Isofraxidin (Fig. 1A) was purchased from Shanghai Yuan Ye Biotechnology Co., Ltd. (Shanghai, China). Isofraxidin was dissolved in dimethyl sulfoxide (DMSO) as the stock solution, which was stored at 4°C. The working solution of isofraxidin was diluted with Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) prior to use (the final DMSO concentration was <0.5% in all cultures).

A549 human lung cancer and BEAS-2B human normal lung epithelial cell lines were purchased from the Cell resource center of Shanghai institute of life sciences, Chinese academy of sciences (Shanghai, China). Cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) and 100 U/ml penicillin and streptomycin in a humidified atmosphere containing 5% CO₂ at 37°C. Cells were cultured to a confluency of 80% and were subsequently digested with 0.25% trypsin and seeded into new plates at the required density.

Cell proliferation was evaluated via a CCK-8 assay (Beyotime Institute of Biotechnology, Haimen, China). A549 and BEAS-2B cells were seeded in 96-well plates at a concentration of 5×10³/100 µl per well and incubated overnight at 37°C. The media were replaced with fresh media containing different concentrations of isofraxidin (0–160 µM) and cells were cultured for 24, 48 and 72 h at 37°C. Subsequently, 10 µl CCK-8 was added and cells were incubated for 4 h in the dark at 37°C. The optical density (OD) at 450 nm was recorded by a microplate reader. The inhibitory rate (IR) of isofraxidin on cell proliferation was calculated using the following formula: IR (%)=(1-OD_treatedgroup/OD_control)×100.

Flow cytometric analysis was performed to determine the effects of isofraxidin on the cell cycle of A549 cells. A total of 4.5×10⁵ A549 cells per 6 cm dish were exposed to isofraxidin (0, 10, 20 and 40 µM) at 37°C for 48 h and subsequently digested with EDTA-free trypsin. Cells were collected by centrifugation (1,000 × g, 4°C, 5 min), fixed with cold 75% ethanol and incubated overnight at 4°C. Cells were then stained with propidium iodide [100 µg/ml; Multi Sciences (Lianke) Biotech, Co., Ltd., Hangzhou, China] at 25°C for 30 min and flow cytometric analysis was performed immediately after staining (First-generation Attune flow cytometer, Thermo Fisher Scientific, Inc.; Attune cytometric software, v2.1.0, Thermo Fisher Scientific, Inc.).

Hoechst 33342 (Beyotime Institute of Biotechnology, Shanghai, China) staining was used to detect the apoptosis of A549 cells treated with isofraxidin. A total of 1.5×10⁵ A549 cells per well of 6-well plate were exposed to isofraxidin (0, 10, 20 and 40 µM) at 37°C for 48 h, followed by fixing with 4% formaldehyde for 20 min at room temperature. Cells were subsequently stained with Hoechst 33342 (10 µg/ml) at 25°C in the dark for 20 min and cell apoptosis was immediately evaluated with a fluorescent microscope (magnification, ×100). Three fields of view were employed for analysis of apoptotic rate.

Transwell inserts (EMD Millipore, Billerica, MA, USA) were utilized for A549 cell migration and invasion assays. Cells were diluted with serum-free DMEM at the logarithmic growth phase and 5×10⁴ cells in 200 µl serum-free DMEM were added in each upper chamber. A total of 500 µl DMEM medium containing 20% FBS was added into the lower chambers. In the invasion assay, Matrigel was also added to the upper well according manufacturer's protocol. After 1 h at 37°C, isofraxidin was added to reach a concentration of 0, 2.5, 5 and 10 µM. The concentrations of isofraxidin in upper and lower wells were in parallel. Following incubation at 37°C for 48 h, the cells on the upper membrane were removed by cotton swab. The cells at the bottom of the membrane were fixed with 4% formaldehyde at 25°C for 20 min, stained with 10% crystal violet at 25°C for 10–15 min and photographed under a light microscope (magnification, ×200). Three fields of view were analyzed per sample.

RT-qPCR was used to determine the transcript levels of the invasion-associated proteins MMP-2, MMP-9. A549 cells were exposed to isofraxidin (0, 10, 20 and 40 µM) for 48 h at 37°C and the total RNA of cells was extracted using TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA). RT of isolated RNA was conducted (PrimeScript™ RT reagent kit; cat. no. RR037A, Takara Biotechnology Co., Ltd., Beijing, China) and amplified using the qPCR [ABI 7900HT Fast Real-Time PCR, (Applied Biosystems; Thermo Fisher Scientific, Inc.); SYBR-Green qPCR Master mix, cat. no. 638320; Takara Biotechnology Co., Ltd.]. Thermocycling conditions for qPCR: 95°C, 5 sec; 55°C, 20 sec; 72°C, 30 sec; 45 cycles. The 2^−ΔΔCq method was employed for normalization. GAPDH was used as the control. Primer sequences used were as follows: MMP-2 forward, 5′-AGCGAGTGGATGCCGCCTTTAA-3′ and reverse, 5′-CATTCCAGGCATCTGCGATGAG-3′; MMP-9 forward, 5′-GCCACTACTGTGCCTTTGAGTC-3′ and reverse, 5′-CCCTCAGAGAATCGCCAGTACT-3′; and GAPDH forward, 5′-GTCTCCTCTGACTTCAACAGCG-3′ and reverse, 5′-ACCACCCTGTTGCTGTAGCCAA-3′.

A549 cells were treated with isofraxidin (0, 10, 20 and 40 µM) for 48 h at 37°C and collected. Cells lysis was conducted on ice for 30 min using lysis buffer, which consisted of a proteinase inhibitor mix, phosphatase inhibitor, phenylmethylsulfonyl fluoride and radioimmunoprecipitation assay buffer (1:1:1:100; Beyotime institute of Biotechnology, Shanghai, China). The supernatant was collected following centrifugation, 10,000 × g, 30 min, 4°C, the loading buffer was added and the mixture was boiled for 5 min. The BCA method was used to determine the protein concentration and 10 µg protein was loaded per loading well. Samples were analyzed by 10% SDS-PAGE and transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 10% skim milk solution at 25°C for 1 h and incubated overnight at 4°C with the following primary antibodies: Rabbit anti-epidermal growth factor receptor (EGFR) antibody (cat. no. 4267S), rabbit anti-phosphorylated (p)-EGFR (pY1068) antibody (cat. no. 3777S), mouse anti-AKT antibody (cat. no. 2920S), mouse anti-p-AKT (pS473) antibody (cat. no. 4060S), rabbit anti-extracellular signal-regulated kinase (ERK) antibody (cat. no. 4695S), rabbit anti-p-ERK (pT202/Y204) antibody (cat. no. 4370S), rabbit anti-Bcl-2-associated X (Bax) antibody (cat. no. 5023T), rabbit anti-Bcl-2 antibody (cat. no. 4223S), rabbit anti-MMP-9 antibody (cat. no. 13667T), rabbit anti-MMP2 antibody (cat. no. 40994S), rabbit anti-GAPDH antibody (HRP Conjugate; cat. no. 8884), which were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA). All primary antibodies were diluted in 1:1,000 proportion. GAPDH was used as the loading control. Following washing with TBST buffer (with 0.1% Tween-20), the PVDF membranes were incubated with species-specific horseradish peroxidase-conjugated secondary antibodies [anti-mouse IgG, horseradish peroxidase (HRP)-linked antibody; cat. no. 7076; anti-rabbit IgG, HRP-linked antibody; cat. no. 7074; Cell Signaling Technology, Inc., (Danvers, MA, USA), 1:5,000 dilution] at 25°C for 1 h. The membranes were visualized using Pierce™ ECL Plus Western Blotting Substrate (Thermo Fisher Scientific, Inc.). Image Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) was used for densitometry.

All procedures involving animals were approved by the Ethics Committee of the Jingzhou Central Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology (Jingzhou, China). A total of 18 male BALB/c nude mice were purchased from the CAVENS Laboratory Animal Company (Changzhou, China; http://www.cavens.com.cn/). The average weight of the mice was ~18 g. They were maintained under specific pathogen-free conditions at 20–25°C (Humidity: 50–60%; 12-h light dark cycle). A549 cells in PBS at 4×10⁶/150 µl per mouse were injected subcutaneously into the 4-week-old nude mice. The mice were returned to their cages for continuous feeding. Ad libitum food and water was available. The tumor formation was regularly analyzed. The visible subcutaneous tumor was inspected by the naked eye. The tumor-bearing mice were separated into three groups: Negative control group (n=6 mice), isofraxidin-treated group (5 mg/kg, n=6 mice) and cisplatin-treated (Shanghai Yuanyeshengwu, Shanghai, China) positive control group (5 mg/kg, n=6 mice). Drugs were delivered daily by intraperitoneal injection for 21 days. All mice were sacrificed following the treatment period and the body weights and tumor volumes were measured. The expression levels of p-EGFR, p-AKT and p-ERK were detected by immunohistochemical staining as described previously (14).

All the assays were repeated in triplicate, data were processed using SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA) and the results are presented as the mean ± standard deviation. The quantitative experiments were analyzed using analysis of variance. Dunnett's test was used as a post-hoc test. Statistical graphs were produced using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

To determine the effect of isofraxidin on the proliferation of A549 cells and the optimal dosage, the cells were treated with isofraxidin at concentrations of 0, 5, 10, 20, 40, 80 and 160 µM for 24, 48 and 72 h. The results revealed that isofraxidin exhibited a dose- and time-dependent inhibitory effect on the proliferation of A549 cells (Fig. 1B), with half-maximal inhibitory concentration (IC₅₀) values of 28.76±3.16, 42.71±4.05 and 75.16±3.42 µM following 72, 48 and 24 h of treatment, respectively. Furthermore, a lower rate of inhibition was observed in BEAS-2B normal lung epithelial cells following isofraxidin treatment compared with A549 cells (Fig. 1C), with an IC₅₀ value of 85.32±2.34 µM after 48 h of treatment, indicating that within a certain dosage range, isofraxidin exhibited a lower toxicity towards normal lung epithelial cells compared with cancerous cells. Additionally, alterations in the cell cycle were analyzed by flow cytometry, the results of which demonstrated that following treatment with isofraxidin for 48 h, the cell cycle of A549 cells was markedly altered, particularly at concentrations of 20 and 40 µM, compared with the control cells (Fig. 1D and E). The percentage of cells in S phase increased in a dose-dependent manner (Fig. 1D and E), indicating that isofraxidin inhibited the proliferation of A549 cells by arresting cells in S phase.

Drug-induced apoptosis was microscopically observed by Hoechst 33342 staining. A549 cells exposed to isofraxidin (0, 10, 20 and 40 µM) for 48 h were fixed, stained and observed under a microscope. The results demonstrated that the nuclei of cells in the control group were uniformly stained and the fluorescence intensity was not as strong compared with cells treated with isofraxidin. Increased fluorescence intensity in isofraxidin-treated cells appeared to be dose-dependent (Fig. 2A). When treated with 40 µM isofraxidin, nuclear pyknosis and crescent-shaped nuclei were observed, indicating that isofraxidin may induce the apoptosis of A549 cells in a dose-dependent manner (Fig. 2A). To determine the expression of two apoptosis-associated proteins, Bcl-2 and Bax, western blot analysis was performed. The ill-defined band of lower molecular weight is assumed to be nonspecific. The results revealed that the expression of Bax was significantly upregulated, while the expression Bcl-2 was significantly downregulated, in response to isofraxidin treatment in a dose-dependent manner (Fig. 2B). This result further indicated that isofraxidin may induce the apoptosis of A549 cells.

Metastasis is closely associated with the prognosis and recurrence of cancer (15,16). To confirm the effects of isofraxidin on cell motility, Transwell migration and invasion assays were conducted. A549 cells were treated with isofraxidin at relatively low concentrations (0, 2.5, 5 and 10 µM) for 48 h, low concentrations of isofraxidin have little inhibitory effect on cell proliferation, which may affect the cell migration and invasion. The results revealed that the increase of isofraxidin concentration led to a decreased number of migratory/invasive cells, indicating that the migration and invasion of A549 cells was significantly inhibited compared with the corresponding controls (Fig. 3A). Western blot and RT-qPCR analyses of the expression of two invasion-associated genes, MMP-2 and MMP-9, revealed that the expression levels of MMP-2 and MMP-9 were significantly reduced following treatment with isofraxidin compared with control cells (Fig. 3B and C), which further confirmed the inhibitory effects of isofraxidin on the invasion of A549 cells.

Phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK)/ERK signaling pathways are important in the proliferation, apoptosis, invasion and migration of tumor cells (17–19). The alterations in p-AKT and p-ERK expression levels in A549 cells following isofraxidin treatment were detected by western blotting. The results demonstrated that the expression levels of p-AKT and p-ERK following treatment with isofraxidin was significantly inhibited in a dose-dependent manner (Fig. 4A and B). PI3K/AKT and MAPK/ERK signaling pathways are primarily regulated by receptor tyrosine kinases (RTKs) and EGFR is the principal member of the RTKs. Western blot analysis revealed that following treatment with isofraxidin, the expression levels of p-EGFR were significantly downregulated (Fig. 4C). Therefore, isofraxidin may inhibit the PI3K/AKT and MAPK/ERK signaling pathways by reducing the levels p-EGFR to hinder the development of lung cancer cells.

To determine whether isofraxidin has potential therapeutic value for the treatment of lung carcinoma, the effects of isofraxidin on A549 cells were investigated in vivo in the present study using a xenograft mouse model. The weights of tumor-bearing nude mice are presented in Fig. 5A. Compared with in the positive control group, isofraxidin treatment reduced the body weight of nude mice less, with body weights more similar to those in the negative control group, indicating the potentially low toxicity and side effects of isofraxidin. The volume of the xenografts of samples collected after 21 days of treatment via intraperitoneal injection are presented in Fig. 5B. Compared with in the negative control group, isofraxidin significantly reduced the volume of the xenografts. Furthermore, Immunohistochemical detection demonstrated that following treatment with isofraxidin, the expression levels of p-EGFR, p-AKT and p-ERK were significantly downregulated compared with in the control group (Fig. 5C), which was consistent with the aforementioned in vitro results of the current study. These data indicated that isofraxidin suppressed the growth of xenografts in mice.