Curcumin inhibits esophageal cancer cell proliferation.

<p>(A) Curcumin inhibits proliferation of esophageal cancer cells. Cells were incubated with increasing doses of curcumin (0–50 µM) for up to 72 h and analyzed for cell proliferation. Curcumin treatment resulted in a significant dose- and time-dependent decrease in cell proliferation in all three cells when compared with untreated controls. (B) Curcumin inhibits colony formation. Esophageal cancer cells were incubated with 30 µM of curcumin for 24 h and allowed to grow into colonies for 10 days. Incubation with curcumin inhibits colony formation. Results are representative of three independent experiments. (C) Cyclin D1 is one the cell cycle regulatory protein and which is involved in the cell cycle arrest. RNA from TE-7 incubated with 30 µM curcumin was subjected to Real Time PCR for cyclin D1 mRNA expression. Curcumin treatment significantly inhibits cyclin D1 mRNA expression (*p<0.05). (D) Lysates from TE-7 incubated with 30 µM curcumin were analyzed by western blotting for cyclin D1 expression levels using mouse anti-cyclin D1 antibody. Curcumin treatment inhibits cyclin D1 protein expression.</p

Curcumin inhibits notch signaling and its downstream targets proteins.

<p>(A) real-time reverse transcription-PCR analysis of total RNA from TE-7 cells following 30 µM of curcumin treatment for 24 h showed reduction in the expression of Notch-1, its ligand Jagged-1 and its target gene Hes-1 mRNA (*p<0.05). (B) lysates from curcumin treatment caused significant reduction in the expression of cleaved Notch-1, its ligand Jagged-1and its target gene Hes-1 protein levels in TE-7 cells. (C) TE-7 cells treated with 30 µM of curcumin for 24 h were subjected to immuno-fluorescent staining using anti-Notch-1, anti-Jagged-1 and anti-Hes-1 antibodies. Curcumin treatment resulted in lower levels of Notch-1 protein in the nucleus and reduced Jagged-1 and Hes-1 expression in TE-7 cells.</p

Curcumin treatment inhibits oncomiR miRNA in esophageal cancer cells.

<p>(A) real-time reverse transcription-PCR analysis of total miRNA from TE-7 cells following 30 µM of curcumin treatment for 24 h. Curcumin treatment significantly inhibit oncomiR miRNA expression in TE-7 cells (*<i>P</i><0.05). (B) Curcumin treatment significantly up-regulated tumor suppressor let-7a miRNA expression in TE-7 cells.</p

Curcumin induces cell death and apoptosis.

<p>(A) Cell cycle analysis of curcumin treated cells. TE-7 cells were treated with 30 µM of curcumin for 12 and 24 h, examined by flow cytometry following propidium iodide staining for DNA content. Curcumin treatment leads to increased number of dead cells. Graphs are representative of data collected from three experiments. (B) Curcumin induces caspase-3, an apoptosis mediator. TE-7 cells incubated with 30 µM of curcumin were analyzed for apoptosis by caspase-3 and-7 activation. Curcumin treatment increased the number of cells undergoing apoptosis compared to untreated controls (*p<0.05). (C) Lysates from TE-7 cells incubated with 30 µM of curcumin were analyzed by western blotting for caspase-3 protein levels using rabbit anti-caspase-3 antibody. Curcumin treatment resulted in decreased procaspase-3. (D) Lysates from TE-7 cells incubated with 30 µM of curcumin were analyzed by western blotting for Bcl2, BclxL, and Bax proteins. Curcumin reduces expression of anti-apoptotic proteins Bcl2 and BclxL, whereas increased expression of pro-apoptotic proteins in treated cells when compared to untreated cells.</p

Curcumin inhibits γ-secretase complex proteins.

<p>(A) real-time reverse transcription-PCR analysis of total RNA from TE-7 cells following 30 µM of curcumin treatment for 24 h showed reduction in the expression of Presenilin 1 and Nicastrin mRNA (*p<0.05). (B) lysates from curcumin treatment caused significant reduction in the expression of γ-secretase complex proteins Presenilin 1 and 2, Nicastrin, APH1 and Pen2 protein levels in TE-7 cells. (C) TE-7 cells treated with 30 µM of curcumin for 24 h were subjected to immuno-fluorescent staining using anti-Presenilin 1 and anti-Nicastrin antibodies. Curcumin treatment resulted in lower levels of Presenilin 1 and Nicastrin expression in TE-7cells.</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 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 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 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

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