The human lung cancer cell line A549 and cisplatin resistant cell line A549/DDP were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained at 37°C in a humidified atmosphere with 5% CO₂. The culture medium was Eagle’s minimal essential medium supplemented with 10% fetal bovine serum (FBS; Gibco-BRL, Carlsbad, CA, USA). The present study was approved by the Ethics Committee of Weifang Medical University (Weifang, China).

The Sorcin siRNA and blank vector were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and the transfection of siRNA was performed according to the manufacturer’s instructions. Briefly, 4×10⁵ A549/DDP cells were seeded into 6-well plates for 24 h in the culture medium. Then, Sorcin siRNA and the blank vector were transfected with Lipofectamine 2000. Following 24 h of incubation, normal growth medium containing 15% FBS was added and the cells were incubated for another 18 h. The medium was aspirated and replaced with fresh normal growth medium (10% FBS) and was added after 72 h with 400 μg/ml G418 to generate Sorcin silencing and negative control sublines. Sorcin expression was measured by western blotting and quantitative polymerase chain reaction (qPCR).

The cells (0.1 ml) were seeded into each well of the 96-well microplate at a density of 1×10⁵/ml and incubated overnight to allow the adherence of cells. Then, different concentrations of cisplatin (Sigma-Aldrich, St. Louis, MO, USA) were added to each well and the cells were cultured for another 72 h. At the end of the incubation, CellTiter 96 Aqueous One Solution Reagent (Promega Corporation, Madison, WI, USA) was employed according to the manufacturer’s instructions. After 4 h, the cell viability was determined by measuring the absorbance at 490 nm using a microplate reader (US Biotek Laboratories, Seattle, WA, USA).

Approximately 3×10⁵ cells were seeded into each well of the 6-well microplate and incubated with 10 μM cisplatin for 24 h. Cells were then harvested and the apoptosis rate was determined using an Annexin V-FITC Apoptosis kit purchased from BD Biosciences (Franklin Lakes, NJ, USA). Briefly, 20 μl of Annexin V-FITC and propidium iodide (PI) were added to the suspension and incubated for 15 min in the dark at room temperature. Subsequently, the cells were washed twice with phosphate-buffered saline (PBS), resuspended with PBS and analyzed by flow cytometry at an excitation wavelength of 488 nm.

Approximately 1×10⁶ cells were harvested and fixed in cold alcohol at 4°C overnight. The cells were washed twice with PBS, stained with 10 μM PI with DnaseA for 30 min and analyzed using flow cytometry (FACSCalibur; BD Biosciences) at an excitation wavelength of 488 nm.

Approximately 1×10⁶ cells were harvested and resuspended in 0.1 ml culture medium. The cells were stained with 10 μM Rhod-123 (Sigma, St. Louis, MO, USA) for 1 h and the intracellular concentration was determined by flow cytometry (FACSCalibur; BD Biosciences) at an excitation wavelength of 488 nm.

The expression of P-gp on the cell surface was determined using a direct immunofluorescence staining kit purchased from BD Biosciences. Briefly, 20 μl of reagent containing anti-P-gp-PE was added to the suspension and incubated for 30 min in the dark at room temperature. Cells were then washed twice with PBS, resuspended with 0.5 ml of 1% paraformaldehyde and analyzed by flow cytometry at an excitation wavelength of 488 nm.

The intracellular GSH was measured using a Total Glutathione Assay kit (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China). Analysis was performed according to the manufacturer’s instructions. Briefly, cells were incubated with cisplatin for 4 h. Cell lysates were harvested and reacted with assay solution for 5 min at 25°C. The absorbance at 412 nm was measured on a Spectra Max M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Approximately 3×10⁶ cells were harvested for qPCR analysis. Total mRNA was extracted from the cells using a Dynabeads mRNA Direct kit (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total mRNA was then reverse transcribed for 1 h at 42°C in incubation buffer containing 250 μM of each deoxynucleotide triphosphate, 5 μM oligo (dT)20, 25 units of RNase inhibitor and 20 units of avian myeloblastosis virus reverse transcriptase (Roche Diagnostics, Mannheim, Germany). The transcription level of MDR1, MRP1, ABCA2, ABCA5 and Bcl-2 was detected by semiquantitative PCR using the iCycler iQ detection system (Bio-Rad, Hercules, CA, USA). The PCR conditions were as follows: decontamination at 65°C for 2 min, denaturation at 95°C for 2 min, followed by 40 cycles at 95°C for 20 sec and at 65°C for 40 sec. β-actin was used as the internal control. The full details are shown in Table I.

Cells were eliminated by trypsinization and the whole proteins were obtained using radioimmunoprecipitation assay lysis buffer (Millipore, Billerica, MA, USA) extraction and centrifugation at 12,000 × g for 10 min. Total protein concentrations of the supernatants were measured using the bicinchoninic acid kit (Sigma-Aldrich, St. Louis, MO, USA). Proteins (20 μg) were separated on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were blocked for 2 h at room temperature in Tris-NaCl-Tween (TNT) buffer (10 mM Tris-HCl and 150 mM NaCl, pH 7.4, 0.1% Tween-20) with 5% non-fat dried milk, followed by incubation overnight at 4°C with rabbit anti-MDR1, MRP1, LRP, GST-π, ABCA2, ABCA5, Bcl-2, p-ERK, p-Akt and β-actin antibody (Santa Cruz Biotechnology, Inc.). All primary antibodies were diluted according to the manufacturer’s instructions. The membranes were washed and incubated for 1 h with peroxidase-labelled anti-rabbit IgG (Santa Cruz Biotechnology, Inc., diluted at 1:2,000). Finally, the membranes were washed three times in TNT buffer and exposed to the Immobilon™ western chemiluminescent horseradish peroxidase substrate (Millipore) for 1 min, and then exposed to autoradiography film for 2–3 min in the dark. β-actin was used as the internal control.

The activity of NF-κB was determined by the reporter gene system (Promega Corporation) according to the manufacturer’s instructions with moderate modification. Briefly, 1×10⁴ cells were seeded into each well of a 96-well plate and incubated overnight to allow the adherence of cells. Then, the activity of transcription factors was measured using a TransAM transcription factor activity ELISA kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Data are expressed as the mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance and P<0.05 was considered to indicate a statistically significant difference.

As shown in Fig. 1, the Sorcin level in A549/DDP cells was elevated significantly compared with the parental A549 cells. After A549/DDP cells were transfected with Sorcin siRNA, the Sorcin level in A549/DDP Sorcin silenced cells was decreased significantly compared with the normal A549/DDP cells. With different silencing levels of Sorcin, A549/DDP cells were divided into the Sorcin siRNA group 1 and Sorcin siRNA group 2. A549/DDP cells transfected with control siRNA were the negative group, A549/DDP cells without treatment were the control group and A549 cells were the parent group.

The present study determined the sensitivity of the cells to cisplatin following Sorcin silencing using an MTS assay to examine whether Sorcin contributed to the cisplatin resistance of A549/DDP. The drug sensitivity of each group of cells was represented by the IC₅₀. As shown in Fig. 2, the IC₅₀ values of the Sorcin silenced cells were lower than that of the parental A549/DDP cells. The IC₅₀ of the Sorcin siRNA group 1 and the Sorcin siRNA group 2 were 36.4 and 17.6 μM, respectively, however, the IC₅₀ of the control group and the negative group were 46.8 and 45.6 μM, respectively, suggesting that the reversal fold of Sorcin silencing were 1.29 and 2.66 fold, respectively [Reversal fold = IC₅₀ (resistance cells) / IC₅₀ (reversal cells)]. These results indicated that Sorcin has a positive function in the cisplatin resistance of the cells.

It was possible that the restoration of cisplatin sensitivity by Sorcin silencing was associated with the regulation of accumulation of intracellular drug, since increasing drug efflux is a major drug resistance mechanism. Rhod-123 is a fluorescent pigment that shares the same membrane transporter protein with cisplatin and is able to reflect the intracellular drug accumulation potency. The results from the flow cytometry demonstrated that the Sorcin silenced cells have a greater intracellular fluorescent activity, which indicated that more Rhod-123 was retained in the cell. In addition, flow cytometric analysis demonstrated that Sorcin silencing was able to downregulate the expression of P-gp, increase cell apoptosis and arrest the cell cycle in G2/M phase, indicating that Sorcin silencing was able to restore cisplatin sensitivity through regulating the drug pump and the cell cycle in A549/DDP cells (Fig. 3).

MDR1, LRP and MRP1 are major membrane transporter proteins that lead to MDR due to their efflux activities. The present study was interested in whether transporters from other sub families of ABC, including ABCA2 and ABCA5 were also involved in the drug resistance. GST-π is a major antioxidant molecule leading to MDR in tumor cells. Bcl-2 has been implicated in several types of cancer and is also considered to be involved in resistance to conventional cancer treatment. Western blot analysis demonstrated that A549/DDP cells expressed a high level of MDR1, MRP1, LRP, GST-π, ABCA2, ABCA5 and Bcl-2. It was then investigated whether Sorcin was able to modulate the expression of these proteins. The qPCR and western blotting results demonstrated that, when compared with the high expression level observed in the A549/DDP cells, the expression of MDR1, MRP1, LRP, GST-π, ABCA2, ABCA5 and Bcl-2 decreased in the Sorcin silenced cells (Fig. 4).

Highly cisplatin resistant cancer cells are frequently associated with a marked increase of GSH synthesis. The reduced intracellular GSH was able to rescue the cells from cytotoxicity. The results demonstrated that the intracellular concentration of GSH was decreased in Sorcin-silenced A549/DDP cells compared with the control and negative groups. The intracellular concentration of GSH of the Sorcin siRNA group 1 and Sorcin siRNA group 2 were 52.7 and 31.6% compared with the control group, respectively (Fig. 5).

The present study aimed to investigate whether the PI3K/AKT and MEK/ERK pathways acted as linkers between Sorcin and drug resistance. The western blotting assays demonstrated that the phosphorylation level of AKT and ERK was downregulated in Sorcin-silenced A549/DDP cells (Fig. 6A).

NF-κB is activated by PI3K/AKT and its activation can lead to the expression of drug resistance-associated genes, including MDR1 and MRP1. STAT3, STAT5 and NFAT are also shown to control EMT and the transition is reported to contribute to the chemoresistance of cancer cells. The present study aimed to elucidate whether NF-κB, STAT3, STAT5 and NFAT transcriptional activities were also modified by Sorcin. The reporter gene system assay demonstrated that the transcriptional activities of NF-κB, STAT3, STAT5 and NFAT were significantly decreased following Sorcin silencing (Fig. 6B).