A normal human bronchial epithelioid (HBE) cell line was purchased from the Cell Bank of Typical Culture Preservation Committee of the Chinese Academy of Sciences (Shanghai, China), and was maintained in KM medium (ScienCell Research Laboratories, Inc., San Diego, CA, USA). NSCLC cells (PC-9, sensitive to gefitinib; PC-9/GR, resistant to gefitinib) were maintained in Dulbecco's modified Eagle's medium (DMEM; Biological Industries, Kibbutz Beit Haemek, Israel). The cell lines PC-9 and PC-9/GR were kindly provided by Guangdong Lung Cancer Institute (Guangdong, China) and were described in a previous study (16).

Cells were equally seeded into 96-well plates at 37°C for incubation overnight. Various fatty acids (C14:0, C15:0, C16:0, C17:0, C18:0, C18:1 or C20:0) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) dissolved in anhydrous ethanol and filtered (17) were added at the indicated concentrations (10, 50, 90, 130, 170, 210 or 250 µM), followed by incubation for 48 h. The cells were then incubated with MTT for 4 h at room temperature and cell proliferation was determined using a spectrophotometer (Molecular Devices, LLC, Sunnyvale, CA, USA) by determining the optical density at OD 490 nm. For apoptosis detection, an equal number of cells was washed with PBS and re-suspended in binding buffer contained in the Annexin V-fluorescein isothiocyanate (FITC) kit (TransGen Biotech Co., Ltd., Beijing, China). The cell suspension was then transferred to a new reaction tube and stained by Annexin V-FITC and propidium iodide (PI) according to the manufacturer's protocol. After incubation in the dark for 15 min at 25°C, cells were re-suspended in binding buffer and subjected to apoptosis measurement by flow cytometry.

Cells were seeded in 6-well plates and cultured at 37°C with 5% CO_2. until the concentration reached 95%. Cells were scratched with a sterile pipette tip to generate a cell-free path (width, ~1 mm) and cells were washed with PBS two times, followed by culture in DMEM (Biological Industries) containing the indicated fatty acids (C16:0, C17:0 or C18:0). After 48 h of incubation 37°C, images of the cells were captured through a fluorescent microscope at low-magnification. The wound gaps were measured and the mean healing ratios were quantified with ImageJ software (National Institutes of Health, Bethesda, MA, USA).

PC-9 and PC-9/GR cells were seeded into a 6-well plate at a density of 300 cells/well and incubated for 48 h. The cells were refreshed with culture media with or without C17:0 (final concentraion, 50 µM) every 48 h. Approximately 12–14 days later cells were fixed with 4% paraformaldehyde (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) at 4°C for 30 min and stained with 0.1% crystal violet at 25°C for 10 min (Beijing Solarbio Science & Technology Co., Ltd.) and cell images were captured with a fluorescence microscope and the number of cell colonies (>50 colonies) was counted.

Equal amounts of PC-9 and PC-9/GR cells were seeded for incubation overnight. Cells were incubated with or without C17:0 (final concentration, 100 µM) for 48 h. The cells were then collected and washed with PBS three times. Cells were counted and a cell suspension was prepared with 3 ml HCl-methanol (v/v=5%), followed by the addition of 10 µl of C15:0 internal standard (C15:0 in chloroform, the final concentration, 5 mg/ml). The tubes were sealed and heated in an incubator at 90°C for 4 h. After natural cooling to room temperature, 4 ml Na₂CO₃ (w/w=10%) was added. Subsequently 1 ml n-hexane was added, followed by agitation for 5 min and centrifugation at 1,000 × g for 10 min. A total of 400 µl of supernatant containing methyl esters of fatty acids was transferred to an automatic injector vial and subjected to a GC-MS assay according to the manufacturer's protocol. In brief, the compositions of methyl esters of fatty acids in the cells were analyzed by a 30-m polar capillary column (0.32 mm internal diameter; SH-FameWax) on a Shimadzu gas chromatograph containing a flame ionization detector (GCMS-QP2010 Ultra; Shimadzu, Kyoto, Japan). Of the samples, 1 µl was injected (injector temperature, 240°C). The column temperature was held at 130°C for 1 min, and increased at a rate of 3°C/min to 200°C, then increased at a rate of 2°C/min to 220°C and maintained for 10 min. Fatty acids in cells were quantified by an internal standard method using C15:0 methyl ester.

Cells were collected and incubated with RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China), followed by centrifugation at 10,000 × g, 4°C for 10 min. Supernatants were harvested and total protein was quantified by a BCA assay. Total protein (30–60 µg) was subjected to 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes, followed by blocking with TBST (10 mM Tris-HCl, pH 7.4, 150 mM NaCl and 0.1% Tween-20) containing 5% dried skimmed milk for 2 h at room temperature. Then the membranes were blotted with primary antibodies against GLUT1 (dilution 1:5,000; cat. no. ab115730; Abcam, Cambridge, UK), GLUT4 (dilution 1:2,000; cat. no. ab654; Abcam), p-S6K (dilution 1:1,000; cat. no. sc-8416; Santa Cruz Biotechnology, Dallas, TX, USA), S6K (dilution 1:1,000; cat. no. sc-8418; Santa Cruz Biotechnology), AKT (dilution 1:5,000; cat. no. ab179463; Abcam), p-AKT (dilution 1:5,000; cat. no. 81283; Abcam) and GAPDH (dilution 1:5,000; cat. no. ab181602; Abcam) at 4°C, for overnight. After washing with TBST for three times, membranes were further incubated with secondary antibody goat anti-rabbit IgG-HRP (dilution 1:5,000; cat. no. AS014; ABclonal) or goat anti-mouse IgG-HRP (dilution 1:5,000; cat. no. AS003; ABclonal) for 1 h at room temperature. At the end, the membranes were incubated with a ECL kit (cwbiotech) and subjected to exposure with X-ray films in dark room.

Differences between two groups were statistically analyzed by one-way analysis of variance with a Tukey's post hoc test using SPSS 19.0 software (IBM Corp., Armonk, NY, USA). All experiments were performed at least three times. Results were expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

Dietary saturated fatty acids may exert antitumor effects, and screening functional fatty acids may provide candidates that may serve as adjuvants in cancer therapy. Beef tallow and mutton tallow are common dietary animal oils. C16:0, C18:0 and C18:1 are the three dominant fatty acids in beef and mutton tallow. In a preliminary analysis, the content of C17:0 was determined to be 2.09% in beef tallow and 2.05% in mutton tallow (data not shown).

To investigate the impact of major dietary fatty acids from ruminant animal oils on lung cancer cells, a cell proliferation assay was performed. C14:0, C15:0, C16:0, C17:0, C18:0, C18:1 or C20:0 were directly added into HBE, PC-9 and PC-9/GR cells. As presented in Fig. 1A and B, the results of the MTT assay indicated that these fatty acids did not affect the proliferation of HBE cells, while 4 types of fatty acids (C16:0, C17:0, C18:0 and C20:0) significantly inhibited the proliferation of PC-9 and PC-9/GR cells. Furthermore, C17:0 was identified to be the most effective fatty acid against PC-9 and PC-9/GR cells, and its effects were dose-dependent (Fig. 1C and D). The effect of C17:0 in other lung cancer cells, including A549 and H1975, was also examined. Similarly, the results revealed that C17:0 inhibited cell proliferation in A549 and H1975 (data not shown). These results indicated that C17:0 may be a fatty acid suitable for lung cancer treatment.

The present study then focused on the functional role of C17:0 in lung cancer cells. A wound healing assay indicated that C16:0, C17:0 and C18:0 inhibited the wound closure ratio of PC-9 and PC-9/GR cells (Fig. 2A and B). Furthermore, the impact of C17:0 on cell migration was the greatest among all fatty acids tested. The apoptosis ratio was also determined by flow cytometry. As presented in Fig. 3A, C17:0 induced 16.62 and 29.63% apoptosis in PC-9 cells and PC-9/GR cells respectively. C18:0 also performed similarly in the two cell line variants. In addition, the results of the cell clone formation assay indicated that C17:0 suppressed the proliferation of PC-9 and PC-9/GR cells (Fig. 3B). These results indicated that C17:0 treatment in PC-9 and PC-9/GR cells was able to induce lung cancer cell death.

EGFR-TKIs are widely used in lung cancer therapy and exhibit marked efficacy. To investigate the additive/enhancing effect of C17:0 on EGFR-TKIs, an MTT assay was also performed on PC-9 and PC-9/GR cells treated with gefitinib plus C17:0. As presented in Fig. 4A and B, C17:0 promoted the inhibitory effect of gefitinib in PC-9 and PC-9/GR cells in a dose-dependent manner. The greatest inhibition ratio in the two cell variants reached ~80%. These results indicated that C17:0 at increasing concentrations enhanced the effect of gefitinib on NSCLC cells in vitro.

Changes in the fatty acid composition of PC-9 and PC-9/GR cells in response to C17:0 treatment was assessed by GC-MS and C15:0 was used as the internal standard. As presented in Fig. 5 and Table I, the contents of various fatty acids ranging from C14:0 to C24:0 were analyzed. In HBE, PC-9 and PC-9/GR cells, the addition of C17:0 increased the content of 10-cis-heptadecenoic acid (C17:1). The concentration of C17:1 in PC-9 and PC-9/GR cells was higher than that in HBE cells. Furthermore, the concentration of C18:1 in HBE cells was lower than that in PC-9 and PC-9/GR cells. In response to the addition of C17:0, C18:1 was identified to be downregulated in HBE cells, while it was not altered in PC-9 cells and only slightly upregulated in PC-9/GR cells. In HBE cells, the total fatty acid composition was not altered during incubation with C17:0. However, the ratio of total fatty acids in PC-9 and PC-9/GR cells was higher than that in HBE cells, indicating that these lung cancer cells have a higher lipid synthesis activity and accumulation (Fig. 6). The increase of C17:1 in PC-9/GR cells may contribute to lung cancer cell death.

To further explore the molecular mechanisms associated with the inhibition of C17:0 on PC-9 and PC-9/GR cells, the activation of relevant signaling pathways was detected. The PI3K/Akt signaling pathway is one pivotal pathway regulating cell proliferation and tumor development. Therefore, its activation was detected in PC-9 and PC-9/GR cells in response to treatment with C17:0. As presented in Fig. 7, the phosphorylation of Akt and S6K was inhibited. In particular, the levels of p-S6K in PC-9/GR cells were markedly downregulated by C17:0 in a dose-dependent manner. In general, cancer cells may upregulate the expression or enhance the activation of glucose transporters, including GLUT1 and GLUT4, to increase glucose import from the extracellular environment into the cytoplasm to compensate ATP production (18). Thus, the expression of GLUT1 and GLUT4 in the two cell line variants in the absence or presence of C17:0 was also detected. The results indicated that treatment of PC-9 and PC-9/GR cells with C17:0 caused downregulation of GLUT1 and GLUT4. Collectively, the present results indicated that addition of C17:0 to PC-9 and PC-9/GR cells inhibited the activation of PI3K/Akt signaling and glucose consumption.