Role of the GATA3 Transcription Factor on AR Signalling in Breast Cancer

Almost 80 % of all breast cancers (BCs) are dependent on the estrogen receptor alpha (ER) for their growth (termed ER+ BC), while the rest are negative for the expression of ER (ER- BC) (~ 20-25 %). Most ER+ BCs (more than 90 %) and around 50 % of ER- BCs express the androgen receptor (AR). AR is an important BC biomarker, with prognostic and therapeutic potential. AR is a tumor-suppressor in ER+ BC, where it inhibits estrogen-stimulated growth of tumours through down-regulation of key ER-regulated cell cycle genes and activation of good outcome genes. AR has been suggested to play a role in promoting a basal to luminal lineage transition in normal mouse mammary epithelial cells. Despite the role of AR in ER- BCs being controversial, AR positivity has been shown to be associated with improved disease-free survival and more benign clinical and pathologic factors (e.g. lower tumor grade and smaller tumor size) in ER- BCs. In the recent past, the GATA3 transcription factor (TF) has been characterized as an important regulator of ER signalling and mediates normal mammary gland development and cell lineage determination. GATA3 is expressed exclusively in the luminal epithelial cell population, plays an essential role in mammary development and specification, and actively maintains the luminal epithelial differentiation in the adult mammary gland. To date, the role of GATA3 in AR signalling in the context of BC, in the presence or absence of ER, has not been investigated. Therefore, the aim of this thesis was to further investigate and characterise the cross-talk between AR and GATA3 in the presence or absence of ER, using a variety of cell-line models, clinical samples and where possible, patient-derived xenografts (PDXs). I first investigated the functional interplay between GATA3 and AR in inducing the luminal epithelial lineage in breast cancer cells regardless of ER expression. Since the inhibitory role of AR in ER+ BCs is well established, I then investigated whether GATA3 is involved in AR-induced growth inhibition in ER+ BC models in response to ER and/or AR activation. Findings of this PhD thesis showed: • GATA3 is a novel AR interacting protein independent of ER expression in normal mammary tissues and different BC subtypes. • Stimulation of BC cells with estrogen or androgen hormones reprograms the GATA3 cistrome in ER+ and ER- BC cell lines and ER+ PDX models. • GATA3 and AR co-regulate the expression of essential luminal epithelial markers in both ER+ and ER- cell lines and ER+ PDXs, indicating a key collaborative role for GATA3 and AR in luminal epithelial differentiation of the breast cells regardless of the ER expression. • AR requires the presence of GATA3 at genomic loci associated with AR-mediated growth inhibition in ER+ BC to inhibit the E2-stimulated growth in these models. Collectively, the generated data from this thesis suggest a cooperative role for AR and GATA3 TFs in suppressing the tumour growth of ER+ BCs and in driving the luminal-lineage identity in mammary glands and BC contexts. Also, the findings of this thesis provide novel insight into the cross-talk between ER, GATA3, and AR in BC, highlight the signalling complexity of TFs in this disease and provide the basis for further investigations into AR and GATA3 co-operative genomic activity and the direct consequences of this for BC progression and differentiation.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 202

Synthesis of hybridized benzylthio-1,3,4-thiadiazol-isatin derivatives and in vitro cytotoxicity evaluation

Introduction: In this research synthesis of hybridized benzylthio-thiadiazol-isatin derivatives has been reported and then the effects of the synthesized compounds were  investigated on cancer cell lines and molecular docking was also studied on proposed receptor. Methods and Results: This project was done in 2 steps that includes the synthesis of new hybrids of thiadiazole-isatin derivatives and characterized by various spectroscopy methods such as "Mass spectroscopy, Infrared spectroscopy, and 1H NMR". To study cytotoxic effects of the compounds, different concentrations of synthesized derivatives were  prepared and tested on the three rank 7 cellular MCF-7 "breast cancer", PC3 "Prostate carcinoma", and SKNMC "Norobelastoma". The method used was MTT that after various stages of the solution and added MTT, the color was measured by the producted formazan during measurements suitable wave. The color ratio was  as equal as  the number of living cells. For comparing the  cytotoxicity we  used doxorubicin as control drug. Conclusions: The most potent of the compounds were 3b, 4c, and 4d against MCF7 cell line, 3b, 4h against PC3 cell line, and 3b,4f, and 4h against SKNMC cell line which seems to be the best ones relative to the control drug. Also we found that treatment with 3b led to  decrease in IC50 and significantly increased cytotoxicity effects of the compound in PC3, SKNMC and MCF7 cells lines

Preparation and physicochemical characterization of prazosin conjugated PLGA nanoparticles for drug delivery of flutamide

In the current work, a sustained drug delivery system of flutamide (FLT) was developed using Poly(D,L‑lactide-co-glycolide) (PLGA) decorated bypoly(ethylene glycol) (PEG) grafted prazosin (PLGA-PEG-Praz) as a targeting moiety. In a multi-step reaction, PLGA was linked to PEG and prazosin. The structure of the synthesized polymers was confirmed by FTIR and 1 H-NMR. Flutamide-loaded nanoparticles were prepared by quasi-emulsion solvent diffusion technique. The nanoparticles were evaluated for size, zeta potential, polydispersity index, drug crystallinity, loading efficiency, and release properties. Also, the physicochemical properties of the nanoparticles were analyzed using Scanning Electron Microscopy (SEM), Differential Scanning Calorimetry, and Powder X-Ray Diffractometry (XRD). The particle size of nanoparticles was ranged between 191 and 249 nm. Loading efficiency of nanoparticles was about 43%-69%. Results showed a steady release rate for nanoparticles compared to that of a pure drug powder. SEM characterization confirmed that particles were in nanosize range. DSC and XRPD results verified a decrease in drug crystallinity in the prepared formulations. In conclusion, the results of this study showed that PLGA-PEG-Praz nanoparticles could be a good choice to improve the physicochemical properties of the drug and these formulations can increase Flutamide efficac

Acute and subchronic toxicological evaluation of Echinophora platyloba DC (Apiaceae) total extract in Wistar rats

OBJECTIVE: Echinophora platyloba DC is a widely used herbal medicine and food seasoning in Iran. It is claimed to exert antimicrobial, antifungal, and antispasmodic effects. Despite the prevalent use of this plant as a food and medicine, there are no reports on its possible toxic effects. To evaluate the safety of E. platyloba, we tested its acute and sub-chronic toxicity in male and female Wistar rats. METHODS: Rats were orally treated with four different single doses of E. platyloba total extract and screened for signs of toxicity two weeks after administration. In the sub-chronic toxicity study, E. platyloba was administered for 45 days. Mortality, clinical signs, body weight changes, hematological and biochemical parameters, gross findings, organ weights, and histological markers were monitored during the study. RESULTS: We found no mortality and no abnormality in clinical signs, body weight, or necropsy findings in any of the animals in the acute study. The results of the subchronic study showed no significant difference in hematological parameters in either sex. There was a significant increase in lactate dehydrogenase in the female groups. A significant increase in the relative lung weight of female rats was noted at 500 mg/kg. Histopathological examinations revealed intra-alveolar hemorrhage in the male rats (500 mg/kg). In the females, congestion of the alveolar capillaries (at 500 mg/kg) and liver bridging necrosis (at 200 mg/kg) were significantly increased. CONCLUSION: The no observed adverse effect level of E. platyloba was determined to be 200 and 50 mg/kg for male and female rats, respectively

Evaluation of antioxidant and cytoprotective activities of Artemisia ciniformis extracts on PC12 cells

Objective(s): In the current study antioxidant capacities of five different extracts of Artemisia ciniformis aerial parts were evaluated by cell-free methods. Then seven fractions of the potent extract were selected and their antioxidant capacity was assayed by cell free and cell based methods. Materials andMethods: Antioxidant ability was measured using the: 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging test, β-carotene bleaching (BCB) method and ferrous ion chelating (FIC) assay. Total phenolic contents (TPC) of all the samples also were determined. The cytoprotective effect of fractions was evaluated by measuring the viability of cells after exposure to doxorubicin (DOX). The mechanism of action was studied by investigating caspase-3, mitochondrial membrane potential (MMP), the level of super-oxide dismutase (SOD) and intracellular reactive oxygen species (ROS). Results: Hydroethanolic extract exhibited a notably higher antioxidant activity and phenolic content. Among the fractions (A to G) of hydroethanolic extract, the highest antioxidant capacity was observed in the Fraction E. Moreover, 24 hr pretreatment of PC12 cells with fractions B, C and D decreased DOX-induced cytotoxicity. In addition, pre-treatment of cells with fraction B resulted in significant decrease in generation of the reactive oxygen species (ROS) and increase in the activity of SOD. We were able to demonstrate remarkable reduction in the activity of caspase-3 and increase in MMP in PC12 cells following pretreatment with fraction B. Conclusion: Our observations indicated that the fraction B of A. ciniformis hydroetanolic extract possessed protective effect on oxidative stress and apoptosis induced by DOX in PC12 cells

Evaluation of the Effect of Six Terpenoids and Phenolics from Echinophora Cinerea against Cisplatin-Induced Oxidative Stress and Apoptosis in PC12 Cell Line

Introduction:Echinophoracinerea is a plant from Apiaceae family and it is used as vegetable, yogurt and cheese seasoning and is used for gasteric ailments in ChaharMahalBakhtiari province. It is a rich source of antioxidant constituents, hence it canpotentially  have protective effects. So, its phytochemical investigation seems to be crucial. Methods and Results:Plant material was extracted. The latter extract was fractionated with VLC and further purified using reversed phase HPLC. The structures of pure compounds were elucidated using spectroscopic methods such as 1HNMR and mass. Cytotoxic effects of cisplatin alone and with other fractions were tested. The effects of isolated compounds against apoptosis induced by CIS were investigated through the measurement of mitochondrial membrane potential, Bax and Bcl2 and caspase-3 activation. We also assessed the oxidative stress by measuring reactive oxygen species. Six compounds (quercetin-3-O-β-D-glucopyranoside, Kaempferol glycoside, osthol, verbenone, isoimperatorin and echinophorin B) were purified and identified. Treatment of cells with QUE and OST before exposure to the CIS increased cell viability. These compounds protected the cells against CIS–induced cytotoxicity. In addition, pretreatment with QUE and OST decreased CIS- induced apoptosis through up-regulation of Bcl2, inhibition of caspase-3 activity and increasing of mitochondrial membrane potential. As well, OST decreased ROS generation. Conclusion:Given that flavonoids are the most important groups of phenolic compounds found in nature, and due to their antioxidant and antiapoptotic effect these could be considered as neuroprotective agent

Investigating the Cytotoxicity of Folate-Conjugated Bismuth Oxide Nanoparticles on KB and A549 Cell Lines

Purpose: Lately, bismuth-based nanomaterials have been widely utilized in medical researches such as imaging, drug delivery and radio-sensitization. Despite their advantages, bismuth-based compounds have shown toxic effects in humans. There are few studies on cytotoxicity effects of bismuth oxide (Bi2O3) nanoparticles (NPs) in-vitro. In this study, we aimed to investigate cytotoxicity of bare and also folate and 5-aminolevulinic acid (5-ALA)-conjugated Bi2O3 NPs on nasopharyngeal carcinoma (KB) and lung cancer (A549) cell lines. Methods: Bi2O3 NPs were synthesized and conjugated with folate and 5-ALA. KB and A549 cells were cultured and incubated with 10, 20, 50 and 100 μg/ml concentrations of bare and folate-5-ALA-conjugated NPs. The survival rates were obtained after 2 and 24 hours incubation of the cells with NPs using MTT assay. Also, apoptosis and ROS generation induced by the NPs in the treated cells were obtained using Caspases-3 activity assay and flow cytometry analysis, respectively. Results: Bi2O3 NPs were successfully synthesized with average size of 19.2 ± 6.5 nm, then conjugated with 5-ALA and folate. Either naked or folate-conjugated NPs were easily taken up by the cells in a concentration-dependent manner and showed cytotoxic effects. The significant cell death was noted at the concentrations more than 50 μg/ml for both compounds. Conclusion: Results indicated low cytotoxicity of the prepared NPs at lower incubation periods, which is very important for their further applications. However, 24 hours incubation of the cells with both forms of NPs caused more cell killing and the cytotoxicity increased with increasing concentrations of the NPs

Protective effect of bioactive compounds from Echinophora cinerea against cisplatin-induced oxidative stress and apoptosis in the PC12 cell line

Objective(s): The present study aims to evaluate the protective effect of the compounds isolated from Echinophora cinerea (E. cinerea) against oxidative stress and apoptosis induced by cisplatin (CIS) in PC12 cells. Materials and Methods: Six compounds were isolated as quercetrin-3-O-β-D-glucopyranoside (QUE), osthol (OST), verbenone-5-O-β-D-glycopyranoside (VER), Isoimperatorin (ISO), kaempferol-3-O-β-D-glucopyranoside (KAM), and echinophorin B (ECH). For this study, we used MTT reduction assay for detection of protective effects of isolated compounds on CIS-induced cytotoxicity in PC12 cells. The effects of isolated compounds against apoptosis induced by CIS were investigated through the measurement of mitochondrial membrane potential (MMP), Bax and Bcl2 mRNA expression, and caspase-3 activation. We also assessed oxidative stress by measuring reactive oxygen species (ROS) generation with 2′, 7′-dichlorofluorescein diacetate (DCFH-DA). Results: Treatment of cells with QUE and OST before exposure to the CIS increased cell viability, i.e., these compounds protected the cells against CIS -induced cytotoxicity. In addition, pre-treatment with QUE and OST decreased CIS-induced apoptosis through up-regulation of Bcl-2, inhibition of caspase-3 activity, and mitochondrial membrane potential (MMP) increase. OST decreased ROS generation induced by CIS, as well. Conclusion: Our in vitro experiment showed that QUE and OST are apoptotic inhibitors that effectively block CIS-induced neurotoxicity predicting their therapeutic potential in the prevention of chemotherapy-induced neurotoxicity

Expression of Recombinant Human Coagulation Factor VII by the Lizard Leishmania Expression System

The variety of recombinant protein expression systems have been developed as a resource of FVII gene expression. In the current study, the authors used a novel protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host for expression of FVII. Plasmid containing cDNA encoding full-length human FVII was introduced into Lizard Leishmania and positive transfectants were analyzed by SDS-PAGE and Western blot analysis. Furthermore, biological activity of purified protein was detected by PT assay. The recombinant strain harboring a construct was analyzed for expression of FVII at the mRNA and protein level. Purified rFVII was obtained and in order to confirm the purified compound was in fact rFVII. Western blot analysis was carried out. Clotting time in PT assay was reduced about 30 seconds with the purified rFVII. In Conclusion, this study has demonstrated, for the first time, that Leishmania cells can be used as an expression system for producing recombinant FVII