Cancer cell‑specific anticancer effects of Coptis chinensis on gefitinib‑resistant lung cancer cells are mediated through the suppression of Mcl‑1 and Bcl‑2

  • Authors:
    • Jae Hwan Kim
    • Eun Sun Ko
    • Dasom Kim
    • Seong‑Hee Park
    • Eun‑Jung Kim
    • Jinkyung Rho
    • Hyemin Seo
    • Min Jung Kim
    • Woong Mo Yang
    • In Jin Ha
    • Myung‑Jin Park
    • Ji‑Yun Lee
  • View Affiliations

  • Published online on: March 24, 2020     https://doi.org/10.3892/ijo.2020.5025
  • Pages: 1540-1550
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Abstract

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 Coptis chinensis (ECC) against gefitinib‑resistant (GR) NSCLC cells were investigated in vitro and in vivo. ECC inhibited the viability, migration and invasion, and effectively induced the apoptosis of GR cells. These effects were associated with the suppression of EGFR/AKT signaling and the expression of anti‑apoptotic proteins, Mcl‑1 and Bcl‑2, which were overexpressed in GR NSCLC cells. Combination treatment with ECC and gefitinib enhanced the sensitivity of GR cells to gefitinib in vitro, but not in vivo. However, ECC increased the survival of individual zebrafish without affecting the anticancer effect to cancer cells in vivo, which indicated a specific cytotoxic effect of ECC on cancer cells, but not on normal cells; this is an important property for the development of novel anticancer drugs. On the whole, the findings of the present study indicate the potential of ECC for use in the treatment of NSCLC, particularly in combination with EGFR‑TKI therapy, in EGFR‑TKI‑resistant cancers.

Introduction

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 (1,2). Understanding apoptotic resistance may assist in the development of strategies with which to restore the sensitivity of cancer cells to apoptosis and, ultimately, may improve the efficacy of cancer therapy. In lung cancer, various epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) resistance mechanisms have been identified, such as the second-site EGFR mutation, T790M, the activation of the bypass path-ways, MET and AXL, and histological transformation, and several efforts have been made to overcome these resistance mechanisms (3,4). A common apoptosis-associated EGFR-TKI mechanism in lung cancer is an intrinsic deletion polymorphism in the gene encoding BIM, although the findings regarding this are contradictory (5-7). BIM is a pro-apoptotic member of the Bcl-2 family and plays an essential role in the induction of cell apoptosis and tumor metastasis (2). The upregulation of BIM is required for apoptosis induced by EGFR and EGFR-TKIs in tumors harboring EGFR mutations (8). Consequently, BIM has become the focus of attention as a potential target for cancer chemotherapy. Furthermore, the overexpression of anti-apoptotic Bcl-2 family proteins, including Mcl-1 and Bcl-2, has been investigated and has been identified to be associated with chemoresistance and the prognosis of various types of cancer, including lung cancer (9-12); however, there are a limited number of studies on EGFR-TKI-resistant lung cancer.

The herb Coptis chinensis (known as goldthread; CC) is widely used in Traditional Chinese medicine; moreover, its alkaloid component, berberine, has been studied for its multiple pharmacological activities, including anti-infectious, anti-inflammatory and anticancer effects (13). In addition, efforts have been made to examine the potential therapeutic and biological functions of CC, not as a single compound, but as multi-compounds, for cancer treatment. CC has been shown to exert an anticancer effect through the downregulation of signal transducer and activator of transcription (STAT)2 phosphorylation by reducing the level of histone deacetylase 2 (HDAC2) in glioma cells and inhibiting hepatocellular carcinoma cell growth through non-steroidal anti-inflammatory drug (NSAID) activated gene (NAG-1) activation (14,15). In non-small cell lung cancer (NSCLC) cells, CC has been shown to inhibit growth and metastasis, and to induce cell apoptosis (16). However, neither its effects on EGFR-TKI resistant lung cancer nor its efficacy in combination with gefitinib have been elucidated to date, at least to the best of our knowledge.

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.

Materials and methods

Cell cultures and reagents

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) (Data S1, Fig. S1 and Table SI). ECC was re-dissolved in RPMI to a concentration of 1,000 µg/ml for the in vitro experiments and in DMSO (Daejunga) to a concentration of 6 mg/ml for the in vivo experiments.

Cell viability assays

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 (www.combosyn.com). CI values of <1, 1, and >1 indicated synergism, additive effects and antagonism, respectively. Cell viability assay for the co-treatment was performed with selected concentrations of of gefitinib (PC9GR and HCC827GRKU cells, 1 µM; A549GR cells, 2 µM) and ECC (PC9GR and HCC827GRKU cells, 10 µg/ml; A549GR cells, 5 µg/ml) for 72 h based on the CI values. The results were representative of a minimum of 3 independent experiments, and the error bars represent the standard deviation (SD).

Transwell invasion assays

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 assay

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).

Western blot analysis

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.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

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: Bcl-2 sense, 5′-AAG GGG GAA ACA CCA GAA TC-3′ and antisense, 5′-ATC CTT CCC AGA GGA AAA GC-3′; Mcl-1 sense, 5′-TGC TGG AGT AGG AGC TGG TT-3′ and antisense, 5′-CCT CTT GCC ACT TGC TTT TC-3′; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sense, 5′-GCC ATC GTC ACC AAC TGG GAC-3′ and antisense, 5′-CGA TTT CCC GCT CGG CCG TGG-3′. The PCR thermocycling conditions consisted of 95°C for 5 min, followed by 40 cycles of 95°C for 10 sec and 62°C for 30 sec. The Ct values of the target genes were normalized to those of an endogenous reference gene (GAPDH) using the ΔΔCq method (17). Each gene was analyzed in triplicate in 2 independent experiments.

Cell cycle analysis

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.

Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay

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).

In vivo zebrafish tumor model

Zebrafish (Danio rerio) and embryos were bred and maintained according to standard procedures. All animal experimental protocols were approved by the Committee for Ethics of Animal Experimentation of the Sookmyung Women's University and performed as previously described (18). Approximately 50 fluorescent cell tracker CM-Dil-labeled HCC827 or HCC827GRKU cells were injected into the yolk sac of each zebrafish embryo (100 embryos for each treatment), after which the embryos were maintained at 33°C and drug treatments were administered every 24 h for 4 days. Fluorescence image acquisition was performed using a Zeiss LSM700 confocal microscope (Carl Zeiss AG). The area penetrated by the CM-Dil-labeled cancer cells was quantified using ImageJ software (ver. 1.52n, NIH) and normalized to the cancer cells (100%) in non-treated zebrafish embryos for each group.

Dose selection of ECC

For the in vitro assay, the IC50 value of 3 days for the PC9GR (32.73 µg/ml) and A549GR (20 µg/ml) cells and an approximately 1.5-fold higher concentration than the IC50 value of 3 days were selected. For the HCC827GRKU, one concentration was selected, which was approximately 1.5-fold higher than the IC50 value of 3 days. For the in vivo assay, the toxicity of ECC was tested on zebrafish embryo development at a series of concentrations of ECC (0.1, 1 and 5 µl/ml); the embryos were observed until 7 days post-fertilization (dpf) and the concentration which did not affect the survival of the zebrafish was selected. The zebrafish embryos died from overall necrosis, a terminated heart beat and no movement from mechanical stimulation at 4 dpf following treatment with 5 µl/ml of ECC, but survived and developed normally following treatment with 0.1 and 1 µl/ml of ECC. Subsequently, further tests were performed to select the ECC concentration which was most effective against cancer cells from a series of ECC concentrations (0.1, 0.2 and 1.0 µl/ml), which was 0.2 µl/ml.

Statistical analysis

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.

Results

ECC inhibits the viability and mobility of the GR NSCLC cell lines, PC9GR, A549GR and HCC827GR

Given that, as previously demonstrated, the GR cell lines, PC9GR, A549GR and HCC827GR, exhibit an enhanced viability upon gefitinib treatment (18-20) and in this study, were found to proliferate more rapidly than their parental cells (Fig. 1A), an MTT assay was performed on these GR NSCLC cells, as well as on their parental cells PC9, A549 and HCC827 and the normal bronchus cell line, BEAS-2B, to examine the effects of ECC on cell viability. The effects of berberine (Data S1), a known alkaloid extracted from CC, on cell viability and cytotoxicity was examined using an MTT assay in order to compare its effects to those of ECC, which is a multi-compound formulation. ECC suppressed the viability of the GR cells more effectively than that of the parental cells and exerted minimal cytotoxic effects on the BEAS-2B cells (Fig. 1B and Table I). However, berberine was less toxic to the PC9GR cells than the PC9 cells, and exerted significant cytotoxic effects on the BEAS-2B cells; moreover, the BEAS-2B cells were even more sensitive to berberine than the HCC827 lung cancer cells (Fig. S2). The effects of ECC on the migratory and the invasive potential of the PC9GR, A549GR and HCC827GR cells were examined using in vitro migration and invasion assays. Treatment with ECC inhibited the migration and invasion of the GR cells in a dose- and time-dependent manner (Fig. 2); however, a limitation of the present study should be stated here in that 10% FBS may have affected cell proliferation. The invasion assay could not be performed for the A549GR cells, as the cells do not attach effectively on Matrigel. Collectively, these results revealed that ECC exerted anticancer effects on the GR cells.

Table I

IC50 values of ECC, gefitinib and berberine in GR and parental cells.

Table I

IC50 values of ECC, gefitinib and berberine in GR and parental cells.

TreatmentCells lines and IC50 values
PC9PC9GRA549A549GRHCC827HCC827GRKUBEAS-2B
Gefitinib (µM)0.0088.7915.3418.650.0110N/A
ECC (µg/ml)69.8032.73302085.3319.07178.08
Berberine (µM)2.817.7313.99<148.113.333.01

[i] ECC, extract of Coptis chinensis; GR, gefitinib-resistant.

ECC induces the apoptosis of GR NSCLC cells (PC9GR, and A549GR cells)

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 (Fig. 3A-C). Apoptosis induced by ECC was confirmed by TUNEL assay, which revealed an increase in the number of TUNEL-positive cells upon ECC treatment (Fig. 3D). Thus, these data indicated that the anticancer effects of ECC on GR cells resulted from the induction of cell cycle arrest and cell death.

ECC suppresses the EGFR-AKT pathway and the expression of the anti-apoptotic proteins, Mcl-1 and Bcl-2

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 (Figs. 4A and B, and S3A). As ECC exerted anti-survival and pro-apoptotic effects, the effects of ECC on the expression of AKT/p-AKT, Mcl-1 and Bcl-2 were then examined. ECC treatment resulted in the suppression of the expression of these molecules (Figs. 4C and S3B). The effects of ECC on the expression of caspase-3 and PARP, which act as Mcl-1/Bcl-2 downstream effectors in the apoptotic pathway in GR cells were then further examined. The expression of Mcl-1 and Bcl-2 was decreased, and the cleaved forms of caspase-3 and PARP were increased in a time- and dose-dependent manner (Fig. 4C). The suppression of the expression of Mcl-1 and Bcl-2 by ECC was confirmed by RT-qPCR (Figs. 4D and S3C).

ECC synergistically enhances the activity of gefitinib in GR NSCLC cells in vitro

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 (Fig. 5A). To elucidate the mechanisms through which ECC restores the antitumor activities of EGFR-TKIs, the activities of EGFR and its downstream molecule, AKT, as well as the anti-apoptotic proteins Bcl-2 and Mcl-1, were examined in GR cells. As expected, combination treatment resulted in the most effective inhibitory effects (Fig. 5B). It should be noted that in the PC9GR cells, combination treatment only decreased Bcl-2 expression at 48 h, not at 24 h (Fig. 5B). The reasons for this are not clear. In addition, whether the combination treatment was able to enhance the inhibitory effects gefitinib on cell viability through a synergistic effect was examined. This was determined by the Fa-CI plot (Chu-Talalay Plot; www.combosyn.com) median effect analysis, which revealed that the combination index (CI) was smaller than 1 (Fig. 5C) (21), indicating synergistic growth inhibition of the GR cells by this treatment combination.

Cancer cell-specific/sensitive toxicity of ECC in vivo

The suppression of tumorigenicity in vivo by ECC treatment in lung cancer cells was examined in xenograft zebrafish models. CM-Dil-labeled HCC827 or HCC827GRKU cells (red) were grafted into Tg(flk1:EGFP) zebrafish embryos and either DMSO, 0.5 µM gefitinib, 0.2 µl/ml ECC, or the combination of gefitinib and ECC were added to the embryo culture water, and refreshed every 24 h for 5 days (Fig. 6A). The anticancer effects of ECC were confirmed in both the HCC827 and HCC827GRKU cells. This result was consistent with the in vitro results, in which the HCC827GRKU cells were more sensitive than the HCC827 cells to ECC (Fig. 6B). In addition, the gefitinib-, ECC-, and the gefitinib and ECC combination-treated embryos were compared and found to have significantly fewer cancer cells than the control group; however, no significant differences were observed between the treatment groups (Fig. 6C). Unexpectedly, the treatment of zebrafish with ECC alone or in combination with gefitinib resulted in the significantly increased survival of the zebrafish compared to treatment with gefitinib alone (Fig. 6D). This result indicated that ECC had a more specific toxicity against cancer cells than normal cells, consistent with the in vitro results.

Discussion

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 (22,23). The anti-apoptotic proteins, Mcl-1 and Bcl-2, play important roles in the maintenance of cell viability and survival, but not proliferation, through the interaction of several other regulators of apoptosis. The overexpression of Mcl-1 and/or Bcl-2 has been shown to facilitate chemoresistance in various types of cancer (24-26), and has been suggested as a therapeutic target. Therefore, efforts have been made to develop drugs targeting Mcl-1 and Bcl-2 to induce chemosensitization and overcome chemoresistance (27-29). Mcl-1 has been suggested to be a critical survival factor in lung cancer as it promotes cancer cell migration ability and epithelial-mesenchymal transition (11,30-32). A previous study demonstrated that the overexpression of Mcl-1 increased the viability of cancer cells following exposure to cytotoxic chemotherapeutic agents and EGFR-TKIs (30).

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 (26,30,33). This highlights the role of Mcl-1 and Bcl-2 in EGFR-TKI resistance, and indicates that combination treatment comprising EGFR-TKI and Mcl-1 and/or Bcl-2 may exert synergistic effects.

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 (34,35), which results in a variety of resistance mechanisms to EGFR-TKIs. In the present study, cell viability, migration and invasion assays were thus performed to investigate the physiological anticancer effects of ECC in GR cells. ECC effectively inhibited GR cell viability, migration and invasion. Simultaneously, ECC suppressed EGFR signaling through AKT, regardless of the EGFR mutation status, resulting in the inhibition of GR cell survival. However, ECC exerted minimal toxicity on the normal bronchial cell line, BEAS-2B, unlike the alkaloid extract, berberine; this was confirmed in the in vivo model. The dose of ECC in the in vivo model was selected, such that it did not pathophysiologically affect survival or induce damage in the zebrafish themselves, but only affected the cancer cells. Therefore, even if the anticancer effect of ECC was not greater than that of gefitinib alone or the combination treatment in vivo, the increased survival of zebrafish indicated the specific toxicity of ECC against cancer cells, but not against normal cells, which is crucial for the development of novel anticancer drugs. Cell cycle and TUNEL assays revealed that ECC induced the apoptosis of GR cells through the suppression of Mcl-1, Bcl-2 and Bcl-xl, and the promotion of the expression of cleaved caspase-3 and PARP. As ECC suppressed anti-apoptotic and EGFR/AKT signaling, its synergistic effects with the EGFR-TKI, gefitinib, in GR cells to overcome EGFR-TKI resistance, were evaluated in cells treated with both gefitinib and ECC, and the CI was calculated. The results revealed that ECC treatment re-sensitized the GR cells to gefitinib synergistically through the inhibition of EGFR-AKT signaling and the anti-apoptotic proteins, Bcl-2 and Mcl-1.

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 in vitro and increased the viability of normal cells and survival of zebrafish in vivo. These results indicated the potential role of ECC in the treatment of EGFR-TKI-resistant NSCLCs, particularly in combination with EGFR-TKI therapy, with minimal side-effects.

Supplementary Data

Acknowledgements

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.

Funding

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.

Availability of data and materials

All data generated or analyzed during the study are included in this published article or are available from the corresponding author upon reasonable request.

Authors' contributions

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.

Ethics approval and consent to participate

All animal experimental protocols were approved by the Committee for Ethics of Animal Experimentation of Sookmyung Women's University.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References

1 

Hanahan D and Weinberg RA: Hallmarks of cancer: The next generation. Cell. 144:646–674. 2011. View Article : Google Scholar : PubMed/NCBI

2 

Fernald K and Kurokawa M: Evading apoptosis in cancer. Trends Cell Biol. 23:620–633. 2013. View Article : Google Scholar : PubMed/NCBI

3 

Lin Y, Wang X and Jin H: EGFR-TKI resistance in NSCLC patients: Mechanisms and strategies. Am J Cancer Res. 4:411–435. 2014.PubMed/NCBI

4 

Huang L and Fu L: Mechanisms of resistance to EGFR tyrosine kinase inhibitors. Acta Pharm Sin B. 5:390–401. 2015. View Article : Google Scholar : PubMed/NCBI

5 

Ng KP, Hillmer AM, Chuah CT, Juan WC, Ko TK, Teo AS, Ariyaratne PN, Takahashi N, Sawada K, Fei Y, et al: A common BIM deletion polymorphism mediates intrinsic resistance and inferior responses to tyrosine kinase inhibitors in cancer. Nat Med. 18:521–528. 2012. View Article : Google Scholar : PubMed/NCBI

6 

Zhao M, Zhang Y, Cai W, Li J, Zhou F, Cheng N, Ren R, Zhao C, Li X, Ren S, et al: The Bim deletion polymorphism clinical profile and its relation with tyrosine kinase inhibitor resistance in Chinese patients with non-small cell lung cancer. Cancer. 120:2299–2307. 2014. View Article : Google Scholar : PubMed/NCBI

7 

Wu SG, Liu YN, Yu CJ, Yang PC and Shih JY: Association of BIM deletion polymorphism with intrinsic resistance to EGFR tyrosine kinase inhibitors in patients with lung adenocarcinoma. JAMA Oncol. 2:826–828. 2016. View Article : Google Scholar : PubMed/NCBI

8 

Costa DB, Halmos B, Kumar A, Schumer ST, Huberman MS, Boggon TJ, Tenen DG and Kobayashi S: BIM mediates EGFR tyrosine kinase inhibitor-induced apoptosis in lung cancers with oncogenic EGFR mutations. PLoS Med. 4:1669–1680. 2007. View Article : Google Scholar : PubMed/NCBI

9 

Pandey MK, Prasad S, Tyagi AK, Deb L, Huang J, Karelia DN, Amin SG and Aggarwal BB: Targeting cell survival proteins for cancer cell death. Pharmaceuticals (Basel). 9. pii: E11. 2016, View Article : Google Scholar

10 

Delbridge AR and Strasser A: The BCL-2 protein family, BH3-mimetics and cancer therapy. Cell Death Differ. 22:1071–1080. 2015. View Article : Google Scholar : PubMed/NCBI

11 

Yang TM, Barbone D, Fennell DA and Broaddus VC: Bcl-2 family proteins contribute to apoptotic resistance in lung cancer multicellular spheroids. Am J Respir Cell Mol Biol. 41:14–23. 2009. View Article : Google Scholar :

12 

Martin B, Paesmans M, Berghmans T, Branle F, Ghisdal L, Mascaux C, Meert AP, Steels E, Vallot F, Verdebout JM, et al: Role of Bcl-2 as a prognostic factor for survival in lung cancer: A systematic review of the literature with meta-analysis. Br J Cancer. 89:55–64. 2003. View Article : Google Scholar : PubMed/NCBI

13 

Tang F, Mei W, Tian D and Huang D: An evidence-based perspective of Coptis chinensis (Chinese Goldthread) for cancer patients. Evidence-based anticancer materia medica. Cho WCS: Springer; NY: pp. 111–130. 2011, View Article : Google Scholar

14 

Li J, Ni L, Li B, Wang M, Ding Z, Xiong C and Lu X: Coptis chinensis affects the function of glioma cells through the down-regulation of phosphorylation of STAT by reducing HDAC3. BMC Complement Altern Med. 17:5242017. View Article : Google Scholar

15 

Auyeung K and Ko J: Coptis chinensis inhibits hepatocellular carcinoma cell growth through nonsteroidal anti-inflammatory drug-activated gene activation. Int J Mol Med. 24:571–577. 2009.PubMed/NCBI

16 

Lulu N, Jiangan L, Weixing Z, Zhiyi Z, Hui L, Jianhui T, Haizhou L and Hongli R: Coptis chinensis inhibits growth and metastasis and induces cell apoptosis in non-small cell lung cancer cells. Int J Clin Exp Med. 10:16037–16048. 2017.

17 

Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar

18 

Park SH, Kim JH, Ko E, Kim JY, Park MJ, Kim MJ, Seo H, Li S and Lee JY: Resistance to gefitinib and cross-resistance to irreversible EGFR-TKIs mediated by disruption of the Keap1-Nrf2 pathway in human lung cancer cells. FASEB J. 32:5862–5873. 2018. View Article : Google Scholar

19 

Rho JK, Choi YJ, Lee JK, Ryoo BY, Na II, Yang SH, Kim CH and Lee JC: Epithelial to mesenchymal transition derived from repeated exposure to gefitinib determines the sensitivity to EGFR inhibitors in A549, a non-small cell lung cancer cell line. Lung Cancer. 63:219–226. 2009. View Article : Google Scholar

20 

Rho JK, Choi YJ, Lee JK, Ryoo BY, Na II, Yang SH, Lee SS, Kim CH, Yoo YD and Lee JC: The role of MET activation in determining the sensitivity to epidermal growth factor receptor tyrosine kinase inhibitors. Mol Cancer Res. 7:1736–1743. 2009. View Article : Google Scholar : PubMed/NCBI

21 

Chou TC and Martin N: CompuSyn software for drug combinations and for general dose-effect analysis, and user's guide. ComboSyn, Inc.; Paramus, NJ: 2007

22 

Czabotar PE, Lessene G, Strasser A and Adams JM: Control of apoptosis by the BCL-2 protein family: Implications for physiology and therapy. Nat Rev Mol Cell Biol. 15:49–63. 2014. View Article : Google Scholar

23 

Wertz IE, Kusam S, Lam C, Okamoto T, Sandoval W, Anderson DJ, Helgason E, Ernst JA, Eby M, Liu J, et al: Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7. Nature. 471:110–114. 2011. View Article : Google Scholar : PubMed/NCBI

24 

Wei G, Margolin AA, Haery L, Brown E, Cucolo L, Julian B, Shehata S, Kung AL, Beroukhim R and Golub TR: Chemical genomics identifies small-molecule MCL1 repressors and BCL-xL as a predictor of MCL1 dependency. Cancer Cell. 21:547–562. 2012. View Article : Google Scholar : PubMed/NCBI

25 

Karnak D and Xu L: Chemosensitization of prostate cancer by modulating Bcl-2 family proteins. Curr Drug Targets. 11:699–707. 2010. View Article : Google Scholar : PubMed/NCBI

26 

Wu DW, Chen CY, Chu CL and Lee H: Paxillin confers resistance to tyrosine kinase inhibitors in EGFR-mutant lung cancers via modulating BIM and Mcl-1 protein stability. Oncogene. 35:621–630. 2016. View Article : Google Scholar

27 

Belmar J and Fesik SW: Small molecule Mcl-1 inhibitors for the treatment of cancer. Pharmacol Ther. 145:76–84. 2015. View Article : Google Scholar :

28 

Brumatti G and Ekert PG: Seeking a MCL-1 inhibitor. Cell Death Differ. 20:1440–1441. 2013. View Article : Google Scholar : PubMed/NCBI

29 

Pan R, Ruvolo VR, Wei J, Konopleva M, Reed JC, Pellecchia M, Andreeff M and Ruvolo PP: Inhibition of Mcl-1 with the pan-Bcl-2 family inhibitor (-)BI97D6 overcomes ABT-737 resistance in acute myeloid leukemia. Blood. 126:363–372. 2015. View Article : Google Scholar : PubMed/NCBI

30 

Song L, Coppola D, Livingston S, Cress D and Haura EB: Mcl-1 regulates survival and sensitivity to diverse apoptotic stimuli in human non-small cell lung cancer cells. Cancer Biol Ther. 4:267–276. 2005. View Article : Google Scholar : PubMed/NCBI

31 

Toge M, Yokoyama S, Kato S, Sakurai H, Senda K, Doki Y, Hayakawa Y, Yoshimura N and Saiki I: Critical contribution of MCL-1 in EMT-associated chemo-resistance in A549 non-small cell lung cancer. Int J Oncol. 46:1844–1848. 2015. View Article : Google Scholar : PubMed/NCBI

32 

Zhang H, Guttikonda S, Roberts L, Uziel T, Semizarov D, Elmore SW, Leverson JD and Lam LT: Mcl-1 is critical for survival in a subgroup of non-small-cell lung cancer cell lines. Oncogene. 30:1963–1968. 2011. View Article : Google Scholar

33 

Zhang J, Wang S, Wang L, Wang R, Chen S, Pan B, Sun Y and Chen H: Prognostic value of Bcl-2 expression in patients with non-small-cell lung cancer: A meta-analysis and systemic review. Onco Targets Ther. 8:3361–3369. 2015. View Article : Google Scholar : PubMed/NCBI

34 

Lohinai Z, Hoda MA, Fabian K, Ostoros G, Raso E, Barbai T, Timar J, Kovalszky I, Cserepes M, Rozsas A, et al: Distinct epidemiology and clinical consequence of classic versus rare EGFR mutations in lung adenocarcinoma. J Thorac Oncol. 10:738–746. 2015. View Article : Google Scholar : PubMed/NCBI

35 

Li K, Yang M, Liang N and Li S: Determining EGFR-TKI sensitivity of G719X and other uncommon EGFR mutations in non-small cell lung cancer: Perplexity and solution (Review). Oncol Rep. 37:1347–1358. 2017. View Article : Google Scholar : PubMed/NCBI

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June-2020
Volume 56 Issue 6

Print ISSN: 1019-6439
Online ISSN:1791-2423

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Copy and paste a formatted citation
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Spandidos Publications style
Kim J, Ko E, Kim D, Park SH, Kim EJ, Rho J, Seo H, Kim M, Yang W, Ha I, Ha I, et al: Cancer cell‑specific anticancer effects of Coptis chinensis on gefitinib‑resistant lung cancer cells are mediated through the suppression of Mcl‑1 and Bcl‑2. Int J Oncol 56: 1540-1550, 2020
APA
Kim, J., Ko, E., Kim, D., Park, S., Kim, E., Rho, J. ... Lee, J. (2020). Cancer cell‑specific anticancer effects of Coptis chinensis on gefitinib‑resistant lung cancer cells are mediated through the suppression of Mcl‑1 and Bcl‑2. International Journal of Oncology, 56, 1540-1550. https://doi.org/10.3892/ijo.2020.5025
MLA
Kim, J., Ko, E., Kim, D., Park, S., Kim, E., Rho, J., Seo, H., Kim, M., Yang, W., Ha, I., Park, M., Lee, J."Cancer cell‑specific anticancer effects of Coptis chinensis on gefitinib‑resistant lung cancer cells are mediated through the suppression of Mcl‑1 and Bcl‑2". International Journal of Oncology 56.6 (2020): 1540-1550.
Chicago
Kim, J., Ko, E., Kim, D., Park, S., Kim, E., Rho, J., Seo, H., Kim, M., Yang, W., Ha, I., Park, M., Lee, J."Cancer cell‑specific anticancer effects of Coptis chinensis on gefitinib‑resistant lung cancer cells are mediated through the suppression of Mcl‑1 and Bcl‑2". International Journal of Oncology 56, no. 6 (2020): 1540-1550. https://doi.org/10.3892/ijo.2020.5025