Schematic diagram of the effect of curcumin on Notch signaling in esophageal cancer.

<p>Our studies demonstrate that curcumin inhibits the expression of Jagged-1 and the Notch-1 receptor. Curcumin also inhibits γ-secretase complex proteins, thereby inhibiting cleavage of the Notch receptor. As a result, the Notch intracellular domain (NICD) is not released and therefore does not translocate to the nucleus to activate the downstream target genes c-myc and cyclin D1. This results in inhibition of cell proliferation and of stem cell regeneration, while at the same time the induction of apoptosis.</p

Combination of Curcumin and DAPT further inhibit proliferation and induce apoptosis.

<p>(A) TE cells treated with DAPT (50 µM) and curcumin (30 µM) alone and in combination for 24 h. Lysates were analyzed by western blotting. Hes-1 and Cyclin D1 proteins were further decreased with the combination of the two compounds. (B) TE cells treated with DAPT (50 µM) and curcumin (30 µM) alone and in combination for 48 h. Cell proliferation was significantly inhibited following treatment with the combination of DAPT and curcumin when compared to each curcumin alone using hexosaminidase enzyme assay (*<i>P</i><0.05). (C) Apoptosis was significantly induced following treatment with the combination of DAPT and curcumin when compared to each curcumin alone using Apo-one Homogeneous Caspase-3/7 Assay kit (*<i>P</i><0.05).</p

Curcumin treatment inhibits esophageal cancer cells spheroid formation.

<p>(A) TE-7 and TE cells were grown in low adherent plates and treated with increasing concentrations of curcumin (0–50 µM) and performed for the spheroid assay. After one week, the spheroids were photographed. (B) Spheroid was counted and performed bar diagram. Curcumin treatment significantly inhibited esophageal cancer cells spheroids (*p<0.05). (C) The primary spheroids were collected and separated into single cells and replated. Curcumin treatment significantly inhibited esophageal cancer cells secondary spheroids (*p<0.05).</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 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 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 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