Biomolecules & Therapeutics 2024; 32(1): 104-114  https://doi.org/10.4062/biomolther.2023.167
Licochalcone C Inhibits the Growth of Human Colorectal Cancer HCT116 Cells Resistant to Oxaliplatin
Seung-On Lee1,†, Sang Hoon Joo2,†, Jin-Young Lee3, Ah-Won Kwak4, Ki-Taek Kim1,5, Seung-Sik Cho1,5, Goo Yoon5, Yung Hyun Choi6, Jin Woo Park1,5,* and Jung-Hyun Shim1,5,7,*
1Department of Biomedicine, Health & Life Convergence Sciences, BK21 Four, College of Pharmacy, Mokpo National University, Muan 58554,
2College of Pharmacy, Daegu Catholic University, Gyeongsan 38430,
3Department of Biological Sciences, Keimyung University, Daegu 42601,
4Biosystem Research Group, Department of Predictive Toxicology, Korea Institute of Toxicology, Daejeon 34114,
5Department of Pharmacy, College of Pharmacy, Mokpo National University, Muan 58554,
6Department of Biochemistry, College of Korean Medicine, Dong-Eui University, Busan 47227, Republic of Korea
7The China-US (Henan) Hormel Cancer Institute, Zhengzhou, Henan 450008, China
*E-mail: s1004jh@gmail.com (Shim JH), jwpark@mokpo.ac.kr (Park JW)
Tel: +82-61-450-2684 (Shim JH), +82-61-450-2704 (Park JW)
Fax: +82-61-450-2689 (Shim JH), +82-61-450-2689 (Park JW)
The first two authors contributed equally to this work.
Received: September 19, 2023; Revised: October 12, 2023; Accepted: October 19, 2023; Published online: January 1, 2024.
© The Korean Society of Applied Pharmacology. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Licochalcone C (LCC; PubChem CID:9840805), a chalcone compound originating from the root of Glycyrrhiza inflata, has shown anticancer activity against skin cancer, esophageal squamous cell carcinoma, and oral squamous cell carcinoma. However, the therapeutic potential of LCC in treating colorectal cancer (CRC) and its underlying molecular mechanisms remain unclear. Chemotherapy for CRC is challenging because of the development of drug resistance. In this study, we examined the antiproliferative activity of LCC in human colorectal carcinoma HCT116 cells, oxaliplatin (Ox) sensitive and Ox-resistant HCT116 cells (HCT116-OxR). LCC significantly and selectively inhibited the growth of HCT116 and HCT116-OxR cells. An in vitro kinase assay showed that LCC inhibited the kinase activities of EGFR and AKT. Molecular docking simulations using AutoDock Vina indicated that LCC could be in ATP-binding pockets. Decreased phosphorylation of EGFR and AKT was observed in the LCC-treated cells. In addition, LCC induced cell cycle arrest by modulating the expression of cell cycle regulators p21, p27, cyclin B1, and cdc2. LCC treatment induced ROS generation in CRC cells, and the ROS induction was accompanied by the phosphorylation of JNK and p38 kinases. Moreover, LCC dysregulated mitochondrial membrane potential (MMP), and the disruption of MMP resulted in the release of cytochrome c into the cytoplasm and activation of caspases to execute apoptosis. Overall, LCC showed anticancer activity against both Ox-sensitive and Ox-resistant CRC cells by targeting EGFR and AKT, inducing ROS generation and disrupting MMP. Thus, LCC may be potential therapeutic agents for the treatment of Ox-resistant CRC cells.
Keywords: Licochalcone C (LCC), Oxaliplatin, Colorectal cancer, EGFR, AKT, Apoptosis
INTRODUCTION

Colorectal cancer (CRC) is a significant threat to global health owing to its frequent diagnosis and high mortality rates (Morgan et al., 2023; Siegel et al., 2023). In the USA, the incidence of CRC nearly doubled from 11% to 20% between 1995 and 2019, and by 2023, CRC is expected to be the cause of more than fifty thousand deaths. CRC can be treated with chemotherapy before or after surgery using 5-fluorouracil (5-FU), oxaliplatin (Ox), irinotecan, and other chemotherapeutics in combination with regimens such as FOLFOX, FOLFIRI, and FOLFRINOX (Tharin et al., 2021). Nonetheless, acquired resistance to these chemotherapies hampers CRC treatment and new strategies are needed to deal with resistance (Martinez-Balibrea et al., 2015). In addition to chemotherapy, targeted therapies targeting molecular targets can also be used to treat CRC. For example, anti-EGFR antibodies, such as cetuximab and panitumumab, target EGFR, a transmembrane receptor tyrosine kinase that can be used to treat CRC (De Mattia et al., 2015). EGFR activation leads to the activation of downstream signaling pathways, including AKT and ERK, and promotes cell proliferation and survival (Lin et al., 2019a). Targeting EGFR and downstream signaling pathways may be a good strategy to overcome resistance to chemotherapeutic agents, and combining oxaliplatin with anti-EGFR antibodies has shown improved outcomes in some patient groups (Folprecht et al., 2022). Glycyrrhiza inflata, commonly known as Chinese licorice, has been used for a wide range of purposes since ancient times owing to its antimicrobial and anti-inflammatory properties (Chiu et al., 2018; van Dinteren et al., 2022; Wang et al., 2022). Several compounds such as glycyrrhizin, chalcones, and flavanones have been isolated from licorice (Tanemoto et al., 2015). Among these, licochalcone C (LCC) has shown biological activities, including anti-inflammatory activity (Franceschelli et al., 2011), antiviral activity (Dao et al., 2011), and cardioprotective (Zhou et al., 2015) and cytotoxic effects in bladder cancer cells (Wang et al., 2015). Previously, we reported the anticancer effects of LCC on esophageal and oral squamous carcinoma cells (Oh et al., 2018; Kwak et al., 2020). The apoptotic effect of LCC involves regulation of the JAK2/STAT3 signaling pathway and activation of the ROS/MAPK signaling pathway. The anticancer activity of LCC against CRC has not been evaluated yet. Moreover, the direct molecular targets of LCC have not been elucidated. In the present study, we focused on the anticancer activity of LCC against CRC HCT116 cells. We established that LCC could induce apoptosis in human CRC cells and modulate the kinase activity of EGFR and AKT.

MATERIALS AND METHODS

Materials

LCC was purified from powdered Glycyrrhiza inflata as previously described (Oh et al., 2018). Cell culture medias RPMI-1640, Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium (MEM), and other supplements, including sodium pyruvate, MEM vitamin solution, and fetal bovine serum (FBS), were purchased from Invitrogen (Karlsruhe, Germany). The MEM non-essential amino acid solution was obtained from Corning (NY, USA). RIPA buffer, phosphate-buffered saline (PBS), and Tris-glycine-sodium dodecyl sulfate buffer were purchased from BioSolution (Seoul, Korea). Penicillin/streptomycin and trypsin were purchased from HyClone (Logan, UT, USA). The Basal Medium Eagle, 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), dimethyl sulfoxide (DMSO), N-acetyl-L-cysteine (NAC), and N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-FMK; pan caspase inhibitor) were obtained from Sigma-Aldrich (St. Louis, USA). Primary antibodies used to detect actin, apoptotic protease activating factor-1 (Apaf-1), Bax, Bcl-2, Bcl-xL, Bid, caspase-3, cleaved poly (ADP-ribose) polymerase (cPARP), cyclin D1, cytochrome c (cyto c), cytochrome c oxidase subunit 4 (COX4), Mcl-1, phosphorylated EGFR (pEGFR), EGFR, phosphorylated AKT, AKT, phosphorylated p38 (pp38), p21, phosphorylated JNK (pJNK), JNK, cdc2, p27, and α-tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against Bim were obtained from Cell Signaling Technology (Danvers, MA, USA).

Cell culture

The human CRC cell line HCT116, human keratinocyte HaCaT, and mouse epidermal cell line JB6 were purchased from American Type Culture Collection (Manassas, VA, USA), and the Ox-resistant HCT116 cell line (HCT116-OxR) was obtained from the University of Texas MD Anderson Cancer Center (Bose et al., 2011). HCT116 and HaCaT cells were maintained in complete RPMI-1640 and DMEM supplemented with 10% FBS and 1% penicillin/streptomycin, respectively. HCT116-OxR cells were cultured in MEM supplemented with 10% FBS, 1% penicillin/streptomycin, 1% sodium pyruvate, 1% MEM non-essential amino acid solution, 1% MEM vitamin solution, and 2 μM Ox. JB6 cells were cultured in MEM supplemented with 5% FBS and 1% penicillin/streptomycin. All cells were incubated in a CO2 incubator under humidified air (5% CO2) at 37°C.

MTT cell viability assay

To assess the cytotoxicity of LCC, we performed MTT cell viability assay. HCT116 (5.0×103 cells per well), HCT116-OxR (4.0×103 cells per well), HaCaT (8.0×103 cells per well), and JB6 (5.5×103 cells per well) cells were seeded in 96-well culture plates and incubated for 24 h. Following this, the cells were treated with 0, 5, 10, and 20 μM LCC dissolved in 0.1% DMSO for 24 or 48 h. To determine the cell viability, 30 μL of MTT solution (5 mg/mL) was added to each well and the cells were incubated at 37°C for 1 h. The medium was removed after incubation, and formazan crystals were dissolved in 100 μL of DMSO. The absorbance of each well was measured at 570 nm using a microplate spectrophotometer (Thermo Fisher Scientific, Vantaa, Finland).

Soft agar assay

To evaluate anchorage-independent growth, a soft agar assay was performed. For the bottom agar layer, 1 mL of Basal Medium Eagle containing 0.6% agar, 10% FBS, 2 mM L-glutamine, 5 μg/mL gentamicin, and various concentrations of drugs dissolved in DMSO were poured into each well of the six-well plate. Once the bottom layer was solidified, 1 mL of cells (8.0×103 cells per well) suspended in the culture media containing 0.3% agar and drugs were poured into the top agar layer. Colony formation was assessed using a light microscope (Leica, Wetzlar, Germany) and photographs were captured. Colonies >50 μm in diameter were counted.

Annexin V/7-aminoactinomycin D (7-AAD) double staining assay

To detect apoptosis induced by LCC, the Muse™ Annexin V & Dead Cell Kit from EMD Millipore (Billerica, MA, USA) was used, following manufacturer’s instructions. Briefly, the cells were treated with various concentrations of LCC (0, 5, 10, and 20 μM dissolved in DMSO) for 48 h, harvested, washed with 1×PBS, and stained with Muse Annexin V & Dead Cell Reagent. The fluorescence intensity was measured via flow cytometry using a Muse™ Cell Analyzer (EMD Millipore). The cells were classified as live, early apoptotic, late apoptotic, or necrotic cells. The total number of apoptotic cells was determined by adding the early and late apoptotic cells.

Cell cycle analysis

To analyze the cell cycle distribution after treatment with LCC, the Muse™ cell cycle reagent (EMD Millipore) was used for flow cytometry. Briefly, cells treated with various concentrations of LCC were fixed with 70% ethanol and incubated at -20°C overnight. The fixed cells were washed with PBS and resuspended in Muse™ Cell Cycle Reagent. The DNA content was assessed by measuring the fluorescence intensity using a Muse™ Cell Analyzer.

Intracellular ROS detection

The generation of intracellular ROS was monitored using a Muse™ Oxidative Stress Kit (EMD Millipore) following manufacturer’s protocol. In short, cells treated with LCC were washed with PBS and stained with Muse® Oxidative Stress Reagent working solution at 37°C for 30 min in the dark. ROS levels were assessed by measuring fluorescence using a Muse™ Cell Analyzer.

Next, we measured the mitochondrial membrane potential (MMP, Δψm). To evaluate the changes in MMP in cells treated with LCC, a Muse™ MitoPotential Kit (EMD Millipore) was used. Briefly, cells treated with LCC were harvested, washed with 1×assay buffer, and resuspended in Muse™ MitoPotential working solution at 37°C in the dark for 20 min. The Muse™ MitoPotential 7-AAD reagent was then added and incubated at room temperature (RT) for 5 min. Changes in MMP were determined via flow cytometry.

Multi-caspase assay

To determine the activation of multiple caspases, including caspase-1, -3, -4, -5, -6, -7, -8, and -9, the Muse™ Multi-caspase Kit (EMD Millipore) was used according to manufacturer’s instructions. Briefly, cells treated with LCC were harvested, washed with 1×caspase buffer, and resuspended in Muse™ Multi-caspase Reagent working solution at 37°C for 30 min. Subsequently, Muse™ Caspase 7-AAD working solution was added and incubated at RT for 5 min. The multicaspase activity of each sample was determined using flow cytometry.

Western blotting

Cells treated with LCC were lysed using RIPA buffer, equal amounts of protein samples (20-40 µg/lane) were separated on 8-15% SDS-polyacrylamide gels, and the resolved proteins were transferred to polyvinylidene difluoride membranes (EMD Millipore). The membranes were blocked with 5% skim milk in PBS containing 1% Tween-20 (PBST) at RT for 2 h and incubated with specific primary antibodies. The primary antibodies were diluted at 1:1,000 in 1×PBST and incubated at RT at 4°C either for 2 h or overnight. Next, the membranes were washed three times with PBST and incubated with secondary antibodies conjugated with horseradish peroxidase. Western blots were visualized using western blotting luminol reagent (Santa Cruz Biotechnology) and detected using ImageQuant LAS 500 (GE Healthcare, Uppsala, Sweden). ImageJ (Schneider et al., 2012) was used to quantify the protein levels.

Kinase assay

To assess if LCC inhibits the kinases EGFR, AKT1, and AKT2, we conducted in vitro kinase assays based on ADP-Glo™ kinase Assay Kit (Promega, Madison, WI, USA). Briefly, active kinases were resuspended in a 384-well plate; mixed with various concentrations of LCC (0, 5, 10, 15, and 20 μM), 0.2 μg/μL of substrates, 5 μM of ATP, and kinase reaction buffer containing 0.1 mg/mL BSA, 50 μM DTT, 20 mM MgCl2, 2 mM MnCl2, 100 μM sodium vanadate, and 40 mM Tris (PH 7.5) for 1 h; and incubated at RT. Gefitinib (1 μM) or MK-2206 (65 nM) was used as control. After kinase reaction, 5 μL of ADP-Glo™ Reagent was added to each well and incubated at RT for 40 min. Next, 10 μL of kinase detection reagent was added to each well for luminescence detection with a Centro LB 960 microplate luminometer (Berthold Technologies, Dettenheim, Germany) for 0.5 s.

Molecular modelling

To predict the binding modes between LCC and the kinases EGFR, AKT1, and AKT2, we conducted molecular docking simulations. To prepare for docking, the protein structure files 1M17, 6CCY, and 3D0E were downloaded from the Protein Data Bank for EGFR, AKT1, and AKT2, respectively. The search grids were set to 60° in each axis for unbiased search results, and AutoDock Vina was used as previously described (Trott and Olson, 2010). Structural depictions were prepared using the best modes reported by AutoDock Vina.

Statistical analysis

Data are expressed as mean ± standard deviation (SD). Each experiment was conducted in triplicates. To conduct multiple comparisons, we used Prism 5.0 by GraphPad software (Boston, MA, USA) and one-way or two-way analysis of variance (ANOVA). Statistical significance was determined by p-values less than 0.05, 0.01, and 0.001, which are denoted by asterisks.

RESULTS

LCC inhibits the proliferation of both Ox-sensitive and -resistant CRC cells

We determined whether LCC had an antiproliferative effect on CRC cells. The MTT cell viability assay showed that the viability of both Ox-sensitive and Ox-resistant HCT116 cells in concentration- (0 to 20 μM) and time- (24 h and 48 h) dependent manners (Fig. 1A). LCC inhibited the growth of HCT116 human CRC cells with IC50 values 16.6 μM for HCT116 and 19.6 μM for HCT116 cells resistant to Ox. In contrast, LCC did not show noticeable cytotoxicity to HaCaT and JB6 cells at 20 μM, implying that the cytotoxicity of LCC is more or less selective to cancer cells. The soft agar assay to evaluate colony formation showed that LCC inhibited colony formation, and both colony counts and sizes decreased following LCC treatment in a concentration-dependent manner (Fig. 1B, 1C).

Figure 1. Antiproliferative effect of LCC in CRC cells. (A) Cell viability determined via MTT assay. HCT116, HCT116-OxR, HaCaT, and JB6 cells treated with LCC (5, 10, and 20 μM) or Ox (2 μM) for 24 and 48 h were compared to untreated cells. (B) Soft agar assay to determine the anchorage-independent colony growth. HCT116 and HCT116-OxR cells treated with LCC or Ox were incubated for 14 days before colony formation analysis under microscope. Histogram of colony number. All data are expressed as mean ± SD. *p<0.05, **p<0.01, and ***p<0.001 vs. control.

LCC inhibits the kinase activity of EGFR, AKT1, and AKT2

To determine whether LCC can inhibit kinase activity, we performed in vitro kinase assays for EGFR, ATK1, and AKT2. We observed that the enzymatic activities of all three kinases were inhibited by the presence of LCC in a dose-dependent manner (0, 5, 10, and 20 μM; Fig. 2A, 2B). Molecular modeling based on the AutoDock Vina simulation (Trott and Olson, 2010) indicated that LCC could be located in the ATP-binding pockets of these three kinases (Fig. 2C). In the interaction between EGFR and LCC, the backbone amino group of Gly772 and side-chain amino group of Lys721 formed hydrogen bonds. Furthermore, several aliphatic residues of EGFR, such as Leu156 and Leu181, were in proximity to possible hydrophobic interactions. Similarly, we mapped residues such as Glu191 in AKT1 (Glu193 in AKT2) and Phe161 in AKT1 (Phe163 in AKT2) for possible hydrogen bonding and hydrophobic interactions between LCC and AKT1 or AKT2.

Figure 2. LCC inhibits the kinase activity of EGR and AKT. (A) In vitro kinase assay for EGFR was performed in the presence of LCC (0, 5, 10, 15, and 20 μM). Gefitinib (GEF) at 1 μM was used as a positive control. (B) In vitro kinase assays for ATK1 and AKT2. MK2206 (65 nM) was used as the positive control. *p<0.05, **p<0.01, and ***p<0.001 vs. control. (C) Docking simulation of LCC located in the ATP-binding pockets of EGFR, AKT1, and AKT2. Top. Surface representation of each kinase with LCC is shown as pink spheres. Bottom. Zoomed-in image of ATP-binding pockets. Amino acids within 4 Å of LCC are displayed as sticks. Those amino acids with possible hydrophobic interactions are circled.

LCC decreases the phosphorylation of EGFR and AKT

We performed western blot analysis to determine whether the phosphorylation level was modulated by LCC treatment. EGFR and AKT levels remained relatively unchanged. However, we observed a decrease in the levels of phosphorylation of both EGFR (Thr1068) and AKT (Ser473 for AKT1 and Ser474 for AKT2) with increasing LCC concentrations (Fig. 3A, 3B).

Figure 3. LCC induces the decrease in the phosphorylation of EGFR and AKT kinases. (A) Cells treated with LCC (0, 5, 10, and 20 μM) for 48 h were analyzed via western blotting for the level of proteins pEGFR (Tyr1068), EGFR, pAKT (Ser473), and AKT. Actin was used as the control. (B) Histogram of the ratio of phosphorylated to total EGFR and AKT proteins. *p<0.05, **p<0.01, and ***p<0.001 vs. control.

LCC induces cell cycle arrest in HCT116 cells

Flow cytometry was performed to analyze cell cycle distribution in HCT116 cells treated with LCC. The treatment of cells with LCC (5, 10, and 20 μM) increased the subG1 population from 4.23 ± 0.06 to 4.97 ± 0.35, 7.73 ± 0.49, and 49.20 ± 2.03% in HCT116 cells and from 4.53 ± 0.40% to 6.80 ± 0.17, 10.27 ± 0.38, and 43.63 ± 0.21% in Ox-resistant HCT116 cells (Fig. 4A, 4B). The levels of proteins involved in G2/M phase regulation, including cyclin B1, cdc2, p21, and p27, were analyzed by western blotting. As shown in Fig. 4C, LCC reduced the protein levels of cyclin B1 and cdc2 in HCT116 cells in a dose-dependent manner and increased the levels of both p21 and p27 compared with those of the controls. These results suggested that LCC induced G2/M cell cycle arrest and exerted an antiproliferative effect in HCT116 cells.

Figure 4. LCC induces cell cycle arrest in CRC cells. (A) Cells treated with LCC (0, 5, 10, and 20 μM) for 48 h were analyzed with a Muse® Cell Analyzer to determine cell cycle distributions. (B) Histogram of the sub-G1 population. *p<0.05 and ***p<0.001 vs. control. (C) Cell lysates were analyzed via western blotting for the levels of proteins related to the cell cycle: p21, p27, cyclin B1, and cdc2. Actin was used as the control.

LCC increases ROS generation and activates JNK and p38 signaling pathway

Next, we examined whether LCC induces an increase in ROS generation in CRC cells using a cell analyzer system. The ROS level was increased from 6.88% ± 0.40 to 21.32 ± 0.56, 29.83 ± 1.09, and 46.00 ± 0.63% in HCT116 cells and from 12.88 ± 1.35% to 14.72 ± 0.67, 26.54 ± 1.14, and 44.39 ± 0.42% in Ox-resistant HCT116 cells following LCC treatment (5, 10, and 20 μM) (Fig. 5A, 5B). We then conducted western blot analysis to determine whether the increase in ROS generation affected the protein level and phosphorylation of proteins involved in the MAPK signaling pathway (Fig. 5C, 5D). We observed an increase in phosphorylation induced by LCC treatment whereas the levels of JNK and p38 were unaffected. To verify that the induction of ROS mediates the anticancer activity of LCC, we pretreated HCT116 cells with the ROS scavenger, NAC, and measured cell viability. Pretreatment with NAC slightly rescued HCT116 cells treated with LCC (Fig. 5E). The viability of HCT116 cells decreased by 67.22% after treatment with LCC whereas it decreased by 17.49% in cells pretreated with NAC. Pretreatment also decreased the phosphorylation of JNK and p38 (Fig. 5F), suggesting that ROS induction leads to the phosphorylation of JNK and p38. In addition to the activation of MAPK signaling, we observed activation of caspase-3 following treatment with LCC. Taken together, these results suggest that the cytotoxicity of LCC in HCT116 cells is mediated by ROS induction.

Figure 5. LCC induces ROS generation in CRC cells. (A) Cells treated with LCC (0, 5, 10, and 20 μM) for 48 h were analyzed using a Muse Cell Analyzer to determine ROS generation. (B) Histogram of cells with high ROS levels. ***p<0.001 vs. control. (C-D) Cell lysates were analyzed via western blotting to determine the levels of pJNK (Thr183/Tyr185), JNK, pp38 (Thr180/Tyr182), and p38. Actin was used as the control. *p<0.05, **p<0.01, and ***p<0.001 vs. control. (E) Cell viability was measured in LCC-treated CRC cells with or without NAC pretreatment. **p<0.01 and ***p<0.001 vs. control. ###p<0.001 compared to cells treated with LCC alone (20 μM). (F) Western blotting of pJNK, pp38, and caspase-3 in cell lysates after treatment of HCT116 and HCT116-OxR cells with or without NAC pretreatment. Actin was used as the control.

LCC disrupts MMP in CRC cells

To determine whether LCC modulates mitochondrial function in HCT116 cells, we measured the MMP using the MitoPotential Kit (EMD Millipore) in HCT116 and HCT116-OxR cells treated with LCC. LCC induced depolarization of the mitochondrial membrane compared to the controls (Fig. 6A, 6B). LCC treatment at 5, 10, and 20 μM increased the ratio depolarized cells to 9.23 ± 1.03, 19.05 ± 1.12, and 55.35 ± 1.23% from 4.95 ± 0.21% in HCT116 cells, and to 13.18 ± 1.52, 23.96 ± 2.07, 42.57 ± 0.26% from 4.51 ± 0.51% in HCT116-OxR cells, respectively. After measuring MMPs, we monitored the levels of proteins involved in mitochondrial apoptosis (Bim, Mcl-1, Bid, Bax, Bcl-xL, and Bcl-2) (Fig. 6C, left). The levels of pro-apoptotic proteins Bim and Bax increased whereas those of anti-apoptotic proteins Mcl-1, Bid, Bcl-xL, and Bcl-2 decreased with LCC treatment in a dose-dependent manner. To determine whether the shift in balance between anti-apoptotic and pro-apoptotic proteins results in the initiation of apoptosis, we performed western blot analysis. We observed the release of cyto c from the mitochondria into the cytoplasm and an increase in Apaf-1 and cleaved PARP proteins whereas full-length caspase-3 decreased with LCC treatment (Fig. 6C, right). Flow cytometry analysis after double staining with Annexin V/7-AAD indicated that LCC treatment (5, 10, and 20 μM) increased the ratio of cell populations undergoing apoptosis from 6.41 ± 0.31% to 11.92 ± 1.51, 26.23 ± 0.52, and 37.38 ± 0.55% in HCT116 cells and from 5.28 ± 0.38% to 19.75 ± 0.48, 32.42 ± 1.09, and 41.02 ± 0.64% in HCT116-OxR cells (Fig. 6D, 6E). Taken together, LCC induced apoptosis in CRC cells by disrupting MMP.

Figure 6. LCC induce mitochondrial membrane depolarization in CRC cells. (A) Cells treated with LCC (0, 5, 10, and 20 μM) for 48 h were analyzed using a Muse® Cell Analyzer to measure MMP. (B) Histogram of the ratio of total depolarized cells. *p<0.05, **p<0.01, and ***p<0.001 vs. control. (C) Cell lysates were analyzed via western blotting for the levels of Bim, Mcl-1, Bid, Bax, Bcl-xL, Bcl-2, cyto c, α-tubulin, COX4, Apaf-1, cPARP, and caspase-3. Actin was used as the control. (D, E) Cells treated with LCC (0, 5, 10, and 20 μM) for 48 h were analyzed using a Muse Cell Analyzer after Annexin V/7-AAD double staining to determine the ratio of cells undergoing apoptosis. **p<0.01 and ***p<0.001 vs. control.

LCC activates multi-caspase to induce the apoptosis in CRC cells

We measured caspase activity in HCT116 and HCT116-OxR cells treated with LCC (Fig. 7A). The ratio of cell populations with multi-caspase activity increased by LCC treatment (5, 10, and 20 μM) from 5.42 ± 0.26% to 10.00 ± 0.23, 15.93 ± 0.71, and 43.22 ± 0.67% in HCT116 cells and from 4.30 ± 0.18% to 9.30 ± 0.35, 12.78 ± 0.50, and 43.03 ± 1.16% in HCT116-OxR cells, respectively (Fig. 7B). Furthermore, pretreatment of CRC cells with Z-VAD-FMK, a pan-caspase inhibitor, at 4 μM prevented LCC-induced apoptosis compared to LCC-only treatment at 20 M (Fig. 7C). These results suggested that LCC induced apoptosis in CRC cells by activating caspases.

Figure 7. LCC induces the activation of multi-caspases. (A, B) Cells treated with LCC (0, 5, 10, and 20 μM) for 48 h were analyzed with a Muse® Cell Analyzer to determine the activity of multi-caspases. Histogram showing the ratio of cells with multicaspase activation. **p<0.01 and ***p<0.001 vs. control. (C) Viability of LCC-treated CRC cells with or without Z-VAD-FMK pretreatment (4 μM). ***p<0.001 vs. control. ###p<0.001 compared to cells treated with LCC alone (20 μM).
DISCUSSION

LCC is a phenolic compound isolated from Chinese licorice, which has been used as a sweetener in traditional medicine. The reported biological activities of LCC imply that they are relatively safe. It has anti-inflammatory (Franceschelli et al., 2011), antiviral activity (Dao et al., 2011), and cardioprotective properties (Zhou et al., 2015). Encouraged by previous studies indicating the anticancer activity of LCC in various cancer cells (Wang et al., 2015; Oh et al., 2018; Kwak et al., 2020), we investigated whether LCC exhibited anticancer activity in CRC. CRC is a notorious cancer with a high mortality rate throughout the world. While the overall incidence is decreasing with the help of endoscopy-based screening, the incidence in younger generations is increasing, probably due to risk factors such as sedentary lifestyles and high consumption of red meat and processed meat. First-line chemotherapeutic regimens for treating CRC are well established, and the FOLFOX, FOLFIRI, and FOLFRINOX regimens are well-known. These regimens utilize a combination of several therapeutics such as 5-fluorouracil (5-FU), oxaliplatin (Ox), and irinotecan. Ox, a platinum-based antineoplastic agent, exerts its anticancer activity by forming platinum-DNA adducts, thereby damaging the DNA of cancer cells. It is a third-generation platinum-based drug that overcomes the shortcomings of cisplatin and carboplatin, and combination therapy with other chemotherapeutics has significantly improved the treatment significantly. However, prolonged administration of Ox-based combination therapy leads to the development of resistance in CRC cells, and combination therapeutic regimens have become less effective. Therefore, it is necessary to find therapeutics that work not only on CRC cells but also on CRC cells resistant to Ox. Our initial results indicated that LCC is cytotoxic with selectivity to CRC cells but does not inhibit the growth of mouse epidermal cells JB6. Cytotoxicity was observed in both HCT116 and HCT116-OxR cells (Fig. 1). There may be several molecular targets for the treatment of Ox-resistant CRC cells. For example, targeting the DNA repair enzyme PARP may sensitize cancer cells to Ox because Ox resistance can develop through enhanced DNA repair mechanisms. Olaparib, a PARP inhibitor, in combination with Ox, suppressed Ox-resistant gastric cancer organoids (Li et al., 2021). Inhibition of drug efflux can be another strategy to overcome Ox resistance, allowing Ox to remain in the cancer cell longer. Modulation of the drug efflux protein P-gp resulted in the reversal of Ox resistance in HCT116 cells (Su et al., 2021). Among several molecular targets, EGFR may be a good target for the treatment of Ox-resistant cancer cells (Balin-Gauthier et al., 2006). It has been reported that the activation or overexpression of EGFR is observed in Ox-resistant cancer cells with concurrent activation of downstream kinases such as AKT (Ekblad and Johnsson, 2012). In addition to the EGFR level, the activation of EGFR by EGF-EGFR binding is a key interaction that induces the growth of cancer cells. This process is involved in conformational changes and autophosphorylation of EGFR (Purba et al., 2017). Following EGFR activation, several protein kinases such as phosphatidylinositol 3-kinase (PI3K) (Efferth, 2012), Ras, and ERK (Koveitypour et al., 2019), are activated to relay EGFR signaling. Gao et al. (2021) showed that licochalcone A, an analog of LCC, suppressed the growth of non-small cell lung cancer cells by inhibiting EGFR. We performed both an in vitro kinase assay and a docking study to determine whether LCC could inhibit kinase activity. LCC inhibited the kinase activities of EGFR, AKT1, and AKT2 in vitro (Fig. 2A, 2B). Furthermore, docking simulation identified LCC in the ATP-binding pockets (Fig. 2C). Additionally, the phosphorylation levels of both EGFR and AKT increased whereas the protein levels remained relatively unchanged (Fig. 3). The alteration of cell cycle distribution after treatment with LCC in both HCT116 and HCT116-OxR cells indicated that the regulation of cell cycle was perturbed by LCC (Fig. 4A), and the increase in SubG1 population suggested that LCC induced cytotoxicity (Fig. 4B). The EGFR signaling pathway is expected to be closely linked to cell cycle progression (Lo and Hung, 2006), and we observed changes in the levels of cell cycle regulators such as cyclin B1, cdc2, p21, and p27 (Malumbres and Barbacid, 2009) (Fig. 4C). Increased ROS levels promote colorectal development (Sreevalsan and Safe, 2013; Chun and Joo, 2022). This relationship is also valid for other cancer types, such as esophageal and lung cancers (Kim et al., 2021; Kwak et al., 2021). High levels, not excessive, seem to sustain the EGFR-mediated signaling pathway (Weng et al., 2018). However, the generation of sufficient levels of oxidative stress could be a therapeutic strategy for treating cancer cells by inducing apoptosis (Moloney and Cotter, 2018). A previous study on esophageal cancer cells indicated that LCC induce the generation of ROS to promote MAPK signaling (Kwak et al., 2020). Experimental data confirmed that ROS increased in CRC cells after treatment with LCC, and the increase in ROS levels was accompanied by phosphorylation of JNK and p38. Further, induction of caspase-3 was observed, which implies the induction of apoptosis by LCC treatment (Fig. 5). Disruption of the mitochondrial membrane potential is closely related to ROS generation (Suski et al., 2012), and other chalcone compounds, such as licochalcone A, have been shown to induce apoptosis by reducing the mitochondrial membrane potential (Lin et al., 2019b). LCC treatment depolarized the mitochondrial membrane, and the release of cytochrome c was observed with a concurrent increase in Apaf-1, cleaved PARP, and cleaved caspase-3. The Annexin V/7-aminoactinomycin D double staining assay indicated an increase in total apoptotic cells as the LCC concentration increased (Fig. 6). The activation of multiple caspases was observed after LCC treatment in both Ox-sensitive and Ox-resistant HCT116 cells, and pretreatment with Z-VAD-FMK prevented cell death induced by LCC. This suggests that LCC induce apoptosis through caspase activation (Fig. 7). In conclusion, our results indicated that LCC inhibited the growth of both Ox-sensitive and Ox-resistant CRC HCT116 cells. LCC inhibited the kinase activities of EGFR and AKT in vitro, and docking simulations supported the inhibition of kinases by LCC (Fig. 8). LCC exert their cytotoxicity by arresting cell cycle progression, inhibiting MAPKs through ROS generation, disrupting mitochondrial membrane potential, and activating multiple caspases. LCC could be a chemotherapeutic agent for treating Ox-resistant CRC cells, and further studies will improve our understanding of chemotherapy.

Figure 8. Schematic representation of LCC effects on Ox-sensitive and -resistant CRC cells.
ACKNOWLEDGMENTS

This study was funded by the Basic Science Research Program of the National Research Foundation of Korea (NRF) (No. 2019R1A2C1005899, 2021R1I1A3058531), and an NRF grant from the Korean Government (MSIT) (No. 2022R1A5A8033794).

CONFLICT OF INTEREST

The authors have no conflicts of interest relevant to this study to disclose.

AUTHOR CONTRIBUTIONS

Seung-On Lee: Conceptualization, Data curation, Formal analysis, Methodology, Validation, Visualization, Investigation, Writing–original draft, Writing–review, and Editing. Sang Hoon Joo: Conceptualization, Data curation, Formal analysis, Methodology, Validation, Visualization, Investigation, Writing–original draft, Writing–review, and Editing. Jin-Young Lee: Conceptualization, Data curation, Formal analysis, Methodology, Validation, Investigation, Software, and Resources. Ah-Won Kwak: Conceptualization, Data curation, Formal analysis, Methodology, Validation, Investigation, Software, and Resources. Ki-Taek Kim: Conceptualization, Data curation, Formal analysis, Methodology, Validation, Investigation, Software, and Resources. Seung-Sik Cho: Conceptualization, Data curation, Formal analysis, Methodology, Validation, Investigation, Software, and Resources. Goo Yoon: Conceptualization, Data curation, Formal analysis, Methodology, Validation, Investigation, Software, and Resources. Yung Hyun Choi: Conceptualization, Data curation, Formal analysis, Methodology, Validation, Investigation, Software, and Resources. Jin Woo Park: Project administration, Resources, Supervision, and Funding acquisition. Jung-Hyun Shim: Project administration, Resources, Supervision, and Funding acquisition. All the data were generated in-house. All authors agree to be accountable for all aspects of the work and to ensure their integrity and accuracy.

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