Differential roles and activation of mammalian target of rapamycin complexes 1 and 2 during cell migration in prostate cancer cells.

BACKGROUND: Mammalian target of rapamycin (mTOR) is a downstream substrate activated by PI3K/AKT pathway and it is essential for cell migration. It exists as two complexes: mTORC1 and mTORC2. mTORC1 is known to be regulated by active AKT, but the activation of mTORC2 is poorly understood. In this study, we investigated the roles and differential activation of the two mTOR complexes during cell migration in prostate cancer cells. METHODS: We used small interfering RNA to silence the expression of Rac1 and the main components of mTOR complexes (regulatory associated protein of mTOR [RAPTOR] and rapamycin-insensitive companion of mTOR [RICTOR]) in LNCaP, DU145, and PC3 prostate cancer cell lines. We performed transwell migration assay to evaluate the migratory capability of the cells, and Western blot analysis to study the activation levels of mTOR complexes. RESULTS: Specific knockdown of RAPTOR and RICTOR caused a decrease of cell migration, suggesting their essential role in prostate cancer cell movement. Furthermore, epidermal growth factor (EGF) treatments induced the activation of both the mTOR complexes. Lack of Rac1 activity in prostate cancer cells blocked EGF-induced activation of mTORC2, but had no effect on mTORC1 activation. Furthermore, the overexpression of constitutively active Rac1 resulted in significant increase in cell migration and activation of mTORC2 in PC3 cells, but had no effect on mTORC1 activation. Active Rac1 was localized in the plasma membrane and was found to be in a protein complex, with RICTOR, but not RAPTOR. CONCLUSION: We suggest that EGF-induced activation of Rac1 causes the activation of mTORC2 via RICTOR. This mechanism plays a critical role in prostate cancer cell migration.

Novel role of Giα2 in cell migration: Downstream of PI3-kinase-AKT and Rac1 in prostate cancer cells.

Tumor cell motility is the essential step in cancer metastasis. Previously, we showed that oxytocin and epidermal growth factor (EGF) effects on cell migration in prostate cancer cells require Giα2 protein. In the current study, we investigated the interactions among G-protein coupled receptor (GPCR), Giα2, PI3-kinase, and Rac1 activation in the induction of migratory and invasive behavior by diverse stimuli. Knockdown and knockout of endogenous Giα2 in PC3 cells resulted in attenuation of transforming growth factor ß1 (TGFß1), oxytocin, SDF-1α, and EGF effects on cell migration and invasion. In addition, knockdown of Giα2 in E006AA cells attenuated cell migration and overexpression of Giα2 in LNCaP cells caused significant increase in basal and EGF-stimulated cell migration. Pretreatment of PC3 cells with Pertussis toxin resulted in attenuation of TGFß1- and oxytocin-induced migratory behavior and PI3-kinase activation without affecting EGF-induced PI3-kinase activation and cell migration. Basal- and EGF-induced activation of Rac1 in PC3 and DU145 cells were not affected in cells after Giα2 knockdown. On the other hand, Giα2 knockdown abolished the migratory capability of PC3 cells overexpressing constitutively active Rac1. The knockdown or knockout of Giα2 resulted in impaired formation of lamellipodia at the leading edge of the migrating cells. We conclude that Giα2 protein acts at two different levels which are both dependent and independent of GPCR signaling to induce cell migration and invasion in prostate cancer cells and its action is downstream of PI3-kinase-AKT-Rac1 axis.

Psoralea glandulosa as a potential source of anticancer agents for melanoma treatment.

With the aim of identifying novel agents with antigrowth and pro-apoptotic activity on melanoma cancer, the present study was undertaken to investigate the biological activity of the resinous exudate of aerial parts from Psoralea glandulosa, and its active components (bakuchiol (1), 3-hydroxy-bakuchiol (2) and 12-hydroxy-iso-bakuchiol (3)) against melanoma cells (A2058). In addition, the effect in cancer cells of bakuchiol acetate (4), a semi-synthetic derivative of bakuchiol, was examined. The results obtained show that the resinous exudate inhibited the growth of cancer cells with IC50 value of 10.5 µg/mL after 48 h of treatment, while, for pure compounds, the most active was the semi-synthetic compound 4. Our data also demonstrate that resin is able to induce apoptotic cell death, which could be related to an overall action of the meroterpenes present. In addition, our data seem to indicate that the apoptosis correlated to the tested products appears, at least in part, to be associated with an increase of reactive oxygen species (ROS) production. In summary, our study provides the first evidence that P. glandulosa may be considered a source of useful molecules in the development of analogues with more potent efficacy against melanoma cells.

Development and validation of a sensitive LC-MS/MS assay of GT-14, a novel Gαi2 inhibitor, in rat plasma, and its application in pharmacokinetic study.

A sensitive and selective LC-MS/MS method was developed and validated for the quantitation of a novel Gαi2 inhibitor, GT-14, in rat plasma using a SCIEX 6500+ triple QUAD LC-MS system equipped with an ExionLC UHPLC unit. GT-14 (m/z 265.2 → 134.1) and griseofulvin (Internal Standard, IS) (m/z 353.1 → 285.1) were detected in a positive mode by electrospray ionization (ESI) using multiple reaction monitoring (MRM). The assay was linear in the concentration range of 0.78-1000 ng/mL in rat plasma. Both accuracy and precision values were within the acceptance criteria of ±15 %, as established by FDA guidance. The matrix effect was negligible from plasma, with signal percentages of 98.5-106.9 %. The mean recovery was 104.5 %, indicating complete extraction of GT-14 from plasma. GT-14 was found to be stable under different experimental conditions. The validated method was successfully applied to evaluate plasma protein binding and in vivo pharmacokinetics of GT-14 in rats.

Gαi2 Protein Inhibition Blocks Chemotherapy- and Anti-Androgen-Induced Prostate Cancer Cell Migration.

We have previously shown that heterotrimeric G-protein subunit alphai2 (Gαi2) is essential for cell migration and invasion in prostate, ovarian and breast cancer cells, and novel small molecule inhibitors targeting Gαi2 block its effects on migratory and invasive behavior. In this study, we have identified potent, metabolically stable, second generation Gαi2 inhibitors which inhibit cell migration in prostate cancer cells. Recent studies have shown that chemotherapy can induce the cancer cells to migrate to distant sites to form metastases. In the present study, we determined the effects of taxanes (docetaxel), anti-androgens (enzalutamide and bicalutamide) and histone deacetylase (HDAC) inhibitors (SAHA and SBI-I-19) on cell migration in prostate cancer cells. All treatments induced cell migration, and simultaneous treatments with new Gαi2 inhibitors blocked their effects on cell migration. We concluded that a combination treatment of Gαi2 inhibitors and chemotherapy could blunt the capability of cancer cells to migrate and form metastases.

Decrease of apoptosis markers during adipogenic differentiation of mesenchymal stem cells from human adipose tissue.

Although the proliferation and differentiation of mesenchymal stem cells (MSCs) from adipose tissue (AT) have been widely studied, relatively little information is available on the underlying mechanism of apoptosis during the adipogenic differentiation. Thus, the aim of this study was to analyze how the expression of some apoptotic markers is affected by in vitro expansion during adipogenic differentiation of AT-MSCs. The cultures incubated or not with adipogenic medium were investigated by Western blot at 7, 14, 21, and 28 days for the production of p53, AKT, pAKT, Bax, PDCD4 and PTEN. MSCs were recognized for their immunoreactivity to MSC-specific cell types markers by immunocytochemical procedure. The effectiveness of adipogenic differentiation was assessed by staining with Sudan III and examination of adipogenic markers expression, such as PPAR-γ and FABP, at different time points by Western blot. The adipogenic differentiation medium led to the appearance, after 7 days, of larger rounded cells presenting numerous vacuoles containing lipids in which it was evident a red-orange staining, that increased in size in a time-dependent manner, parallel to an increase of the levels of expression of PPAR-γ and FABP. More than 50 % of human MSCs were fully differentiated into adipocytes within the four-week induction period. The results showed that during adipogenic differentiation of AT-MSCs the PI3K/AKT signaling pathway is activated and that p53, PTEN, PDCD4, and Bax proteins are down-regulated in time-dependent manner. Our data provide new information on the behavior of some apoptotic markers during adipogenic differentiation of AT-MSCs to apply for tissues repair and regeneration.

Sodium L-lactate differently affects brain-derived neurothrophic factor, inducible nitric oxide synthase, and heat shock protein 70 kDa production in human astrocytes and SH-SY5Y cultures.

The present study analyzed the in vitro effects induced by sodium L-lactate on human astrocytes and the SH-SY5Y cell line, when added at concentrations of 5, 10, and 25 mmol/liter. Expression of brain-derived neurotrophic factor (BDNF), inducible nitric oxide synthase (iNOS), and heat shock protein 70 kDa (HSP70) was evaluated by Western blot analysis. Cell viability with MTT, release of nitric oxide (NO) through the Griess reaction, and production of BDNF by enzyme-linked immunoassay was determined. Data indicate that, in SH-SY5Y as well as in cortical astrocytes, after 4 hr sodium L-lactate increases the expression and release of BDNF, iNOS, and NO; after 24 hr, it turns is ineffective for the production of the neurotrophin in SH-SY5Y and not in astrocytes, but the expression of iNOS and release of NO appear to be further increased compared with those after 4 hr. Sodium L-lactate influences differently the expression of HSP70 in SH-SY5Y compared with astrocytes. We propose, based on these findings, that sodium L-lactate affects the expression of BDNF in SH-SY5Y and astrocytes in a different manner: high levels of iNOS and NO expressed in SH-SY5Y have a profound inhibitory effect on the release of BDNF related to a more limited production of HSP70 by SH-SY5Y. In conclusion, the results demonstrate differences in the responses of SH-SY5Y and astrocytes to stimulation by high levels of sodium L-lactate. Sodium L-lactate differently and dose and time dependently influences the expression and release of BDNF, iNOS, NO, and HSP70 depending on the cell type.

Phytochemical profile and apoptotic activity of Onopordum cynarocephalum.

A phytochemical investigation of acetone and chloroform extracts of the aerial parts of Onopordum cynarocephalum Boiss. et Blanche was carried out. It led to the isolation of two new sesquiterpenes, the elemane aldehyde (2) and the eudesmane (11), together with 15 known compounds: two lignans (1 and 15) and 13 sesquiterpenes (3-10, 12-14, 16, 17). The structures were elucidated by spectroscopic analyses, especially 1D and 2D NMR spectra. The anti-growth effect against three human melanoma cell lines, M14, A375, and A2058, of the different extracts and compounds of O. cynarocephalum was also investigated. Among them, the chloroform extract exhibited the strongest biological activity, while the most active compounds were the lignan arctigenin (1), and the sesquiterpenes, compounds 3, 5, and 6 belonging to the elemane type, and 7 belonging to the eudesmane type. Our data also demonstrate that acetone and chloroform extracts induce, in the A375 cell line, apoptotic cell death that could be related to an overall action of the compounds present, but in particular to the lignans arctigenin (1) and the sesquiterpenes compounds 3-8 and 16. In fact, these molecules were able to induce a high DNA fragmentation, correlated to a significant increase of the caspase-3 enzyme activity. Furthermore, apoptosis appears to be mediated, at least in part, via PTEN activity and the inhibition of Hsp70 expression.

Modulation of YY1 and p53 expression by transforming growth factor-ß3 in prostate cell lines.

Transforming growth factor-ß (TGF-ß) is the prototype of a family of secreted polypeptide growth factors. These cytokines play very important roles during development, as well as in normal physiological and disease processes, by regulating a wide array of cellular processes, such as cell growth, differentiation, migration, apoptosis, and extracellular matrix production. TGF-ß utilizes a multitude of intracellular signalling pathways in addition to Smads with actions that are dependent on circumstances, including dose, target cell type, and context. The aims of this research were (i) to verify the effects of dose-dependent TGF-ß3 treatment on YY1 and p53 expression, in BPH-1 cell line, human benign prostate hyperplasia, and two prostate cancer cell lines, LNCaP, which is androgen-sensitive, and DU-145, which is androgen-non responsive, (ii) establish a correlation between p53 and YY1 and (iii) determine the expression of a number of important intracellular signalling pathways in TGF-ß3-treated prostate cell lines. The expression of YY1, p53, PI3K, AKT, pAKT, PTEN, Bcl-2, Bax, and iNOS was evaluated through Western blot analysis on BPH-1, LNCaP, and DU-145 cultures treated with 10 and 50 ng/ml of TGF-ß3 for 24 h. The production of nitric oxide (NO) was determined by Griess reagent and cell viability through MTT assay. The results of this research demonstrated profound differences in the responses of the BPH-1, LNCaP, and DU-145 cell lines to TGF-ß3 stimulation. We believe that the findings could be important because of the clinical relevance that they may assume and the therapeutic implications for TGF-ß treatment of prostate cancer.

Mineral fibre toxicity: expression of retinoblastoma (Rb) and phospho-retinoblastoma (pRb) protein in alveolar epithelial and mesothelial cell lines exposed to fluoro-edenite fibres.

Several asbestos-like mineral fibres, including fluoro-edenite, may cause lung cancer and/or other lung diseases. However, biological and molecular mechanisms linked to cancer development after mineral fibre exposure have not been fully investigated. In the present study, human non-malignant mesothelial (MeT-5A) and human bronchoalveolar alveolar epithelial (A549) cell lines were incubated with rising concentrations of fluoro-edenite to evaluate the expression of retinoblastoma (Rb) protein, which has been demonstrated to play an important role in cell cycle control and tumour progression. Intriguingly, these results show that Rb expression was unchanged, while the level of the phosphorylated protein increased significantly in a dose-dependent manner, suggesting an involvement of this regulator protein in the pathogenesis of the lung diseases induced by mineral fibres. In conclusion, fluoro-edenite regulates the expression of phospho-retinoblastoma to trigger a network of signals strictly connected with cell proliferation and neoplastic cell transformation.

Dehydroxymethylepoxyquinomicin inhibits expression and production of inflammatory mediators in interleukin-1beta-induced human chondrocytes.

The present research was carried out to determine the effects of a nuclear factor-kappaB (NF-kappaB) inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), derivative of the antibiotic epoxyquinomicin C, on normal human chondrocytes treated with interleukin-1beta (IL-1beta). This is a cell model particularly useful to reproduce the mechanisms involved in degenerative arthropathies, where oxidative-inflammatory stress determines a progressive destruction of the articular cartilaginous tissue. The expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and inter-cellular adhesion molecule (ICAM)-1 was evaluated through Western blot analysis. The release of chemokines like monocyte chemoattractant protein-1 (MCP-1), regulated upon normal activation T-cell expressed and secreted (RANTES), and interleukin-8 (IL-8) were determined by ELISA assays. DHMEQ acts as a potent inhibitor of iNOS and COX-2 gene expression while also suppressing the production of nitrite in human chondrocytes. In addition, DHMEQ induces a significant dose-dependent decrease in ICAM expression, MCP-1, RANTES, and IL-8 release. DHMEQ helps to decrease the expression and production of pro-inflammatory mediators in IL-1beta-induced chondrocytes. DHMEQ may become a therapeutic agent for treatment of chondro-degenerative diseases.

Dehydroxymethylepoxyquinomicin, a novel nuclear factor-kappaB inhibitor, prevents inflammatory injury induced by interferon-gamma and histamine in NCTC 2544 keratinocytes.

1. The novel nuclear factor (NF)-kappaB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) is a derivative of the antibiotic epoxyquinomicin C from Amycolatopsis sp. that has been found to inhibit tumour necrosis factor (TNF)-alpha-induced activation of NF-kappaB by suppressing nuclear translocation of NF-kappaB. The aim of the present study was to determine the effects of DHMEQ on interferon (IFN)-gamma- and histamine-activated NCTC 2544 keratinocytes. 2. Keratinocytes were stimulated or not with 200 U/mL IFN-gamma and 10(-4) mol/L histamine in the absence or presence of different concentrations of DHMEQ (1, 5 and 10 microg/mL) or hydrocortisone (10(-5) mol/L), which was used as a reference anti-inflammatory drug. After 48 h, each sample was tested for the presence of intercellular adhesion molecule (ICAM)-1 by western blot analysis, as well as for the release of monocyte chemoattractant protein (MCP)-1, RANTES and interleukin (IL)-8 using specific sandwich ELISAs. To verify the effect of DHMEQ on cell viability of non-stimulated NCTC 2544 keratinocytes, the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used. 3. The results showed that 10 microg/mL DHMEQ potently inhibited ICAM-1 production (by 50%), as well as the release of MCP-1 (to 25% of control), RANTES (to 5% of control) and IL-8 (to 2% of control). The results of the MTT assay indicated that DHMEQ has no effect on cell viability. 4. In conclusion, DHMEQ inhibits the IFN-gamma- and histamine-induced activation of the keratinocyte cell line NCTC 2544. The anti-inflammatory effects of DHMEQ could be exploited by applying the drug topically alone or in combination with sub-toxic concentrations of anti-inflammatory drugs to producer a synergistic effect.

Small Molecule Inhibitors Targeting Gαi2 Protein Attenuate Migration of Cancer Cells.

Heterotrimeric G-proteins are ubiquitously expressed in several cancers, and they transduce signals from activated G-protein coupled receptors. These proteins have numerous biological functions, and they are becoming interesting target molecules in cancer therapy. Previously, we have shown that heterotrimeric G-protein subunit alphai2 (Gαi2) has an essential role in the migration and invasion of prostate cancer cells. Using a structure-based approach, we have synthesized optimized small molecule inhibitors that are able to prevent specifically the activation of the Gαi2 subunit, keeping the protein in its inactive GDP-bound state. We observed that two of the compounds (13 and 14) at 10 µΜ significantly inhibited the migratory behavior of the PC3 and DU145 prostate cancer cell lines. Additionally, compound 14 at 10 µΜ blocked the activation of Gαi2 in oxytocin-stimulated prostate cancer PC3 cells, and inhibited the migratory capability of DU145 cells overexpressing the constitutively active form of Gαi2, under basal and EGF-stimulated conditions. We also observed that the knockdown or inhibition of Gαi2 negatively regulated migration of renal and ovarian cancer cell lines. Our results suggest that small molecule inhibitors of Gαi2 have potential as leads for discovering novel anti-metastatic agents for attenuating the capability of cancer cells to spread and invade to distant sites.

Raf kinase inhibitor protein (RKIP) and phospho-RKIP expression in melanomas.

Melanoma, a cancer notorious for its high potential to metastasize, arises from melanocytes, cells dedicated to melanin production and located in the basal layer of the epidermis. Raf-1 kinase inhibitor protein (RKIP) is an inhibitory molecule that down-regulates the effects of the Ras/Raf/MEK/ERK signaling pathway. The aim of this study was to examine the expression of RKIP and pRKIP in melanomas at different stages. We evaluated the RKIP and pRKIP protein by immunohistochemistry in control skin, pigmented nevi and melanomas, and through Western blotting in human normal melanocytes and in four different melanoma-derived cell lines (WM35, A375, M14, and A2058). Our results demonstrated a correlation between the expression of RKIP and pRKIP, and metastatic ability in melanoma cells. This raises the possibility to analyze both RKIP and pRKIP in all melanomas. Down-regulation of both RKIP and pRKIP expression could represent a useful marker of metastatic melanoma. On the contrary for non-metastatic melanoma, especially in Clark I and II, low RKIP and high pRKIP expression could be indicative. In conclusion, the observed negative correlation of the RKIP and pRKIP expression in metastatic melanomas indicates that expression of these proteins may become a prognostic marker for the progression of human cutaneous melanoma. We propose that the investigation of both RKIP and pRKIP may provide a useful tool indicative for metastatic or non-metastatic melanoma in different Clark's level melanomas. Further studies are required to verify the molecular background of the observed RKIP and pRKIP variations.

Evaluation of monooleine aqueous dispersions as tools for topical administration of curcumin: characterization, in vitro and ex-vivo studies.

Curcumin (CUR) is a well-known natural compound showing antioxidant, anti-inflammatory, and antitumor abilities but characterized by poor bioavailability and chemical instability, which drastically reduce its application in the treatment of chronic diseases such as osteoarthritis. The aim of the present study is the design and evaluation of monooleine aqueous dispersion (MAD) as novel carriers for the topical administration of CUR. CUR-loaded MAD was formulated using two different emulsifier systems, namely poloxamer 407 (MAD-A) and sodium cholate-sodium caseinate (MAD-B). These vehicles were characterized, and their influence on in vitro percutaneous absorption of CUR was also evaluated. Furthermore, an oxygen radical absorbance capacity assay was used to determine their antioxidant activity, and a Western blot analysis was performed to evaluate the inhibitory effect of the formulations on inducible nitric oxide synthase and cyclooxygenase 2 expressions. From the obtained results, CUR encapsulation efficiency was higher than 98% for MAD-A and 82% for MAD-B. Shelf-life studies showed that MAD-A maintains CUR stability better than MAD-B, and both vehicles demonstrated, in vitro, control of drug diffusion through the skin. Finally, MAD-A and MAD-B were able to extend the antioxidant/anti-inflammatory effects of CUR, also confirming the protective effect toward CUR chemical stability.

A new jasmonic acid stereoisomeric derivative induces apoptosis via reactive oxygen species in human prostate cancer cells.

With the aim of identifying novel agents with antigrowth and pro-apoptotic activity on prostate cancer cells, in the present study, we evaluated the effect of a (-)-jasmonic acid derivative, the 3-hydroxy-2(S)-(2Z-butenyl)-cyclopentane-1(S)-acetic acid, obtained by biotransformation, on cell growth in androgen-sensitive (LNCaP) and androgen-insensitive (DU-145) human prostate cancer cells. The results obtained show that the new compound was able to inhibit the growth of both prostate cancer cells. In addition, our data seem to indicate that the apoptosis evocated by this new molecule, at least in part, appears to be associated with an increase of reactive oxygen species (ROS) production.

Boldo prevents UV light and nitric oxide-mediated plasmid DNA damage and reduces the expression of Hsp70 protein in melanoma cancer cells.

OBJECTIVES: This study was designed to investigate the potential protective effect of a methanolic extract of Peumus boldus leaves on UV light and nitric oxide (NO)-mediated DNA damage. In addition, we investigated the growth inhibitory activity of this natural product against human melanoma cells (M14). METHODS: Boldine, catechin, quercetin and rutin were identified using a HPLC method. The extract was incubated with plasmid DNA and, before irradiating the samples with UV-R, H(2) O(2) was added. For analysis of DNA single-strand breaks induced by NO, the experiments were performed by incubating the extract with Angeli's salt. In the study on M14 cell line, cell viability was measured using MTT assay. Release of lactate dehydrogenase, a marker of membrane breakdown, was also measured. For the detection of apoptosis, the evaluation of DNA fragmentation (COMET assay) and caspase-3 activity assay were employed. The expression of heat shock protein 70 (Hsp70) was detected by Western blot analysis. Generation of reactive oxygen species was measured by using a fluorescent probe. KEY FINDINGS: The extract (demonstrating the synergistic effect of the constituents boldine and flavonoids), showed a protective effect on plasmid DNA and selectively inhibited the growth of melanoma cells. But a novel finding was that apoptosis evoked by this natural product in M14 cells, appears to be mediated, at least in part, via the inhibition of Hsp70 expression, which may be correlated with a modulation of redox-sensitive mechanisms. CONCLUSIONS: These results confirm the promising biological properties of Peumus boldus and encourage in-vivo investigations into its potential anti-cancer activity.

Mesenchymal stem cells from adipose tissue which have been differentiated into chondrocytes in three-dimensional culture express lubricin.

The present study focused on the isolation, cultivation and characterization of human mesenchymal stem cells (MSCs) from adipose tissue and on their differentiation into chondrocytes through the NH ChondroDiff medium. The main aim was to investigate some markers of biomechanical quality of cartilage, such as lubricin, and collagen type I and II. Little is known, in fact, about the ability of chondrocytes from human MSCs of adipose tissue to generate lubricin in three-dimensional (3D) culture. Lubricin, a 227.5-kDa mucinous glycoprotein, is known to play an important role in articular joint physiology, and the loss of accumulation of lubricin is thought to play a role in the pathology of osteoarthritis. Adipose tissue is an alternative source for the isolation of multipotent MSCs, which allows them to be obtained by a less invasive method and in larger quantities than from other sources. These cells can be isolated from cosmetic liposuctions in large numbers and easily grown under standard tissue culture conditions. 3D chondrocytes were assessed by histology (hematoxylin and eosin) and histochemistry (Alcian blue and Safranin-O/fast green staining). Collagen type I, II and lubricin expression was determined through immunohistochemistry and Western blot. The results showed that, compared with control cartilage and monolayer chondrocytes showing just collagen type I, chondrocytes from MSCs (CD44-, CD90- and CD105- positive; CD45-, CD14- and CD34-negative) of adipose tissue grown in nodules were able to express lubricin, and collagen type I and II, indicative of hyaline cartilage formation. Based on the function of lubricin in the joint cavity and disease and as a potential therapeutic agent, our results suggest that MSCs from adipose tissue are a promising cell source for tissue engineering of cartilage. Our results suggest that chondrocyte nodules producing lubricin could be a novel biotherapeutic approach for the treatment of cartilage abnormalities.

New stereoisomeric derivatives of jasmonic acid generated by biotransformation with the fungus Gibberella fujikuroi affect the viability of human cancer cells

Background: Several studies have shown that (-)-Jasmonic acid, (+)-7-iso-Jasmonic acid and its methyl ester, methyl jasmonate, have anti-cancer activity in vitro and in vivo, exhibiting selective cytotoxicity towards cancer cells. The degree of activity of these molecules is strongly related to their stereochemistry. The biotransformation of known compounds, natural or synthesized, related to interesting biological activities, generates new molecules displaying new improved properties compared with the original ones, increasing its value and providing new more effective products. Therefore, based on the above rationales and observations, in this work a biotransformation protocol to modify the chemical structure of the plant hormone jasmonic acid by using the fungus Gibberella fujikuroi was established. Results: The three jasmonic acid derivatives obtained, 3(S)-Hydroxy-2(R)-(2Z-pentenyl)-cyclopentane-1(R)-acetic acid (1), 3(R)-Hydroxy-2(R)-(2Z-pentenyl)-cyclopentane-1(R)-acetic acid (2), 3-Hydroxy-2(S)-(2Z-pentenyl)-cyclopentane-1(S)-acetic acid (3), were tested for cell-growth inhibition activity towards the human cancer epithelial cell line, the oral squamous carcinoma cells (KB). The results obtained show that jasmonic acid derivatives (1-3) are active on human cancer cells examined in different concentration ranges, with IC50 value less than of 25 uM. The compound 3, with the same molecular structure of compounds 1 and 2, but with different stereochemistry, was more active confirming that the activity of jasmonate compounds is related to their stereochemistry and to substituents in the cyclopentane ring. In this study, we also tested the potential proapoptotic activity of compound 3, and our data suggest that it, as other jasmonate compounds, is able to trigger apoptotic death in cancer cells. This event may be correlated at an elevation of reactive oxygen species (ROS). Administration of N-acetylcysteine (NAC) prevented compound 3 cytotoxicity...