Abstract 2128: The effects of Quercetin on prostate cancer
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Abstract Prostate cancer (PCa) affects nearly 80% of men worldwide and is the second leading killer after lung cancer. The efficacy of current treatments offered in the clinics is highly compromised due to indolent nature of PCa, which provides large window of opportunity for prevention. Hence, the major focus of this study is to determine the chemo-preventive effects of Quercetin, a bioflavonoid, on prostate cancer. Effect of Quercetin on cell viability and IC50 was determined by MTT assay. The effect of Quercetin on cell motility was determined by wound healing assay. Potential role of Quercetin on cell cycle, apoptosis as well as genes involved in cell motility and invasion was determined using flow cytometry, Real-time qPCR and ELISA. Furthermore, Quercetin induced changes in signaling molecules involved in cell survival/apoptosis, cell cycle and cytoskeletal rearrangement was determined using antibody microarray. Prostate cancer cells treated with Quercetin showed dose and time dependent inhibition of proliferation/viability, and induction of apoptosis as compared to normal prostatic epithelial cells and untreated controls. Prostate cancer cell motility was inhibited in Quercetin treated cells. Prostate cancer cells were arrested in G2 phase of the cell cycle following Quercetin treatment. In addition to these, we found differential expression of caspases, matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs in different PCa cell lines compared to untreated controls. Furthermore, antibody microarray analysis demonstrated selective modulation of genes and associated signaling cascades responsible for apoptosis induction, cellular motility, adhesion and invasion in Quercetin treated cells compared to controls. These findings suggest Quercetin as a potent chemo-preventive agent. In addition to this it can be also used with chemotherapeutic agents directed to G2 phase of the cell cycle, which may improve the efficacy of chemotherapeutics offered in clinics to treat advance prostate cancer. Citation Format: Ashley B. Ward, Pranav Gupta, Gurpreet Kaur, Hina Mir, James W. Lillard, Shailesh Singh. The effects of Quercetin on prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2128. doi:10.1158/1538-7445.AM2014-2128Keywords:
Viability assay
MTT assay
Background : Cell viability is an important factor in radiaon therapy and thus is a method to quanfy the effect of the therapy. Materials and Methods : The viability of human hepatoma (HepG2) cells exposed to radiaon was evaluated by both the MTT and Trypan blue assays. The cells were seeded on 96 well-plates at a density of 1 x 10 4 cells/well, incubated overnight, and irradiated with 1-100 Gy. Results: The cell viability was decreased in a dose- andme- dependent manner when using the Trypan blue assay, but no significant changes in the response to dose could be detected using the MTT assay. It indicated that the MTT assay was not efficient at a cell density of 1 x 10 4 cells/well on 96 well-plates to determine cell viability. Subsequently, the relaonship between cell viability and lower cell density (1 x 10 3 , 3 x 10 3 , and 5 x 10 3 cells/well) was invesgated. A cell density of 1 x 10 3 was found to be the most effecve when using the MTT assay. Results show that the cell density is most important when using the MTT assay in 96 well-plates to follow in radiaon effects. Furthermore, the radiao n-induced cell viability dependent on cell density was confirmed by using the tradional Clonogenic assay. Conclusion: Our results suggest that the MTT and Trypan blue assays are rapid methods to detect radiaon-induced cell viability of HepG2 cells in about 3 days as compared with 14 days of assayme in the Clonogenic assay. To obtain accurate cell viability measures using both rapid assays, an incubaonme of at least 3 days is needed a6er irr adiaon.
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To evaluate the expression of uncoupling protein 2 (UCP2) in a retinal pigment epithelium cell line (ARPE-19), under oxidative stress (OS).ARPE-19 cells were divided into groups treated with various concentrations of hydrogen peroxide (H2O2; 0, 150, 300, 500, 700, and 900 µmol/L) for 24h, to induce oxidative damage and cell viability was assessed by MTT assay. UCP2 mRNA expression in cells treated with H2O2 was investigated by reverse transcription-polymerase chain reaction (RT-PCR). UCP2 protein expression was assessed by Western blotting and ROS levels analyzed by flow cytometry (FCM). Further, UCP2-siRNA treated cultures were exposed to H2O2 (0, 75, 150, and 300 µmol/L) for 2h and cell viability determined by MTT assay.Cells treated with higher concentrations of H2O2 appeared shrunken; their adhesion to adjacent cells was disrupted, and the number of dead cells increased. The results of cell viability assays demonstrated that the numbers of cells were decreased in a dose-dependent manner following treatment with H2O2. Compared with untreated controls, cell viability was significantly reduced after treatment with >300 µmol/L H2O2 (P<0.05). Cell metabolic activity was decreased with increased concentrations of H2O2 as detected by MTT assay. Levels of OS were further decreased in cells treated with UCP2-siRNA compared with those treated with H2O2 alone (P<0.05). The results of RT-PCR and Western blotting demonstrated that UCP2 expression was reduced in H2O2-treated groups compared with controls (P<0.05). FCM analysis showed that cell reactive oxygen species (ROS) levels were increased in H2O2-treated groups and further upregulated by UCP2-siRNA treatment (P<0.05).Expression levels of UCP2 are decreased in ARPE-19 cells treated with H2O2. ROS levels are further increased in cells treated with UCP2-siRNA relative to those treated with H2O2 alone. UCP2 may have a protective role in ARPE-19 cells during oxidative injury.
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Chlorogenic Acid
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Objective To assess the role of MTT colorimetric assay in measuring mitochondrial succinate-dehydrogenase(SDH)and establish a new way to determine pretransplantational cell viability.Methods Human or rat testicular Leydig cell suspensions were incubated with MTT medium,isopropanol was used to dissolve MTT fornlazan,then the absorbance of supertants was measured at 563 nm wavelength.Cell culture and testosterone production were used to aaseas the reliability of MTT colorimetric assay with conlparimn with Trypan bluedye.Results 1 g/L MTT concentration,3 h incubation,and impropanol as a solvent were the best optimum for the cell culture in vitro and testosterone determination showed that MTT colorimetric assay was more sensitive and accurate than Trypan bluedye.MTT colorimetric assay was employed to successfully measure cell viability for 10 cases of human testicular Leydig cell transplantation.Conelusion MTT colorimetric assay provided a reliable,objective,accurate method for determining the pretransplantational cell viability.
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MTT colorimetric assay; Cell/viability; Cell/transplantation; Trypan btuedye
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Abstract Metabolic viability based high throughput assays like MTT and MTS are widely used in assessing the cell viability. However, alteration in both mitochondrial content and metabolism can influence the metabolic viability of cells and radiation is a potential mitochondrial biogenesis inducer. Therefore, we tested if MTT assay is a true measure of radiation induced cell death in widely used cell lines. Radiation induced cellular growth inhibition was performed by enumerating cell numbers and metabolic viability using MTT assay at 24 and 48 hours (hrs) after exposure. The extent of radiation induced reduction in cell number was found to be larger than the decrease in MTT reduction in all the cell lines tested. We demonstrated that radiation induces PGC-1α and TFAM to stimulate mitochondrial biogenesis leading to increased levels of SDH-A and enhanced metabolic viability. Radiation induced disturbance in calcium (Ca 2+ ) homeostasis also plays a crucial role by making the mitochondria hyperactive. These findings suggest that radiation induces mitochondrial biogenesis and hyperactivation leading to increased metabolic viability and MTT reduction. Therefore, conclusions drawn on radiation induced growth inhibition based on metabolic viability assays are likely to be erroneous as it may not correlate with growth inhibition and/or loss of clonogenic survival.
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Amyloid beta
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Amyloid (mycology)
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The purpose of this experiment is to investigate the effects of forskolin-mediated cAMP activation on the viability of LPS-treated Schwann cells. The immortalized rat RT4-D6P2T (ATCC #CRL-2768) and S16 (ATCC #CRL-2941) cell lines were cultured and received one of the following treatments: 0.1, 1, or 10 μg/mL of LPS, in N2 media (control) or N2 media supplemented with 2 µM of forskolin, for 1, 3, 12, or 24 hours, and the CyQUANT MTT Cell Viability Assay Kit (Thermo Fisher) was used to perform the viability assay.
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