The NSCLC cell lines A549, H460, H1299, H1650 and H1975 were purchased from American Type Culture Collection (Manassas, VA, USA). All NSCLC cell lines were maintained in RPMI-1640 medium with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml of streptomycin and 100 µg/ml of penicillin. Oxfendazole was purchased from Selleck Chemicals (Houston, TX, USA). Cisplatin was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

A549 and H1299 cells (1×10⁶ cells/well) were seeded in 6-well plates overnight. Then, the cells were starved overnight in serum-free medium. Subsequently, starved A549 and H1299 cells were incubated with 0, 10 or 20 µM of OFD for 6 h at 37°C, then stimulated with 10 ng/ml EGF (P5552; Beyotime Institute of Biotechnology, Haimen, China) for 20 min at 37°C.

Immunoblotting was conducted as previously described (12,13). Briefly, cells (1×10⁶ cells/well) were lysed by RIPA lysis (P0013B; Beyotime Institute of Biotechnology), and whole cell lysates were extracted. Then, proteins were determined by BCA method (BCA kit, P0011; Beyotime Institute of Biotechnology) and 30 µg of total proteins were subjected to SDS-PAGE separation by using 10% or 12% acrylamide gel, and the proteins were transferred onto 0.2 µm polyvinylidene difluoride (PVDF) membrane (1620177; Bio-Rad Laboratories, Inc., Hercules, CA, USA), followed by immunoblotting with specific antibodies. The primary antibodies phosphorylated (p)-c-Src (Tyr416) (6943; 1:1,000), c-Src (2109; 1:1,000), CDK4 (12790; 1:1,000), CDK6 (13331; 1:1,000), p-Rb (Ser807/811) (8516; 1:1,000), E2F1 (3742; 1:1,000), p53 (2527; 1:1,000) and p21 (2947; 1:1,000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-GAPDH antibody (AM1020A; 1:5.000) was purchased from Abgent Biotech Co., Ltd. (Suzhou, China). Anti-mouse (A0216; 1:1,000) and anti-rabbit (A0208; 1:1,000) immunoglobulin G (IgG) horseradish peroxidase-conjugated antibodies were purchased from Beyotime Institute of Biotechnology (Haimen, Nantong, China). All immunoblotting signals were further assessed by using ECL (BeyoECL Plus kit, P0018M; Beyotime Institute of Biotechnology) and analyzed with Quality One software (version number: 4.0.1; Bio-Rad Laboratories, Inc.).

Viable cells (8,000 cells/well) were analyzed by Cell Counting Kit-8 (CCK-8; BioTools, Inc., Jupiter, FL, USA) assay according to the manufacturer's instruction, as described previously (14). To assess the effect of OFD on the survival rates of different NSCLC cell lines, 5 NSCLC cell lines were treated with 0, 5, 10 or 20 µM of OFD for 24 h at 37°C, or cells were treated by 5 µM OFD for 0, 24, 48 or 72 h at 37°C, followed by a CCK-8 assay. For the transfected cells, A549 or H1299 cells were treated with 0, 2.5, 5 or 10 µM of OFD for 24 h at 37°C. In addition, A549 and H1299 cells were treated with 5 µM cisplatin for 24 h at 37°C in the presence or absence of 10 µM OFD, and then the cells were assessed by a CCK-8 assay.

The human c-Src gene was generated and cloned into the pcDNA3.1 vector as previously described (15,16). The primers for c-Src amplification were as follows: Forward, 5′-ATGGGTAGCAACAAGAGCAAGC-3′ and reverse, 5′-CTAGAGGTTCTCCCCGGGCTGGTA-3′. A549 or H1299 cells were transfected with 1 µg/µl empty vector (pcDNA3.1 vector) or 1 µg/µl c-Src plasmids for 24 h by Lipofectamine^® 2000™ (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.

Cell cycle analysis was performed as previously described (17). A549 and H1299 cells (1×10⁶ cells/well) were treated with 0, 5 or 10 µM of oxfendazole for 24 h at 37°C prior to cell cycle analysis. Then, cells were fixed with 70% cold ethanol overnight at −20°C and washed with cold PBS, followed by being resuspended in 100 µl PBS containing 100 µg/ml RnaseA (Beijing Solarbio Science & Technology, Co., Ltd., Beijing, China) for 30 min at 37°C. Cells were then washed with cold PBS and incubated with propidium iodide (PI) for 5 min at room temperature by using the Cell Cycle Detection kit (C1052; Beyotime Institute of Biotechnology). Cell cycle distribution was analyzed on a flow cytometer (Attune^® NxT; Thermo Fisher Scientific, Inc.) and the data was analyzed by FlowJo 7.6.1 (FlowJo LLC, Ashland, OR, USA).

Statistical analysis was performed using Statistical Package for Social Sciences 13.0 for Windows (SPSS, Inc., Chicago, IL, USA). The results were presented as the mean ± standard deviation. One-way analysis of variance with Bonferroni post hoc tests were used to determine significance. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the effects of oxfendazole on c-Src activation in NSCLC cells, a panel of NSCLC cell lines were treated with oxfendazole for 24 h, followed by immunoblotting against p-c-Src (Tyr416) and c-Src. As shown in Fig. 1A and B, c-Src was activated in all five NSCLC cell lines, and oxfendazole markedly inhibited the phosphorylation of c-Src. In addition, oxfendazole downregulated the phosphorylation of c-Src in a concentration-dependent manner in A549 and H1299 cells (Fig. 1C). Next, A549 and H1299 cells were starved overnight in serum-free medium followed by oxfendazole treatment and epidermal growth factor (EGF) stimulation. As shown in Fig. 1D, EGF, a key trigger of c-Src signaling, markedly activated c-Src, but this action was suppressed by oxfendazole in a concentration-dependent manner. These results suggested that oxfendazole inhibited the activation of c-Src in NSCLC cells, and that it may be a novel c-Src inhibitor.

It was previously reported that the activation of c-Src promoted the cell survival of tumor cells (9). In the present study, to evaluate the effects of oxfendazole on NSCLC cell survival, a panel of NSCLC cell lines were treated with increased concentrations of oxfendazole for 24 h, followed by a CCK-8 assay. As shown in Fig. 2A, oxfendazole decreased NSCLC cell viability in a concentration-dependent manner. In addition, oxfendazole also inhibited the cell survival of A549 and H1299 cells in a time-dependent manner (Fig. 2B). Furthermore, when A549 and H1299 cells were transfected with c-Src plasmids, oxfendazole-induced cell death was significantly attenuated (Fig. 2C and D). For example, in A549 cells treated with 10 µM of oxfendazole, the fraction of surviving cells was increased from 50% in vector-transfected cells to 60% in c-Src-transfected cells (Fig. 2C). In H1299 cells treated with 10 µM of oxfendazole, the fraction of surviving cells was increased from 55% in vector-transfected cells to 65% in c-Src-transfected cells (Fig. 2D). These results indicated that c-Src signaling may be involved in the underlying mechanism of the action of oxfendazole.

It has been previously reported that c-Src activation regulated the cell growth and cell cycle of tumor cells (18,19). Therefore, the present study evaluated whether oxfendazole regulated the cell cycle of NSCLC cells. As shown in Fig. 3, flow cytometry revealed that oxfendazole induced cell cycle arrest at the G₀/G₁ phase in A549 and H1299 cells. In A549 cells, the fraction of the G₀/G₁ cells was increased from 60.05% in the control to 88.30% in cells treated with 10 µM oxfendazole, coupled with a decrease in the fraction of S phase cells from 22.80 to 3.42% (Fig. 3; upper panel). In H1299 cells, the percentage of G₀/G₁ cells was increased from 64.65 to 86.23% following treatment with 10 µM oxfendazole, coupled with a decrease in the fraction of S phase cells from 16.91 to 5.34% (Fig. 3; lower panel).

D-cyclins combine with CDK4/6 to phosphorylate p-Rb, allowing for the release of E2F transcription factors, which activate G1/S-phase gene expression (20). Thus, the present study next evaluated the effects of oxfendazole on the expression of these proteins. As shown in Fig. 4A and B, oxfendazole downregulated the expression levels of CDK4, CDK6, p-Rb and E2F1 in a concentration-dependent manner.

The p53 tumor suppressor protein serves a major role in arresting cell cycle progression (21). To determine whether p53 was associated with oxfendazole cytotoxicity in cell survival, the present study then examined the expression of p53. As shown in Fig. 4C, oxfendazole upregulated p53 expression. The expression of p21 expression was also detected, which is a potent CDK inhibitor and a target of p53 (22). As shown in Fig. 4C, oxfendazole markedly upregulated p21 expression.

These results indicated that oxfendazole inhibited cell cycle progression by suppressing CDK signaling in NSCLC cells, and its activity in survival inhibition in NSCLC cells was observed.

Cisplatin is a common chemotherapeutic drug for NSCLC therapy, but resistance is frequently observed during the process of NSCLC treatment in the clinic (23–25). It has been reported that overactivation of c-Src could result in drug resistance to NSCLC treatment (10,26); thus, the present study then evaluated whether oxfendazole could enhance the effect of cisplatin in the treatment of NSCLC cells. As shown in Fig. 5A, the CCK-8 assay demonstrated that oxfendazole enhanced the cytotoxicity of cisplatin against A549 and H1299 cells. In addition, the immunoblotting analysis revealed that the combination of oxfendazole and cisplatin could enhance the inhibition of c-Src activation, as well as the upregulation of p53 (Fig. 5B). Therefore, oxfendazole enhanced the cytotoxic activity of cisplatin by suppressing c-Src activation, and oxfendazole could utilized for anti-NSCLC therapy in the clinic in combination with other antitumor drugs, such as cisplatin.