Angelica polymorpha Maxim Induces Apoptosis of Human SH-SY5Y Neuroblastoma Cells by Regulating an Intrinsic Caspase Pathway

Abstract

Angelica polymorpha Maxim root extract (APRE) is a popular herbal medicine used for treating stomachache, abdominal pain, stomach ulcers, and rheumatism; however the effect of APRE on cancer cells has not yet been explored. Here, we examined APRE cytotoxicity seen on target neuroblastoma cells (NB) using cell viability assays, DAPI visualization of fragmented DNA, and Western blotting analysis of candidate signaling pathways involved in proliferation and apoptosis. We demonstrated that APRE reduced cell viability in NB to a greater extent than in fibroblast cells. In addition, we found that APRE could inhibit the three classes of MAPK proteins and could also down-regulate the PI3K/AKT/GSK-3β activity all being relevant for proliferation and survival. APRE could also up-regulate Bax expression and down-regulate Bcl-2 and Mcl-1. With APRE treatment, depolarization of mitochondria membrane potential and activation of caspase-3 was demonstrated in the SH-SY5Y cells. We could not found increased activity of death receptor and caspase-8 as markers of the extrinsic apoptosis pathway for the APRE treated cells. In presence of a caspase-3 siRNA and a pan-caspase inhibitor, APRE could not reduce the viability of NB cells to a significant degree. So we predicted that with APRE, the intrinsic pathway was solely responsible for inducing apoptosis as we also showed that the non-caspase autophagy pathway or ER stress-ROS mediated pathways were not involved. These findings demonstrate that an intrinsic mitochondria-mediated apoptosis pathway mediates the apoptotic effects of APRE on SH-SY5Y cells, and that APRE shows promise as a novel agent for neuroblastoma therapy.

Keywords: Angelica polymorpha; Bax/Bcl-2 ratio; apoptosis; caspase; neuroblastoma.

Fig. 1.

Anti-proliferative effects of APRE in different cell lines. (A) SH-SY5Y, B103, Rat-2 and NIH 3T3 cells were cultured in 96-well culture dishes to 50–60% confluence in DMEM containing 10% FBS. The cells were treated with various concentrations of APRE. Cell death was determined by using the cytotoxicity assay kit (CCK-8, Dojindo Lab). Each point is mean ± SE of quintuple of samples. Data is composed of the mean of three independent experiments in which the activity in absence of APRE compared to presence of APRE was significantly different (n = 5, ^*P < 0.05, ^**P < 0.01). (B) APRE dose of 6 μg/ml was added in a time course experiment. Results are mean ± SEM and representatives of three independent experiments are shown. Significant differences compared with untreated and APRE added for different times are indicated (n = 5, ^*P < 0.05).

Fig. 2.

Effects of APRE on AKT/GSK-3β and MAPK signaling pathway. (A, C) SH-SY5Y cells were cultured and treated with APRE for 24 h. Whole cell lysates were subjected to 10% SDS–PAGE and the levels of p-AKT Ser473, AKT, p-GSK-3αSer21, p-GSK-3βSer9, GSK-3β, p-p38, p38, p-ERK1/2, ERK1/2, p-JNK and JNK proteins were detected by Western blotting. (B, D) The protein band intensities were determined by densitometric scanning and analyzed by Bio-Profil software and the expression levels were normalized to beta-actin. Results are expressed as mean ± SE and representatives of three independent experiments are shown (n = 3, ^*P < 0.05, ^**P < 0.01)

Fig. 3.

ROS and ES stress-mediated pathways were not involved in neuroblastoma cell death. (A) ROS production was determined by flow cytometry using DCFHDA. (B) Statistical analysis of ROS production from flow cytometry was done with cyflogic software. “Results” are expressed as mean ± SE with representatives of three independent reads shown (n = 3). (C) Cells were pretreated with 5 mM NAC for 1 h followed by in another 24 h by APRE and identification of ROS using oxidized DCFHDA by flow-cytometry. (D) Statistical analysis of ROS was determined by using cyflogic software. (E) The expression levels of ER stress protein IRE1α, Ero1-Lα, BiP and PERK were detected by Western blotting. (F) The expression of the proteins were analyzed by densitometric scanning. Results are expressed as mean ± SE and representatives of three independent experiments are shown (n = 3, ^**P < 0.01).

Fig. 4.

APRE induced cellular morphology changes in SH-SY5Y typical of apoptosis induction. (A, B) Cells were grown in 24-well culture dishes to 50% confluence and then treated with 0 to 8 μg/ml APRE for 24 h for a dose-dependent experiment and with 6 μg/ml APRE for a time-dependent experiment and morphology was observed by bright-field microscopy. (C) Apoptotic nuclear morphology was visualized by DAPI staining (arrow indicates fragmented DNA). (D) The percentages of DAPI stained cells were counted in three independent random areas. Results are mean ± SE and representatives of three independent experiments are shown (n = 3, ^*P<0.05). (E, F) Cells were stained with Annexin V-FITC/7-AAD before cell apoptosis analysis by flow cytometry. Results are expressed as mean ± SE and representatives of three independent experiments (n = 3, ^***P < 0.001).

Fig. 5.

APRE apoptosis involved intrinsic apoptotic signaling pathways. (A) Depolarization of mitochondrial membrane was measured by flow cytometry using Rhodamine-123 dye. (B) Statistical analysis of depolarization was determined by using cyflogic software. (C) Whole cell lysates were subjected to 15% SDS-PAGE and the levels of Bax and Bcl-2 were detected by Western blotting. (D) The intensity of the Bax and Bcl-2 bands were determined by densitometric scanning and analyzed by Bio-Profil software. Data was composed of the mean ± SE and representatives of three independent experiments are shown (n = 3, ^*P < 0.05). (E) Whole cell lysates were subjected to 10% SDS-PAGE and the levels of Fas, FasL, and caspase-8 proteins were detected by Western blotting. (F) The expression of the proteins was analyzed by densitometric scanning.

Fig. 6.

APRE altered the expression of apoptosis-related protein caspase-3. (A) Whole cell lysates were subjected to 15% SDS-PAGE and the levels of caspase-3 proteins were detected by Western blotting. (B) The intensity of the cleaved caspase-3 bands were determined by densitometric scanning and analyzed by Bio-Profil software. Data was composed of the mean ± SE and representatives of three independent experiments are shown (n = 3, ^*P < 0.05). (C) Red fluorescence (Alexa Fluor 568 goat anti-rabbit) indicates cleaved caspase-3 expression, whereas the nucleus is stained blue (DAPI). (D) The percentages of cleaved caspase-3 contents cells were counted in three independent random areas (n = 3, ^*p < 0.05). (E) Cells were exposed to 0 and 6 μg/ml APRE for 24 h and autophagy was analyzed by flow cytometry following acridine orange (AO) staining. (F) Whole cell lysates were subjected to 15% SDS-PAGE and the levels of LC3 was detected by Western blotting.

Fig. 7.

APRE-induced apoptosis in SH-SY5Y cells was caspase-3 dependent. (A) Cells were pretreated with 10 μM DEVD-FMK for 1 h just before exposure to 6 μg/ml of APRE for 24 h and viable cells’ number was determined by using the cytotoxicity assay kit. Results are means ± SE and representatives of three independent experiments are shown (n = 3, ^*P < 0.05). (B) Whole cell lysates were subjected to 15% SDS-PAGE and the levels of cleaved caspase-3 were detected by Western blotting. (C) The band intensity of cleaved caspase-3 was determined by densitometric scanning and analyzed by Bio-Profil software. Data was composed of the mean ± SE and representatives of three independent experiments are shown (n = 3, ^**P < 0.01). (D) Cells were grown in 24-well culture dishes to near confluence 50% and after treatment, morphology was observed by Bright-Field Microscopy. (E) SH-SY5Y cells were transfected with control and caspase-3 siRNA for 48 h followed by treatment with 6 μg/ml of APRE for 24 h. Cell number was quantified by CCK-8 kit. Results are expressed as mean ± SE and representatives of five independent experiments (n = 5, ^*P < 0.05). (E) The levels of cleaved caspase-3 were detected by Western blotting using 15% SDS-PAGE.