Contributed equally
The epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), gefitinib, is an effective therapeutic drug used in the treatment of non-small cell lung cancers (NSCLCs) harboring EGFR mutations. However, acquired resistance significantly limits the efficacy of EGFR-TKIs and consequently, the current chemotherapeutic strategies for NSCLCs. It is, therefore, necessary to overcome this resistance. In the present study, the anticancer potential of natural extracts of
The ability to evade apoptosis is one of the hallmarks of cancer and is a crucial property of cancer cells that confers them resistance to chemotherapeutic agents (
The herb
Therefore, the present study examined the expression of Mcl-1 and Bcl-2 in order to determine the effects of the extract of CC (ECC) on apoptosis. The anticancer effects of ECC, as well as combination treatment with ECC and gefitinib on gefitinib-resistant (GR) NSCLC cells (PC9GR, A549GR and HCC827GR) were also investigated.
BEAS-2B, and the GR human lung cancer cell lines, PC9GR and A549GR, were gifts from Dr J.K. Rho, Ulsan University, Asan Hospital. The HCC827GRKU cell line was established from HCC827 cells treated with 2 µM gefitinib for >6 months (data not shown). All cell lines were grown in RPMI (Welgene, Inc.) supplemented with 10% fetal bovine serum (Welgene, Inc.) and 1% penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO2 for all the experiments. Gefitinib was purchased from Selleck Chemicals and berberine was purchased from Sigma-Aldrich; Merck KGaA. Airdried roots of CC were purchased from Dongguk University, Ilsan Korean Medicine Hospital. CC (10 g) was extracted in 100 ml distilled water at room temperature. After 24 h, the solution was heated to 90°C for 4 h. The extract was then filtered, evaporated and lyophilized (yield, 12.6%). The lyophilized extract of CC (ECC) was stored at −20°C until use. The identification of chemical components in ECC was performed by ultra-performance liquid chromatography (UPLC)-quadrupole time-of-flight (QTOF) (
Cell viability was measured by MTT assay. Briefly, 1×103 cells per well were seeded in 96-well culture plates overnight and, subsequently incubated with or without the relevant treatments of ECC, or berberine. After 72 h, 50 µl MTT solution (0.5 mg/ml, Sigma-Aldrich; Merck KGaA) were added to each well. Following incubation at 37°C for a further 4 h, the MTT solution was discarded and DMSO was added. The absorbance at 750 nm was measured using a microplate reader (SpectraMax Plus 384, Molecular Devices, LLC). The fraction affected (Fa) and combination index (CI) values were calculated using CompuSyn (
The invasiveness of the tumor cells was assessed via an invasion assay in Transwell chambers comprising a Transwell membrane (8 µm pore size, 6.5 mm in diameter, Corning Life Science, Inc.) coated with Matrigel (100 µg/ml, 10 µl/well). The cells (1×105) were seeded in the upper chambers in the presence of the indicated concentrations (PC9GR cells: 0, 30 and 50 µg/ml; HCC827GRKU cells: 0 and 30 µg/ml) of ECC. The lower chambers of the Transwell plate were filled with RPMI with 10% FBS The cells were fixed with 70% ethanol for 10 min, stained with hematoxylin and eosin for 5 min at room temperature, and counted under a light microscope (Olympus-IX71, Olympus Corp.) following incubation for 24 h.
Cell migration was assessed using a wound-healing assay. The cells (5×105) were seeded in 6-well plates and incubated at 37°C for 24 h. After the cell monolayer was scraped with a sterile micropipette tip, the wells were washed several times with phosphate-buffered saline (PBS) and cultured with the designated concentrations (PC9GR cells: 0, 30 and 50 µg/ml; A549GR cells: 0, 10 and 20 µg/ml; HCC827GRKU cells: 0 and 30 µg/ml) of ECC. The first image of each scratch from 4 independent areas was acquired at time zero. The image of each scratch at the same location was captured under a light microscope (Olympus-IX71, Olympus Corp) after the indicated incubation times (0, 24 and 48 h). The healed area was measured from the captured images using Image J software (Ver. 1.52n, NIH).
Cell were lysed with ice-cold TNN buffer (1 M Tris-Cl pH 7.4, 0.5% NP40, 5 M NaCl., 0.5 M EDTA pH 8.0) at 4°C for overnight. Cell lysates were centri-fuged at 16,100 × g for 15 min and the supernatants were used as total cellular protein extracts. The protein concentrations were determined by Bradford assay (Microplate reader, model-680, Bio-rad). Protein denaturation (20 µg/lane) was carried out by sodium dodecyl sulfate (SDS) and mercaptoethanol loading and electrophoresed on a 12% acrylamide gel (this excluded caspase-3 which was electrophoresed on a 15% acrylamide gel). This was followed by transfer onto nitrocellulose membranes (GE Healthcare Life Science, Inc.). The membranes were blocked with 5% non-fat dry milk (SK1400.500, BioShop) in TBST (247 mM Tris, 1.37 M NaCl, 27 mM KCl, 1% Tween-20, pH 7.6) at room temperature for 1 h. These membranes were, subsequently, probed with the indicated primary antibodies at 4°C for overnight and incubated with the appropriate goat anti-mouse IgG (1:5,000, sc-2005, Santa Cruz Biotechnology, Inc.) or goat anti-rabbit IgG (1:5,000, sc-2004, Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. Secondary antibodies were conjugated with horseradish peroxidase prior to signal detection using the enhanced chemiluminescence system (Translab) in accordance with the manufacturer's instructions. The primary antibodies (dilution, cat. no.) against EGFR (1:1,000, #2232), AKT (1:1,000, #4691), p-AKT (1:1,000, #4691), caspase-3 (1:1,000, #9662) and poly(ADP-ribose) polymerase (PARP) (1:1,000, #9542) were purchased from Cell Signaling Technology, Inc. The antibodies against p-EGFR (1:1,000, sc-101668), MET (1:1,000, sc-161), Bcl-2 (1:1,000, sc-492), Mcl-1 (1:1,000, sc-819), Bcl-xL (1:1,000, sc-7195) and β-actin (1:20,000, sc-47778) were purchased from Santa Cruz Biotechnology, Inc.
Total RNA was isolated from the cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesized from the total RNA using a reverse transcription kit (LaboPass, Cosmo Genetech) in accordance with the manufacturer's instructions. qPCR was conducted using gene-specific primers with SYBR-Green Q Master (LaboPass) on an ABI 7500 Real Time PCR System (Applied Biosystems). The following PCR primers were used:
GR cells were harvested following treatment with ECC (PC9GR cells, 50 µg/ml; A549GR cells, 30 µg/ml) for the indicated time periods (0, 24 and 48 h) and dissociated into single cells. The cells were fixed with 95% ethanol, incubated at −20°C for at least 1 h, and washed with PBS. The cells were then resuspended in PBS with 0.1 mg/ml RNase A, 50 mg/ml propidium iodide (PI), and 0.05% Triton X-100 for 15 min at room temperature in the dark and washed with PBS. The stained samples were analyzed using a FACS Canto 2 (BD Biosciences) within 1 h of staining. All experiments were performed in triplicate.
Cells were seeded on a coverslip with complete medium and incubated with or without the indicated concentrations (PC9GR cells, 50 µg/ml; A549GR cells, 30 µg/ml) of ECC. Following incubation at 37°C for 24 and 48 h, the cells were fixed with 4% paraformaldehyde for 25 min at 4°C and washed twice with PBS at room temperature. The cells were then permeabilized with 0.2% Triton X-100 in PBS at room temperature for 5 min and washed twice with PBS. TUNEL assay of the nuclei was performed, and the labeled cells were viewed under a fluorescent microscope (Olympus-IX71, Olympus Corp.), as described in the manufacturer's protocol (DeadEnd™ Fluorometric TUNEL System; Promega).
Zebrafish (
For the
The experiments were repeated at least twice. The values are expressed as the means ± standard deviation and were compared using a two-tailed Student's t-test or ANOVA. If the P-value obtained by one-way ANOVA was <0.05, P-values between the groups were compared with a post hoc test, such as the Bonferroni and Tukey's HSD. A value of P≤0.05 was considered to indicate a statistically significant difference.
Given that, as previously demonstrated, the GR cell lines, PC9GR, A549GR and HCC827GR, exhibit an enhanced viability upon gefitinib treatment (
To elucidate the mechanisms through which ECC affects GR cell viability, cell cycle and apoptosis analyses were performed using PI-stained cells through FACS analysis and TUNEL assay. The distribution of GR cells in the cell cycle phase was analyzed following treatment with the indicated concentrations of ECC for 24 and 48 h. ECC treatment increased the percentage of GR cells in the sub-G1 phase (i.e., dead cells) in a time-dependent manner (
Increased cell survival owing to the impairment of an essential pathway for EGFR-TKI-mediated apoptosis has been suggested as a mechanism responsible for resistance to EGFR-TKIs. To investigate this pathway in GR cells, the expression of the EGFR pathway and anti-apoptotic proteins was examined in GR cells and compared with that in their parental cells. The expression of AKT/p-AKT and the anti-apoptotic proteins, Mcl-1 and/or Bcl-2, was increased in the GR cells (
The ability of ECC to enhance the effects of gefitinib on GR cells was evaluated by MTT assay using cells treated with a combination of ECC and gefitinib. Combination treatment reduced GR cell viability in comparison to treatment with gefitinib or ECC alone (
The suppression of tumorigenicity
EGFR-TKIs are some of the most effective therapeutic drugs against NSCLCs with EGFR mutations. However, the various adaptive and acquired resistance mechanisms reported have significantly limited the efficacy of EGFR-TKIs and, consequently, the current chemotherapeutic strategies for NSCLCs. Therefore, there is a need to overcome GR resistance to EGFR-TKIs, as GR resistance in lung cancer results in more aggressive cells with an increased viability, proliferation and metastatic ability.
Apoptosis is the natural process through which the elimination of unwanted or damaged cells that present a threat to the health of an organism occurs. This process is highly controlled, and the Bcl family of proteins, which comprises anti-apoptotic proteins and pro-apoptotic proteins, serves as the main regulator of this process (
Conversely, the inhibition of Bcl-2 by various methods (gene suppression and inhibitors) has been shown to increase the sensitivity of lung cancer cells to EGFR-TKIs (
Therefore, the present study evaluated the expression of the anti-apoptotic molecules, Mcl-1, Bcl-2 and Bcl-xL, as well as EGFR signaling molecules, in established GR lung cancer cell and compared it with the expression in their parental cell PC9, A549 and HCC827. The PC9GR cells exhibited a higher expression of EGFR signaling molecules and Bcl-2, but not of Mcl-1. The A549GR cells exhibited a higher expression of AKT/p-AKT and Mcl-1, but not of EGFR/p-EGFR and Bcl-2. The HCC827GRKU cells exhibited a higher expression of AKT/p-AKT, Bcl-2 and Mcl-1. These data suggested that the cellular response to EGFR-TKIs is dependent on the EGFR mutation status, as described in other studies (
The fact that in the present study, no tumor xenograft mouse model was used validating the anti-tumor effect of ECC addition to the zebrafish tumor model, which can provide more convincing evidence to the present data and the fact that ECC is a multi-component formulation, rather than a single compound, may be a limitation of this study. However, it should considered that the very weak cytotoxic effects of ECC on normal cells compared to those of berberine, a known alkaloid extracted from ECC, may arise due to the multi-component nature of ECC.
Collectively, the present study found that ECC exerted anti-cancer effects through the suppression of EGFR/AKT signaling and induced apoptosis via the suppression of the anti-apoptotic proteins, Mcl-1 and Bcl-2, which were overexpressed in GR cells. Moreover, combination treatment with ECC synergistically enhanced GR cell sensitivity to gefitinib, regardless of the EGFR mutation status
The abstract was presented and published as a poster (no. P-35-049) in supplement (vol. 9, issue S1): The 44th FEBS Congress July 6-11, 2010 in Krakow, Poland.
The present study was supported by the Bio-Synergy Research Project (NRF-2014M3A9C4066487) of the Ministry of Science, ICT and Future Planning through the National Research Foundation and by the Basic Science Research Program Grants (NRF-2017R1A2B4003233 and NRF-2019R1A2C1083909) from the National Research Foundation of Korea, which is funded by the Ministry of Education, Science and Technology, Republic of Korea.
All data generated or analyzed during the study are included in this published article or are available from the corresponding author upon reasonable request.
JYL conceived and designed the experiments; JHK, ESK, DK, SHP, EJK, JR, WMY, IJH, HS and IJH conducted the experiments; JHK, ESK, DK, SP, EJK, JR, MJK, WMY, IJH, MJP, WMY and JYL analyzed and interpreted the results. All authors reviewed the manuscript and all authors read and approved the final manuscript.
All animal experimental protocols were approved by the Committee for Ethics of Animal Experimentation of Sookmyung Women's University.
Not applicable.
The authors declare that they have no competing interests.
ECC inhibits the viability of GR cell lines (PC9GR, A549GR and HCC827GRKU), and their parental cells (PC9, A549 and HCC827) with minimal cytotoxic effects on BEAS-2B cells. (A) Proliferation of GR cells increased in comparison with their parental cells, as determined by MTT assay. (B) Cell viability was determined by MTT assay following treatment with ECC for 3 days and the GR cells were more sensitive to ECC than their parental cells. The results shown are the means ± SD of triplicate experiments. *P<0.05, **P<0.01 and ***P<0.001; the hash symbol (#) indicates that there were no significant differences. ECC, Extract of
ECC inhibits the migration and invasion of GR cells (PC9GR, A549GR and HCC827GRKU). (A) Representative images of the scratched areas of the cultures of cells treated with the indicated concentrations of ECC collected at the indicated time after wounding with a pipette tip. (B) Representative images of cells that migrated through the Transwell and were stained with hematoxylin and eosin. The results are shown as the means ± SD of triplicate experiments. *P<0.05, and ***P<0.001; the hash symbol (#) indicates that there were no significant differences. ECC, extract of
ECC induces the death of GR cell lines (PC9GR and A549GR). (A-C) Cells were treated with the indicated concentrations of ECC for the indicated periods of time. The cell cycle distribution of the harvested cells was analyzed by flow cytometry. Representative (A) histograms and (B) quantification of the analysis of cells in G1/G0, S, G2/M and (C) sub-G1 fractions phases shown in the figure were measured by FACS analysis. (D) Representative images of the immunocytochemistry of treated and untreated GR cells (left panel) examined by TUNEL assay after 24 h. TUNEL-positive nuclei are indicated with white arrows. The nuclei were visualized using 4′,6-diamidino-2-phenylindole (DAPI) staining. Magnification, ×200. The results are the means ± SD of triplicate experiments. *P<0.05, and **P<0.01; the hash symbol (#) indicates that there were no significant differences. ECC, extract of
ECC suppresses the expression of EGFR/AKT and apoptosis-related signalling. (A) Anti-apoptotic proteins Bcl-2 or Mcl-1 were overexpressed in GR cells compared with their parental cells as examined by western blot analysis. (B) Higher expression of Bcl-2 in PC9GR than PC9 and Mcl-1 in A549GR than in A549 cells was confirmed by RT-qPCR. (C) ECC suppressed EGFR/AKT signalling and the anti-apoptotic proteins Bcl-2 and Mcl-1, resulting in increased cleaved caspase-3 and PARP expression in GR cells, as determined by western blot analysis. (D) Suppression of Bcl-2 and Mcl-1 by ECC was confirmed by RT-qPCR. GR cells were treated with ECC with the indicated concentrations for the indicated periods of time. The cells were harvested, and the indicated protein or RNA expression was examined by western blot analysis or RT-qPCR. The results are shown as the means ± SD of triplicate experiments. **P<0.01. ECC, extract of
ECC synergistically enhances the activity of gefitinib in GR cells. (A) Cell viability was assessed by MTT assay following treatment of the PC9GR, A549GR and HCC827GRKU cells with the indicated concentrations of of gefitinib alone, ECC alone, or co-treatment with gefitinib and ECC for 72 h. (B) Western blot analysis was performed with the indicated antibodies following treatment with the indicated concentrations of gefitinib, ECC and the combination of gefitinib and ECC for 24 and/or 48 h. (C) The CI of gefitinib and ECC were calculated and the Fa-CI plots generated by the Chou-Talalay method using data obtained from MTT assay following co-treatment with the indicated concentrations of each drug for 72 h. CI values of <1, 1 and >1 indicate synergism, additive effects and antagonism, respectively. The results are shown as the means ± SD of triplicate experiments. *P<0.05, **P<0.01 and ***P<0.001; the hash symbol (#) indicates that there were no significant differences. ECC, extract of
ECC exhibits specific anticancer cell effects
IC50 values of ECC, gefitinib and berberine in GR and parental cells.
Treatment | Cells lines and IC50 values
| ||||||
---|---|---|---|---|---|---|---|
PC9 | PC9GR | A549 | A549GR | HCC827 | HCC827GRKU | BEAS-2B | |
Gefitinib (µM) | 0.008 | 8.79 | 15.34 | 18.65 | 0.01 | 10 | N/A |
ECC (µg/ml) | 69.80 | 32.73 | 30 | 20 | 85.33 | 19.07 | 178.08 |
Berberine (µM) | 2.81 | 7.73 | 13.99 | <1 | 48.1 | 13.3 | 33.01 |
ECC, extract of