Curcumin inhibits COX-2 and VEGF protein expression in pancreatic cancer cells while inducing CUGBP2 and TIA-1.

<p>(A) mRNA expression. MiaPaCa-2 cells were treated with curcumin for 2 h. Curcumin treatment increased the levels of COX-2, VEGF, CUGBP2 and TIA-1 mRNA. There was no significant change of HuR mRNA expression (* p<0.05). Data from three independent experiments. (B) Western blot analyses demonstrate that lysates with curcumin treated MiaPaCa-2 cells have lower levels of COX-2 and VEGF proteins and increasing levels of CUGBP2 and TIA-1. Representative of three independent experiments. (C) (Left panel), Increased binding of CUGBP2 binding to COX-2 or VEGF mRNA following curcumin treatment. Whole cell extract (T) from curcumin-treated cells were immunoprecipitated with anti-CUGBP2 antibody, and RNA from the immunoprecipitates (P) and supernatant (S) were isolated and subjected to RT-PCR for COX-2 and VEGF mRNA. Data demonstrates increased COX-2 or VEGF mRNA in the pellet of curcumin-treated cells. (Right panel) Data was quantified and there was a clear increase in COX-2 and VEGF mRNA in the pellet of curcumin treated cells. (D) MiaPaCa-2 cells were treated with curcumin for 2 h and the stability of COX-2 and VEGF mRNA were determined following addition of actinomycin D (final concentration: 10 µg/ml). Curcumin increases the half-life of COX-2 mRNA from 30 min to 8 h. Similarly, curcumin increased the half-life of VEGF mRNA from 30 min to 8 h. Data demonstrates curcumin treatment significantly increased stability of COX-2 or VEGF mRNA.</p

Curcumin inhibits COX-2, VEGF and Cyclin D1 expression in the tumors while inducing CUGBP2 and TIA-1.

<p>(A) Total RNA from Pan02 tumor xenografts were subjected to Real time PCR. Curcumin treatment resulted in reduced COX-2, VEGF and cyclin D1 mRNA levels when compared to controls. On the other hand, CUGBP2 and TIA-1 mRNA expression was increased (*p<0.05). Data is an average from xenografts in 5 mice. (B) Western blot analysis demonstrated that tissue lysates from the curcumin treated animals have significantly lower levels of COX-2, VEGF, and cyclin D1 proteins but increased levels of CUGBP2 and TIA-1 proteins. (C) Immunohistochemistry demonstrates that curcumin treatment significantly reduced the expression of COX-2 and VEGF. (D) Immunohistochemistry demonstrates increased expression of CUGBP2 and TIA-1 in the tumor xenografts.</p

Curcumin inhibits pancreatic cancer cell growth.

<p>(A) Curcumin inhibits proliferation of pancreatic cancer cells. MiaPaCa-2, Pan02, AsPC-1 and BxPC-3 cells were incubated with curcumin (1–50 µM) for up to 72 h. Cell proliferation was analyzed using hexosaminidase enzyme activity. Curcumin treatment resulted in a significant dose-and time-dependent decrease in cell proliferation in all the cells when compared with untreated controls. (B) Curcumin inhibits colony formation. MiaPaCa-2 and Pan02 cells were incubated with 30 µM of curcumin for 24 h and colonies were allowed to form by incubating in regular media containing 10% FBS for an additional 10 d. Treatment with curcumin inhibited colony formation. A representative of three independent experiments is shown. (C) Expression of cyclin D1 is suppressed at 24 h. RNA from MiaPaCa-2 and Pan02 cells incubated with 30 µM curcumin were subjected to Real time PCR for cyclin D1 mRNA expression. There was significant suppression of the mRNA at 24 h but not at 12 h (*p<0.05). (D) Lysates from MiaPaCa-2 or Pan02 incubated with 30 µM curcumin were analyzed by western blotting for cyclin D1 expression levels. Curcumin treatment inhibits cyclin D1 mRNA and protein expression.</p

Curcumin mediated COX-2 and VEGF mRNA stability requires CUGBP2.

<p>(A) CUGBP2 is necessary for curcumin-induced COX-2 and VEGF mRNA levels. MiaPaCa-2 cells were transfected with CUGBP2 siRNA for 72 h and subsequently treated with curcumin for 2 h. Knockdown of CUGBP2 significantly decreased curcumin-mediated increase in COX-2 and VEGF mRNA expression (*p<0.05). Data from three independent experiments. (B) Western blot analyses of MiaPaCa-2 cell lysates demonstrate that knockdown of CUGBP2 partially restored the expression of COX-2 and VEGF proteins. (C) Knockdown of CUGBP2 reduces curcumin-induced stability of COX-2 and VEGF mRNA. MiaPaCa-2 cells were treated with curcumin for 2 h and the stability of COX-2 and VEGF mRNA were determined following addition of actinomycin D (10 µg/ml). Curcumin increases the half life of COX-2 and VEGF mRNA. However, knockdown of CUGBP2 using specific siRNA before curcumin treatment decreased the stability of both COX-2 and VEGF mRNA.</p

Curcumin induces mitotic catastrophe.

<p>A) Cell cycle profiles of MiaPaCa-2 cells treated with curcumin for 12 h and 24 h and were determined by flow cytometry using propidium iodide staining for DNA content. Curcumin treatment significantly increased cells in the S and G₂M phase of cell cycle within 12 h. (B) Curcumin treatment induces apoptosis. MiaPaCa-2 and Pan02 cells incubated with 30 µM of curcumin were analyzed for apoptosis by caspase 3/7 activation. Curcumin treatment increased the number of cells undergoing apoptosis compared to untreated controls (*p<0.05). (Inset) Curcumin induces caspase 3, an apoptosis mediator. Lysates from MiaPaCa-2 or Pan02 cells incubated with 30µM curcumin were analyzed by western blotting for caspase 3 protein. Curcumin treated cells shows cleaved (activated) caspase 3 while untreated cells have no cleaved caspase-3. (C) Curcumin treatment increased the levels of cyclin B1, Cdc-2 and phosphorylated checkpoint kinase Chk2. Lysates from curcumin treated MiaPaCa-2 cells (left) and Pan02 tumor xenografts (right) were analyzed by western blotting for phospho Chk2, and cyclin B1 and Cdc-2 protein expression levels. Curcumin treatment phosphorylates checkpoint kinases and increased levels of cyclin B1 and Cdc-2 in both MiaPaCa-2 cells and Pan02 tumor xenografts. (D) Curcumin treatment results in nuclear localization of cyclin B1 and Cdc-2. Immunocytochemistry of curcumin treated with MiaPaCa-2 cells both the cyclinB1 and Cdc-2 protein levels were predominately in the nucleus compared than untreated cells. Phosphorylation of Chk2, coupled with increased expression and nuclear translocation of cyclin B1 and Cdc-2 demonstrates mitotic progression of cells.</p

Curcumin inhibits growth of Pan02 tumor xenografts.

<p>(A) Nude mice carrying Pan02 cell tumor xenografts in the flanks were administered curcumin intraperitoneally for 3 weeks. There was a significant reduction in tumor size from curcumin-treated animals when compared to untreated controls (*p<0.05). (B) Curcumin treatment resulted in significantly lower tumor weight when compared to controls (*p<0.05). (C) Tumor sections were stained for CD31, an endothelial cell specific surface marker and with hematoxylin and eosin (H&E). A representative figure is presented showing significant reduction in microvessels. (D) The number of microvessels in the tumor tissues were counted in 12 high power fields and averaged. Data shows that microvessel density was significantly reduced in the xenografts of curcumin treated animals (*p<0.05).</p

Proteins Associated with Disease and Clinical Course in Pancreas Cancer: A Proteomic Analysis of Plasma in Surgical Patients

New biomarkers for pancreas cancer are needed to improve its detection and management. We surveyed the plasma of patients undergoing surgical resection to identify proteins which change in abundance after complete resection of tumor. Using longitudinally collected specimens from surgical patients, we control for normal inter-individual variation which can confound cross-sectional analysis. Recent refinements in two-dimensional gel electrophoresis allowed us to quantify changes in low abundance plasma proteins with precision. To circumvent the traditional limitations of image analysis in comparing two-dimensional gels, we used fluorometric two-dimensional difference gel electrophoresis to resolve the proteins from pre- and post-surgical plasma from each patient on one physical gel. Furthermore, we increased the ability of our assay to detect low-abundance proteins by depleting the plasma of 12 high-abundance proteins with a multi-affinity column. Informative protein spots from 20 plasma samples across 10 patients were submitted for identification with mass-spectrometry. We identified a group of proteins which change consistently in plasma following complete resection of pancreas tumor. Furthermore, we identified proteins which correlate with post-surgical rapid recurrence of disease. With further identification and validation, the candidate biomarkers which we identify in this study may prove to be useful in the diagnosis, management and prognostication of patients with pancreas cancer. Keywords: pancreas cancer • biomarkers • proteomics • fluorescence two-dimensional difference gel electrophoresi

Proteins Associated with Disease and Clinical Course in Pancreas Cancer: A Proteomic Analysis of Plasma in Surgical Patients

New biomarkers for pancreas cancer are needed to improve its detection and management. We surveyed the plasma of patients undergoing surgical resection to identify proteins which change in abundance after complete resection of tumor. Using longitudinally collected specimens from surgical patients, we control for normal inter-individual variation which can confound cross-sectional analysis. Recent refinements in two-dimensional gel electrophoresis allowed us to quantify changes in low abundance plasma proteins with precision. To circumvent the traditional limitations of image analysis in comparing two-dimensional gels, we used fluorometric two-dimensional difference gel electrophoresis to resolve the proteins from pre- and post-surgical plasma from each patient on one physical gel. Furthermore, we increased the ability of our assay to detect low-abundance proteins by depleting the plasma of 12 high-abundance proteins with a multi-affinity column. Informative protein spots from 20 plasma samples across 10 patients were submitted for identification with mass-spectrometry. We identified a group of proteins which change consistently in plasma following complete resection of pancreas tumor. Furthermore, we identified proteins which correlate with post-surgical rapid recurrence of disease. With further identification and validation, the candidate biomarkers which we identify in this study may prove to be useful in the diagnosis, management and prognostication of patients with pancreas cancer. Keywords: pancreas cancer • biomarkers • proteomics • fluorescence two-dimensional difference gel electrophoresi

Figure S2 from Assessing the Magnitude of Immunogenic Cell Death Following Chemotherapy and Irradiation Reveals a New Strategy to Treat Pancreatic Cancer

Supplemental figure 2</p

Figure S6 from Assessing the Magnitude of Immunogenic Cell Death Following Chemotherapy and Irradiation Reveals a New Strategy to Treat Pancreatic Cancer

Supplemental figure 6</p