The human NSCLC cell lines A549 (cat. no. 60074) and H460 (cat. no. 60373) and the normal lung cell line IMR-90 (cat no. 60204) were purchased from the Bioresource Collection and Research Center of the Food Industry Research and Development Institute. Both A549 and IMR-90 cell lines were cultured in DMEM (HyClone; Cytiva), while the H460 cell line was cultured in RPMI-1640 medium (Corning, Inc.). All media were supplemented with 10% fetal bovine serum (HyClone; Cytiva) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.), and all cells were maintained in a humidified incubator with 5% CO₂ at 37°C.

Erl, purchased from MedChemExpress, was dissolved in dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA) to a concentration of 5 mM as the stock solution and stored at −20°C. A549 cells were seeded in 96-well plates at 3×10³ cells/well and incubated overnight at 37°C before being treated with 0.5, 2.0, 8.0 or 10.0 µM of Erl, TC-HT or a combination treatment. For the combination treatment, TC-HT was applied for 1 h before Erl treatment. After single or combination treatment, the cells were maintained at 37°C in the cell culture humidified incubator for an additional 24, 48 and 72 h for further analyses. The TC-HT parameters employed in this study were based on our previous study involving different cell types (22–25), consisting of a high temperature period for 3 min and a cooling period of 30 sec, and this protocol was repeated continuously for 10 cycles (Fig. 1A). A modified PCR system, which featured waterproof capabilities in the heating area, was utilized to conduct TC-HT (Fig. S1A). A minimal volume of water served as a thermal conduction medium, with cell culture dishes placed directly on the heating area, while the modified PCR apparatus regulated the heating and cooling processes. The actual temperatures sensed by the cells at the bottom of the well were measured by a needle thermocouple (Fig. S1B).

The viability of A549 cells was assessed by MTT assay (Sigma-Aldrich; Merck KGaA). After drug or TC-HT treatment, the medium was replaced by DMEM containing 0.5 mg/ml MTT and incubated for 4 h at 37°C. The formazan crystals were dissolved using 10% SDS (Bioman Scientific Co., Ltd.) at room temperature overnight. Thereafter, the optical density of formazan solution was measured at 570 nm using the Multiskan GO spectrophotometer (Thermo Fisher Scientific, Inc.). Background absorbance at 690 nm was subtracted, and the final absorbance value was expressed as a percentage of the untreated controls to represent the cell viability. The cell morphology of A549 cells under different treatments was observed and imaged using the Zyla 5.5 sCMOS camera (Andor Technology Ltd.).

The SQ was calculated by subtracting the baseline values from all treatment groups and dividing the resulting net effect of the combination by the total of the individual effects as follows: (Erl + TC-HT)/(Erl) + (TC-HT).

After treatment with Erl and/or TC-HT, cells were washed with ice-cold PBS and lysed in RIPA lysis buffer (MilliporeSigma) on ice for 30 min. Cell lysates were clarified by centrifugation at 23,000 × g for 30 min at 4°C and the protein concentration in the supernatant fraction was measured using the Bradford protein assay (cat. no. BRA222; BioShop Canada Inc.). Proteins (20–40 µg) were subjected to 10% SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes (MilliporeSigma). After incubation in a blocking buffer containing 5% bovine serum albumin (BioShop Canada Inc.) in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 h at room temperature, the membranes were immunoblotted with diluted primary antibodies at 4°C overnight. The specific primary antibodies against phosphorylated EGFR (p-EGFR; cat. no. 4407; Cell Signaling Technology, Inc.), poly ADP-ribose polymerase (PARP; cat. no. 9542; Cell Signaling Technology, Inc.), p-JNK (cat. no. 4668; Cell Signaling Technology, Inc.), p-Akt (cat. no. 4060; Cell Signaling Technology, Inc.), total Akt (t-Akt; cat. no. 9272; Cell Signaling Technology, Inc.), MutT homolog 1 (MTH1; cat. no. 43918; Cell Signaling Technology, Inc.), p-p38 (cat. no. GTX133460; GeneTex, Inc.), Cdc2 (cat. no. GTX108120; GeneTex, Inc.) and GAPDH (cat. no. GTX100118; GeneTex, Inc.) were used. After being washed with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (cat. no. 111-035-003; Jackson ImmunoResearch Laboratories, Inc.). All the primary antibodies were diluted at a 1:1,000 concentration and the secondary antibodies were diluted at a 1:10,000 concentration, according to the manufacturer's instructions. The membranes were visualized with an enhanced chemiluminescence substrate (Advansta Inc.) and quantified using an Amersham Imager 600 imaging system (AI600; GE Healthcare).

After treatment with Erl and/or TC-HT, cells were washed and resuspended with PBS followed by staining with 20 nM 3,3′-dihexyloxacarbocyanine iodide (DiOC₆(3); Enzo Life Sciences, Inc.) for 30 min at 37°C in the dark. The fraction of mitochondrial depolarization in cells was indicated by a decrease in fluorescence intensity measured using a flow cytometer (FACSVerse; BD Biosciences) and data were analyzed using FlowJo (version 7.6.1; FlowJo LLC).

A549 cells were seeded and cultured as a confluent monolayer in 35 mm Petri dishes. Wounds were made by scratching straight lines across the cell monolayer using a 10 µl pipette tip. Non-adherent cells and debris were removed by gently rinsing the cells with PBS (HyClone; Cytiva). After PBS was discarded and replaced with fresh medium, the cells were treated with either Erl and/or TC-HT. Each wound was observed and imaged using the Zyla 5.5 sCMOS camera (Andor Technology Ltd.) at 0 h and 24 h post treatment. The distances between wound edges were measured and analyzed using ImageJ (version 1.51j8; National Institutes of Health).

A549 cells were seeded at a density of 300 cells/dish in 35 mm Petri dishes overnight for cell adherence. Subsequently, cells were treated with Erl and/or TC-HT. The cell medium was replaced 24 h after treatment, and the cells were cultured for additional 10 days, with fresh medium replaced every 3 days during the culture period. The cells were fixed with 4% paraformaldehyde (Sigma-Aldrich; Merck KGaA) for 10 min at room temperature and stained with 0.1% crystal violet (Sigma-Aldrich; Merck KGaA) at room temperature for 15 min for visualization and cell counting. A colony was defined as a cluster consisting of ≥50 cells. The colonies were imaged using a light microscope and manually counted, and the number of colonies in each group was normalized to the control group.

After 24 h of treatment with 10 µM Erl and/or TC-HT, the cells were harvested, rinsed with PBS and fixed with 70% ethanol at 4°C for 30 min before staining. The cells were stained for 30 min in the dark at room temperature with propidium iodide and ribonuclease A (Gibco; Thermo Fisher Scientific, Inc.). Subsequently, the stained cells were subject to the cell cycle analysis using a flow cytometer (FACSVerse; BD Biosciences), and data were analyzed using FlowJo (version 7.6.1; FlowJo LLC).

Results were expressed as the mean ± standard deviation (n=3). Statistical analyses were performed using OriginPro 2015 (version 92E; OriginLab Corporation). Statistical significance was determined using one-way ANOVA followed by Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

The thermal cycling treatment was applied with a modified PCR machine as previously described (22,23). The schematic temperature and duration settings are shown in Fig. 1A, where the temperature was increased to the desired high temperature setting and maintained for 3 min, followed by a natural cooling period for 30 sec. In practice, the heating device was switched off in the cooling process, and the low temperature setting was chosen to be 37°C to mimic human body temperature. A single cycle containing a high temperature and a low temperature period was repeated 10 times. Fig. 1B shows the actual temperature in the culture well measured by a thermocouple at moderate temperature TC-HT setting. The cycling temperature came to an equilibrium state after the third heating period, thereafter the temperature cycling between 41.5–43.0°C for moderate temperature TC-HT (Fig. 1B). In the current study, the moderate temperature TC-HT treatment (41.5–43.0°C) was adopted in the subsequent experiments to study the synergistic anticancer effect of Erl and TC-HT in A549 cancer cells.

To determine the effect of the combination of Erl and TC-HT treatment on human NSCLC cell viability, A549 cells were treated with different concentrations of Erl with or without TC-HT. At 72 h post treatment, cell viability was assessed using an MTT assay. Erl exhibited a significant antineoplastic effect in a concentration-dependent manner (Fig. 2A). The IC₅₀ of Erl was ~10 µM in A549 cells after 72 h treatment. Compared with treatment with Erl alone, TC-HT therapy sensitized A549 cells to Erl and reduced the cell viability to ~30% of the control group (Fig. 2A). It is noteworthy that the combination of Erl and TC-HT demonstrated synergistic ability compared with Erl monotherapy, which reduced the IC₅₀ to 0.5 µM. To determine the synergistic effect of TC-HT and Erl, SQ calculations were conducted, where an SQ value >1.0 indicated a synergistic effect (25–27). The SQ calculations of cell viability indicated a synergistic effect when TC-HT was combined with Erl at concentrations of 0.5–8.0 µM (Table SI). The highest SQ value was observed with the combination treatment using 0.5 µM Erl, suggesting that the synergistic effects at lower concentrations of Erl were more pronounced compared with those at higher concentrations. To enhance understanding of the temporal effects on cell viability, the viability of A549 cells was evaluated at 24 and 48 h (Fig. S2A). Additionally, the investigation was expanded to include the H460, another NSCLC cell line, assessing the impact of the combination of Erl and TC-HT on NSCLC (Fig. S2B). These results demonstrated that the combination of Erl and TC-HT led to a significant time- and dose-dependent decrease in cell viability in both A549 and H460 cell lines. To verify the effect of this combined treatment in normal cells, the normal lung cell line IMR-90 was used and the same treatment conditions were applied. The cell viability of IMR-90 normal lung cells decreased to ~90% when treated with 8 or 10 µM Erl, while it remained unaffected by doses <2 µM Erl and/or TC-HT (Fig. 2B). Additionally, Erl and TC-HT combination treatment resulted in notable morphological changes in A549 cells, including shrinkage and fragmentation of cells as well as a decreased number of cells (Fig. 2C). These result suggested that TC-HT may serve as a sensitizer with minimal observed adverse effects to increase the anticancer effect of targeted therapy drugs (22,23).

The signaling pathways involved in the anticancer mechanism of Erl and TC-HT combination treatment were investigated. Despite the application of targeted therapy agents aimed at inhibiting EGFR activity, limited efficacy remains a challenge in certain NSCLC cells with intrinsic resistance to Erl (11–13). In vitro studies have identified A549 cells as resistant to the EGFR-TKI Erl (15,28,29). Consequently, it is imperative to explore innovative strategies to augment the anticancer effects of Erl (14,30–32). In the present study, Erl alone caused a marked decrease in EGFR phosphorylation compared with the control cells (Fig. 3). Moreover, while TC-HT alone was capable of significantly reducing p-EGFR expression compared with the control cells, the combination of Erl and TC-HT resulted in a more substantial inhibitory effect on p-EGFR expression compared with the single treatment and control cells. Additionally, a comparable decrease in p-EGFR expression was also observed in the combination of low-dose Erl and TC-HT, compared with both low-dose Erl alone and the control cells (Fig. S3). Although this combination was less effective in inhibiting p-EGFR compared with higher concentrations of Erl in conjunction with TC-HT, the lower doses of Erl still exhibited significant inhibitory effects when combined with TC-HT. These results suggested that TC-HT may modulate the resistance of A549 cells to Erl.

In the present study, the expression levels of JNK and Akt proteins (33) were examined in A549 cells. JNK, an EGFR downstream protein belonging to the MAPK family, is responsive to stress stimuli and heat shock, with its phosphorylation capable of altering the activities of several proteins that reside in mitochondria or act in the nucleus to trigger apoptosis (34). TC-HT treatment significantly increased the expression levels of the p-JNK protein compared with the control or Erl-treated A549 cells (Fig. 4A). Furthermore, it was demonstrated that the treatment combining Erl and TC-HT was more effective in increasing p-JNK protein expression levels compared with TC-HT alone. On the other hand, another EGFR downstream protein, Akt, is involved in cellular survival pathways by inhibiting the apoptotic process (35). Once activated by phosphorylation, Akt actively regulates several transcription factors facilitating the expression levels of survival proteins. It was shown that the protein expression levels of p-Akt in Erl-treated A549 cells were significantly lower than that compared with untreated cells, while the inhibitory effect on phosphorylation of Akt was further enhanced upon TC-HT treatment and was more pronounced in the combination Erl and TC-HT treatment group (Fig. 4B). Meanwhile, t-Akt exhibited a trend similar to that of the p-Akt protein, suggesting that decrease in the phosphorylation of Akt by these treatments was caused by a reduction in the amount of Akt present, leading to a weakened survival signal. Moreover, it has been reported that both JNK and Akt signaling pathways are important regulators in influencing mitochondrial function. In addition, mitochondria-dependent apoptosis is associated with a loss in MMP, causing the release of cytochrome c and other apoptotic factors (36). To confirm that the apoptosis caused by Erl and TC-HT could be related to mitochondrial disruption, MMP was assessed with DiOC6(3) fluorescence staining using flow cytometry. It was confirmed that Erl and TC-HT individually had no effect on MMP level compared with the control, but the combination treatment caused significant MMP depolarization in A549 cells, indicating mitochondrial dysfunction in their apoptosis mechanism (Fig. S4).

The injured mitochondria release cytochrome c into the cytoplasm, cleaving caspase 9 and thus activating caspase 3 downstream (37), which enters further the nucleus and cleaved PARP. It should be noted that PARP serves an important role in mitochondria-mediated apoptosis, in addition to being a key enzyme for DNA repair. In apoptosis, the PARP protein is typically cleaved and inactivated, thereby suppressing DNA repair and causing programmed cell death (38). To understand the apoptosis mechanism in A549 cells triggered by the combination Erl and TC-HT treatment, the present study evaluated the expression levels of apoptosis-related proteins using western blotting. It was shown that the ratio of cleaved PARP to full length PARP in TC-HT-treated cells was significantly higher compared with that in Erl-treated cells (Fig. 4C). Moreover, it was demonstrated that the ratio of cleaved PARP to full length PARP in combination Erl and TC-HT treatment was significantly higher compared with that in the TC-HT treatment alone, indicating that the combination treatment enhanced apoptosis of A549 cells via the mitochondrial pathway. Furthermore, the MTH1 protein has received increasing attention in cancer treatment, due to its key role in DNA repair (39). High expression level of MTH1 is deemed to be a sign of NSCLC metastasis (40). Therefore, the present study examined MTH1 protein expression levels via western blotting. MTH1 protein expression levels were found to significantly decrease in Erl-treated cells compared with controls, while the inhibitive effect on MTH1 protein expression levels was significantly higher in the TC-HT treatment group, which significantly increased further in the combination Erl and TC-HT treatment group (Fig. 4D). These results suggested that the combination of Erl and TC-HT may prevent MTH1-related DNA repair and induce the death of cancer cells via apoptosis.

To further evaluate the anticancer effects of the combination of Erl and TC-HT treatment on human NSCLC, the cell cycle progression in A549 cells was examined by flow cytometry. Treatment with moderate TC-HT led to a significant increased in cell cycle arrest at the G2/M phase (22.95±0.56%) compared with the group treated with Erl (13.06±1.16%) and the untreated cells (12.66±1.18%; Fig. 5A and B). Meanwhile, the combination of Erl and moderate TC-HT caused significant accumulation of cells in the G2/M phase (33.77±2.31%) compared with the single treatments and the untreated cells, with a concomitant reduction of cells in the G0/G1 phase. Besides, no significant differences in the S phase were observed among the various treatments tested. Next, relevant proteins involved in the G2/M progression were examined to investigate the mechanism of the combined treatment effect on cell cycle distribution. Cdc2 is a core regulator in the cell cycle and it binds to the cyclin B complex guiding G2/M cell cycle transition (41,42). It has been previously reported that the inhibition of Cdc2 expression resulted in G2/M cell cycle arrest (43–45). In the present study, the protein expression levels of Cdc2 were reduced significantly upon treatment with the combination of Erl and TC-HT compared with the single Erl and TC-HT treatments and the control group (Fig. 5C). Meanwhile, as a well-known MAPK member, p38 serves an important role in cell cycle regulation, and numerous studies have reported that activation of the p38 pathway can induce G2/M cell cycle arrest via Cdc2 suppression in human NSCLC cells (46–48). Consistently, it was shown that the protein expression levels of p-p38 were significantly increased by the combination treatment of Erl and moderate TC-HT (Fig. 5D). Taken together, these results suggested that the combined treatment could synergistically induce cell cycle arrest in the G2/M phase by increasing the activation of p38 while reducing Cdc2 expression in cells, thereby suppressing cell cycle progression in human lung cancer A549 cells.

Cancer mortality is associated with cancer recurrence and metastasis, both of which are common clinical issues in NSCLC (49,50). Therefore, it is important to reduce the proliferation and migration ability of cancer cells. To further confirm the anti-proliferative and anti-migration activities of the Erl and TC-HT combination treatment in NSCLC, wound healing (Fig. 6A) and colony formation assays (Fig. 6B) were performed using A549 cells. Erl-treated cells showed a significantly decreased migration capacity compared with the control cells, while cells treated with TC-HT alone exhibited a significant reduction in migration compared with single Erl treatment and the control cells (Fig. 6C). Furthermore, the Erl and TC-HT combination treatment caused a more significant decrease in A549 cell migration compared with that of the single treatments. Compared with the control group, the wound closure percentage in the Erl-treated group and the TC-HT-treated group was 83.8 and 59.2%, respectively, while the wound closure percentage in the combined treatment group was only 35.9%. The results indicated that the migration ability of A549 cells was significantly suppressed after the application of the Erl and TC-HT combination treatment. On the other hand, it was demonstrated that colony formation ability of A549 cells treated with either Erl or TC-HT was significantly decreased compared with the control cells, whereas the colony formation ability of cells treated with the combination treatment was further significantly reduced compared with that of the single treatments (Fig. 6D). Overall, the combination treatment was shown to impede A549 cell proliferation and migration, indicating the potential anticancer efficacy of the Erl and TC-HT combination treatment.