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

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

Selecting Tyrosine Kinase Inhibitors for Gastrointestinal Stromal Tumor with Secondary KIT Activation-Loop Domain Mutations

<div><p>Advanced gastrointestinal stromal tumors (GIST), a <i>KIT</i> oncogene-driven tumor, on imatinib mesylate (IM) treatment may develop secondary <i>KIT</i> mutations to confer IM-resistant phenotype. Second-line sunitinib malate (SU) therapy is largely ineffective for IM-resistant GISTs with secondary exon 17 (activation-loop domain) mutations. We established an <i>in vitro</i> cell-based platform consisting of a series of COS-1 cells expressing <i>KIT</i> cDNA constructs encoding common primary±secondary mutations observed in GISTs, to compare the activity of several commercially available tyrosine kinase inhibitors on inhibiting the phosphorylation of mutant KIT proteins at their clinically achievable plasma steady-state concentration (Css). The inhibitory efficacies on <i>KIT</i> exon 11/17 mutants were further validated by growth inhibition assay on GIST48 cells, and underlying molecular-structure mechanisms were investigated by molecular modeling. Our results showed that SU more effectively inhibited mutant KIT with secondary exon 13 or 14 mutations than those with secondary exon 17 mutations, as clinically indicated. On contrary, at individual Css, nilotinib and sorafenib more profoundly inhibited the phosphorylation of KIT with secondary exon 17 mutations and the growth of GIST48 cells than IM, SU, and dasatinib. Molecular modeling analysis showed fragment deletion of exon 11 and point mutation on exon 17 would lead to a shift of KIT conformational equilibrium toward active form, for which nilotinib and sorafenib bound more stably than IM and SU. In current preclinical study, nilotinib and sorafenib are more active in IM-resistant GISTs with secondary exon 17 mutation than SU that deserve further clinical investigation.</p></div

Rapid progression of IM-resistant tumor after SU treatment.

<p>A patient harboring KIT exon 11^{Val555_Leu576del}/17^Asn822Lys mutated, metastatic GIST within the liver after 3 months of SU at dose of 50 mg/day, 4 weeks-on/2 weeks-off., (a) before and (n) after SU treatment.</p

Effects of TKIs on phosphorylated KIT with mutations on exon 9 or 11/17.

<p>(a) COS-1 cell transfected with KIT double mutants were starved and treated with indicated doses of multiple TKIs for 30 minutes respectively. The total expression and degree of phosphorylation of KIT were determined by western blot analysis. (b) Inhibitory ratios at Css (IR_Css) of multiple TKIs, as the red arrow pointed, on KIT secondary mutations on exon 17 were determined by quantification of phosphorylated KIT/total KIT and estimated from the western blot in (a). The data are expressed as the mean ± SE of three independent experiments.</p

Binding energies and interactions of TKIs to KIT 11^{Val555_Leu576del}/17^Asp820Gly mutations.

<p>(a) Stereo views of IM, nilotinib, and sorafenib binding to KIT showing key hydrogen bonds formed with A599 and R684 in different models. (b) Binding energies of IM, SU, nilotinib, dasatinib, and sorafenib were estimated according docking TKIs to KIT mutants on exon 11^{Val555_Leu576del}/exon 17^Asp820Gly.</p