Fig 1.
Evodiamine (EVO) reduction of viability of human A498 renal cell carcinoma (RCC) cells via apoptosis induction.
(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. ** p<0.01, significantly differs compared to the control (CON) group.
Fig 2.
The c-Jun N-terminal kinase (JNK) inhibitors, SP600125 (SP) and JNKI, protect A498 cells from evodiamine (EVO)-induced apoptosis.
(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. ** p<0.01, significantly differs between the indicated groups. Arrows indicate the chromatin-condensed cells.
Fig 3.
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.
(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. ** p<0.01, significantly differs compared to the EVO-treated group.
Fig 4.
Structure-activity relationship of evodiamine (EVO) and related chemicals on apoptotic events in human A498 renal cell carcinoma (RCC) cells.
(A) The chemical structures of EVO and structurally related chemicals including EVO-1, -6, -7, and -8 are depicted. (B) Alternative morphological changes in EVOs were observed microscopically. A498 cells were treated with the indicated EVOs (4 μM) for 12 h, and the morphology of cells was observed microscopically via Giemsa staining. (C) Differential cytotoxicity elicited by the EVOs in A498 cells. A498 cells were treated with the indicated EVOs (4 μM) for 12 h, and cytotoxicity induced by the EVOs was analyzed by an MTT assay. (D) The JNK inhibitor, SP600126 (SP), inhibited EVO-7- and EVO-8-induced cytotoxicity in A498 cells. Cells were treated with SP (20 μM) for 30 min followed by EVO stimulation for 12 h. Viability of cells under various treatments was examined by an MTT assay. (E) EVO, -7, and -8, but not EVO-1 or -6, induced poly(ADP ribose) polymerase (PARP) cleavage and phosphorylation of the Bcl-2 protein in A498 cells. (F) SP addition reduced PARP cleavage and phosphorylation of the Bcl-2 protein by EVO-7 and -8 in A498 cells. Each data point was calculated from triplicate determinations, and data are displayed as the mean ± S.D. ** p<0.01, significantly differs compared to the control group (C) or between indicated groups (D).
Fig 5.
Activation of protein kinase RNA-like endoplasmic reticular kinase (PERK) contributes to evodiamine (EVO)-induced apoptosis of human A498 renal cell carcinoma (RCC) cells.
(A) The PERK inhibitor, GSK, reversed morphological changes by EVOs including EVO, -7, and -8 in A498 cells. A498 cells were treated with or without GSK (20 mM) for 30 min followed by the indicated EVOs (4 μM) for 12 h. The morphology of A498 cells under different treatments was observed microscopically via Giemsa staining. (B) GSK protects A498 cells from EVO-induced cytotoxicity according to an MTT assay. As described in (A), the viability of cells was detected by an MTT assay. (C) GSK addition inhibits EVO-induced DNA ladder formation in A498 cells. As described in (A), the DNA integrity was examined by agarose electrophoresis. (D) EVO-7 and -8 concentration-dependently increased phosphorylation of PERK at Thr980 (p-PERK Thr980) in A498 cells. Cells were treated with different concentrations of EVO-7 or -8 for 12 h, and expressions of phosphorylated and total PERK were examined by Western blotting using specific antibodies. (E) GSK inhibited EVO-, EVO-7-, and EVO-8-induced phosphorylation of c-Jun N-terminal kinase (JNK), PERK, and Bcl-2 proteins in A498 cells. As described before, expressions of indicated proteins were examined by Western blotting using specific antibodies. Each data point was calculated from triplicate determinations, and data are displayed as the mean ± S.D. ** p<0.01, significantly differs between the indicated groups (B).
Fig 6.
In vivo antitumor activity of evodiamine (EVO) against renal cell carcinoma (RCC) growth elicited by a subcutaneous injection of A498 cells with increased phosphorylated protein kinase RNA-like endoplasmic reticular kinase (PERK) protein.
(A) A representative example of tumors derived from the control (phosphate-buffered saline (PBS); vehicle) and EVO-treated groups at the end the experiments are shown. (B) No difference in the body weight of mice between the control (vehicle) and EVO-treated groups was observed. (C) EVO treatment significantly inhibited in vivo RCC growth in nude mice. The in vivo study is described in "Materials and Methods", and tumor volumes were measured. (D) EVO significantly reduced tumor weights elicited by A498 cells in vivo at the end of the experiment. At the end of the experiment (18 days), tumors were excised and weighed from the control and EVO-treated groups. (E) Tumor specimens were stained with H&E to identify tumor locations, and expressions of total (t-PERK) and phosphorylated PERK (p-PERK) protein in tumor sections of the control and EVO-treated groups were detected by immunohistochemistry using specific antibodies. Each data point was calculated from triplicate determinations, and data are displayed as the mean ± S.D (n = 5). ** p<0.01, significantly differs between the indicated groups (D).
Fig 7.
Effects of EVO on various human renal carcinoma cells including 786-O, ACHN, and Caki-1.
(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.
Fig 8.
A tentative mechanism of evodiamine (EVO)-induced apoptosis in human A498 renal cell carcinoma (RCC) cells is depicted.
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.