Protein Kinase RNA-Like Endoplasmic Reticulum Kinase-Mediated Bcl-2 Protein Phosphorylation Contributes to Evodiamine-Induced Apoptosis of Human Renal Cell Carcinoma Cells

<div><p>We investigated the anticancer mechanism of evodiamine (EVO) against the viability of human A498 renal cell carcinoma (RCC) cells in vitro and in vivo. The in vitro study showed that EVO decreased the viability of A498 cells with the occurrence of apoptotic characteristics such as hypodiploid cells, DNA ladders, chromatin-condensed cells, and cleaved caspase (Casp)-3/poly(ADP ribose) polymerase (PARP) proteins. Pharmacological studies using chemical inhibitors of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) indicated that phosphorylation of the c-Jun N-terminal kinase (JNK) protein participated in EVO-induced cell death of A498 cells, and application of the JNK inhibitor, SP600125 (SP), inhibited EVO-induced cleavage of the Casp-3/PARP proteins and chromatin condensation according to Giemsa staining. EVO disruption of the mitochondrial membrane potential (MMP) with increased protein levels of the phosphorylated Bcl-2 protein (p-Bcl-2) was prevented by JNK inhibitors in A498 cells. A structure-activity relationship study showed that a methyl group at position 14 in EVO was important for its apoptotic effects and increased p-Bcl-2 protein in A498 cells. Furthermore, significant increases in the phosphorylated endoplasmic reticular stress protein, protein kinase RNA-like endoplasmic reticulum kinase (p-PERK at Thr980), by EVO were detected in A498 cells, and the PERK inhibitor, GSK2606414, significantly suppressed EVO-induced apoptosis, p-JNK, p-PERK, and cleaved PARP proteins. The in vivo study showed that EVO significantly reduced RCC growth elicited by a subcutaneous injection of A498 cells, and an increased protein level of p-PERK was observed according to an immunohistochemical analysis. Apoptosis by EVO was also demonstrated in other RCC cells such as 786-O, ACHN, and Caki-1 cells. This is the first study to demonstrate the anti-RCC effect of EVO via apoptosis in vitro and in vivo, and activation of JNK and PERK to induce Bcl-2 protein phosphorylation, which led to disruption of the MMP.</p></div

Palladium-Catalyzed S_N2′-Cyclization of Ambivalent (Bromoalkadienyl)malonates: Preparation of Medium- to Large-Membered Endocyclic Allenes

A palladium-catalyzed reaction for preparing various endocyclic allenes was developed. The substrates for the reaction were readily available ω-(pronucleophile-tethered)-3-bromo-1,3-alkadienes, and a palladium-catalyst facilitated their unimolecular S_N2′-cyclization in the presence of potassium <i>tert</i>-butoxide to give the corresponding 9- to 16-membered endocyclic allenes in fair yields of up to 67% together with the dimeric 16- to 32-membered endocyclic bis-allenes and other oligomeric/polymeric intermolecular reaction products. For higher yields of the monomeric endocyclic allenes, the reaction needed to be conducted under high-dilution conditions. Using a chiral palladium catalyst, axially chiral endocyclic allenes were obtained in up to 70% ee

A tentative mechanism of evodiamine (EVO)-induced apoptosis in human A498 renal cell carcinoma (RCC) cells is depicted.

<p>It indicates that EVO induces phosphorylation of c-Jun N-terminal kinase (JNK) and protein kinase RNA-like endoplasmic reticular kinase (PERK) leading to disruption of the mitochondrial membrane potential which in turn activates caspases to cause the apoptosis of human RCC cells.</p

The c-Jun N-terminal kinase (JNK) inhibitors, SP600125 (SP) and JNKI, protect A498 cells from evodiamine (EVO)-induced apoptosis.

<p>(A) The JNK inhibitors, SP and JNKI, prevented EVO-induced cell death in human A498 renal cell carcinoma (RCC) cells. Cells were treated with the indicated kinase inhibitors (20 mM) for 30 min followed by EVO (4 mM) treatment for 12 h, and viability of cells under different treatments was evaluated by an MTT assay. (B) The JNK inhibitors, SP and JNKI, inhibited EVO-induced cleavages in caspase (Casp)-3 and poly(ADP ribose) polymerase (PARP) protein by Western blotting. (C) EVO-induced alternations in A498 morphology were reversed by the addition of the JNK inhibitors, SP and JNKI. As described above, the morphology of A498 cells was observed microscopically via Giemsa staining. Each data point was calculated from triplicate determinations, and data are displayed as the mean ± S.D. ** <i>p</i><0.01, significantly differs between the indicated groups. Arrows indicate the chromatin-condensed cells.</p

Effects of EVO on various human renal carcinoma cells including 786-O, ACHN, and Caki-1.

<p>(A) The apoptotic morphology of EVO-treated 786-O, ACHN, and Caki-1 cells. These cells were treated with or without EVO (4 μM) in the presence or absence of SP or GSK (20 μM) for 12 h, and the morphology of cells was observed by Giemsa staining under microscopy. (B) SP and GSK inhibited EVO-induced cell death and DNA ladders in 786-O and ACHN cells. Human RCC cells 786-O and ACHN were treated as described in (A), and viability of cells and DNA integrity were analyzed by MTT assay (upper panel) and agarose electrophoresis (lower panel), respectively. (C) SP and GSK inhibited EVO-induced cleavages of PARP protein and phosphorylated PERP protein in 786-O and ACHN cells. As described in (A), expression of PARP, pPERK, and α-TUB protein 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, significantly differs from the control (CON) groups.</p

Evodiamine (EVO) reduction of viability of human A498 renal cell carcinoma (RCC) cells via apoptosis induction.

<p>(A) Alterations in cellular morphology by EVO (0.5, 1, 2, 4, and 8 μM) were observed microscopically via Giemsa staining. A498 cells were treated with different concentrations of EVO (0.5, 1, 2, 4, and 8 μM) for 12 h, and morphology of cells was observed microscopically. (B) EVO reduction of cell viability of A498 cells according to an MTT assay. A498 cells were treated with different concentrations of EVO (0.5, 1, 2, 4, 8, 16, and 32 μM) for 12 h, and viability of cells was examined by an MTT assay. (C) Loss of DNA integrity with increased DNA ladders by EVO (0.5, 1, 2, 4, and 8 μM) was examined by agarose electrophoresis. (D) EVO induction of cleavage of caspase (Casp)-3 and the PARP protein in A498 cells by Western blotting using specific antibodies. (E) Increased percentage of hypodiploid cells by EVO in A498 RCC cells. Cells were treated with EVO (4 and 8 μM) for 12 h, and the percentage of hypodiploid cells was examined by a flow cytometric analysis via propidium iodide (PI) staining. Each data point was calculated from triplicate determinations, and data are displayed as the mean ± S.D. ** <i>p</i><0.01, significantly differs compared to the control (CON) group.</p

Disruption of the mitochondrial membrane potential (MMP) with an increase in the phosphorylation of the Bcl-2 protein in evodiamine (EVO)-treated human A498 renal cell carcinoma (RCC) cells, which was inhibited by adding the JNK inhibitors, SP600125 (SP) and JNKI.

<p>(A) Loss of the MMP by EVO was inhibited by addition of the JNK inhibitors, SP and JNKI, in A498 cells. Cells were treated with or without the JNK inhibitors, SP or JNKI, (20 μM) for 30 min followed by EVO (4 μM) treatment for an additional 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) Altered expressions of Bcl-2 family proteins including Bcl-2, phosphorylated Bcl-2 (Ser-70), Bax, and the Mcl-1 protein by EVO were detected in A498 cells by Western blotting using specific antibodies. As described above, expression of the indicated protein was detected using specific antibodies. Each data point was calculated from triplicate determinations, and data are displayed as the mean ± S.D. ** <i>p</i><0.01, significantly differs compared to the EVO-treated group.</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

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