Anticancer effects of epigallocatechin-3-gallate nanoemulsion on lung cancer cells through the activation of AMP-activated protein kinase signaling pathway

Epigallocatechin-3-gallate (EGCG), a green tea–derived polyphenol, exhibits antitumor activities. An EGCG nanoemulsion (nano-EGCG) was prepared to improve the stability and reduce the side effects of EGCG for treatment of human lung cancer cells, and the antitumor effects were studied. The possible molecular mechanism underlying its antitumor effects on cultured human lung cancer cells was also elucidated. The antitumor effects of EGCG and nano-EGCG were determined using methylthiazolyldiphenyl-tetrazolium bromide (MTT), colony formation, migration, and invasion assays. In addition, changes in the AMP-activated protein kinase (AMPK) signaling pathway were investigated using Western blot analyses. AMPK inhibitors were used to determine the roles of the AMPK signaling pathway involved in the molecular mechanism of the nano-EGCG. Our results showed that both EGCG and nano-EGCG inhibited the growth of H1299 lung cancer cells, with half-maximal inhibitory concentrations of 36.03 and 4.71 μM, respectively. Additionally, nano-EGCG effectively suppressed lung cancer cell colony formation, migration, and invasion in a dose-dependent manner. Nano-EGCG may inhibit lung cancer cell invasion through matrix metalloproteinase (MMP)-2- and MMP-9-independent mechanisms. Furthermore, the expression of several key regulatory proteins in the AMPK signaling pathway was modulated by nano-EGCG. Nano-EGCG may inhibit lung cancer cell proliferation, colony formation, migration, and invasion through the activation of AMPK signaling pathways. This novel mechanism of nano-EGCG suggests its application in lung cancer prevention and treatment. Our results provide an experimental foundation for further research on its potential activities and effects in vivo.

Colony formation assay. For the anchorage-dependent growth assay, 200 cells were resuspended in the RPMI 1640 medium and seeded in six-well plates. The culture media only or nanoemulsion added in media without (controls) or with various concentrations of EGCG or nano-EGCG solutions were changed every 2-3 days. After 7-10 days, the media were removed and the cells were washed and fixed with 4% paraformaldehyde. The fixed cells were stained with 0.05% crystal violet. Colonies >0.8 mm were counted under an inverted microscope. For the anchorage-independent growth assay, the bottom layer contained 0.7% agarose in RPMI 1640, and the top layer contained 0.35% agarose. Cells were seeded at a density of 1,500 cells per well in a six-well plate. The culture media or blank nanoemulsion without (controls) or with various concentrations of EGCG or nano-EGCG solutions were changed every 2-3 days. The plates were incubated at 37 °C with 5% CO 2 for 4 weeks and subsequently stained with crystal violet. Colonies >0.5 mm were counted under an inverted microscope. The colony formation was assessed in duplicate for three independent experiments.
Cell migration assay. The cells were seeded into 6-cm culture dishes at a density of 3 × 10 6 cells per well and cultured for 24 hours in a medium containing 10% FBS. Subsequently, the nearly confluent cell monolayer was www.nature.com/scientificreports www.nature.com/scientificreports/ carefully scratched using a 10-μL pipette tip. Any cellular debris was removed by washing with phosphate-buffered saline. After being wounded, the cultures were incubated with various concentrations of EGCG or nano-EGCG solutions at 37 °C. At the indicated times (0, 4, 8, and 12 hours) after scraping, the cells were washed twice and immediately photographed. The number of cells migrating into the cell-free zone was counted through a light microscope. In addition, the effects of AMPK inhibitor (BML-275; Enzo Life Sciences, Inc., Farmingdale, NY, USA) on cell migration capabilities were evaluated. All experiments were performed in triplicate.

Matrigel invasion assay. The invasiveness of tested cells treated with various concentrations of EGCG
or nano-EGCG solutions was examined in a Transwell assay using chambers (8-μm pore size; Corning Costar, Cambridge, MA, USA) and Transwell filters coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA), as described previously 23 . The number of cells attached to the lower surface of the polycarbonate filters was determined under a light microscope at 400× magnification. All experiments were performed in triplicate.
Gelatin zymography assay. Cells were seeded at a density of 1 × 10 6 cells per well in six-well plates for 24 hours. Subsequently, the tested cells treated with various concentrations of EGCG or nano-EGCG was cultured in serum-free media for another 24 hours. To detect matrix metalloproteinase (MMP)-2/9 activities, conditioned media were prepared without boiling or reduction and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis with gels containing 0.1% gelatin. After electrophoresis, the gels were washed with 2.5% Triton X-100 for 30 min and incubated in a developing buffer (50 mM Tris-HCl, pH 8.0, 0.2 M NaCl, 5 mM CaCl 2 , 0.02% Brij35) at 37 °C for 24 hours. Finally, the gels were stained with Coomassie Brilliant Blue R-250.
Western blot analysis. Western blot analysis was used to examine the expression levels of the affected proteins after nano-EGCG treatments of the tested cell lines. The details of these procedures were described previously 24 . The specific primary antibodies against protein kinase B (Akt), p-Akt, LKB1, p-LKB1, AMPK, p-AMPK, mammalian target of rapamycin (mTOR), p-mTOR, p-P70 (Thr), p-P70 (Ser), and phosphorylated 4E-binding protein 1 (p-4EBP1; Cell Signaling Technology, Danvers, MA, USA) were used for detection, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control. After incubation with the primary antibodies, the membranes were washed three times with a solution of Tris-buffered saline and Tween 20. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, Biotech Inc., CA, USA), and detection was conducted using an enhanced chemiluminescence detection system (ECL, GE Healthcare, NJ, USA).

Statistical analysis.
All experiments were performed in triplicate and analyzed through analysis of variance (Excel, Microsoft) to determine significant differences. Where appropriate, the results are expressed as the mean ± standard deviation (SD). All statistical tests were two-sided, and P values <0.05 were considered statistically significant.

Results
Effects of Nano-EGCG on lung cancer cell proliferation activity. EGCG was reported to exhibit an antiproliferative effect on lung cancer cells 16 . To study the effects of EGCG and nano-EGCG on human lung cancer cells, we first determined whether EGCG or nano-EGCG at the indicated concentrations of treatments for 24, 48, and 72 hours could influence the viabilities of H1299, A549, and BEAS2B cells. After treatment, we conducted an MTT assay to determine the cell viabilities ( Fig. 1). A dose-dependent decrease was demonstrated in the H1299 cell viability after treatment with EGCG or nano-EGCG for 72 hours (Fig. 1A,B). We discovered that EGCG could suppress H1299 cell proliferation at doses higher than 20 μM. However, only 5 μM doses of nano-EGCG could significantly inhibit H1299 cell viability. Comparatively, the half-maximal inhibitory concentration (IC 50 ) of EGCG and nano-EGCG for H1299 lung cancer cells was 36.03 μM and 4.71 μM, respectively. Nano-EGCG exhibited more efficient inhibition than did EGCG of the growth of H1299 cells. In addition, the effects of nano-EGCG on the growth of another lung cancer cell, A549, were determined. As indicated in Fig. 1C, A549 cell viability followed a dose-dependent decline after nano-EGCG treatment for 48 and 72 hours. The IC 50 of nano-EGCG for A549 cells was 16.05 μM. To further clarify whether nano-EGCG could influence the growth of lung epithelial cells, the viability of BEAS2B cells was detected. With a nano-EGCG dose of <5 μM, no significant decrease was exhibited in BEAS2B cell viability after the indicated time periods (Fig. 1D). When the nano-EGCG doses were raised to 5 μM and 10 μM, the viability of BEAS2B cells at 72 hours decreased to 90.17% and 77.72%, respectively. Thus, nano-EGCG exhibited greater antiproliferative activity in H1299 and A549 human lung cancer cells than in BEAS2B cells.

Effects of EGCG and nano-EGCG on lung cancer cell colony formation activity.
Subsequently, the effect of nano-EGCG on the colony formation ability of lung cancer cells was determined using anchorage-independent and -dependent colony formation assays. As displayed in Fig. 2A, both H1299 and A549 cells treated with nano-EGCG formed fewer anchorage-independent colonies than did the control cells. Quantitative data revealed that nano-EGCG inhibited the anchorage-independent colony formation activity of the H1299 and A549 cells in a concentration-dependent manner. Treatment with nano-EGCG at a low dose (1 μM) significantly reduced the number of colonies formed in the A549 cells to 86% relative to the control (P < 0.05), whereas treatment with higher concentrations of nano-EGCG (20 μM) significantly inhibited colony formation in the A549 cells. However, unlike with EGCG, the colony formation activity was significantly inhibited at doses as low as 250 nM nano-EGCG for H1299 cells (P < 0.05). With EGCG doses >5 μM, the colony formation activity of H1299 cells was completely suppressed ( Fig. 2A,B). The results of the anchorage-dependent colony formation assay revealed that nano-EGCG doses of 250 nM and 1 μM could significantly inhibit the colony formation abilities of H1299 and A549 cells, respectively ( www.nature.com/scientificreports www.nature.com/scientificreports/ Effects of nano-EGCG on lung cancer cell migration and invasive activity. Because tumor metastasis is the main impediment to treating lung cancer, we considered whether nano-EGCG could inhibit the migration and invasion capabilities of lung cancer cells. As indicated in Fig. 1B,C, nano-EGCG did not greatly affect the cell viabilities of H1299 and A549 after 24 hours at doses lower than 5 μM and 20 μM, respectively. Thus, the effect of nano-EGCG on cell invasion of H1299 was determined through Transwell invasion assay to be <5 μM. As illustrated in Fig. 3A, nano-EGCG reduced the invasive ability of H1299 cells in a dose-dependent manner. Treatment with nano-EGCG at 2 μM significantly reduced (P < 0.05) the invasion ability of H1299 cells to 19.8% relative to the control. Similar results were discovered in the EGCG groups (Fig. 3B). Higher doses of EGCG (10 and 20 μM) could significantly inhibit the invasion capability of H1299 cells. In addition, the effect of nano-EGCG on lung cancer cell migration was evaluated using a wound-healing assay. Nano-EGCG could inhibit the migration of H1299 cells both in concentration-and time-dependent manners (Fig. 3C). Similar results were also discovered in the EGCG groups (Fig. 3D). However, the effective inhibitory concentration of nano-EGCG for H1299 cell migration ability was lower than that of EGCG. Furthermore, nano-EGCG inhibited the migration and invasion capabilities of A549 cells in a dose-dependent manner, as illustrated in Fig. 3E,F (P < 0.05). These results revealed that nano-EGCG could suppress lung cancer cell migration and invasion at low doses.

Effects of nano-EGCG on MMP-2 and MMP-9 activities in lung cancer cells. A previous study
demonstrated that EGCG could repress the activities of MMP-2 and MMP-9 in highly invasive CL1-5 lung adenocarcinoma cells 25 . Thus, to determine whether nano-EGCG has any effects on MMP-2 and MMP-9 in lung cancer cells, we first collected conditioned media of H1299 and CL1-5 cells treated for 24 hours with various concentrations of EGCG or nano-EGCG and analyzed the media through gelatin zymography assay. In accordance with previous results 25 , the gelatin-degrading activity significantly decreased in the presence of EGCG at 10 μM in CL1-5 cells. In addition, EGCG could reduce the activities of MMP-2 and MMP-9 in H1299 cells in a dose-dependent manner (Fig. 4A). However, nano-EGCG had no significant effects on the activities of MMP-2 and MMP-9 in either H1299 or A549 cells (Fig. 4B,C). These results suggested that nano-EGCG could suppress the invasion activity of lung cancer through MMP-2-and MMP-9-independent mechanisms; this is different from how EGCG acted on lung adenocarcinoma cells. www.nature.com/scientificreports www.nature.com/scientificreports/ Effects of nano-EGCG on AMPK signaling in lung cancer cells. As previously mentioned, nano-EGCG could significantly inhibit the proliferation, colony formation, migration, and invasion activities of the H1299 and A549 cells. However, the signal transduction mechanisms responsible for the inhibitory effects of nano-EGCG remained unknown. The activation of AMPK has been reported to inhibit tumor progression in several types of cancers; therefore, we further assessed the effects of nano-EGCG on the signal transductions of AMPK in lung cancer cells through western blot analysis. The H1299 cells were treated with various concentrations of nano-EGCG for 48 hours. Subsequently, the total protein lysates of each sample were collected and subjected to western blotting with various total protein or phosphoprotein antibodies. As illustrated in Fig. 5, nano-EGCG treatment significantly reduced the phosphorylation of Akt in H1299 cells and significantly increased the LKB1 and AMPK phosphorylation levels. Furthermore, the phosphorylation levels of downstream target proteins of AMPK, such as mTOR, P70, and 4EBP1, were also regulated by nano-EGCG treatment. In summary, the inhibitory effects of nano-EGCG on the proliferation and invasion of the lung cancer cells might result from activation of the AMPK signaling pathway.

Figure 2. Effects of EGCG and nano-EGCG on the colony formation activity in lung cancer cells. (A)
Representative images of the anchorage-independent colony formation assay in H1299 and A549 cells. The graphs are the summarized data of the colony formation assays. Colonies >0.5 mm in diameter were counted and displayed in comparison with the control group. Anchorage-dependent colony formation assays of H1299 cells treated with EGCG (B) and nano-EGCG (C). (D) The effects of nano-EGCG on A549 cells anchoragedependent colony formation abilities were also studied. Colonies >0.8 mm in diameter were counted and displayed in comparison with the control group. Values are reported as means ± SD (n ≥ 3). *P < 0.05 compared with the control group. (2020) 10:5163 | https://doi.org/10.1038/s41598-020-62136-2 www.nature.com/scientificreports www.nature.com/scientificreports/

Effects of nano-EGCG and AMPK inhibitor on migration capability of lung cancer. To further
confirm that AMPK plays a major role in reducing the migration ability regulated by nano-EGCG in H1299 cells, the inhibitor of AMPK, BML-275, was used. H1299 cells were pretreated with 2 μM nano-EGCG, 100 nM AMPK inhibitor, or a combination and then subjected to wound-healing migration assays. As indicated in Fig. 6, 2 μM nano-EGCG could significantly suppress the migration activity of H1299 cells compared with control groups (P < 0.05). However, the AMPK inhibitor treatment could slightly increase the numbers of migrated H1299 cells. Moreover, the treatment of H1299 cells with combined nano-EGCG and AMPK inhibitor could significantly reverse the inhibitory effects of nano-EGCG on cell migration capabilities at the indicated time (P < 0.05). These results indicated that the AMPK signaling pathway might be involved in nano-EGCG regulated antitumor activities in lung cancer cells (Fig. 7).

Discussion
Phytochemicals present in certain fruits, vegetables, and tea have attracted scientific attention as potential agents for cancer treatment and prevention. Among various tea products, green tea has received considerable attention; numerous studies have suggested that EGCG, the most abundant constituent of total catechins in green tea, has anticarcinogenic activities 26 . However, the effects of EGCG on human lung cancer are largely unknown. In the present study, to enhance the stability of EGCG and its ability to target human lung cancer cells, an EGCG nanoemulsion was prepared, and the antitumor effects of nano-EGCG on lung cancer cells were determined. www.nature.com/scientificreports www.nature.com/scientificreports/ Our data demonstrated for the first time that nano-EGCG can play a crucial role in increasing the AMPK phosphorylation expression and suppressing the proliferation, colony formation, migration, and invasion capabilities of lung cancer cells by activating the AMPK signaling pathway. Notably, nano-EGCG could suppress the invasion activity of lung cancer through MMP-2-and MMP-9-independent mechanisms; this differs from how EGCG was reported to exert effects on lung adenocarcinoma cells 25 . Our results were consistent with previous studies on breast cancer cells 27 , according to which the antitumor effectiveness of nano-EGCG is superior to that of EGCG on lung cancer cells. Furthermore, the blockage of activated AMPK in lung cancer cells could  www.nature.com/scientificreports www.nature.com/scientificreports/ significantly reverse the inhibitory effects of nano-EGCG on cell migration activities. Taken together, our results indicate that the activated AMPK signaling pathway induced by nano-EGCG treatment is involved in antitumor activities in human lung cancer cells.  www.nature.com/scientificreports www.nature.com/scientificreports/ Catechins have been reported to have several biological benefits. EGCG is the major constituent of catechins present in green tea. Previous studies have suggested that EGCG exhibits a variety of activities, such as antiobesity, antidiabetes, anti-inflammatory, and antitumor activities 26 . EGCG could inhibit carcinogenic activity, proliferation, angiogenesis, migration, invasion, and tumorigenesis and induce cell death. These antitumor effects are associated with the modulation of several signaling molecules, including reactive oxygen species, NF-κB, Akt, vascular endothelial growth factor, peroxisome proliferator-activated receptor, Bcl-2, and mitogen-activated protein kinases, as well as epigenetic modification 28 . Although numerous studies have demonstrated that EGCG exhibits health-promoting effects at a certain dose, toxicity of EGCG has also been reported. An overdose of green tea or EGCG on rat models caused adverse complications such as hepatotoxicity through liver enzyme alterations 29 . In addition, previous studies have demonstrated that EGCG is lower in plasma than the actual ingested quantity, thus indicating low bioavailability 30 . Therefore, nanotechnology was recently introduced to improve bioavailability and reduce the size of doses of EGCG.
For example, chitosan nanoparticles encapsulating EGCG have been reported to possess numerous significant biological and chemical properties such as biocompatibility, hemostasis, low toxicity, biodegrability, and anticarcinogenesis 31 . Our data revealed that nano-EGCG could achieve similar anticancer to EGCG effects at a lower concentration. The IC 50 of nano-EGCG in inhibiting H1299 cells was much lower than the EGCG standard (4.71 μM vs. 36.03 μM). However, nano-EGCG could not drastically reduce the viability of BEAS2B bronchial epithelial cells at this low dose. In addition, low concentrations of nano-EGCG could significantly inhibit the colony formation, migration, and invasion abilities of lung cancer cells. A previous study suggested that EGCG could inhibit the invasion of CL1-5 lung cancer cells by suppressing MMP-2 expression through c-Jun N-terminal kinase signaling 25 . However, our results demonstrated that nano-EGCG might inhibit lung cancer cell invasion through an MMP-2-and MMP-9-independent pathway. Although EGCG could suppress the MMP-2 and MMP-9 activities in H1299 cells and CL1-5 cells, the inhibitory effects on MMP activity were lost on the H1299 and A549 cells treated with nano-EGCG (Fig. 4). The different characteristics of these two biomaterials require further investigations.
EGCG has been reported to play a role in the management of cancer through the modulation of different cell signaling pathways. For example, previous studies have demonstrated that EGCG could induce the expression of p53 and Bax in prostate cancer and breast cancer cells 32,33 . Other findings have suggested that EGCG could inhibit the activation of Akt in bladder cancer and breast cancer cells 34,35 . Furthermore, the activation and expression of c-Met; MAPK; and transcription factors activator protein 1, NF-kB, and Stat3 were reported to be modulated by EGCG 28 . AMPK is known to be involved in cellular homeostasis, and AMPK plays a crucial role in cell survival and apoptosis. EGCG could suppress colon cancer proliferation by modulating the AMPK activation 36 . The loss of function of LKB1, an upstream kinase of AMPK, in lung adenocarcinoma is 30-50% 37 . Because of the discovery of the antineoplastic effects of AMPK, the number of patents for potential AMPK activators has grown rapidly 38,39 . However, in some cases, AMPK activation may promote tumor survival through mTORC2/PI3K-Akt signaling pathway activation 40 .
The data in the present study revealed that nano-EGCG not only induces LKB1 and AMPK activation but also suppresses the activation of Akt. However, the study also has several limitations. First, the effect of nano-EGCG on Ca 2+ /CaM-dependent protein kinase kinase β, another upstream activator of AMPK, requires investigation in further studies. Moreover, although in vitro experiments were performed in the current study, the effects of nano-EGCG in vivo, such as tumorigenesis and metastasis, should be determined. Finally, whether nano-EGCG could improve the targeting ability and bioavailability in vivo requires further investigation.

Conclusions
Our results demonstrated for the first time that significant inhibition of proliferation, colony formation, migration, and invasion of human lung cancer cells modulated through the activation of the AMPK signaling pathway by low doses of nano-EGCG. Therefore, nano-EGCG could be developed as a potential antitumor candidate targeting AMPK for the prevention and treatment of lung cancer. In addition, the potential clinical use of this agent warrants further evaluation and investigation. The use of low doses of nano-EGCG, which provides low toxicity and high efficacy, in adjuvant therapies with other agents may be more promising for lung cancer therapy.

Data availability
The obtained results of the research are available on reasonable request.