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Introduction

At present, lung cancer is a malignant tumor with the globally highest morbidity and fatality rates. ~80–85% of lung cancers are non-small-cell lung carcinoma (NSCLC). Surgical resection is considered the main treatment for NSCLC, with surgery being indicated for ~20–30% of patients and the postoperative recurrence and the metastasis rate being ≥50% (1). Therefore, for the lung cancer patients who are unable to undergo surgery and those with postoperative recurrence or metastasis, combinations of platinum-containing agents are the primary chemical treatment to extend the lifetime. However, to date, no effective measures have been established for patients who are subject to such regimen control, and who present with new cancer progression. Sorafenib is a small molecule with activity against neoplasms via multiple biological targets. Sorafenib has a dual function of inhibiting tumor cell proliferation and angiogenesis. At present, the Food and Drug Administration (FDA, Silver Spring, MD, USA) has approved sorafenib for the use in the clinical treatment of kidney and liver cancers (2,3). To date, basic research and clinical trials using sorafenib for the prevention and treatment of lung cancer have made positive progress globally (4,5). The present study used the A549/DDP cisplatin-resistant lung adenocarcinoma cell strain to study the biological effects of sorafenib. Effects on proliferation, apoptosis and invasion of the A549/DDP cisplatin-resistant lung adenocarcinoma cell strain was observed, which provided scientific experimental data and a theoretical basis for the application of sorafenib for the subsequent treatment of cisplatin-resistant lung cancer.

Materials and methods

Cell strains

The A549 lung adenocarcinoma cell strain and the A549/DDP cisplatin-resistant lung adenocarcinoma cell strain were purchased from the tumor cell bank of the Chinese Academy of Sciences (Shanghai, China), with a resistance index of 13 times.

Main reagents

Sorafenib was purchased from Bayer (Leverkusen, Germany), RPMI-1640 culture medium and fetal bovine serum (FBS) were from HyClone (Logan, UT, USA), cisplatin (DDP) from Haosen Pharmacy, Ltd. (Ganzhou, China), MTT and dimethylsulfoxide (DMSO) from Sigma-Aldrich (St. Louis, MO, USA), Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis kit from BioVision (Mountain View, CA, USA) and Matrigel® from BD Biosciences (Franklin Lakes, NJ, USA).

Cell culture

The A549 and A549/DDP cells were cultured in RPMI-1640 medium at 37°C with 5% CO2 and 95% saturated humidity. To maintain the stable drug-resistance of A549/DDP cells, 2 μmol/l DPP was added into the RPMI-1640 with 10% FBS for joint culture and the cells were cultured in the medium without DDP for one week prior to beginning the trial.

Experimental groups

The experiment involved five groups: S0 and experimental groups S1, S2, S3 and S4. The control group S0 was incubated with the culture medium RPMI-1640 only; S1–4 were incubated with 2, 4, 8 and 16 μmol/l sorafenib, respectively.

MTT assay

Cells were collected at the exponential growth phase, the cell density was adjusted to 3×104 ml−1 and 100 μl was inoculated into each well on 6 culture plates and 7 repeated wells set for each 96-well plate. One culture plate was removed each day at the same time, MTT reagent (20 μl) was added, placed in a CO2 incubator and incubated for 4 h. Following removal of the medium, DMSO (100 μl) was added to each well and the plate was placed on an agitator for 10 min to fully dissolve the crystals. The optical density (OD) of each well was measured at 490 nm using an ELISA reader (R&D Systems, Minneapolis, MN, USA). The blank controls (containing medium, MTT and DMSO) and control wells (containing cells, medium, MTT and DMSO) were measured. The formula adopted for the calculation of the inhibition rate of cell proliferation is: Inhibition rate = 100% × [(OD value of the control well − OD value of the blank control well) − (OD value of the experimental well − OD value of the blank control well)]/(OD value of control well − OD value of the blank control well). Each experiment was repeated three times.

Flow cytometric analysis of apoptosis

The inoculated cells were cultured in a 5% CO2 incubator for 24 h and the original culture medium was discarded. Different concentrations of sorafenib (2, 4, 8 and 16 μmol/l) were added to the experimental groups at 2 ml/well in RPMI medium. The control group was added with the same amount of culture medium without sorafenib. Three complexes were prepared for each group. The culture plates were incubated for a further 72 h. The cells of each group were digested with EDTA-free pancreatin (Gibco, Grand Island, NY, USA) and collected. Binding buffer (500 μl; KeyGen Biotech, Nanjing, China) was added to prepare a cell suspension. Under the exclusion of light, 5 μl Annexin V-FITC and 5 μl PI was added to each group of cell suspension. The groups were allowed to react for 15 min at room temperature in the dark. Flow cytometric analysis was performed within one hour and the percentage of apoptotic cells was calculated using CellQuest software, version 3.0 (BD Biosciences).

Transwell assay

A549/DDP cells were collected during the exponential growth phase, the concentration was adjusted to 5×105 ml−1 and the cells were inoculated into the upper chamber of a Transwell plate (6.5 mm; 24-well plate, Costar; Corning, NY, USA) at 200 μl/well and three repeated wells were set for each group. Sorafenib was added to the upper chamber with a final concentration of 2, 4, 8 and 16 μmol/l for the experimental groups, and no reagent was added for the control group. At the same time, RPMI-1640 (500 μl) culture medium containing 10% FBS was added into the lower chamber and the plate was cultured for a further 24 h. The cells were removed from the Transwell chamber, washed twice with phosphate-buffered saline and the filter membrane was fixed with 4% paraformaldehyde for 30 min. The filter membrane was subjected to crystal violet staining (Beyotime Institute of Biotechnology, Shanghai, China) for 20 min following air drying and was then rinsed with double distilled water. Five fields of view at upper, lower, left, right and central locations were captured under a light microscope (x400). To assess the number of cells migrated through the membrane to the lower chamber surface, the average values were analyzed. The number of migrated cells reflected the level of tumor cell invasiveness. Accordingly, a reduced number of cells permeating the septum indicated marked inhibition of invasion.

Statistical analysis

Values are presented as the mean ± standard deviation and were determined by the statistical software SPSS 11.0 (SPSS Inc., Chicago, IL, USA). Groups were compared using one-way analysis of variance. Homogeneity of variance was tested by the least significant difference method, abd heterogeneity of variance was tested using Dunnett’s T method. P<0.05 was considered to indicate a statistically significant difference.

Results

Comparison between the growth curves of A549/DDP cisplatin-resistant lung adenocarcinoma cells and A549 parental cells

Microscopic observation indicated that the adherent growth of cisplatin-resistant lung adenocarcinoma cells A549/DDP was marked and vigorous, indicating a satisfying light refraction of cytoplasm. Compared with A549 cells, the A549/DDP cells were slightly larger in volume, growing in fusiform or polygonal structures, with a large cell nucleus. Compared with parental A549 cells, the A549/DDP cisplatin-resistant cells exhibited a higher cell proliferation rate and reached the exponential growth phase 2–4 days following passage, while the A549 cells required 3–5 days to reach the exponential growth phase (Fig. 1).

Figure 1 Comparison between the growth curves of A549/DDP cisplatin-resistant lung adenocarcinoma cells and parental A549 cells.

Sorafenib inhibits the proliferation of A549/DDP cisplatin-resistant lung adenocarcinoma cells

The MTT assay was used to determine the inhibition rate of sorafenib on the proliferation of A549/DDP cisplatin-resistant lung adenocarcinoma cells at different concentrations and time-points. The results showed that, compared with the control group, sorafenib inhibited the proliferation of A549/DDP cisplatin-resistant lung adenocarcinoma cells within the concentration range of 2, 4, 8 and 16 μmol/l. With the extension of the action time and increase of the concentration, the inhibition rate of sorafenib on A549/DDP cisplatin-resistant lung adenocarcinoma cells gradually increased (P<0.05). In addition, the differences in the inhibition rates of sorafenib on A549/DDP cisplatin-resistant lung adenocarcinoma cells at different times and concentrations were statistically significant (P<0.05), as shown in Table I and Figs. 2 and 3.

Figure 2 Concentration-dependent growth inhibition by sorafenib on A549/DDP cisplatin-resistant lung adenocarcinoma cells. S1–4, incubation with sorafenib at 2, 4, 8 and 16 μmol/l.

Figure 3 Time-effect curves of the growth inhibition of A549/DDP cells treated with different concentrations of sorafenib. S1–4, incubation with sorafenib at 2, 4, 8 and 16 μmol/l.

Table I The inhibition rate of sorafenib on A549/DDP cisplatin-resistant lung adenocarcinoma cells at different incubation times (mean ± standard deviation).

Table I

The inhibition rate of sorafenib on A549/DDP cisplatin-resistant lung adenocarcinoma cells at different incubation times (mean ± standard deviation).

Group	24 h	48 h	72 h
S1	4.58±2.81	14.98±2.93a	18.80±2.82b
S2	14.93±2.62	26.28±7.31a	32.71±2.55b
S3	37.58±7.13	63.00±3.05a	75.51±4.73c
S4	58.39±8.15	78.84±3.96a	87.50±3.36c

a P<0.01 vs. 24 h, ;

b P<0.05 vs. 48 h;

c P<0.01 vs. 48 h.

{ label (or @symbol) needed for fn[@id='tfn4-mmr-10-01-0347'] } S1–4, incubation with sorafenib at 2, 4, 8 and 16 μmol/l

Sorafenib induces apoptosis of A549/DDP cisplatin-resistant lung adenocarcinoma cells

The A549/DDP cisplatin-resistant lung adenocarcinoma cells in the experimental groups S1–4 were respectively treated with sorafenib at the concentrations of 2, 4, 8 and 16 μmol/l for 72 h, and the S0 control group was cultured with RPMI-1640 medium only for 72 h. The rate of apoptosis was determined using Annexin V-FITC/PI staining and flow cytometric analysis. It was observed that the rate of apoptosis of the control group S0 was 8.89±0.81% and the rates of apoptosis in the experimental groups S1–4 were 12.84±0.24, 17.27±0.78, 21.98±0.75 and 49.67±1.38%, respectively. Compared with the control group, the differences were statistically significant (P<0.01); the differences of further pairwise comparison in the same group were also statistically significant (P<0.01), as shown in Fig. 4.

Figure 4 Flow cytometric assessment of apoptosis of A549/DDP cisplatin-resistant lung adenocarcinoma cells following 72 h sorafenib treatment and staining with Annexin V-fluorescein isothiocyanate/propidium iodide. S0–4, incubation with sorafenib at 0, 2, 4, 8 and 16 μmol/l. X-axis represents Annexin V-FITC staining and y-axis represents PI staining. Upper left: necrosis cells; upper right: late apoptotic cells; lower left: normal cells; lower right: early apoptotic cells.

Sorafenib inhibits the invasion of A549/DDP cisplatin-resistant lung adenocarcinoma cells

The Transwell assay was used to determine the number of cells permeating the Transwell membrane in each group. The results showed that, following 24 h of incubation, the average number of cells permeating the septum in the normal control group S0 reached 82.7±2.3/high power lens (HP), while the average number of cells permeating the septum in S1–4 in the presence of sorafenib at concentrations of 2, 4, 8 and 16 μmol/l, were decreased to 58.2±2.5, 41.3±1.3, 22.6±2.1 and 14.7±1.1/HP, respectively. Compared with the control group, the difference in the number of cells permeating the Matrigel® of each group was statistically significant (P<0.01). When the concentration increased and the cell permeation of the septum gradually decreased, the differences obtained by pairwise comparisons among the groups also became statistically significant (P<0.01). In addition, observation by optical microscopy following crystal violet staining indicated that the cells in the normal control group which had not been treated with sorafenib were of fusiform, polygonal or oval shaped, with a larger cell volume a dense distribution at the lower surface of the Transwell chamber, while the cells of the experimental groups, which had been treated with sorafenib, gradually became smaller, pseudopodia decreased and the shape of the cells became round and sparsely scattered at the lower surface of the Transwell chamber (Fig. 5).

Figure 5 Sorafenib inhibits the invasion of A549/DDP cisplatin-resistant lung adenocarcinoma cells following incubation for 24 h (S0–4, incubation with sorafenib at 0, 2, 4, 8 and 16 μM/l). Photomicrographs (magnification, ×400) of lower Transwell membranes staned with crystal violet.

Discussion

Sorafenib is an antineoplastic agent with multiple biological targets, which inhibits tumor growth, invasion and metastasis by preventing the proliferation of tumor cells and angiogenesis in tumor tissue. In 2005 and 2007, the FDA approved the use of sorafenib for the clinical treatment of advanced kidney and liver cancer (6,7). At present, basic research and clinical trials using sorafenib for the prevention and treatment of lung cancer are progressing with positive preliminary results (4,5). Dy et al (8) have tested sorafenib in a Phase II clinical trial as a first-line treatment of advanced NSCLC and the results showed that among the 25 patients selected for the study, three went into partial remission and six achieved a stable condition, with progression-free survival (PFS) for 2.8 months, and a certain clinical efficacy was obtained. Spigel et al (9) performed a clinical trial of combining sorafenib with erlotinib, or using only erlotinib as the second-line treatment of patients with advanced NSCLC. A total of 168 patients were involved. The results showed that the disease control rates of the combination group and the group with only erlotinib were 54 and 38%, respectively, and PFSs were 3.38 and 1.94, respectively, indicating that sorafenib has potential as a second-line treatment. In the present study, in order to provide a novel method for the second-line treatment of NSCLC, the A549/DDP cisplatin-resistant lung adenocarcinoma cell strain was chosen to assess the effect of sorafenib on the proliferation, apoptosis and invasion in vitro.

In the present study, the MTT assay was used to determine the activity of sorafenib on the proliferation of A549/DDP cisplatin-resistant lung adenocarcinoma cells and to observe its inhibition effects. The results showed that compared with the parental A549 cells, the A549/DDP cisplatin-resistant lung adenocarcinoma cells were slightly larger in volume, growing in fusiform or polygonal structures, with a large intracellular nucleus. The cell proliferation was faster, reaching the exponential phase within 2–4 days following passage, while A549 cells reached the exponential phase within 3–5 days following passage, indicating that the metabolic activity of A549/DDP cisplatin-resistant lung adenocarcinoma cells was significantly enhanced and marked mitosis was observed following acquisition of drug-resistance characteristics; thus, they were able to enter the logarithmic phase faster than the parental A549 cells. This is consistent with the observations on the growth characteristics of A549/DDP cisplatin-resistant lung adenocarcinoma cells reported by Giard et al (10). A further analysis of the inhibitory effect of sorafenib on the proliferation of A549/DDP cisplatin-resistant lung adenocarcinoma cells was conducted, showing that sorafenib is capable of inhibiting the proliferation of the A549/DDP cisplatin-resistant lung adenocarcinoma cells within the concentration range of 2–16 μmol/l. Furthermore, following treatment of A549/DDP cells with varying concentrations of sorafenib, the inhibition rate of cells was gradually increased with increasing time, while at the same time, the inhibition rate was gradually increased as well with increasing drug concentration. The results of this trial showed that sorafenib was capable of inhibiting the proliferation of A549/DDP cisplatin-resistant lung adenocarcinoma cells and this inhibitory effect was time- and concentration-dependent. The Annexin V-FITC/PI assay was used to determine the rate of apoptosis of A549/DDP cisplatin-resistant lung adenocarcinoma cells and to observe the induction effect of sorafenib on the apoptosis of A549/DDP cells. The results showed that marked apoptosis appeared in the cells in each experimental group following treatment with sorafenib at different concentrations for 72 h. In addition, with the increase of the drug concentration, the rate of apoptosis also increased. The difference from the control group was statistically significant (P<0.05), indicating that sorafenib is capable of promoting the apoptosis of A549/DDP cisplatin-resistant lung adenocarcinoma cells, and this effect was dependent on the concentration. The difference obtained by pairwise comparison among experimental groups was also statistically significant (P<0.05). The results of the present study were similar to those investigating the effects of sorafenib on the proliferation and apoptosis of parental A549 cells reported by Yu et al (11) and Mao et al (12). In the present study, to further determine the influence of sorafenib on the invasiveness of A549/DDP cisplatin-resistant lung adenocarcinoma cells, the Transwell assay was used for each experimental group in the presence of sorafenib for 24 h. The results showed that with increasing drug concentration, the number of cells permeating the basement membrane in each experimental group significantly decreased. The difference between each experimental and the control group was statistically significant (P<0.05), indicating that sorafenib had an in vitro inhibitory effect on the invasion of A549/DDP cisplatin-resistant lung adenocarcinoma cells. At the same time, it was noted that the cells permeating the septum in each experimental group markedly changed their shape, the occurrence of pseudopodia decreased and the cell volume decreased.

In conclusion, sorafenib inhibited the proliferation of A549/DDP cisplatin-resistant lung adenocarcinoma cells in a time- and concentration-dependent manner. Sorafenib also induces apoptosis and reduces the invasiveness of A549/DDP cells, which provided scientific experimental evidence for the theoretical basis of the application of sorafenib in the subsequent treatment of cisplatin-resistant lung cancer and offers a novel and effective strategy for further treatment of advanced lung cancer (13–15).

References