NADPH oxidase is involved in epidermal growth factor (EGF)-induced reactive oxygen species (ROS) production in HT-29 cells.

<p>HT-29 cells were incubated for various time intervals with EGF (10 ng/ml) (A) or incubated with EGF (1∼10 ng/ml) for 20 min, and then ROS production was determined. Data are representative of three experiments, which are presented as the mean ± S.E.M. *<i>p</i><0.05, compared to the control group. C, HT-29 cells were pretreated for 30 min with 10 µM DPI and then stimulated with 10 ng/ml EGF. After 20 min of incubation, ROS production was determined. Data are representative of three experiments, which are presented as the mean ± S.E.M. <i>*p</i><0.05, compared to EGF treatment.</p

Nuclear factor (NF)-κB is involved in epidermal growth factor (EGF)-induced heme oxygenase (HO)-1 expression in HT-29 cells.

<p>A, HT-29 cells were transfected with 0.5 µg pcDNA or 0.5 µg IκBαM for 24 h, and then stimulated with 10 ng/ml EGF for another 18 h. After incubation, HO-1 and α-tubulin protein levels were determined. Immunoblots are representative of three experiments, which are presented as the mean ± SEM. <i>*p</i><0.05, compared to EGF treatment. B, Cells were transiently transfected with either 0.5 µg of pcDNA or 0.5 µg of IκBαM for 24 h. Levels of IκBα and α-tubulin protein expression were determined by a Western blot analysis. Traces represent results from three independent experiments. HT-29 cells were treated with 10 ng/ml EGF for the indicated time intervals (C) or treated with EGF (1∼10 ng/ml) for 20 min (D). The cytosolic and nuclear fractions were then isolated, and the protein levels of p65 in the cytosolic and nuclear fractions were determined by Western blot analysis. Typical traces represent three experiments with similar results. Lamin A/C and α-tubulin were respectively used as nuclear and cytosolic internal controls. E, HT-29 cells were transiently transfected with 0.5 µg of pGL2-ELAM-Luc and 0.5 µg of pBK-CMV-Lac Z for 24 h. Cells were then stimulated with HT-29 (0.3∼10 ng/ml) for another 24 h. Luciferase activities were determined as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104891#s2" target="_blank">Materials and Methods</a>”. The level of induction of luciferase activity was compared to that of cells without EGF treatment. Data are presented as the mean ± SEM. of three experiments performed in duplicate. *<i>p</i><0.05, compared to the control without EGF treatment.</p

NADPH oxidase and reactive oxygen species (ROS) are involved in epidermal growth factor (EGF)-induced heme oxygenase (HO)-1 expression in HT-29 cells.

<p>HT-29 cells were pretreated for 30 min with 3∼10 µM DPI (A) and 3∼10 mM glutathione (D) and then stimulated with 10 ng/ml EGF. After an 18h incubation, HO-1 and α-tubulin protein levels were determined. Immunoblots are representative of three experiments, which are presented as the mean ± SEM. <i>*p</i><0.05, compared to EGF treatment. HT-29 cells were treated with 10 ng/ml EGF for the indicated time intervals (B) or treated with different concentrations of EGF (C). The cytosolic and membrane fractions were then isolated, and protein levels of p47<i>^phox</i> in the cytosolic and membrane fractions were determined by a Western blot analysis. Na⁺/K⁺ ATPase and α-tubulin were respectively used as the membrane and cytosolic internal controls. Typical traces represent three experiments with similar results.</p

Involvement of c-Src, NADPH oxidase, and PI3K in epidermal growth factor (EGF)-induced Akt Ser473 phosphorylation in HT-29 cells.

<p>HT-29 cells were transiently transfected with 0.5 µg of pcDNA or 0.5 µg of a dominant negative mutant of c-Src (c-Src DN) (A) for 24 h, or cells were pretreated for 30 min with 10 µM DPI (B) or 10 µM LY 294002 (C) and then stimulated with 10 ng/ml EGF. After 20 min of stimulation, Akt Ser473 phosphorylation was determined. Immunoblots are representative of three experiments with similar results.</p

Involvement of heme oxygenase (HO)-1 in epidermal growth factor (EGF)-induced proliferation of HT-29 cells.

<p>Cells were pretreated for 30 min with 3 µM Sn(IV)protoporphyrin-1X (snPP) and then stimulated with 10 ng/ml EGF for another 48 h. A BrdU cell proliferation assay was carried out as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104891#s2" target="_blank">Materials and Methods</a>”. Data are presented as the mean ± SEM of three experiments. *<i>p</i><0.05, compared to EGF treatment.</p

PI3K/Akt is involved in epidermal growth factor (EGF)-induced heme oxygenase (HO)-1 expression in HT-29 cells.

<p>HT-29 cells were pretreated for 30 min with 10 µM LY 294002 (A) or were transiently transfected with 0.5 µg of pcDNA or 0.5 µg of a dominant negative mutant of Akt (Akt DN) (B) for 24 h. Cells were then stimulated with EGF (10 ng/ml) for another 18 h. After incubation, HO-1 and α-tubulin protein levels were determined. Immunoblots are representative of three experiments, which are presented as the mean ± SEM. <i>*p</i><0.05, compared to EGF treatment. C, Cells were transiently transfected with either 0.5 µg of pcDNA or 0.5 µg of the Akt DN for 24 h. Levels of Akt or α-tubulin protein expressions were determined by a Western blot analysis. Traces represent results from three independent experiments. D, HT-29 cells were incubated with 10 ng/ml EGF for 0∼60 min. Cell lysates were prepared, and Akt Ser473 phosphorylation was determined by immunoblotting using the phospho-Akt Ser473 antibody. Immunoblots are representative of three experiments with similar results.</p

Schematic summary indicating how signal transduction by epidermal growth factor (EGF) induces HO-1 expression in human HT-29 colon cancer cells.

<p>EGF activates the c-Src, NADPH oxidase, and PI3K/Akt pathways, which in turn induces NF-κB activation and HO-1 expression in human colon cancer cells. Moreover, HO-1 is involved in EGF-induced colon cancer cell proliferation.</p

Activation of JNK Contributes to Evodiamine-Induced Apoptosis and G₂/M Arrest in Human Colorectal Carcinoma Cells: A Structure-Activity Study of Evodiamine

<div><p>Evodiamine (EVO; 8,13,13b,14-tetrahydro-14-methylindolo[2′3′-3,4]pyrido[2,1-b]quinazolin-5-[7H]-one derived from the traditional herbal medicine <i>Evodia rutaecarpa</i> was reported to possess anticancer activity; however, the anticancer mechanism is still unclear. In this study, we investigated the anticancer effects of EVO on human colon COLO205 and HT-29 cells and their potential mechanisms. MTT and lactate dehydrogenase (LDH) release assays showed that the viability of COLOL205 and HT-29 cells was inhibited by EVO at various concentrations in accordance with increases in the percentage of apoptotic cells and cleavage of caspase-3 and poly(ADP ribose) polymerase (PARP) proteins. Disruption of the mitochondrial membrane potential by EVO was accompanied by increased Bax, caspase-9 protein cleavage, and cytochrome (Cyt) c protein translocation in COLO205 and HT-29 cells. Application of the antioxidant N-acetyl-L-cysteine (NAC) inhibited H₂O₂-induced reactive oxygen species (ROS) production and apoptosis, but did not affect EVO-induced apoptosis of COLO205 or HT-29 cells. Significant increases in the G₂/M ratio and cyclinB1/cdc25c protein expression by EVO were respectively identified in colon carcinoma cells via a flow cytometric analysis and Western blotting. Induction of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) protein phosphorylation was detected in EVO-treated cells, and the JNK inhibitor, SP600125, but not the ERK inhibitor, U0126, inhibited EVO-induced phosphorylated JNK protein expression, apoptosis, and G₂/M arrest of colon carcinoma cells. Data of the structure-activity analysis showed that EVO-related chemicals containing an alkyl group at position 14 were able to induce apoptosis, G₂/M arrest associated with increased DNA ladder formation, cleavage of caspase-3 and PARP, and elevated cycB1 and cdc25c protein expressions in COLO205 and HT-29 cells. Evidence supporting JNK activation leading to EVO-induced apoptosis and G₂/M arrest in colon carcinoma cells is provided, and alkylation at position 14 of EVO is a critical substitution for treatment of colonic cancer.</p></div

EVO reduction of viability of colorectal carcinoma COLO205 and HT-29 cells via apoptosis induction.

<p>(A) EVO reduction of cell viability of COLO205, HT-29, NIH3T3, and WI-38 cells by an MTT assay. These cells were treated with indicated concentrations (0.5, 1, 2, 4, and 8 µM) of EVO for 24 h, and cell viability was examined by an MTT assay. (B) EVO induction of lactate dehydrogenase (LDH) release by COLO205 and HT-29 cells according to an LDH release assay. As described in (A), the amount of LDH in the medium was examined by LDH kits. (C) Increased percentages of hypodiploid cells in EVO-treated COLO205 and HT-29 cell lines. Cells were treated with EVO (2 µM) for 24 h, and the percentage of hypodiploid cells was measured by flow cytometric analysis using PI staining. (D) EVO-induced loss of DNA integrity through increased DNA ladder formation. As described in (C), DNA integrity was analyzed by agarose electrophoresis. (E) Induction of caspase-3 (Casp 3) and poly(ADP ribose) polymerase (PARP) protein cleavage by EVO was detected in COLO205 and HT-29 cells by Western blotting using specific antibodies. (F) A significant increase in Casp 3 enzyme activity in EVO-treated colorectal carcinoma cells. As described in (C), activity of Casp 3 was measured by adding the Casp 3-specific colorimetric peptidyl substrate, Ac-DEVD-pNA. Each data point was calculated from three triplicate groups, and data are displayed as the mean ± S.D. ** p<0.01 denotes a significant difference compared to the control (C or CON) group.</p

Structure-activity relationship of EVO and related chemicals on apoptosis and G₂/M arrest elicited by EVO in colorectal carcinoma cells.

<p>(A) The chemical structures of EVO and structurally related chemicals (EVO-1∼12) are depicted. (B) Differential apoptotic effects elicited by EVOs in colorectal carcinoma cells. Cells were treated with the indicated EVOs (2 µM) for 24 h, and DNA integrity was analyzed by agarose electrophoresis. (C) Four EVOs with different substitutions at the position 14 of quinazolin showed differential effects on caspase (Casp) 3/poly(ADP ribose) polymerase (PARP) protein cleavage and cycB1/cdc 25c protein expressions in colorectal carcinoma cells. Cells were treated with the indicated chemicals (2 µM) for 24 h, and expressions of Casp 3/PARP, cycB1/cdc 25c, and α-tubulin (TUB) were detected by Western blotting using specific antibodies. (D) EVO, EVO4 (4), and EVO-8 (8), but not EVO-5 (5), increased the G₂/M ratio of COLO205 and HT-29 cells. As described in (C), the G₂/M ratio of COLO205 and HT-29 cells under different treatments was examined by flow cytometric analysis via PI staining. Each data point was calculated from three triplicate groups, and data are displayed as the mean ± S.D. **p<0.01 denotes a significant difference compared to the control (CON).</p