Human A549 lung carcinoma cells and cisplatin-resistant human A549/DDP lung carcinoma cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in GibcoRPMI-1640 medium (Thermo Fisher Scientific, Inc, Waltham, MA, USA), supplemented with 10% (v/v) dialyzed heat-inactivated bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in 5% CO₂.

Cell viability was determined using the Cell Counting kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). CCK-8 allows very convenient assays by utilizing Dojindo's highly water-soluble tetrazolium salt. WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] produces a water-soluble formazan dye upon reduction in the presence of an electron carrier. CCK-8 allows sensitive colorimetric assays for the determination of the number of viable cells in cell proliferation and cytotoxicity assays. WST-8 is reduced by dehydrogenases in cells to give an orange colored product (formazan), which is soluble in the tissue culture medium. The quantity of formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells. Briefly, cells in the early log phase were trypsinized and plated in 96-well plate at a density of 5×10³ cells per well. Cells were treated with various concentrations of niclosamide (Sigma-Aldrich; Merck KGa, Darmstadt, Germany) and DDP (Haosen Medicine Corp., Liangyungang, China) for 24 h at 37°C. Cell density was measured using the CCK-8 assay according to the manufacturer's instructions. The absorbance of each well was determined at a wavelength of 450 nm using a microplate reader (Thermo Electron Corp., Shanghai, China). The inhibition rate was calculated as follows: Inhibition rate (%)=[1-(T-B)/(U-B)]x100%; where T is the treated cell absorbance, U is the untreated cell absorbance and B is the background absorbance when neither drug nor CCK-8 was added. All experiments were repeated at least three times independently.

To evaluate whether the antitumor effects of niclosamide combined with DDP were synergistic, additive or antagonistic, combination index (CI) value for drug synergy was calculated using the CompuSyn software (Version 2.1, ComboSyn, Inc., Paramus, NJ, USA) as previously described (14). Using data obtained from CCK-8 assays and CompuSyn software, the dose-effect curves for single agents and their combinations were generated, and the CI values for each dose and the corresponding effect level, referred to as the fraction affected (Fa; the fraction of cells inhibited following drug exposure, for example 0.5 when cell growth is inhibited by 50%), were calculated. CI values <1 indicated a synergistic effect, values equal to 1 indicated an additive effect and values >1 indicated an antagonistic effect. Then, to provide a visual illustration of drug interactions, the Fa-CI plot was constructed by simulating CI values over a range of Fa levels from 0.1 to 0.95 (18,19).

Apoptosis was assessed by Annexin V/PI detection as described previously (20). The A549/DDP cells were plated at a density of 1×10⁵ cells per well in six-well plates. The next day, cells were treated with DDP (5 µg/ml), niclosamide (1 µM) or cisplatin (5 µg/ml) combined with niclosamide (1 µM) for 36h. The cells were harvested, and washed three times with phosphate-buffered saline (PBS) at 4°C. Cells were then incubated with 5 µl Annexin V-fluorescein isothiocyanate for 3 min and with 20 ng/ml PI in the dark for 15 min. The suspension was then analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA). All data were collected and analyzed by FACSDiva version 6.1.3 (BD Biosciences). The experiments were repeated three times independently and the results were presented as the mean ± standard deviation.

Subsequent to treatments, the cells were collected and lysed. Lysis buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, sodium pyrophosphate, β-glycerophosphate, EDTA, Na₃VO₄ and leupeptin was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Total protein (~20 µg) was separated on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred to a polyvinylidene fluoride membrane. After blocking with 5% non-fat milk in PBS + 0.1% Tween-20 for 1 h, the membrane was incubated with the appropriate primary antibody: Anti-caspase-3 (#14220; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-LRP (sc-23916; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-c-myc (D84C12, 1:1,000; Cell Signaling Technology, Inc.) and anti-β-tubulin (AT819, 1:2,000; Beyotime Institute of Biotechnology) overnight at 4°C. After washing with PBS three times (10 min each), the membrane was incubated with goat anti-rabbit (A0208; 1:2,000; Beyotime Institute of Biotechnology) or anti-mouse (A0216; 1:2,000; Beyotime Institute of Biotechnology) IgG-horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature and washed with PBS three times. The ECL system (Applygen Technologies, Inc., Beijing, China) was used to detect blotting signals according to the manufacturer's instructions. Detection of β-tubulin served as a loading control.

Continuous data are expressed as the mean ± standard deviation. For two-group comparison, the Student's t-test method was used. SPSS 13.0 software was used to perform all statistical analyses (SPSS, Inc., Chicago, IL, USA). For more than two-group comparison, one-way ANOVA was used. P<0.05 was considered to indicate a statistically significant difference.

In order to confirm the differential sensitivity of A549 and its derivative cisplatin-resistant cell line, A549/DDP, to DDP, cells were treated with different concentrations of DDP as indicated for 24 h, and cell viability was measured by CCK-8 assay. IC₅₀ values were calculated using GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA). The data demonstrated that the IC₅₀ values of DDP in A549 and A549/DDP cells were 6.81±0.78 and 32.5±0.21 µg/ml, respectively (Fig. 1). The resistance index of A549/DDP cells to DDP was the IC₅₀ of A549/DDP cells divided by IC₅₀ of the A549 cells or 32.5/6.81 µg/ml=4.77. These data demonstrate that the A549/DDP cell line has a certain resistance to DDP, which is suitable for drug resistance study.

The A549 and A549/DDP cells were treated with various concentrations of niclosamide as indicated (0, 0.125, 0.25,0.5, 1.0, 2.0 and 4.0 µM) for 24 h. Cell viability was measured using CCK-8 assay. The current results demonstrated that niclosamide significantly suppressed cell growth in a dose-dependent manner in A549 and A549/DDP cells (Fig. 2; P<0.05). The IC₅₀ values after 24 h of niclosamide in the A549 and A549/DDP cells were 2.60±0.21 and 1.15±0.18 µM, respectively. Niclosamide appears to exert a markedly greater inhibitory effect on A549/DDP cells when compared with parental A549 cells.

To examine whether niclosamide combined with DDP exhibits enhanced antitumor effects in cisplatin-resistant lung cancer, A549/DDP cells were treated with 0.5 or 1 µM niclosamide along with 1.25, 2.5, 5, 10 or 20 µg/ml DDP for 24 h. The niclosamide-treated cells demonstrated increased sensitivity to DDP at all concentrations (Fig. 3; P<0.05). According to the combined index calculated with CompuSyn software, the CI value of DDP in combination with niclosamide was <1, indicating that DDP combined with niclosamide exerts a synergistic effect on A549/DDP cells (Fig. 4).

Subsequently, the apoptosis of A549/DDP cells after niclosamide (1 µM) and DDP (5 µg/ml) treatment was evaluated using flow cytometry. Apoptotic cells were detected following treatment with 1 µM niclosamide and/or 5 µg/ml DDP for 36 h. Annexin V/PI analysis indicated that the apoptotic ratios of the control group, niclosamide, DDP and combined treatment group in the A549/DDP cells were 8.36±1.05, 11.0±3.18, 16.5±5.25 and 30.36±4.36%, respectively (Fig. 5A and B). The current data demonstrated that niclosamide in combination with DDP significantly enhanced the tumor killing effect by inducing apoptosis. Furthermore, western blotting was used to detect the activation of caspase-3 protein in A549/DDP cells after the same treatment. The cleavage of caspase-3 was observed to be markedly increased in the combined treatment group compared with the mono-treatment group (Fig. 5C). Therefore, the current results indicate that niclosamide combined with DDP may enhance cytotoxic effects by inducing apoptosis in A549/DDP cells.

To further document the underlying mechanisms by which niclosamide enhanced the inhibitory effect of DDP in A549/DDP cells, western blotting was used to detect the impact of niclosamide on DDP-resistant associated proteins. Initially, the basic expression levels of LRP and c-myc proteins were evaluated in A549 and A549/DDP cells. The expression levels of LRP and c-myc proteins in A549/DDP cells were significantly higher than those of the A549 cells (Fig. 6A). Subsequently, the changes of LRP and c-myc protein expression levels were investigated after treatment with niclosamide and/or DDP in A549/DDP cells. Following treatment with 1.0 µM niclosamide alone, A549/DDP cells demonstrated downregulation of LRP and c-myc protein expression levels, while DDP alone exerted no effect on LRP and c-myc protein expression levels. However, upon the combination treatment of niclosamide and DDP, the expression levels of the two proteins were significantly decreased compared with the controls (Fig. 6B). Due to the roles of LRP and c-myc protein on DDP resistance, it was inferred that niclosamide may enhance the cytotoxic effect of DDP on A549/DDP cells by downregulating the expression level of c-myc protein.