A549, A549/DDP, NCI-H446, and NCI-H446/DDP cells used in this study were purchased from the National Authorized Cell Culture Preservation Center (Chinese Academy of Sciences, Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Grand Island, New York, NY, USA) supplemented with 10% fetal bovine serum (FBS, HyClone, Waltham, MA, USA), 2 mM L-glutamine and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin sulfate). All cell lines were validated by STR profiling and tested negative for mycoplasma. All cells were cultured at 37 °C in a humidified incubator with 5% (v/v) carbon dioxide in the air.

Cells were cultured in a 96-well plate at a density of 1 $\times$ 10${}^3$ cells/well. After treatment for the desired time, the cell viability was examined by using a cell counting kit-8 (CCK-8) (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. Absorbance was read using a Multiskan GO microplate reader (Thermo Scientific, Waltham, MA, USA) with a detection wavelength of 450 nm. Data were expressed as means $\pm$ SD, among which means were calculated based on the average from at least three independent experiments.

Cell migration was examined by performing a Transwell assay. In brief, transfected or treated cells in 100 µL serum-free media were plated on the upper compartment of the chamber; meanwhile 800 µL media with 10% fetal bovine serum were added in the lower compartment of the chamber as a chemoattractant. After 6 h or 12 h of incubation at 37 °C, cells the upper chamber were carefully removed by using a cotton swab. The migrated cells on the lower surface were analyzed by crystal violet staining and photographed with a Leica DMi8 inverted microscope (Deerfield, IL, USA).

Lung cancer cells at the concentration of 10 viable cells/mL in 100 µL media were seeded into ultra-low attachment 96-well plates (Corning) in DMEM medium supplemented with 20 ng/mL epidermal growth factor (EGF), 10 ng/mL basic fibroblast growth factor (bFGF), 5 mg/mL insulin, and 2% B27 and incubated at 37 °C and 5% CO${}_2$ in a humidified atmosphere. Cell media were replenished every other day through replacing the half of existing media with fresh media. Tumor spheres were visualized and calculated by a microscope (Olympus, Tokyo, Japan).

Total RNA was extracted by using a Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA-seq libraries were established with an Illumina TruSeq RNA sample prep kit (San Diego, CA, USA) and sequenced using Illumina HiSeq 4000 (San Diego, CA, USA) with a 150-bp paired-end sequencing strategy. Differential gene expression analysis was performed using HISAT2 and DESeq2, and determined with the cutoff fold change $\ge$1.5 and p-value $\le$ 0.05. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enriched pathway was determined according to ErichR, the web-based gene set enrichment analysis software (http://amp.pharm.mssm.edu/Enrichr).

Protein extracts from cultured cells were collected by using RIPA lysis buffer (Beyotime, Hangzhou, China) with protease inhibitor cocktail, and protein concentrations were determined by a Pierce BCA protein assay (Thermo Fisher Scientific, Waltham, MA, USA). Aliquots of protein extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then, transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The following antibodies used in the study were listed below: rabbit polyclonal anti-ALDH1 antibody (1:1500), rabbit polyclonal anti-ABCG2 antibody (1:1000), rabbit polyclonal anti-Nanog antibody (1:1500) and mouse monoclonal anti-$\beta$-actin antibody (1:1000) from Santa Cruz (Dallas, TX, USA); mouse monoclonal anti-NOTCH1 antibody (1:500) and rabbit polyclonal anti-phospho-STAT3 antibody (1:500), from Thermo Fisher, USA; rabbit polyclonal anti-STAT3 antibody (1:500) and rabbit polyclonal anti-BMAL1 antibody (1:500) from Abcam (Waltham, MA USA).

The lentivirus-mediated BMAL1 knockdown and overexpression system and corresponding controls were constructed by GeneCreate (Wuhan, China). Transfection was performed using the transfection reagent Endo-FectinTM Max transfection (Genecreate) according to the manufacturer’s instructions. Briefly, 5 µg lentivirus were diluted in 250 µL Opti-MEM media (Thermo Fisher Scientific, Inc.), supplemented with 10 µL Endo- Endo-FectinTM Max. After mixed softly, the mixture was stewed at room temperature for 10–20 min. Then, the mixture was added into the cells (1 $\times$ 10${}^5$/500 µL) and for 6 hours; fresh complete medium was used. Transfected cells were harvested at the indicated times for subsequent analysis.

SPSS 24.0 software (SPSS/IBM, Chicago, IL, USA) was used for the statistical analysis. Continuous variables and categorized variables were respectively reported as mean $\pm$ SEM and frequency (n) or proportion (%). The comparisons between different groups were performed by using t-tests or ANOVA. Both the Cox proportional hazard model and the Log-rank test were applied to survival analyses. p 0.05 was considered statistically significant.

To investigate the effects of L-Theanine on the chemosensitivity of lung cancer cells, we examined the changes in the sensitivity of lung cancer cells A549 and NCI-H446 to DDP after L-Theanine treatment. It showed that L-Theanine did not lead to a significantly enhancemed sensitivity of lung cancer cells A549 and NCI-H446 to DDP (Fig. 1A,B). However, it is noteworthy that for DDP-resistant strains (A549/DDP, NCI-H446/DDP), the resistance index of cells to DDP was significantly reduced after L-Theanine treatment (Fig. 1C,D).

Fig. 1.

L-Theanine relieves the chemoresistance to cisplatin in resistant lung cancer cells. (A) A549/DDP cells were treated with increasing concentrations of DDP (0–100 µM). (B) NCI-H446/DDP cells were treated with increasing concentrations of DDP (0–4 µM). (C) A549 cells were treated with increasing concentrations of DDP (0–400 µM). (D) NCI-H446 cells were treated with increasing concentrations of DDP (0–160 µM). After that, CCK-8 analysis showed the viability of the above cells following 48 h DDP treatment. Data are presented as Mean $\pm$ SEM. *indicated a significant difference. *p 0.05, **p 0.01. CCK-8, Cell Counting Kit-8; DDP, cis-diamminedichloroplatinum.

As mentioned above [2], there is a close relationship between cancer stemness and platinum-based chemotherapy resistance. To clarify whether the effect of L-Theanine on drug resistance in lung cancer is related to cancer stemness, we first examined the clone forming ability of lung cancer cells with L-Theanine treatment. The results showed that L-Theanine significantly inhibited the clone formation ability of DDP-resistant lung cancer cells; while no significant effect on the clone formation ability of non-resistant lung cancer cells (Fig. 2A–D). Subsequently, we examined the expression of stemness markers in DDP-resistant and non-resistant lung cancer before and after L-Theanine treatment. The results revealed that L-Theanine led to decreased expressions of stemness markers in DDP-resistant lung cancer cells, whereas no significant reduction in non-resistant lung cancer cells (Fig. 2E–H). In addition, L-Theanine only inhibited the invasion ability of DDP-resistant lung cancer cells (Fig. 2I–L). In summary, L-Theanine may targetedly inhibit the stemness of DDP-resistant lung cancer cells, but no significant effect on the stemness of non-resistant cells, which will be helpful for the personalized development of reversal strategies for chemotherapy-resistant lung cancer.

Fig. 2.

L-Theanine inhibiting the stemness of chemoresistance lung cancer cells. Tumor spheroid formation assay was applied in A549, A549/DDP, NCI-H446, and NCI-H446/DDP cells with or without L-Theanine treatment for 14 days. The number of tumor spheres means colonies/5000 cells. Scale bar 200 µm (A–D). The levels of proteins, including ALDH1, ABCG2, and Nanog in A549, A549/DDP, NCI-H446, and NCI-H446/DDP cells with or without L-Theanine treatment were analyzed by a western blotting assay (E–H). The indicated cells were treated with DDP for 14 days. Images and quantification of transwell migration assays; cells were stained with crystal violet. Scale bar 100 µm (I,K). The bar graphs show the statistical analysis of the number of cells cross membranes (J,L). Data are presented as Mean $\pm$ SEM; n = 3. **p 0.01; ns, no significant.

To clarify the differences in the effects of L-Theanine on DDP-resistant and non-resistant lung cancer cells, we performed a comparative analysis on the RNA-seq data from L-Theanine-treated DDP-resistant lung cancer cells and control cells. For lung cancer cells, a total of 725 differentially expressed genes were identified between the L-Theanine-treated group and the control group, including 278 up-regulated genes and 447 down-regulated genes; for DDP-resistant lung cancer cells, a total of 829 differentially expressed genes were identified between the L-Theanine-treated group and the control group, including 387 up-regulated genes and 442 down-regulated genes. Relative to non-DDP-resistant cells, a total of 316 differentially expressed genes (DEGs) were identified in L-Theanine-treated DDP-resistant lung cancer cells, among 208 up-regulated genes, BMAL1 was significantly upregulated in L-Theanine-treated DDP-resistant lung cancer cells. DEGs in L-Theanine-treated DDP-resistant lung cancer cells relative to non-DDP-resistant lung cancer cells were enriched by using molecular Signature Data Base (mSigDb) markers and the Gene Ontology of Biological Processes (GO). The result showed they are statistically enriched in some biosynthesis (fatty acids, glycosyl-phosphatidylinositol, etc.), signaling transduction, transcription factors, and virus infection (Fig. 3A). Subsequent KEGG and Reactome enrichment analysis identified 239 genes enriched in cancer-related signaling pathways, including the dominant STAT3/NOTCH1 signaling pathway and the IL-17 signaling pathway (Fig. 3B,C).

Fig. 3.

Involvement of STAT3/NOTCH1 signaling in L-Theanine relieving chemoresistance in lung cancer. Different expressed genes in L-Theanine-treated DDP-resistant lung cancer cells relative to control cells were statistically enriched in molecular Signature Data Base (mSigDb) markers and in the Gene Ontology of Biological Processes (GO) (A), which are statistically enriched for genes associated with biosynthesis (fatty acids, glycosylphosphatidylinositol, etc.), signaling transduction, transcription factors and virus infection (B); subsequent Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome enrichment analysis identified 239 genes enriched in cancer-related signaling pathways, including the STAT3/NOTCH1 signaling pathway and the IL-17 signaling pathway (C).

Based on the analysis of transcriptomics data, we further explored the effect of L-Theanine on STAT3/NOTCH1 signaling in lung cancer cells. The results showed that L-Theanine treatment resulted in a significant decrease in phosphorylated STAT3 and Notch1 levels in DDP-resistant lung cancer cells, which was not observed in lung cancer cells (Fig. 4A–D). It was also observed that BMAL1 expression was significantly reduced in L-Theanine-treated DDP-resistant lung cancer cells, which was relatively not considerably altered in L-Theanine-treated lung cancer cells (Fig. 4E–H). It has been reported that STAT3/NOTCH1 signaling activation in cervical cancer can upregulate BMAL1 expression [11], which is the dominant changed gene in our transcriptomics data. To further clarify the regulation of BMAL1 expression induced by STAT3/NOTCH1 signaling in lung cancer cells, we treated DDP-resistant lung cancer cells with Stattic, a specific inhibitor of STAT3. It was found that BMAL1 protein expression was significantly reduced when phosphorylated STAT3 was inhibited considerably, suggesting that the expression of BMAL1 was affected by STAT3/NOTCH1 signaling. In addition, the expression of cell stemness markers was significantly reduced (Fig. 4I–L), after the effective knockdown of BMAL1 in DDP-resistant lung cancer cells (Fig. 4M); meanwhile the clone formation and invasive migration ability of cells were also significantly inhibited after BMAL1 knockdown (Fig. 4N–Q). The above results suggest that L-Theanine can inhibit BMAL1 expression in DDP-resistant lung cancer cells by regulating STAT3/NOTCH1 signaling, inhibiting cancer stemness.

Fig. 4.

Involvement of BMAL1 in the regulation of lung cancer cells stemness as the downstream of STAT3/NOTCH1 signaling. The protein levels of Notch1, BMAL1, and p-STAT3/t-STAT3 in A549, A549/DDP, NCI-H446, and NCI-H446/DDP cells with or without L-Theanine treatment were detected by western blotting (A–D). Moreover, the protein levels of BMAL1, p-STAT3/t-STAT3, and stemness-related factors, including ALDH1, ABCG2, and Nanog in A549, A549/DDP, NCI-H446 and NCI-H446/DDP cells with or without static treatment were analyzed by a western blotting assay (E–L). After BMAL1 knockdown (M), tumor sphere formation assay was performed in A549/DDP and NCI-H446/DDP cells with or without shBMAL1 knockdown for 14 days. The number of tumor spheres means colonies per 5000 cells. Scale bar 200 µm (N,O). In addition, images and quantification of transwell migration assays were presented in A549/DDP and NCI-H446/DDP cells with or without shBMAL1 knockdown. Scale bar 100 µm (P,Q). Error bars indicate SEM; n = 3. *p 0.05; ** p 0.01; ns, no significant.

Based on the above findings, we further explored the possibility of reversing drug resistance in lung cancer cells by combining L-Theanine with STAT3 blockade. Firstly, the changes in the cellular resistance index of DDP-resistant lung cancer cells to DDP after L-Theanine combined with STAT3 blockade treatment were investigated. The results showed that the combination treatment synergistically reduced the resistance index of DDP-resistant lung cancer cells relative to monotherapy (Fig. 5A,B). Secondly, it was also found that L-Theanine combined with STAT3 blockade treatment significantly inhibited the clone formation ability and the invasive migration ability of DDP-resistant lung cancer cells (Fig. 5C,D). Finally, L-Theanine combined with STAT3 blockade treatment effectively reduced the expression levels of stemness markers in DDP-resistant lung cancer cells (Fig. 5E–H). These results suggest that L-Theanine combined with STAT3 blockade synergistically inhibits the stemness of DDP-resistant lung cancer cells, thus overcoming platinum-based chemotherapy resistance in lung cancer cells.

Fig. 5.

Combination of L-Theanine and STAT3 blockade improved synergistically the chemoresistance in lung cancer. The changes in the cellular resistance index to DDP after L-Theanine combined with STAT3 blockade treatment of A549/DDP and NCI-H446/DDP cells (A,B). Moreover, number of colonies per 5000 A549/DDP and NCI-H446/DDP cells was quantified (C); meanwhile, number of migrated cells (A549/DDP and NCI-H446/DDP cells) was calculated (D). In addition, the levels of proteins including ALDH1, ABCG2, and Nanog in A549, A549/DDP, NCI-H446, and NCI-H446/DDP cells with or without combination treatment were analyzed by western blotting (E–H). Error bars indicate SEM; n = 3. * vs control, p 0.05; ${}^{\mathrm{\#}}$ vs Stattic, p 0.05.