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

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

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

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

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

Drugs included in the study.

<p>Drugs included in the study.</p

Cumulative incidence of vascular access dysfunction stratified by drugs.

<p>(a) ACEI & AVF (b) ACEI & AVG (c) ARB & AVF (d) ARB & AVG (e) CCB & AVF (f) CCB & AVG.</p

Patient characteristics and association with using arteriovenous fistula and arteriovenous graft among end-stage renal disease hemodialysis patients.

<p>Patient characteristics and association with using arteriovenous fistula and arteriovenous graft among end-stage renal disease hemodialysis patients.</p

Cumulative incidence of vascular access dysfunction.

<p>Cumulative incidence of vascular access dysfunction.</p