OAR@UM Collection:

Osteogenic bioactivity and oestrogen growth factor response induced by extracts from Ceratonia siliqua

Fri, 01 Jan 2010 00:00:00 GMT

Title: Osteogenic bioactivity and oestrogen growth factor response induced by extracts from Ceratonia siliqua Abstract: The main aim of the study was the production of an extract from a local indigenous plant that induces the differentiation of the osteoblast cell line MC3T3-El without inducing an estrogen growth factor response in the breast cancer cell line MCF-7, therefore being a suitable candidate for the substitution of the currently utilized hormone replacement therapy. A wide range of different methodologies have been used. Methanol extraction has been utilized for the extraction process. Titrations were performed so as to obtain the working concentrations for 17 β-oestradiol, the extracts, and a commercialized product (Lignan, Brevail®). To determine the cytotoxicity, XTT assays were performed. Alizarin Red staining measurements were utilized to directly measure the osteogenesis rate. Real time-PCR was utilized for expression studies of identified oestradiol response genes. The working concentrations of the test compounds have been identified and XTT assays indicated us that none of the test compounds tested (17β-oestradiol 10-4 M, lignan 5000 ppm, and carob pod extract 32750 ppm, carob flower extract 6000 ppm) was cytotoxic in the highest concentrations tested. The osteogenesis rate has been quantified and after 16 days, a 30-fold increase in osteogenesis has been obtained with lignan and a 20-fold increase in osteogenesis has been obtained with carob flower extract, when compared with osteogenesis induction medium. For the investigation of oestrogen growth factor response on the MCF-7 breast cancer cell line, 241 direct ERα target genes were selected utilizing a published dataset and utilizing another published dataset, 11 genes from the previous 241 were selected that contain estrogen response elements (EREs). Utilizing current literature and the involvement in breast cancer, a total of 3 out of 11 genes were selected; gene regulated by estrogen in breast cancer protein (GREBl), nuclear receptor interacting protein 1 (NRIPl), and insulin-like growth factor binding protein 4 (Igfbp4). Whilst ERα expression is downregulated in MCF-7 cells at both time points in all the test compounds utilized, expression of NRIPl and Igfbp4 is transient with the carob flower extract. Thus we concluded that the carob flower extract, is a potential candidate for the substitution of the currently utilized hormone replacement therapy. Description: M.SC.PATHOLOGY

Enhanced efficacy of bioactive compounds: targeting isoprenylation in cancer cells to mediate apoptosis

Fri, 01 Jan 2010 00:00:00 GMT

Title: Enhanced efficacy of bioactive compounds: targeting isoprenylation in cancer cells to mediate apoptosis Abstract: The study aimed to establish a combinatory treatment of low-dose isoprenoids with Rapamycin, an mTOR (mammalian target of Rapamycin) inhibitor, and a potential chemotherapeutic sensitizer. Isoprenylation of proteins has been involved in survival and transformation of cancer cells. Isoprenoids decrease the pool of isoprene precursor molecules, downregulating signal transduction mechanisms. The combinatory treatment served to sensitize tumour cells for induction of apoptosis by isoprenoids, enhancing the therapeutic index of isoprenoids. Three cell lines, a prostate carcinoma (PC3), melanoma (C32) and non small cell lung carcinoma (AS49) were chosen from a panel of cell lines. Isoprenoids (Limonene, Perillyl alcohol and α-pinene) were added alone or in combination with Rapamycin. Cytotoxicity assays (XTT) showed that Perillyl Alcohol was the most effective isoprenoid on all cell lines and addition of 50ng/ml Rapamycin sensitized PC3 and C32 cell lines resulting in a reduced dosage of α-pinene and Limonene to reach the IC50. The sensitivity of PC3 and C32 to rapamycin is supported by the presence of mutations in PTEN known to increase the activity of PI3K/Akt/mTOR pathway. Annexin V assay was used for apoptotic quantification by flow cytometry. Interestingly PC3 and C32 showed no evidence of apoptosis at the dosages required to reach IC50s, which may be caused by highly expressed anti-apoptotic proteins in such cell lines and could be possibly induced by rapamycin treatment. Increased apoptosis induction was observed in AS49 upon exposure to Perillyl alcohol and Limonene in combination with rapamycin. We hypothesize that the combination promotes an early G 1 block in the cell cycle of AS49 cell-line through Ras/MAPK and PI3K/Akt/mTOR pathways. The results obtained in this study can be utilized in further studies to characterize mechanisms of apoptosis induction. Of interest, the cytotoxic effect of isoprenoids alone and in combination using rapamycin sensitive cell lines shall give insights into other possible mechanisms of cell death such as autophagy. Description: M.SC.PATHOLOGY

Identification of natural polyphenols and plant extracts as potent inhibitors of lipid membrane destabilisation by amyloid-beta peptide (1-42)

Fri, 01 Jan 2010 00:00:00 GMT

Title: Identification of natural polyphenols and plant extracts as potent inhibitors of lipid membrane destabilisation by amyloid-beta peptide (1-42) Abstract: Amyloid-beta (Aβ) aggregation is a recognised key process in the pathogenesis of Alzheimer's Disease {AD}, the most common age-related neurodegenerative disorder. Misfolded Aβ peptides self-assemble sequentially into oligomers, protofibrils and fibrils; current evidence points to oligomers as being the primary neurotoxic Aβ species, especially because they are highly disruptive to lipid membranes. Hence the aim was to explore whether small-molecule compounds and bioactive natural extracts can inhibit aggregated Aβ from damaging lipid membranes. Using a combination of fluorophore-based lipid vesicle assays, Thioflavin T assays and immunoblotting, a robust protocol for Aβ(1-42) peptide aggregation into a range of ollgomers (20-220 kDa) having 30-40% liposomal toxicity was first established. Assays using liposomes loaded with FITC-dextran molecules of different sizes confirmed that the Aβ oligomers efficiently permeabilised the vesicle membranes. Next, 15 small-molecule polyphenolic compounds, 8 N'-benzylidene-benzohydrazide (NBB) compounds, 7 diphenylpyrazole (DPP) compounds, and 4 plant extracts were assessed for their ability to antagonise liposome permeabilisation by the Aβ(1-42) oligomers. Essentially, it was found that black tea extract, apigenin, baicalein and nordihydroguaiaretic acid (NDGA) potently inhibited lipid membrane damage by aggregated Aβ to < 30% of the control value (permeabilisation caused by Aβ). Solubilised extract from the marine plant Padina pavonica, scutellarein and synthetic DPP compound nr. 15 inhibited liposome permeabilisation by Aβ aggregates to "'40%. Interestingly, apigenin, baicalein and scutellarein are flavones and both NDGA and DPP nr. 15 are symmetrical molecules of similar length having a benzene ring at each end of the molecule. Synergism was found when pairwise combinations of DPP15, apigenin and NDGA were performed. Indeed, detailed structure-function analysis of our data could assist in the identification of common chemical scaffolds for inhibition of oligomer toxicity in neurodegenerative amyloidoses. Description: M.SC.PATHOLOGY