The human squamous cell lung carcinoma cell lines NCI-H1703 and NCI-H2170 were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin/streptomycin at 37°C in an atmosphere containing 5% CO₂.

According to the experimental method applied in a previous study (16), NCI-H1703 and NCI-H2170 cell suspensions (1×10⁴ cells/well) were seeded into 96-well plates overnight. Cells were then divided into four experimental groups and treated as follows: Control (CON) group, which was not treated; MW group, treated with the novel microwave applicator alone with a frequency of 433 MHz [±5 KHz; patent no. CN-204824903-U (15)] at 42°C for 60 min; GEM group, treated with GEM (a duration of 24 h; Lilly France, Neuilly-sur-Seine, France) alone at a dose of 5 µmol/l; and MW + GEM group, in which cells were exposed to GEM (a duration of 24 h and a dose of 5 µmol/l), and then treated with MWHT (performed as mentioned in the MW group) at 42°C for 60 min. Following the various treatments, the cells were immediately returned to the cell incubator and incubated at 37°C for 24 h prior to further experiments.

The effects of the different treatments on cell viability were determined by the Cell Counting Kit-8 (CCK-8) assay (MedChem Express, Monmouth Junction, NJ, USA) according to the manufacturer's protocol. Briefly, 10 µl CCK-8 solution was added to cells (1×10⁴ cells/well) in 96-well plates, and then the cells were incubated at 37°C for 1–3 h. Subsequently, the cell viability in each group was measured at 450 nm using a Multiskan Spectrum spectrophotometer (Thermo Fisher Scientific, Inc.).

Cells were seeded in 6-well plates at a density of 1×10⁶ cells/well, and then divided into the four experimental groups (CON, MW, GEM and MW + GEM). After 24 h of treatment, the cells were harvested, washed with phosphate-buffered saline (PBS) and fixed with 70% ice-cold ethanol at −20°C overnight. The cells were then washed further with PBS and stained using the CycletestPlus DNA Reagent kit (BD Biosciences, San Jose, CA, USA), according to the manufacturer's protocol. The cell cycle distribution was analyzed by a flow cytometer (BD Biosciences).

Cells were cultured in 6-well plates at a density of 5×10⁵ cells/well and separated into the four experimental groups (CON, MW, GEM and MW + GEM). After 24 h of treatment, cells from the different groups were collected and incubated with 50 nM LysoTracker Red (Beyotime Institute of Biotechnology, Haimen, China) for 30 min at 37°C in the dark. Samples were then analyzed using a fluorescence microscope (BX61; Olympus Corp., Tokyo, Japan) in order to examine autophagy.

Detection of intracellular ROS production was performed by fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining (Beyotime Institute of Biotechnology). Briefly, cells were seeded into 6-well plates at a density of 5×10⁵ cells/well in a volume of 2 ml and divided into the four experimental groups (CON, MW, GEM and MW + GEM). After 24 h of treatment, the cells were preloaded with 10 µM DCFH-DA in FBS-free RPMI-1640 medium for 30 min. Subsequent to washing three times with PBS, the cells were mounted under a fluorescence microscope (BX61) at an excitation wavelength of 488 nm and emission wavelength of 525 nm. Using Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD, USA), the mean fluorescence intensity of the images was assessed and normalized to obtain relative ratios, which were compared among the experimental groups.

NCI-H1703 and NCI-H2170 cells in the logarithmic growth phase were collected and seeded at a density of 5×10⁵ cells/well in 6-well plates. Following treatment according to the four experimental groups (CON, MW, GEM and MW + GEM), the cells were then lysed in ice-cold radioimmunoprecipitation assay lysis buffer for 30 min and then centrifuged at 20,000 × g for 10 min at 4°C to obtain the total protein. The protein concentration was determined by the BCA assay (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Equal amounts (40 µg) of cell lysates were separated by 8–12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Subsequent to blocking with 5% non-fat milk for 1–2 h at room temperature, the membranes were incubated with specific primary antibodies at 4°C overnight. The following antibodies were used: Anti-microtubule-associated protein light chain 3 (LC3; dilution 1:1,000; cat. no. sc-398822; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-sequestosome1/p62 (SQSTM1/p62; dilution 1:1,000; cat. no. sc-48402; Santa Cruz Biotechnology), anti-phosphatidylinositol 3-kinase (PI3K; dilution 1:1,000; cat. no. 4249; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-phosphorylated-phosphatidylinositol 3-kinase (p-PI3K; dilution 1:1,000; cat. no. 4228; Cell Signaling Technology, Inc.), anti-protein kinase B (AKT; dilution 1:1,000; cat. no. 2920; Cell Signaling Technology, Inc.), anti-phosphorylated-protein kinase B (p-AKT; dilution 1:1,000; cat. no. 4060; Cell Signaling Technology, Inc.), anti-mammalian target of rapamycin (mTOR; dilution 1:1,000; cat. no. 2983; Cell Signaling Technology, Inc.), anti-phosphorylated-mammalian target of rapamycin (p-mTOR; dilution 1:1,000; cat. no. 5536; Cell Signaling Technology, Inc.), anti-phosphorylated-S6 (pS6; dilution 1:1,000; cat. no. 4858; Cell Signaling Technology, Inc.), anti-ribosomal protein S6 kinase (p70S6k; dilution 1:1,000; cat. no. 2708; Cell Signaling Technology, Inc.) and anti-β-actin (dilution 1:100; cat. no. sc-47778; Santa Cruz Biotechnology). Next, the membranes were washed three times with Tris-buffered saline and Tween 20, and then incubated with horseradish peroxidase-conjugated goat anti-rabbit/mouse IgG secondary antibodies (anti-rabbit IgG; dilution 1:5,000; cat. no. sc-2357; and anti-mouse IgG; dilution 1:5,000; cat. no. sc-2005; from Santa Cruz Biotechnology) at room temperature for a further 1–2 h. An enhanced chemiluminescence detection reagent (Beyotime Institute of Biotechnology) was used for signal detection, and images were captured with an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). The relative expression level of the total protein is represented by the ratio of the gray value of the total protein band to the gray value of the β-actin band of the internal reference. The ratio of the gray value of the phosphorylated protein band to the corresponding gray value of the total protein band indicates the relative expression of the phosphorylated protein (ImageJ Software; v.1.8.0; National Institutes of Health, Bethesda, MD, USA). All experiments were repeated at least three times.

Cells were pretreated for 1 h with 5 mmol/l NAC (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), a ROS scavenger, and then treated with MWHT and GEM as described earlier. Then, the CCK-8 experiment and the detection of autophagy-related proteins and PI3K/AKT/mTOR pathway proteins were performed.

Cells were pretreated for 1 h with 5 mmol/l 3-MA (Selleck Chemicals, Houston, TX, USA), a selective PI3K inhibitor, which was dissolved in PBS, followed by treatment with MWHT and GEM as described earlier. Subsequently, DCFH-DA staining was performed to examine generation of cellular ROS after treatment with autophagy inhibitors, while the expression levels of autophagy-associated and PI3K/AKT/mTOR proteins were detected by western blot analysis.

The statistical analysis was performed using SPSS (version 17.0; SPSS, Inc., Chicago, IL, USA) and GraphPad Prism software (version 7.00 for Macintosh; GraphPad Software, Inc., La Jolla, CA, USA). The one-way ANOVA followed by Tukey's post hoc test was used for multiple comparisons. The results were considered statistically significant when P<0.05. All analyses represented at least three independent in vitro experiments, and the results are expressed as the mean ± standard deviation.

Our previous study investigated whether MWHT subsequent to treatment with GEM can synergistically inhibit the proliferation of human squamous lung carcinoma cells in vitro (16). To further elucidate the underlying mechanism, the cells were divided into four groups (CON, MW, GEM and MW + GEM), and a CCK-8 assay was conducted to evaluate cell viability. The results revealed that, compared with the CON group, cell viability significantly decreased in the MW, GEM and MW + GEM groups (P<0.001) in the two cell lines (Fig. 1A). Compared with the GEM or MW alone groups, the cell viability in the combination group was also significantly decreased (P<0.001). To verify the causal association of cell proliferation inhibition with cell cycle arrest, the cell cycle distribution was analyzed by flow cytometry. As shown in Fig. 1B and C, MW + GEM treatment induced G0/G1 phase arrest, as the cell population at this phase was significantly increased compared with the other three groups in NCI-H1703 cells (P<0.01 or P<0.05). By contrast, the percentages of cells in the S and G2/M phases were significantly decreased (P<0.05 or P<0.01) compared with the CON group. Similar effects were also observed in NCI-H2170 cells. These cell cycle changes may have caused the marked reduction in NCI-H1703 and NCI-H2170 cell proliferation.

As autophagy contributes to cell death, the present study then investigated whether MWHT in combination with GEM induces autophagy in lung cancer cells. Autophagy is characterized by the formation of numerous acidic vesicular organelles, which is correlated with an increased number of autophagosomes and can be detected by LysoTracker Red staining (19,24). LysoTracker Red staining was thus conducted in the present study and was quantified using a fluorescence microscope. The results demonstrated that, compared with GEM or MWHT treatment alone, cell autophagy was significantly increased (P<0.001) after combined treatment with MWHT and GEM (Fig. 2A). To verify these findings, the expression of several marker proteins of autophagy was further tested by western blotting. The results revealed an increased light chain 3 (LC3)-II/LC3-I ratio and reduced p62 expression in cells receiving combined treatment as compared with those receiving single treatment (Fig. 2B). To further elucidate the mechanism underlying autophagy induction by the combined treatment with MWHT and GEM, 3-MA was used to block autophagy. Consistently, the administration of 3-MA in MW + GEM cells decreased the ratio of LC3-II/LC3-I protein and increased the expression of p62, as compared with those in cells treated with 3-MA alone (Fig. 2C). Taken together, these findings indicated the crucial role of autophagy in the treatment of squamous cell lung carcinoma with MWHT and GEM.

ROS signaling serves an important role in several biochemical functions, including cell autophagy. Therefore, the production of ROS was detected by DCFH-DA staining and analyzed by fluorescence microscopy. As shown in Fig. 3A and B, the results demonstrated that ROS production increased significantly in the MW + GEM group when compared with the CON group (5.06-fold in NCI-H1703 cells and 5.25-fold in NCI-H2170 cells), while there was no marked increase in ROS production in the MW (1.39-fold in NCI-H1703 cells and 1.23-fold in NCI-H2170 cells) or GEM group alone (1.40-fold in NCI-H1703 cells and 1.39-fold in NCI-H2170 cells). Subsequently, in order to study the effect of ROS accumulation on the antiproliferative potential of MWHT combined with GEM, a CCK-8 assay was performed. As expected, the cell viability in the MW + GEM group was significantly decreased (P<0.001), whereas pretreatment with NAC markedly prevented cell death (P<0.001; Fig. 3C). These observations further confirmed that MWHT combined with GEM induced NCI-H1703 and NCI-H2170 cell death via the generation of ROS.

Several studies have reported that PI3K/AKT/mTOR signaling is involved in the regulation of autophagy. Therefore, the present study investigated the effects of MWHT and GEM on PI3K/AKT/mTOR signaling. As shown in Fig. 4A, compared with the CON and single treatment groups, the protein expression levels of PI3K, phosphorylated (p)-PI3K, AKT, p-AKT, mTOR, p-mTOR, PS6 and p70 S6 kinase (P70S6K) in the MW + GEM group were significantly decreased in the two cell lines (P<0.05, P<0.01 or P<0.001). To further examine the association between autophagy and the PI3K/AKT/mTOR signaling pathway, the autophagy inhibitor 3-MA was used. It was revealed that the PI3K/AKT/mTOR pathway was upregulated (P<0.05, or P<0.001) (Fig. 4B). Taken together, these data indicate that MWHT combined with GEM induce cell autophagy though activation of PI3K/AKT/mTOR signaling. In addition, ROS has been demonstrated to be an inducer for the activation of the PI3K/AKT/mTOR signaling pathway; therefore, the present study further assessed the effect of ROS production on the PI3K/AKT/mTOR pathway using the ROS scavenger NAC. The results demonstrated that pretreatment with NAC, followed by MWHT and GEM treatment, significantly reversed the phosphorylation of PI3K, AKT and mTOR, as compared with MW + GEM group (P<0.01 or P<0.001; Fig. 5) (35). These changes in the protein expression levels indicated that ROS production was upstream of the PI3K/AKT/mTOR signaling pathway.

To elucidate the interplay between ROS and autophagy, the underlying molecular mechanism was further investigated using western blot analysis. Notably, the protein expression levels of p62 were found to be significantly increased following pretreatment with NAC in the MW + GEM group for 24 h, as compared with the group without NAC (P<0.001; Fig. 6A). By contrast, the ratio of the LC3-II/LC3-I protein level was markedly decreased in the NAC pretreated cells, as compared with the MW + GEM group (P<0.001; Fig. 6A). Furthermore, when the autophagy inhibitor 3-MA was used, no significant change was observed in ROS production (Fig. 6B). Taken together, these results indicate that MWHT in combination with GEM induced autophagy through ROS-mediated PI3K/AKT/mTOR signaling in the two human squamous cell lung carcinoma cell lines.