Human lung cancer cell lines obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (PAA, A15-101) and 10 U/ml penicillin–streptomycin (Gibco/Invitrogen, 15140-122) at 37°C in a humidified atmosphere with 5% CO₂. Erlotinib (S7786), bosutinib (S1014), and trametinib (S2673) were purchased from Selleck China.

The erlotinib-resistant HCC827 lung cancer cell line was generated as described previously (16). Briefly, the HCC827 cells were cultured in growth medium in the presence of vehicle (0.1% DMSO) or gradually increasing concentrations of erlotinib to result in the resistant cell line that was designated HCC827-ER.

Cell proliferation was detected as described before (17). Briefly, cell line was seeded into a 96-well plate; after attachment, the cells were then treated with different doses of trametinib or bosutinib, alone or in combination for 24 h. After treatment, 10 μl of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) solution was added to each well and incubated with the cells for 3 h. After removing the medium, 100 μl of DMSO was added and then the absorbance at 570 nm was measured. The cell viability was normalized and is presented as a percentage of the control.

The interaction effect between bosutinib and trametinib was analyzed using the combination index (CI) (18, 19). The CI value is defined by the Loewe Additivity equation: CI = (D)1/(Dx)1 + (D)2/(Dx)2, where (Dx)1 and (Dx)2 are the concentrations for D1 (bosutinib) and D2 (trametinib) alone that inhibit x% cell growth, and (D)1 and (D)2 are the concentrations of bosutinib and trametinib in combination that result in identical cell growth inhibition. Each CI value is the mean value of three independent experiments.

Cells were seeded at specific cell densities in six-well plates (1 × 10⁵, 2 × 10⁵, and 1.5 × 10⁵ for H1650, HCC827, and HCC827 ER cells, respectively), and the plates were incubated overnight. The cells were then treated with different doses of trametinib or bosutinib, alone or in combination, for the indicated periods. Cells were washed with PBS and then cultured in fresh medium for 14 days. Subsequently, the cells were fixed and stained with 0.1% crystal violet. Colonies of at least 50 cells observed under a microscope were counted as one positive colony.

After treatment, the cells were harvested, washed twice with ice-cold PBS, and then stained using an Annexin V-FITC/PI Cell Apoptosis Detection Kit (BD Biosciences) according to the manufacturer's instructions. In brief, 1 × 10⁶ cells were resuspended in 400 μl of binding buffer, and 5 μl of 2 mg/ml Annexin V and 5 μl of 20 μg/ml PI were then added. After 15-min incubation in the dark, the stained cells were quantified by a FACScan flow cytometer, and the data were analyzed using FACSCalibur software. The cells in the early stage of apoptosis were Annexin V positive and PI negative, whereas the cells in the late stage of apoptosis were both Annexin V and PI positive.

The equivalent amounts of protein were fractionated using 10% SDS–PAGE, and then the proteins were transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 0.1% TBS/T (0.1%) containing 5% non-fat milk. After blocking for 1 h, membranes were incubated with different antibodies diluted in 3% bovine serum albumin in washing buffer. The following primary antibodies were used in this study: Y419-phospho-SRC (Abcam#ab185617), Y529-phospho-SRC (Abcam#ab4817), SRC (Abcam#ab109381), S235/236-phospho-S6 (CST#4856), S240/244-phospho-S6 (CST#5364), S6 (CST#2217), Y1068-phospho-EGFR (CST#2236), Y854-phospho-EGFR (CST#6963), EGFR (CST#2236), p44/42 (CST#9107), phospho-p44/42 (CST#4370), AKT (CST#9272), and phospho-AKT (CST#9271). Unless indicated otherwise, all antibodies were used at a 1:1,000 dilution.

Afterward, the membrane was washed and probed with the HRP-conjugated secondary antibodies at room temperature for 1 h. The protein bands were detected using an ECL kit (Pierce, Rockford, IL, USA).

This study was carried out in accordance with the recommendations of the Guidelines for the Care and Use of Laboratory Animals, and the protocols were approved by the Institutional Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine. Specific pathogen-free BALB/c male nude mice (4 weeks old) were purchased from the Experimental Animal Center of the Chinese Academy of Science (Shanghai, China). HCC827 cells (5 × 10⁶ cells per mouse) were re-suspended in 200 μl of Matrigel (BD, USA) and then were subcutaneously inoculated into the dorsal flank of nude mice. After tumors had established (~75 mm³), the mice were randomly divided into four groups (six mice per group) for intragastric administration with either vehicle control (1% Tween−80 in saline) (Group 1), trametinib at 0.4 mg/kg every day for 2 weeks (Group 2), bosutinib at 60 mg/kg every day for 2 weeks (Group 3), or a combination of trametinib and bosutinib. The body weight and tumor sizes of all mice were recorded every 2 days. The length and width of the tumor were measured by a digital caliper, and the tumor volumes were calculated according to the formula: [(shortest diameter)² × (longest diameter)]/2. After 14 days of treatment, the mice were humanely killed, and their tumors were resected, weighed, and photographed.

Data are presented as means ± SD from three independent experiments. Student's two-tailed t-test was used for comparison between two different groups. ANOVA was used for multiple comparisons. All P-values < 0.05 were considered to be statistically significant.

The proliferation inhibition effects of bosutinib and trametinib as single drugs and as a combination on a collection of 22 lung cancer cell lines were evaluated. For single drug treatments, the results indicated that trametinib showed overall a better inhibition effect than bosutinib. After treatment, 11 out of 22 cell lines (50%) were observed to be sensitive to trametinib (cell proliferation IC₅₀ < 2 μM), while 6 cell lines (27%) were considered to be resistant to it (cell proliferation IC₅₀ > 10 μM) (Figure 1A). For bosutinib single-drug treatment, we found that only six cell lines (27%) were sensitive. The percentage of resistant cell lines was also 27% (Figure 1B). Next, the effect of the combination of bosutinib and trametinib (1:1 molar ratio) on the proliferation of these 22 cell lines was investigated (Figure 1C). The drug combination indices (CI) for the 50–80% growth inhibition ranges were then calculated using the Loewe Additivity model (Figure 1D). We found that 19 of the cell lines showed synergistic effects with CI values < 1. Among these cell lines, the HCC827 cell line showed the strongest synergistic effect (CI valve: 0.14), and this effect was also observed in the erlotinib-resistant HCC827 (HCC827ER) cell line. The synergistic effect of these two compounds was further studied on both HCC827 and HCC827ER cells. Another cell line with an EGFR mutation, the H1650 cell line, was also used in our study later.

Based on the screening results of the 22 lung cancer cell lines, the cytotoxic effects of the two drugs and their combinations were further confirmed in three NSCLC lines (HCC827, HCC827ER, and H1650) using the MTT assay after treatment for 24 h. Based on the IC₅₀ value of these two compounds on the three cell lines, here the dosage we used for H1650 was 10 μM, while the dosage for both HCC827 and HCC827ER was 5 μM. As shown in Figures 2A–C, the inhibition percentage for single treatment of bosutinib was around 10–20% in all three cell lines, while the cells were more sensitive to trametinib, for which the rate was around 30 to 40%. As compared with single treatment, the combination of the drugs greatly increased the cell growth inhibition rate. This rate reached nearly 60% in both HCC827 and HCC827ER cells, and similar results were observed in H1650 cells.

In order to study the potential effect on long-term proliferation, a colony formation assay was used in our study. Cells were seeded and treated with different concentrations of bosutinib and trametinib, or their combination for 24 h. The clonogenic assays showed that bosutinib and trametinib had a synergetic effect on colony formation on both HCC827 and HCC827 ER cells (Figures 2D–I). Interestingly, a combination of 0.625 μM bosutinib and 0.625 μM trametinib completely blocked colony formation in HCC827 ER cells, while either agent alone showed no such effect. The synergistic inhibitory effect of these two drugs was also observed in H1650 cells (Figures 2F–I). Colony formation was greatly inhibited after treatment with 0.5 μM of bosutinib and trametinib for 24 h, and was only slightly inhibited by either agent alone.

To investigate the effect of bosutinib and trametinib on NSCLC, an apoptosis assay was performed using flow cytometric analysis. The cells were treated with different concentrations of bosutinib and trametinib, or their combination. After treatment for 24 h, cells were harvested and stained with Annexin V/PI for analysis. As shown in Figure 2, the number of apoptotic cells in both the HCC827 and HCC827 ER cells was slightly increased by treatment with bosutinib and trametinib alone, whereas combined treatment with bosutinib and trametinib increased the percentage of apoptotic cells to 32.6% in HCC827 and 41.6% HCC827 ER cells (Figures 3B,C). Our results showed that when the two drugs were used together, half of the dosage could give a promising result (Figures 3D–F). Similar results were also observed in H1650 cells (Figure 3A). Additionally, the apoptotic effect of the drug was further confirmed by Western blotting analysis (Supplementary Figure 1). The activities of caspase-dependent pathway markers, including caspase-3, caspase-9, and PARP, were investigated. The activity of caspases-3 and-9 in the combined treatment cells was significantly higher than that in the single treatment cells, since they contained less mount of pro-caspases-3 and-9. As a result of the activation of the caspase-signaling pathway, the amount of cleaved PARP was significantly increased in the combined treatment cells. Consistent with the results of flow cytometric analysis, the combination of bosutinib and trametinib also showed synergistic effects in Western blot analysis.

We further studied the mechanism of combined bosutinib and trametinib on H1650, HCC827, and HCC827ER cell lines. Since bosutinib and trametinib are inhibitors of SRC and MEK, respectively, the phosphorylation and total protein levels of SRC and its downstream signals STAT3, AKT, ERK, and S6 were analyzed after bosutinib, trametinib, or combination treatment. As shown in Figure 4 and Supplementary Figure 2, bosutinib treatment dramatically decreased the phosphorylation level of SRC and the downstream phosphorylated proteins, such as STAT3, AKT, and S6 in all three cell lines, while trametinib specifically inhibited the expression level of phosphorylated ERK. Co-treatment with these two drugs synergistically decreased the phosphorylation levels of SRC Y419 and S6.

It is well-known that Src can phosphorylate EGFR on Y845, while Y1068 is one of the major autophosphorylation sites of EGFR, so we studied the effect of combined bosutinib and trametinib on these two sites. The results indicated that bosutinib treatment significantly decreased the phosphorylation level of EGFR on both Y845 and Y1068, and this effect was intensified by co-treatment with trametinib in these three cell lines. Specifically, we carefully compared the effects of the co-treatments on phosphorylation of EGFR in the HCC827 and HCC827ER cell lines. Our study showed that HCC827ER seemed to be more sensitive than HCC827 cells to co-treatment. As shown in Figures 4B,C, a combination of bosutinib and trametinib showed synergistic effects on down-regulation of EGFR Y1068 at both higher (5 μM) and lower (0.5 μM) dosages, while this effect was seen only at higher dosage in HCC827 cells.

To further investigate the inhibition effect of bosutinib and trametinib on NSCLC growth and development, HCC827 cells were injected subcutaneously into the dorsal flank of nude mice. Bosutinib (60.0 mg/kg, p.o.), trametinib (0.4 mg/kg, p.o.), or a combination of these was administered every day for 14 days. The mice were treated for 14 days in our study, since the tumor volume was around 1,000 mm³ in the control group, and it is thought unethical if the tumor volume exceeds 1,000 mm³. The result showed that there was no significant difference in body weight between the vehicle and the three drug-treatment groups (Figure 5A). Treatment with bosutinib and trametinib did not alter cell morphology, nor did it cause bleeding in the heart, lung, kidney, or liver (Supplementary Figure 3). Compared with the vehicle control group, the combination of bosutinib and trametinib could significantly reduce the tumor volume of the mice, as seen on day 14. The mean tumor volumes measured on day 14 for the vehicle control, bosutinib, and trametinib groups were 916 ± 260, 513 ± 185, and 408 ± 167 mm³, respectively (Figure 5B). The tumor inhibition effect of the combination was significantly stronger than single treatment with bosutinib and trametinib. As shown in Figure 5, the tumor volume was dramatically decreased to 163 ± 185 mm³ after treatment for 14 days. A similar inhibitory effect was observed in the growth of tumors (Figure 5C). The results demonstrated that the tumor weight for mice treated with the combination of the two drugs was reduced by 82% compared with the control group, while the decreases with bosutinib and trametinib were 38 and 53%, respectively (Figure 5D).

To better understand the molecular mechanism of bosutinib and trametinib action on tumorigenesis in vivo, the phosphorylation levels of related proteins in tumor tissue were detected using Western blot analysis. As shown in Figure 5E and Supplementary Figure 4, the expression level of phosphorylated ERK was obviously inhibited by trametinib treatment, while the bosutinib administration down-regulated the level of phosphorylated EGFR and SRC as compared with the vehicle group. This result was reasonable, since bosutinib and trametinib are inhibitors of SRC and MEK, respectively. The combination treatment showed better effects on inhibition of these proteins in tumor tissue. The expression levels of the proteins in the co-treatment group were decreased significantly as compared with the single treatment group to the point of being hard to detect by Western blot. These results further supported our conclusion that the combination of bosutinib and trametinib showed better effects in a xenograft model as compared with single treatment.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.