and R. Meanwhile, the Michaelis constant changed mainly in groups fed with the MA and restricted diet, these changes are related to the best substrate affinity by enzyme. Moreover, in the Western-blot result, we found that MA increased both protein levels studied, this behaviour being consistent with the regulation of the number of enzyme molecules. All results, indicate that MA, independently of the fed regimen, could potentially be a nutritional additive for fish as it improved the metabolic state of fish, as consequence of increased activity and expression of NADP-IDH and ME enzymes.

Maslinic Acid, a Natural Triterpene, Induces a Death Receptor-Mediated Apoptotic Mechanism in Caco-2 p53-Deficient Colon Adenocarcinoma Cells.

Maslinic acid (MA) is a natural triterpene present in high concentrations in the waxy skin of olives. We have previously reported that MA induces apoptotic cell death via the mitochondrial apoptotic pathway in HT29 colon cancer cells. Here, we show that MA induces apoptosis in Caco-2 colon cancer cells via the extrinsic apoptotic pathway in a dose-dependent manner. MA triggered a series of effects associated with apoptosis, including the cleavage of caspases -8 and -3, and increased the levels of t-Bid within a few hours of its addition to the culture medium. MA had no effect on the expression of the Bax protein, release of cytochrome-c or on the mitochondrial membrane potential. This suggests that MA triggered the extrinsic apoptotic pathway in this cell type, as opposed to the intrinsic pathway found in the HT29 colon-cancer cell line. Our results suggest that the apoptotic mechanism induced in Caco-2 may be different from that found in HT29 colon-cancer cells, and that in Caco-2 cells MA seems to work independently of p53. Natural antitumoral agents capable of activating both the extrinsic and intrinsic apoptotic pathways could be of great use in treating colon-cancer of whatever origin.

Target molecules in 3T3-L1 adipocytes differentiation are regulated by maslinic acid, a natural triterpene from Olea europaea.

BACKGROUND: Metabolic syndrome is a set of pathologies among which stand out the obesity, which is related to the lipid droplet accumulation and changes to cellular morphology regulated by several molecules and transcription factors. Maslinic acid (MA) is a natural product with demonstrated pharmacological functions including anti-inflammation, anti-tumor and anti-oxidation, among others. PURPOSE: Here we report the effects of MA on the adipogenesis process in 3T3-L1 cells. METHODS: Cell viability, glucose uptake, cytoplasmic triglyceride droplets, triglycerides quantification, gene transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ) and adipocyte fatty acid-binding protein (aP2) and intracellular Ca2+ levels were determined in pre-adipocytes and adipocytes of 3T3-L1 cells. RESULTS: MA increased glucose uptake. MA also decreased lipid droplets and triglyceride levels, which is in concordance with the down-regulation of PPARγ and aP2. Finally, MA increased the intracellular Ca2+ concentration, which could also be involved in the demonstrated antiadipogenic effect of this triterpene. CONCLUSION: MA has been demonstrated as potential antiadipogenic compound in 3T3-L1 cells.

Maslinic Acid, a Triterpene from Olive, Affects the Antioxidant and Mitochondrial Status of B16F10 Melanoma Cells Grown under Stressful Conditions.

Maslinic acid (MA) is a natural compound whose structure corresponds to a pentacyclic triterpene. It is abundant in the cuticular lipid layer of olives. MA has many biological and therapeutic properties related to health, including antitumor, anti-inflammatory, antimicrobial, antiparasitic, antihypertensive, and antioxidant activities. However, no studies have been performed to understand the molecular mechanism induced by this compound in melanoma cancer. The objective of this study was to examine the effect of MA in melanoma (B16F10) cells grown in the presence or absence of fetal bovine serum (FBS). We performed cell proliferation measurements, and the reactive oxygen species (ROS) measurements using dihydrorhodamine 123 (DHR 123) and activities of catalase, glucose 6-phosphate dehydrogenase, glutathione S-transferase, and superoxide dismutase. These changes were corroborated by expression assays. FBS absence reduced cell viability decreasing IC50 values of MA. The DHR 123 data showed an increase in the ROS level in the absence of FBS. Furthermore, MA had an antioxidant effect at lower assayed levels measured as DHR and antioxidant defense. However, at higher dosages MA induced cellular damage by apoptosis as seen in the results obtained.

Solid-phase library synthesis of bi-functional derivatives of oleanolic and maslinic acids and their cytotoxicity on three cancer cell lines.

A wide set of 264 compounds has been semisynthesized with high yields and purities. These compounds have been obtained through easy synthetic processes based on a solid-phase combinatorial methodology. All the members of this library have one central core of a natural pentacyclic triterpene (oleanolic or maslinic acid) and differ by 6 amino acids, coupled with the carboxyl group at C-28 of the triterpenoid skeleton, and by 10 different acyl groups attached to the hydroxyl groups of the A-ring of these molecules. According to the literature on the outstanding and promising pharmacological activities of other similar terpene derivatives, some of these compounds have been tested for their cytotoxic effects on the proliferation of three cancer cell lines: B16-F10, HT29, and Hep G2. In general, we have found that around 70% of the compounds tested show cytotoxicity in all three of the cell lines selected; around 60% of the cytotoxic compounds are more effective than their corresponding precursors, that is, oleanolic (OA) or maslinic (MA) acids; and nearly 50% of the cytotoxic derivatives have IC50 values between 2- to 320-fold lower than their corresponding precursor (OA or MA).

Maslinic acid, a triterpenic anti-tumoural agent, interferes with cytoskeleton protein expression in HT29 human colon-cancer cells.

Maslinic acid (MA) is an anti-tumoural agent which shows potent anti-proliferative properties against the HT29 colon-cancer cells. To shed light upon the active mechanism of MA we have investigated its effects upon the cytoskeleton. We used a proteomics procedure based on two-dimensional gel electrophoresis, mass analysis and peptide mass fingerprinting. The incubation of HT29 cells with MA led to G1 cell-cycle arrest. After 24hours' exposure to 3.7µM (IC50/8) and 30µM (IC50) MA fourteen differentially expressed cytoskeletal proteins could be discerned. One group of these proteins, made up of keratin 2, keratin 8, keratin type II cytoskeletal 8, keratin type I cytoskeletal 9, keratin type I cytoskeletal 18, cytokeratins 18 and 19, and ß-actin, exert a structural function, whilst another group, made up of lamin B1, gelsolin 1, septin 2, villin 1, actin-related protein 2 and moesin, is related to the nucleation of actin and cytoskeleton formation. Changes in the expression of moesin, villin 1 and ß-actin identified by the proteomics techniques were corroborated by Western blotting. This is the first evidence obtained of the regulatory effects of MA on the cytoskeleton, which may prove to be one of the bases of its anti-proliferative effect against colon-cancer cells. BIOLOGICAL SIGNIFICANCE: In this paper we describe the changes in the expression of different cytoskeleton proteins identified by the proteomics techniques and corroborated by Western blotting. This is the first evidence obtained of the regulatory effects of MA on the cytoskeleton, which may prove to be one of the bases of its anti-proliferative effect against colon-cancer cells.