Pseudo-G-Rh2 (Fig. 1) was provided by Professor Chen (College of Chemistry, Jilin University, Changchun, China). The purity of pseudo-G-Rh2 used in experiments was >95% as assessed by high-performance liquid chromatography using Agilent 1100 with a Zoebax Extent C18 column (250×4.6 mm, 5 µm) (both, Agilent Technologies, Inc., Santa Clara, CA, USA) at 25°C. Methanol and water (90:10) was used as the mobile phase, the flow rate was 1.2 ml/min and the sample quantity was 10 µl). Antibodies against procaspase-3 (cat. no. 19677-1-AP), procaspase-9 (cat. no. 66169-1-Ig) and β-actin (cat. no. 20536-1-AP) were purchased from Wuhan Sanying Biotechnology (Wuhan, China). Antibodies against PARP (cat. no. 9542), Bcl-2 (cat. no. 2876), Bax (cat. no. 2772), Ras (cat. no. 3965), phosphorylated (p)-Raf (cat. no. 9427), Raf (cat. no. 9422), p53 (cat. no. 9282), ERK (cat. no. 4695), c-Jun N-terminal kinase (JNK) (cat. no. 9258), p38 (cat. no. 8690), p-ERK (cat. no. 4370), p-JNK (cat. no. 4668) and p-p38 (cat. no. 4511) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The BCA protein assay reagent kit, DAPI staining kit, reactive oxygen species (ROS) assay kit (cat. no. S0033) and Rhodamine 123 were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). An Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit was obtained from Tianjin Sungene Biotech Co., Ltd. (Tianjin, China). MTT and all other reagents were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

Lung adenocarcinoma A549 cells were obtained from the Shanghai Institute of Cell Biology, Chinese Academic of Sciences (Shanghai, China). A549 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd., Huzhou, China) under standard culture conditions (37°C in an atmosphere containing 5% CO₂). Culture medium was replaced every 3 days with fresh complete medium and maintained in log phase growth. Pseudo-G-Rh2 was dissolved in dimethyl sulfoxide (DMSO) at room temperature and 104 µM was the maximum solubility. Then Pseudo-G-Rh2 was added to the culture media at the final concentrations. The final concentration of DMSO was <0.1%. Cells were treated with different concentrations of Pseudo-G-Rh2 (40, 56, 72, 88 or 104 µM) for 24, 48 and 72 h at 37°C, prior to an MTT assay. The cells were administered with 24, 48 or 96 µM Pseudo-G-Rh2 for 24 h at 37°C prior to DAPI staining, flow cytometry and western blot analysis.

Cell viability was assessed using an MTT assay as previously described (19). Briefly, A549 cells were seeded into 96-well plates (5×10⁴/ml) following treatment with 40, 56, 72, 88 or 104 µM pseudo-G-Rh2. Cells were incubated for 20 h at 37°C. A total of 10 µl of MTT solution (5 mg/ml in PBS) was added to each well and plates were incubated for a further 4 h at 37°C. A total of 100 µl DMSO was added to each well and plates were agitated for 10 min. Absorbance was measured at 570 nm using a microplate reader (SpectraMax 340PC384; Molecular Devices LLC, Sunnyvale, CA, USA). Cell viability was calculated as a fraction of the untreated cells. The half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism v5 (GraphPad Software, Inc., La Jolla, CA, USA).

DAPI staining was performed as previously described (20). Briefly, A549 cells were seeded on 6-well plates (9×10⁴/ml) and treated with 24, 48 and 96 µM pseudo-G-Rh2 at 37°C for 24 h. Cells were collected and washed twice with PBS, permeabilized with 0.1% Triton X-100 and stained with 2 µg/ml DAPI for 10 min at room temperature. Cells were subsequently observed using a fluorescence microscope (magnification, ×100; Nikon TE-2000U; Nikon Corporation, Tokyo, Japan).

To assess pseudo-G-Rh2-induced apoptosis in A549 cells, Annexin V-FITC/PI staining and flow cytometry were performed as previously described with an (19). Briefly, following pseudo-G-Rh2 treatment, A549 cells were collected, washed twice with PBS and resuspended in 300 µl of binding buffer containing PI and Annexin V-FITC. The samples were incubated for 15 min at room temperature in dark and subsequently detected by flow cytometry and CellQuest™ Pro (version 6.0) software (BD Biosciences, Franklin Lakes, NJ, USA).

The activities of caspase-3 and caspase-9 were measured using a caspase 3 Activity Assay kit (cat. no. C1115) and caspase 9 Activity Assay kit (cat. no. C1157) according to the manufacturer's protocol (both Beyotime Institute of Biotechnology). Briefly, cell lysates were incubated in a radioimmunoprecipitation lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) for 15 min on ice and centrifuged at 18,000 × g for 10 min at 4°C. Supernatants were collected and total protein was quantified using the Bradford method. Protein lysates was mixed with reaction buffer (Ac-DEVD-pNA for caspase-3, Ac-LEHD-pNA for caspase-9) and incubated at 37°C for 2 h in the dark. Cell absorbance was subsequently measured at 405 nm using a microplate reader. Results were reported as the percentage activity change compared with the control.

The ΔΨm was measured using the cationic dye Rhodamine 123 (cat. no. C2007; Beyotime Institute of Biotechnology) as previously described (21). Briefly, A549 cells were treated with 24, 48 and 96 µM pseudo-G-Rh2 for 24 h and incubated with Rhodamine 123 at 37°C for 30 min. Fluorescence intensities were analyzed by flow cytometry and CellQuest™ Pro software as above.

ROS levels were assessed using the nonfluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Beyotime Institute of Biotechnology) as previously described (22). DCFH-DA permeates into cells and is deacetylated by nonspecific esterase in the cell to form 2′,7′-dichlorofluorescein, which is nonfluorescent. The cellular oxidizing agent oxidize DCFH to dichlorofluorescein, which is a highly fluorescent compound (23). As such, the amount of ROS produced in the cells is detected by measuring the fluorescence intensity. Following pseudo-G-Rh2 treatment, A549 cells were collected, washed twice with PBS and incubated with 10 µM DCFH-DA at 37°C for 20 min in the dark. Cells were subsequently washed three times with PBS. Fluorescence was observed under a Nikon TE-2000U fluorescence microscope (magnification, ×100) and measured using flow cytometry as above.

Western blotting was performed to assess protein expression. Following treatment with pseudo-G-Rh2, A549 cells were harvested and lysed in radioimmunoprecipitation assay buffer (Beyotime Biotechnology, Jiangsu, China) for 30 min on ice. The protein concentration was determined using a BCA protein assay kit according to the manufacturer's protocol. Proteins (20 µl) were separated by 12% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane, which was subsequently blocked with 5% (w/v) non-fat milk for 1 h at room temperature. Membranes were incubated with the appropriate primary antibodies against procaspase-3, procaspase-9, PARP, Bcl-2, Bax, Ras, p-Raf, Raf, p53, ERK, c-JNK, p38, p-ERK, p-JNK, p-p38 and β-actin at 1:1,000 dilution at 4°C overnight. Primary antibody binding was detected by incubation with a secondary antibody conjugated to horseradish peroxidase. The goat-anti-mouse (cat. no. IH-0031) and goat-anti-rabbit (cat. no. IH-0011) secondary antibodies (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China) were used at 1:5,000 dilution at room temperature for 1 h. The bands were visualized using a BeyoECL Plus enhanced chemiluminescence kit (Beyotime Institute of Biotechnology). ImageJ software (version 1.5.0.26; National Institutes of Health, Bethesda, MD, USA) was used for analysis.

The results are expressed as the mean + standard deviation of three independent experiments. Statistical differences were evaluated using one-way analysis of variance with Tukey's post hoc test. P<0.05 was considered to indicated a statistically significant difference.

To evaluate the antiproliferative effects of pseudo-G-Rh2 in vitro, A549 cells were treated with 40, 56, 72, 88 or 140 µM pseudo-G-Rh2 for 24, 48 or 72 h, following which cell viability was assessed by MTT assay. The highest dose of pseudo-G-Rh2 selected was 104 µM, as this is the maximum solubility of this compound in DMSO. The results demonstrated that pseudo-G-Rh2 significantly decreased the viability of A549 cells in a concentration-dependent manner compared with the control (Fig. 2). The IC50 value was 74.5 µM for 24 h. Based on these results, dosages of 24, 48 and 96 µM pseudo-G-Rh2 were selected for further experiments

To elucidate whether pseudo-G-Rh2 induces apoptosis, DAPI staining was performed. DAPI staining revealed an increase in the number of apoptotic bodies containing nuclear fragmentations in cells treated with pseudo-G-Rh2 compared with the control group (Fig. 3A and B), which indicates that pseudo-G-Rh2 induces apoptosis in A549 cells. The apoptotic rate was measured using Annexin V-FITC/PI staining and the percentage of apoptotic cells was revealed to be 2.65, 4.44, 14.1 and 48.56% in A549 cells treated with 0, 24, 48 and 96 µM of pseudo-G-Rh2, respectively (Fig. 3C and D). To further investigate the mechanism by which pseudo-G-Rh2 induces apoptosis in A549 cells, the ΔΨm was examined using Rhodamine 123 (Fig. 4A). The ΔΨm (percentage of strong fluorescent cells) in A549 cells treated with 0, 15, 30 and 60 µg/ml pseudo-G-Rh2 for 24 h was 91.35, 88.85, 87.18 and 74.28%, respectively (Fig. 4A and B). In addition, western blotting revealed that the expression of B-cell lymphoma 2 (Bcl-2) was decreased compared with the control, whereas that of Bcl-2-associated X protein (Bax) was increased (Fig. 4C-E). Western blotting revealed that precursors of caspase-9 and caspase-3 were decreased in A549 cells following treatment with pseudo-G-Rh2 treatment compared with the control (Fig. 5A-D). Upregulated cleaved poly ADP-ribose polymerase (PARP), a product of caspase-3, was observed following pseudo-G-Rh2 treatment compared with the control. Furthermore, spectrophotometry results revealed that pseudo-G-Rh2 enhanced the activity of caspase-9 and caspase-3 compared with the control (Fig. 5E). These results suggest that pseudo-G-Rh2 induces apoptosis via the mitochondrial pathway in A549 cells.

It has previously been reported that ROS serve important roles in mediating cancer initiation, promotion and apoptosis (24,25). To determine whether pseudo-G-Rh2 affects ROS production, A549 cells were treated with 24, 48 or 96 µM pseudo-G-Rh2 for 24 h and subjected to DCFH-DA fluorescence analysis. Pseudo-G-Rh2 increased the intensity of green fluorescence, which indicates ROS-positive cells, in a dose dependent manner compared with the control (Fig. 6A). The results of flow cytometry revealed that ROS expression (percentage of strong fluorescent cells) was 5.84, 7.86, 11.61 and 35.68% in cells treated with 0, 24, 48 or 96 µM pseudo-G-Rh2 for 24 h, respectively (Fig. 6B). These results indicate that pseudo-G-Rh2 induces ROS production in A549 cells, which may lead to apoptosis.

ROS is known to be a mediator of mitogen-activated protein kinases (MAPKs) and a number of studies have demonstrated that ROS is able to maintain the activation of Ras, Raf and ERK, resulting in apoptosis and autophagy (9,26,27). In the present study, the expression of Ras, p-Raf and Raf proteins were assessed using western blotting (Fig. 7A). Pseudo-G-Rh2 increased the expression of p-Raf (Fig. 7B), Ras (Fig. 7C) and p53 (Fig. 7D) compared with the control in a dose-dependent manner; meanwhile, the ratio of p-Raf and p-ERK was significantly increased. The expression of MAPK family proteins was also assessed (Fig. 7E) and the results demonstrated that p-ERK (Fig. 7F) was upregulated by pseudo-G-Rh2 in a dose-dependent manner compared with the control. However, pseudo-G-Rh2 had no significant effect on the ratio of p-p38/p38 or p-JNK/JNK. Increased activation of the Ras/Raf/ERK pathway signaling is able to enhance the stability and activity of tumor suppressor p53, which is known to serve an important role in mediating apoptosis (11,28).