A549 cells (American Type Culture Collection, Manassas, VA, USA) were maintained in 90% Dulbecco's modified Eagle's medium (Corning, Manassas, VA, USA) containing 10% fetal bovine serum and cultured in an incubator at 37°C and 5% CO₂ with saturated humidity conditions. The cells were digested with 0.25% trypsin-EDTA for passaging. All experiments used cells in the logarithmic growth phase.

Cells were seeded at 5×10³ cells/well in 96-well plates. Evodiamine (Sigma-Aldrich, St. Louis, MO, USA) was added to obtain final concentrations of 0, 2.5, 5, 10, 25, 50 and 100 µM. The plates were then placed in an incubator for routine culture under 37°C and 5% CO₂ conditions. Samples were collected at 72 h to determine the optical density (OD). Inhibition rate = (1 − OD of experimental group / OD of control group) × 100. The fitting curve was plotted using logarithmic concentration of evodiamine as abscissa and inhibition rate as ordinate. The compound concentration corresponding to 50% inhibition rate was the IC₅₀. Phosphate-buffered saline (PBS) was used as the negative control.

Tumor cells in the logarithmic growth phase were seeded in 6-well plates at a density of 5×10⁵ and cultured for 24 h. PBS or 1 or 2.5 µM evodiamine were separately added to the test wells and cells were cultured for 24 h. The cells were harvested and incubated with 10 µl Annexin V-fluorescein isothiocyanate/propidium iodide (PI) away from light for 15 min prior to flow cytometry using a FACSAria flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) in order to determine the effect of evodiamine on the apoptosis of tumor cells. Next, PBS or 1 and 2.5 µM evodiamine were added to the test wells and cells were cultured for 24 h. The cells were harvested and incubated with 25 µl PI away from light for 15 min. Flow cytometry was used to determine the effect of evodiamine on the cell cycle of tumor cells. PBS or 1 or 2.5 µM evodiamine were added to the test wells to continue the culture for 24 h. The cells were harvested and incubated with 20 µl dihydroethidium away from light for 15 min prior to the use of flow cytometry to determine the effect of evodiamine on ROS expression in tumor cells. N-acetyl-L-cysteine (NAC; 20 mM; Sigma-Aldrich), which protects against oxidation, was added to determine whether evodiamine affects oxidative stress. DIVA 6.1.3 software (BD Biosciences) was used to analyze flow cytometry results.

Tumor cells in logarithmic growth phase were seeded in 6-well plates at a density of 5×10⁵ and cultured for 24 h. PBS or 1 or 2.5 µM evodiamine were separately added to the test wells and were cultured for 24 h. The cells were harvested and lysed to extract the proteins by protein extraction kit (Beyotime Institute of Biotechnology, Shanghai, China). The protein content in cell lysate was determined with a bicinchoninic acid assay. Then, caspase-3 and caspase-8 activity test reagent was added in accordance with the kit (Promega Corporation, Madison, WI, USA) instructions to incubate at room temperature for 30 min. The caspase-3/8 activity in these cells was determined with a microplate reader by luminescent signal.

Tumor cells (5×10⁵) in logarithmic growth phase were seeded in 6-well plates and cultured for 24 h. PBS or 1 or 2.5 µM evodiamine were separately added to the test wells and cultured for 24 h. Once the total RNA was extracted with TRIzol (Invitrogen, Thermo Fisher Scientific, Inc.) from each group, the real-time PCR kit [Takara Biotechnology (Dalian) Co., Ltd., Dalian, China] was used for reverse transcription to obtain the cDNA (2 µl). The cDNA was then amplified by specific primers using the ABI7500 system (Applied Biosystems, Waltham, MA, USA). The primer pairs were as follows: Survivin, forward (F) 5′-CGAGGCTGGCTTCATCCACT-3′ and reverse (R) 5′-ACGGCGCACTTTCTTCGCA-3′; Bcl-2, F 5′-GGCTGGGATGCCTTTGTG-3′ and R 5′-GCCAGGAGAAATCAAACAGAGG-3′; cyclin B1, F 5′-TCTGGATAATGGTGAATGGACA-3′ and R 5′-CGATGTGGCATACTTGTTCTTG-3′; Sonic hedgehog (SHH), F 5′-GTGGCCGAGAAGACCCTA-3′ and R 5′-CAAAGCGTTCAACTTGTCCTTA-3′; GLI family zinc finger 1 (GLI1), F 5′-AGCGTGAGCCTGAATCTGTG-3′ and R 5′-CAGCATGTACTGGGCTTTGAA-3′; β-actin, F 5′-CTCGCTGTCCACCTTCCA-3′ and R 5′-GCTGTCACCTTCACCGTTC-3′. The PCR conditions were 95°C for 5 min, and then 40 cycles at 95°C for 35 sec, 60°C for 35 sec, and 72°C for 65 sec, and extension step at 72°C for 10 min. The 2^−ΔΔCq method was used for relative quantifications (9).

Tumor cells in logarithmic growth phase (5×10⁵) were seeded in 6-well plates and cultured for 24 h. PBS or 1 or 2.5 µM evodiamine were separately added to the test wells and cultured for 24 h. The cells were harvested and lysed to extract the proteins by protein extraction kit (Beyotime Institute of Biotechnology). Next, 100 µl total protein was applied in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis for separation by electrophoresis. The separated proteins were transferred onto a polyvinylidene difluoride membrane. The membrane was blocked in blocking solution containing 5% skimmed milk powder for 2 h. The membranes were then incubated with monoclonal antibodies against Survivin (cat. no. sc-8807; 1:1,500); Bcl-2 (cat. no. sc-492; 1:1,500); cyclin B1, (cat. no. sc-752; 1:1,500); AKT (cat. no. sc-5298; 1:1,000); p-AKT (cat. no. sc-135650; 1:1,000); nuclear factor (NF)-κB p65 (cat. no. sc-372; 1:1,000); p-p65, (cat. no. sc-293111; 1:1,000); SHH (cat. no. sc-373779; 1:1,000) and GLI1 (cat. no. sc-20687; 1:1,000) (all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight. The membrane was washed 3 times with tris-buffered saline and Tween-20 (TBST) for 15 min each time. Horseradish peroxidase-labeled goat anti-rabbit secondary antibody (cat. no. sc-516087; 1:4,000) was added and incubated at room temperature for 1.5 h. The membrane was washed again 3 times with TBST for 15 min per wash. Finally, enhanced chemiluminescence developing agent was used for coloration and fixation.

Tumor cells (5×10⁵) in logarithmic growth phase were seeded in 6-well plates and cultured for 24 h. PBS or 1 or 2.5 µM evodiamine were separately added to the test wells and cultured for 24 h. Reduced GSH in the cells was quantified using a commercial GSH determination kit (Jiancheng Institute of Biotechnology, Nanjing, China), following the manufacturer's protocol. The result was detected by spectrophotometry at 420 nm.

Tumor cells (5×10⁵) in logarithmic growth phase were seeded in 6-well plates and cultured for 24 h. PBS or 1 or 2.5 µM evodiamine were separately added to the test wells and cells were cultured for 24 h. Cell pellets were washed twice with cold PBS, and the cells were then lysed with an appropriate volume of the provided lysis buffer from the protein extraction kit (Beyotime Institute of Biotechnology). After 30 min of incubation on ice, the suspension was centrifuged for 30 min at 4°C and 12,000 × g. The supernatant (2 µl) was then used to to assess the telomerase activity. The telomerase activity was determined by SYBR Green real-time PCR.

The data are presented as the mean ± standard deviation and analyzed with SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). Data were compared with one-way analysis of variance followed by the Bonferroni test. P<0.05 was considered to indicate a statistically significant difference.

An MTS assay was used to investigate the effect of evodiamine treatment for 72 h on in vitro proliferation of A549 cells. The results indicated that evodiamine significantly inhibited the in vitro proliferation of A549 cells (Fig. 1).

When treated with 1 and 2.5 µM evodiamine, the distribution of cells in different phases of the cell cycle changed significantly compared with control, with the majority of cells arrested at the G2/M phase (Fig. 2A). Evodiamine at 1 and 2.5 µM was used to determine its effect on apoptosis. Incubation with 1 and 2.5 µM evodiamine for 24 h significantly induced apoptosis (P<0.01; Fig. 2B). Following the incubation of the tumor cells with 1 and 2.5 µM evodiamine for 24 h, the ROS level was 217.3% and 354.2% compared with the control group (P<0.05; Fig. 2C). However, no significant difference was identified in ROS production in the NAC group (data not shown); therefore, evodiamine-induced tumor cell apoptosis may be associated with oxidative injury in these cells.

To further investigate the inhibitory effect of evodiamine and its mechanism of action on tumor cell proliferation, the changes in proliferation-associated gene expression were examined. The results indicated that the protein and mRNA expression levels of survivin, Bcl-2, cyclin B1, p-Src, SHH and GLI1 proteins were downregulated following incubated with 1 and 2.5 µM evodiamine for 24 h (Fig. 3).

In order to evaluate the role of evodiamine in the regulation of telomerase activity in A549 cells, the cells were cultured with 1 µM evodiamine for increasing time periods. Addition of evodiamine to A549 cells substantially reduced the telomerase activity (P<0.01; Fig. 4).

GSH is important for the antioxidant defense of cells. Under oxidative stress conditions, ROS are reduced by GSH by the concomitant formation of glutathione disulfide (GSSG). The intracellular concentration of GSH for detoxifying ROS significantly decreased following treatment with 1 and 2.5 µM evodiamine for 24 h (P<0.01; Fig. 5). No significant difference in ROS production with 1 and 2.5 µM evodiamine by NAC treatment when compared with the control.

SHH is a member of the family of hedgehog proteins. It is critical for oligodendrocyte development, including induction, survival, proliferation and migration of oligodendrocytes and control of axon growth (10,11). SHH binds to the transmembrane receptor protein, patched, to activate the transmembrane receptor, smoothened, and induces a complex series of intracellular reactions that target the GLI family of transcription factors. GLI1 is the principal effector of SHH signaling in neural progenitor cells (12,13). To assess the effect of evodiamine on the SHH/GLI1 signaling pathway, A549 cells were treated with 1 and 2.5 µM evodiamine. The protein and mRNA expression levels of GLI1 and SHH were reduced following evodiamine treatment (Fig. 6A and B). These findings suggest that evodiamine decreases the cellular levels of SHH and GLI1.

Recent studies have demonstrated that phosphoinositide 3-kinase (PI3K)/AKT induction of cell survival signals is mediated, in part, through the activation of the NF-κB transcription factor (14–16). NF-κB is important for tumorigenesis and tumor progression. It is associated with various signal transduction pathways and transcription activation events that mediate cell proliferation, cell migration and angiogenesis. Aberrant or constitutively activated NF-κB has been detected in human cancer (17,18). To investigate the contribution of the AKT/NF-κB signaling pathway to the inhibition of the SHH/GLI1 pathway, AKT and NF-κB phosphorylation was analyzed by immunoblotting. AKT and NF-κB phosphorylation levels were decreased following treatment with evodiamine (Fig. 6C). These results suggested that evodiamine inhibited cell proliferation via inhibition of SHH/GLI1/AKT/NF-κB signaling in A549 cells.