SH003 induces apoptosis of DU145 prostate cancer cells by inhibiting ERK-involved pathway

Abstract

Background: Herbal medicines have been used in cancer treatment, with many exhibiting favorable side effect and toxicity profiles compared with conventional chemotherapeutic agents. SH003 is a novel extract from Astragalus membranaceus, Angelica gigas, and Trichosanthes Kirilowii Maximowicz combined at a 1:1:1 ratio that impairs the growth of breast cancer cells. This study investigates anti-cancer effects of SH003 in prostate cancer cells.

Methods: SH003 extract in 30% ethanol was used to treat the prostate cancer cell lines DU145, LNCaP, and PC-3. Cell viability was determined by MTT and BrdU incorporation assays. Next, apoptotic cell death was determined by Annexin V and 7-AAD double staining methods. Western blotting was conducted to measure protein expression levels of components of cell death and signaling pathways. Intracellular reactive oxygen species (ROS) levels were measured using H₂DCF-DA. Plasmid-mediated ERK2 overexpression in DU145 cells was used to examine the effect of rescuing ERK2 function. Results were analyzed using the Student's t-test and P-values < 0.05 were considered to indicate statistically-significant differences.

Results: Our data demonstrate that SH003 induced apoptosis in DU145 prostate cancer cells by inhibiting ERK signaling. SH003 induced apoptosis of prostate cancer cells in dose-dependent manner, which was independent of androgen dependency. SH003 also increased intracellular ROS levels but this is not associated with its pro-apoptotic effects. SH003 inhibited phosphorylation of Ras/Raf1/MEK/ERK/p90RSK in androgen-independent DU145 cells, but not androgen-dependent LNCaP and PC-3 cells. Moreover, ERK2 overexpression rescued SH003-induced apoptosis in DU145 cells.

Conclusions: SH003 induces apoptotic cell death of DU145 prostate cancer cells by inhibiting ERK2-mediated signaling.

Keywords: Anticancer effect; Apoptosis; DU145 human prostate cancer cells; ERK pathway; Herbal medicine; SH003.

Fig. 1

SH003 induces apoptosis in DU145 cells. a DU145 cells were exposed to the indicated concentrations of SH003 for 72 h or treated with 30% ethanol as control, and cell viability was measured by the MTT assay. Experiments were performed three times independently and data are presented as the means ± SD.*P < 0.05. b Cells were treated with SH003 for 48 h and double-stained with anti-BrdU and propidium iodide (PI). Cell proliferation was determined using cells stained with BrdU for DNA synthesis and DNA content was detected by PI. The dot blots display BrdU incorporation (Y-axis) and DNA content (X-axis). c DU145 cells were exposed to SH003 for 48 h and then stained with Annexin V and 7-AAD before flow cytometry. Data are presented as the means ± SD and experiments were replicated three times. *P < 0.05. d Levels of apoptosis-related proteins were detected by western blotting with indicated antibodies after treatment with SH003 for 24 h. β-actin was used as a loading control. Black triangle indicates increasing concentrations of SH003 (50, 250, and 500 μg/mL)

Fig. 2

Effects of intracellular ROS generation on apoptotic cell death of DU145. a Cells were treated with 50, 250, or 500 μg/mL SH003 and H₂DCF-DA dye for 1 h at 37 °C. b Cell were pretreated with 3 mM NAC for 30 min and then treated with 500 μg/mL of SH003. ROS generation in DU145 cells was measured by flow cytometry. Experiments were repeated three times. *P < 0.05. c Cells were pretreated with 3 mM of NAC for 1 h and then treated with 500 μg/mL SH003 for another 48 h. Apoptosis was detected by flow cytometry and data are presented as the means ± SD. Experiments were performed in triplicate. *P < 0.05. d Apoptosis-related protein levels were detected by western blotting

Fig. 3

Effects of SH003 on the activation of intracellular signaling pathways and induction of apoptosis by JNK phosphorylation in DU145 cells. DU145 cells were exposed to 50, 250, or 500 μg/mL SH003 for 15 min and western blotting was used to determine expression levels of a ERK, JNK, and p38 related MAPK components, b SRC-STAT3 signaling pathway components, or c PI3K-AKT pathway proteins. β-actin served as the internal control. d Cells were pretreated with 10 μM SP600125 for 30 min and then treated with 500 μg/mL SH003 for 24 h. Levels of apoptosis-related proteins and p-JNK were measured by western blotting. e Cells were pretreated with 10 μM of SP600125 for 30 min and then treated with 500 μg/mL SH003 for 48 h before staining with Annexin V and 7-AAD at room temperature in the dark. Levels of apoptosis were analyzed by flow cytometry. *P < 0.05

Fig. 4

SH003 induces apoptotic cell death in DU145 cells via the ERK signaling pathway. a Western blotting of ERK signaling-related protein expression levels in DU145 cells treated with indicated concentrations of SH003. b Western blotting of ERK2 and PARP levels in DU145 cells transfected with ERK2-expressing or control vectors and treated with indicated concentration of SH003 for 24 h. c Flow cytometry analysis of apoptotic cell death of DU145 cells transfected with the ERK2-expressing or control vectors. Results are presented as the means ± SD of three independent experiments. *P < 0.05 comparison of control vector-transfected cells with ERK2-transfected cells treated with SH003 in late apoptosis, #P < 0.05 comparison of ERK2-transfected cell with SH003 treatment in early apoptotic cells. d Schematic of the hypothetical system regulated by SH003. SH003 induces apoptosis in prostate cancer cells and inactivates ERK signaling