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

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

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

c-Jun N-terminal kinase (JNK) activation participates in EVO-induced apoptosis of COLO205 and HT-29 cells.

<p>(A) Induction of extracellular signal-regulated kinase (ERK) and JNK protein phosphorylation by EVO in colorectal carcinoma cells. Both cell lines were treated with EVO (2 µM) for different times, and expressions of phosphorylated (p)ERK/(p)JNK and total (t)ERK/(t)JNK were detected by Western blotting using specific antibodies. (B) The JNK inhibitor, SP600125 (SP; 20 µM), but not the ERK inhibitor, U0126 (U0; 20 µM), protected COLO205 and HT-29 cells from EVO-induced cytotoxicity according to an MTT assay. (C) SP600125 attenuates EVO-induced DNA ladder formation in colorectal carcinoma cells. Cells were treated with SP600125 (10 µM) for 30 min followed by EVO stimulation for an additional 24 h, and DNA integrity was examined by agarose electrophoresis. (D) SP600125 inhibited EVO-induced JNK protein phosphorylation and caspase (Casp) 3/poly(ADP ribose) polymerase (PARP) protein cleavage; however, U0126 inhibited EVO-induced ERK protein phosphorylation without affecting EVO-induced Casp 3/PARP protein cleavage in both cell lines. Both cell lines were treated with different concentrations of SP600125 or U0126 for 30 min followed by EVO stimulation for 30 min (for ERK and JNK protein expressions) or 24 h (for Casp 3 and PARP protein expressions) via Western blotting. 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 between indicated groups.</p

Disruption of the mitochondrial membrane potential (MMP) with increased Bax protein and cytosolic cytochrome (Cyt) c protein expressions, and caspase-9 (Casp 9) protein cleavage in EVO-treated COLO205 and HT-29 cells.

<p>(A) Loss of the MMP by EVO and H₂O₂ in COLO205 and HT-29 cells. Cells were treated with EVO (2 µM) or H₂O₂ (100 µM) for 12 h, and the MMP was detected by a flow cytometric analysis using DiOC6 as a fluorescent dye. (upper) A representative example of flow cytometric data is shown; (lower) quantification of the M₁ ratio from three independent experiments is shown. (B) Alternative Bcl-2 family protein expression by EVO was detected by Western blotting using specific antibodies. Cells were treated with different concentrations of EVO for 24 h, and expressions of indicated proteins were detected by Western blotting. (C) EVO induction of Casp 9 protein cleavage and cytosolic Cyt c protein in COLO205 and HT-29 cells. As described in (C), expressions of Casp 9, cytosolic Cyt C, and mitochondrial Cyt c proteins were examined by Western blotting using specific antibodies. (D) The peptidyl Casp 9 inhibitor, Ac-YVAD-FMK (YVAD; 100 µM), inhibited EVO-induced DNA ladder formation by COLO205 and HT-29 cells. Cells were incubated with Ac-YVAD-FMK (100 µM) for 2 h followed by EVO (2 µM) treatment for 24 h, and DNA integrity was examined by agarose electrophoresis. (E) A significant increase in Casp 9, but not Casp 8, enzyme activity in EVO-treated colorectal carcinoma cells. As described in (C), activities of Casp 9 and 8 were respectively measured by adding the Casp 9-specific colorimetric peptidyl substrate, Ac-DEVD-pNA, or the Casp 8-specific colorimetric peptidyl substrate, Ac-IETD-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. The intensity of each band was examined by a densitometric analysis (Imag J), and expressed as multiples of the control.</p

Role of reactive oxygen species (ROS) in EVO-induced apoptosis of colorectal carcinoma cells.

<p>(A) EVO shows no effect on intracellular peroxide production in COLO205 and HT-29 cells. Both cells were treated with EVO (2 µM) or H₂O₂ (100 µM) for 3 h followed by adding a fluorescent dye (DCHF-DA) to examine intracellular peroxide levels via a flow cytometric analysis. (upper) A representative example of flow cytometric data is shown; (lower) quantification of the M₁ ratio from three independent experiments is shown. (B) N-Acetyl-L-cysteine (NAC; N; 20 mM) protected cells from H₂O₂-induced cell death and DNA ladder formation, but had no effect on EVO-induced apoptosis. Both cells were treated with NAC (20 mM) for 30 min followed by EVO (2 µM) or H₂O₂ (100 µM) treatment for 24 h. DNA integrity (upper panel) and the viability (lower panel) of cells under different treatments are examined by agarose electrophoresis and MTT assay, respectively. (C) NAC inhibited H₂O₂-induced caspase (Casp) 3 and poly(ADP ribose) polymerase (PARP) protein cleavage, but did not affect EVO-induced events in COLO205 and HT-29 cells. As described in (B), the indicated protein expression was examined by Western blotting using specific antibodies. 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) in (A) or between indicated groups (B).</p

Risk analysis of use of different classes of antidepressants on subsequent dementia: A nationwide cohort study in Taiwan

<div><p>Depression and dementia are common mental health problems and are associated in several ways. Early-life depression is associated with increased risk of later life dementia, and depression can present as a preclinical symptom or consequence of dementia. Despite the plausible relationship between these two clinical entities, the potential association between antidepressant medication and dementia has rarely been investigated. We conducted a 9-year retrospective analysis of Taiwan’s National Health Insurance Research Database (NHIRD), enrolling 5819 cases who had received prescriptions of antidepressants between 2003 and 2006, and 23,276 (with ratio of 1:4) age, sex, and index date-matched controls. The hazard ratio (HR) of dementia among antidepressant users with depression was 2.42 (95% confidence interval (CI): 1.15–5.10), for those without depression was 4.05 (95% CI: 3.19–5.15), compared to antidepressant non-users respectively. Among the 6 classes of common antidepressants used in Taiwan, the adjusted HRs were 3.66 (95% CI: 2.62–5.09) for SSRIs, 4.73 (95% CI: 2.54–8.80) for SNRI, 3.26 (95% CI: 2.30–4.63) for TCAs, 6.62 (95% CI: 3.34–13.13) for TeCA, 4.94 (95% CI: 2.17–11.24) for MAOI, and 4.48 (95% CI: 3.13–6.40) for SARI. Furthermore, the multivariate analysis result showed that the adjusted HRs of cumulative defined daily doses (cDDDs) were 3.74 (95% CI: 2.91–4.82), 3.73 (95% CI: 2.39–5.80) and 5.22 (95% CI: 3.35–8.14) for those who had cDDDs of <90, 90–180 and >180 compared to those who had taken no antidepressant medication. This is a retrospective study based on secondary data, hence, we could not claim causality between antidepressant medication and dementia. However, a potential association between antidepressant and occurrence of dementia after controlling for the status of depression was observed. Lack of patients’ data about smoking status and body mass index in NHIRD, which are considered related to dementia, was also a limitation in this study. In this study, we concluded that antidepressant medication is a potential risk factor for dementia, independent from any effect of depression itself.</p></div

Assessment of risk factors for dementia using cox proportional hazards regression model.

<p>Assessment of risk factors for dementia using cox proportional hazards regression model.</p

Examination of the risk factors of dementia in terms of the antidepressant cDDD and its side effects using cox proportional hazards regression.

<p>Examination of the risk factors of dementia in terms of the antidepressant cDDD and its side effects using cox proportional hazards regression.</p

Flowchart of subjects selection.

<p>Flowchart of subjects selection.</p