Project description:DU 145 prostate cancer cells were treated with and without 100?g/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland). Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation, the first time point (0 min) was harvested for both treated and untreated cells. Simultaneously, Actinomycin D (Sigma-Aldrich, St Louis, MO) with the final concentration of 5ug/ml, was added to the remaining treated and untreated cells to stop new transcription. Time points of 10min, 30min, 1h, 2h, 4h and 8h were harvested in both groups for most of the cell lines. Cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturer’s instructions. For each time point, The untreated sample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000). Image analysis was done by DeArray software. Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction18. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997). The data were quality filtered with ratio quality method (Chen et al, 2002), which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis. Keywords: time-course
Project description:The first experiment involved the DU-145 tumor cell samples cultured with or without macrophages. This experiment was planned with an aim to evaluate the gene expression changes that might occur in DU-145 cells when grown in proximity to macrophages as compared to DU145 cells being grown alone. Similarly, the second experiment, involved the macrophage samples cultured with or without the DU-145 cells. This experiment was planned with an aim to evaluate the gene expression changes that might occur in Macrophages when grown in proximity to DU-145 cells .
Project description:Diarylpentanoids exhibit a high degree of anti-cancer activity and stability in vitro over curcumin in prostate cancer cells. Hence, this study aims to investigate the effects of a diarylpentanoid, 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadiene-3-one (MS13) on cytotoxicity, anti-proliferative, apoptosis-inducing, anti-migration properties, and the underlying molecular mechanisms on treated androgen-independent prostate cancer cells, DU 145 and PC-3. A cell viability assay has shown greater cytotoxicity effects of MS13-treated DU 145 cells (EC50 7.57 ± 0.2 µM) and PC-3 cells (EC50 7.80 ± 0.7 µM) compared to curcumin (EC50: DU 145; 34.25 ± 2.7 µM and PC-3; 27.77± 6.4 µM). In addition, MS13 exhibited significant anti-proliferative activity against AIPC cells compared to curcumin in a dose- and time-dependent manner. Morphological observation, increased caspase-3 activity, and reduced Bcl-2 protein levels in these cells indicated that MS13 induces apoptosis in a time- and dose-dependent. Moreover, MS13 effectively inhibited the migration of DU 145 and PC-3 cells. Our results suggest that cell cycle-apoptosis and PI3K pathways were the topmost significant pathways impacted by MS13 activity. Our findings suggest that MS13 may demonstrate the anti-cancer activity by modulating DEGs associated with the cell cycle-apoptosis and PI3K pathways, thus inhibiting cell proliferation and cell migration and inducing apoptosis in AIPC cells.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:The first experiment involved the DU-145 tumor cell samples cultured with or without macrophages. This experiment was planned with an aim to evaluate the gene expression changes that might occur in DU-145 cells when grown in proximity to macrophages as compared to DU145 cells being grown alone. Similarly, the second experiment, involved the macrophage samples cultured with or without the DU-145 cells. This experiment was planned with an aim to evaluate the gene expression changes that might occur in Macrophages when grown in proximity to DU-145 cells . Agilent one-color experiment, Organism: Human, Agilent-Custom Whole Genome Human 8x60k designed by Genotypic Technology Pvt. Ltd. (AMADID: 027114), Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)