Reverse transcription was performed using the PrimeScript RT reagent kit with gDNA Eraser (cat. no. RR047A) and Real-time polymerase chain reaction (PCR) was performed using the SYBR PrimeScript (Perfect Real Time) kit (cat. no. RR086A; Takara, Dalian, China). MAPK Family bodies Sampler kit (cat. no. 9926), Phospho-MAPK Family Antibody Sampler kit (cat. no. 9910) and Human GAPDH antibody (cat. no. 686613) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA), and anti-PCNA antibody (cat. no. ab18197), anti-Nrf2 antibody (cat. no. ab62352), anti-ABCC1 antibody (cat. no. ab24102) and anti-GSTA1 antibody (cat. no. ab111947) were purchased from Abcam (Cambridge, MA, USA). Primary Antibody Dilution Buffer was purchased from Beyotime Institute of Biotechnology (P0023A; Haimen, China). Nrf2-specific shRNA and control shRNA was obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China) (Table I). The Nuclear and cytoplasmic protein Extraction kit was purchased from Beyotime Institute of Biotechnology (P0027). Metformin was obtained from Sangon Biotech Co., Ltd (Shanghai, China). Primer synthesis was performed by Biocolors Biological Technology Co., Ltd. (Shanghai, China). PI3K-specific inhibitor LY294002, ERK-specific inhibitor PD98059, JNK-specific inhibitor SP600125 and p38 kinase-specific inhibitor SB203580 were purchased from BioVision Inc. (Milpitas, CA, USA).

The A549 human lung adenocarcinoma cell line and cisplatin-resistant A549/DDP cells were obtained from GAS Shanghai Life Sciences Cell (Shanghai, China). Cells were cultured in RPMI-1640 dry powder culture medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with fetal bovine serum (FBS; Hyclone; GE Healthcare, Logan, UT, USA). Cell transfections were performed with Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) A549 and A549/DDP cells were cultured in RPMI-1640 with 10% FBS, benzylpenicillin (100 U/ml) and streptomycin (100 mg/ml), and cultured at 37°C in a humidified atmosphere of air with 5% CO₂.

A549/DDP cells were cultured in 24-well cell culture microplates at 1×10⁵ cells/well (0.5 ml medium per well, for PCR) or 6-well cell culture microplates at 5×10⁴ cells/well (2 ml medium per well, for western blot) for 24 h. The shRNA transfection was performed with Lipofectamine 2000, according to the manufacturer's protocol. The shRNA/medium mixture was incubated at room temperature for 5 min, then evenly mixed with Lipofectamine 2000 and placed at room temperature for 20 min for shRNA reagent/Lipofectamine complex formation. The 100-µl mixture was then added to the cells. After 12 h, the medium was replaced complete RPMI-1640 medium and the cells were cultured for another 48 h. Cells were then harvested for analysis. To screen for the most effective Nrf2 shRNA, the number of transfected cells was counted under the fluorescence microscope, and The knockdown efficiency was further detected by PCR and western blot analysis, which indicated that >70% of the cells were successfully transfected. The shRNA sequences are listed in Table I.

A549 and A549/DDP cells were cultured in six-well plates at a concentration of 1×10⁵ cells/well for 24 h. Total RNA was extracted with TRIzol reagent and reverse transcribed to complementary DNA with the PrimeScript RT reagent kit with gDNA Eraser, according to the manufacturer's protocol. Quantitative PCR was performed using the SYBR Green PCR system on the CFX96 PCR instrument (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The thermocycling conditions were as follows: 1 min at 95°C, followed by 40 cycles of 5 sec at 95°C and 30 sec at 60°C. The relative expression of each gene was used to perform relative fold change between treated and control groups using the 2^−∆∆Cq method (38). The primer sequences are listed in Table II.

Intracellular protein was extracted with radioimmunoprecipitation assay buffer. The protein concentration of the cell extract was determined by bicinchoninic acid protein assay, and 25 µg loaded sample amounts of total protein were separated by 10% SDS-PAGE. Proteins were then transferred to a polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA USA), which was blocked in 5% dry milk for 2 h. Membranes were then probed with the specific primary antibodies: Akt (pan) rabbit mAb (cat. no. 4685; 1:1,000 dilution); phospho-Akt (Ser473) rabbit mAb (cat. no. 4060; 1:1,000 dilution); p44/p42 MAPK(ERK1/2) rabbit mAb (cat. no. 4695; 1:1,000 dilution); phospho-p44/p42 MAPK(ERK1/2) (Thr202/Tyr204) rabbit mAb (cat. no. 4370; 1:1,000 dilution); p38 MAPK Rabbit mAb (cat. no. 8690; 1:1,000 dilution); phospho-p38 MAPK (Thr180/Tyr182) rabbit mAb (cat. no. 4511; 1:1,000 dilution); SAPK/JNK antibody (cat. no. 9252; 1:1,000 dilution); phospho-SAPK/JNK (Thr183/Tyr185) rabbit mAb (cat. no. 4668, 1:1,000 dilution); The aforementioned antibodies were all obtained by Cell Signaling Technology, Inc.; anti-PCNA antibody (1:1,000 dilution); anti-Nrf2 antibody (1:2,000 dilution); anti-ABCC1 antibody (1:1,000 dilution); anti-GSTA1 antibody (1:2,000 dilution); and GAPDH antibody (cat. no. 686613, 1:1,000 dilution; Cell Signaling Technology, Inc. MA, USA) was used as a loading control at 4°C overnight. The membrane was washed three times with Tris-buffered saline containing 1% Tween-20 (TBST), followed by incubation with peroxidase-labeled secondary antibodies (anti-rabbit IgG; cat. no. 7074; dilution, 1:5,000; Cell Signaling Technology, Inc.) for 2 h at room temperature. After 24 h incubation with 5 mM Metformin and the different inhibitors (10–40 µM PI3K-specific inhibitor LY294002; 10–40 µM ERK-specific inhibitor PD98059; 1–20 µM JNK-specific inhibitor SP600125; and 0.1–10 µM p38 kinase-specific inhibitor SB203580), the Nrf2 protein expression was evaluated by western blot analysis. The blots were visualized with the enhanced chemiluminescence plus kit purchased from Engreen Biosystem (Auckland, New Zealand) and the Bio-Rad ChemiDoc MP Gel imaging analysis system (Bio-Rad Laboratories, Inc.), and protein levels were analyzed with ImageJ software v1.8.0 (National Institutes of Health, Bethesda, VA, USA).

Nuclear protein extracts were prepared using the Nuclear and cytoplasmic protein Extraction kit, according to the manufacturer's protocol. The protein concentration of the cell extract was determined by BCA protein assay. A total of 25 µg protein were electrophoretically separated and other steps were identical to that aforementioned.

Transfected cells were cultured in 96-well plate at a density of 1×10⁴ cells/well with various concentrations of metformin (0, 1, 5 or 10 mM) for various durations. Next, 10 µl CCK8 was added to each well, followed by incubation for 4 h. The absorbance at the wavelength of 450 nm was recorded for each well using a FlexStation 3 microplate reader (Molecular Devices, Sunnyvale, CA, USA), and the cell viability was then evaluated according to the manufacturer's protocol.

For apoptosis detection, transfected cells were seeded in a 24-well plate at 5×10⁴ cells/ml. After treatment with metformin at different concentrations for 24 or 48 h, the cells were collected by trypsin digestion and re-suspended in binding buffer. Cells were incubated with 5 µl Annexin V-fluorescein isothiocyanate (20 µg/ml) and 5 µl propidium iodide (5 µg/ml) in 100 µl volume. Apoptosis was detected using a BD FACS Aria II flow cytometer (BD Biosciences, San Jose, CA, USA).

Values are expressed as the mean ± standard error of the mean. All analyses were performed with SPSS software version 13.0 (SPSS Inc., Chicago, IL, USA). Student's t-test was used for comparison of data between two groups. When multiple groups were compared, one-way analysis of variance followed by the least-significant differences post-hoc test was used for data with a normal distribution, as assessed by the Shapiro-Wilk test. A two-sided P<0.05 was considered to indicate a statistically significant difference.

First, the mRNA and protein expression of GSTA1, ABCC1 and Nrf2 was compared between A459 and A549/DDP cells (Fig. 1). The mRNA and protein expression of Nrf2, ABCC1 and GSTA1 in A549/DDP cells were significantly higher than those in A549 cells (P<0.01) (Fig. 1A). Furthermore, the response to different concentrations of metformin (1, 5 and 10 mM) was examined. In A549 and A549/DDP cells, the mRNA and protein expression of Nrf2, GSTA1 and ABCC1 were decreased by metformin in a concentration-dependent manner, with a significant reduction in mRNA expression achieved with metformin concentrations of 5 and 10 mM (P<0.01) (Fig. 1B).

Cell proliferation and apoptotic rates were then assessed using a CCK-8 assay and flow cytometry, respectively (Fig. 2). A549 cells treated with 5 and 10 mM metformin exhibited decreased proliferation from 24–72 h, and most prominently by 10 mM metformin (Fig. 2A), while A549/DDP cells only displayed a significant decrease in proliferation ability in the presence of 10 mM metformin at 48 h (P<0.01) (Fig. 2B). The apoptotic rate of A549 treated with 5 and 10 mM metformin was elevated at 24 h (Fig. 2C) and further increased at 48 h (P<0.01) (Fig. 2E). However, in A549/DDP cells, the apoptotic rate was low at 24 h and had slightly risen at 48 h (P<0.05) (Fig. 2D and F). These results indicate that metformin induced the largest amount of apoptosis in drug-resistant cells at 48 h. Thus, the subsequent experiments were performed using an incubation time of 48 h.

The Nrf2 shRNA sequence no. 2 produced a more pronounced decrease of Nrf2 mRNA and protein expression than the other two shRNAs (P<0.01) (Fig. 3A); thus, this shRNA was selected for the subsequent experiments. After transfection of A549/DDP cells with Nrf2 shRNA, the gene expression of GST and ABCC1 was decreased (Fig. 3B). It is likely that this downregulation of GSTA1 and ABCC1 may in part influence biological functions including cell proliferation and apoptosis. In A549/DDP cells without transfection, the proliferation was only inhibited by 10 mM metformin; however, the sensitivity of the cells to metformin was enhanced by transfection of Nrf2 shRNA, and a significant reduction in proliferation was achieved by metformin for 48 h at only 5 mM (Fig. 3C). Additionally, at 72 h, 10 mM metformin had a significant inhibition effect, compared with the control (P<0.01). Furthermore, after transfection with Nrf2 shRNA and culture with metformin for 48 h, the apoptotic rate of A549/DDP cells increased from 4.24 to 27.47% (P<0.01) (Fig. 3C). The apoptotic rate was similar to that of A549 cells treated with metformin for 48 h, which indicated that knockdown of Nrf2 sensitized A549/DDP cells to metformin and abrogates their acquired drug resistance.

A549/DDP cells are the cells pretreated by cisplatin, which is a interference factor that may activate some downstream signalling pathways to regulate Nrf2. In addition, the results (Fig. 2) identified that the sensitivity of A549/DDP cells to metformin is not as good as that of A549 cells. Therefore the native A549 cell line was selected to study the signaling pathway involved in the process. In A549 cells cultured with 5 and 10 mM metformin, the phosphorylation of Akt and ERK1/2 was decreased, while the phosphorylation of p38 MAPK and phospho-p46 subunit of p-JNK was sharply increased (Fig. 4A) (39). These results indicated that metformin inhibits the Akt and ERK1/2 pathways in A549 and activates the p38 MAPK and JNK pathways. Furthermore, in the presence of metformin, inhibitors of the p38 MAPK and JNK signaling pathway at different concentrations did not affect the levels of Nrf2 (Fig. 4B). However, inhibitors of the Akt and ERK1/2 pathway reduced the expression of Nrf2 (P<0.01, Fig. 4B). This suggests that metformin reduces the mRNA and protein expression of Nrf2 by inhibiting the PI3K/Akt and ERK1/2 signaling pathways to affect proliferation and apoptosis.