2023 Impact Factor
2023 Impact Factor
According to the International Agency for Research on Cancer (IARC), there were an estimated 18.1 million new cases of cancer in 2020 worldwide and approximately 9.9 million cancer-related deaths, excluding non-melanoma skin cancer. Due to factors such as population growth and aging, the incidence and mortality rates of cancer have been continuously increasing. By 2040, an estimated 28.4 million new cases of cancer are projected worldwide, representing a 47% increase compared to 2020 (Sung et al., 2021). Therefore, a deep understanding of the causes and mechanisms of cancer and developing new treatment strategies are essential.
Natural compounds possess various properties, and some of them have been reported to exhibit physiological activities, such as antioxidant, anti-inflammatory, antibacterial, and anticancer effects (Prabhu et al., 2018; Dias et al., 2021; Liskova et al., 2021; Kimura et al., 2022). Recent years have seen a steady increase in research on natural compounds, leading to high expectations for improving cancer prevention and treatment strategies (Scaria et al., 2020; Islam et al., 2022; Pathak et al., 2022). Natural compounds, especially those extracted from plants, offer advantages over synthetic drugs in terms of their relatively low side effects and multiple pathway targets involved in cancer development and progression (Siddiqui et al., 2022). Well-known examples of natural compounds effective in cancer research include curcumin (Giordano and Tommonaro, 2019), resveratrol (Ren et al., 2021), epigallocatechin gallate (Aggarwal et al., 2022), and quercetin (Shafabakhsh and Asemi, 2019). Natural compounds are often used in combination with synthetic compounds, demonstrating complementary or enhanced effects (Uzoigwe and Sauter, 2012).
Licochalcone D (LicoD, Fig. 1A), used in this study, is a compound extracted from licorice (Glycyrrhiza). Licochalcones A, B, C, D, and E are the major licochalcones found in licorice extract and are known to possess potent biological activities, including anticancer, anti-microbial, anti-inflammatory, and antioxidant effects (Fu et al., 2013; Maria Pia et al., 2019). Recent studies reported that LicoD demonstrated anticancer effects in several types of cancer cells, including human skin cancer, lung cancer, and oral squamous cell carcinoma (Si et al., 2018; Seo et al., 2019; Oh et al., 2020). However, compared to other licochalcone compounds, current research on LicoD is limited, and studies investigating mechanisms related to cancer treatment and prevention are also scarce.
Figure 1. LicoD structure and its effects on cell viability on EGF- or TPA-induced cell proliferation. (A) The structure of LicoD, a natural com-pound isolated from licorice root, is shown. (B) Inhibition of EGF-induced JB6 cell proliferation by LicoD. EGF was treated at a concentration of 10 ng/mL, and various concentrations of LicoD were applied for 24 and 48 h. Statistical analysis was performed to evaluate the signifi-cance of the results, and any differences were considered statistically significant when p<0.05. (C) The morphological changes in EGF-induced cells by LicoD. The cells were treated with EGF (10 ng/mL) at various concentrations of LicoD (2.5, 5, 10 μM) in serum-free MEM for 48 h. (D) MK2206 inhibited EGF-induced JB6 cell proliferation. EGF (10ng/mL) and MK2206 (0, 1, 3, and 10 μM) were treated and incubated for 24 and 48 h. (E) The effect of MK2206 on morphological changes in EGF-induced transformed cells. JB6 cells were treated with EGF with 0, 1, 3, and 10 μM MK2206 and then incubated for 48 h. The morphological changes in the cells were observed using a microscope. Statistical analysis was performed to evaluate the significance of the results, and significantly different at: ###p<0.001 compared to the control; ***p<0.001 compared to group treated with EGF only.
Cell transformation is the process by which normal cells undergo alterations, leading to abnormal cell proliferation and changes in cell morphology similar to those of cancer cells. The process is primarily induced by external factors, and epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) are well-known factors that induce cell transformation. Cell transformation induced by EGF or TPA is helpful in understanding the molecular mechanisms that contribute to cancer initiation and progression (Nomura et al., 2005) and will ultimately lead to the advancement of cancer prevention and treatment strategies.
The AKT signaling pathway is closely associated with several aspects of cancer progression, including cell proliferation, survival, angiogenesis, migration, and invasion. Abnormal activation of these pathways was identified as a common feature of cancer and is an attractive target for anticancer drug development (Hwang et al., 2020; Tewari et al., 2022). Numerous previous studies reported the anticancer effects of licochalcones by targeting various signaling molecules associated with the PI3K/AKT/mTOR pathway (Oh et al., 2020; Deng et al., 2023).
With increasing interest in the AKT signaling pathway as a target for cancer prevention, our study aimed to investigate the impact of LicoD in modulating the AKT pathway in EGF and TPA-induced cell transformation.
Eagle’s minimum essential medium (MEM), Medium 199, Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin (P/S), MEM non-essential amino acid, sodium pyruvate, L-glutamine, and trypsin-EDTA were purchased from Gibco (Grand Island, NY, USA). Dimethyl-sulfoxide (DMSO), EGF, TPA, 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide (MTT), 5-fluorouracil (5-Fu), and Basal Medium Eagle (BME) were obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA). The antibodies against p21, phosphorylated NFκB (p-NFκB), NFκB, caspase-3, caspase-7, and Bax were purchased from Santa Cruz (Santa Cruz Biotechnology, CA, USA). The antibodies against phosphorylated AKT (p-AKT, Ser 473) and panAKT were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against actin, cyclin D1, phosphorylated GSK3β (p-GSK3β), GSK3β, phosphorylated mTOR (p-mTOR), mTOR, and β-actin were purchased from Invitrogen (Beverly, MA, USA). LicoD (Fig. 1A) was fortunately provided by Professor Yoon (Wang et al., 2013).
JB6 Cl41 (JB6), mouse epithelial cell line, were procured from American Type Culture Collection (ATCC, Manassas, VA, USA). JB6 cells were cultured in MEM supplying 5% FBS, 100 U/mL P/S, 1X MEM non-essential amino acid, and 1X sodium pyruvate. The cells were cultured at 37°C under 5% CO2, and the medium were changed every 2-3 days.
To investigate the effect of the LicoD on the proliferation of EGF- or TPA-induced transformed cells, MTT assay was performed. JB6 (8×103 cells/well) cells were seeded in a 96-well plate. After 24 h, the medium was removed, replaced with serum-free MEM, and cultured for 18 h. The cells were treated with various concentrations of LicoD in the presence of EGF (10 ng/mL) in serum-free MEM. Each of the cells was incubated for the indicated time, and then 10 μL of MTT (5 mg/mL) was added to each well. The formed formazan crystals were dissolved in 100 μL of DMSO, and then the absorbance was measured at 570 nm using a Multiskan SkyHigh spectrophotometer (ThermoScientific, Vantaa, Finland). The viability of LicoD-treated cells was normalized to untreated cells.
After overnight culture with 2×105 JB6 cells per well in 6-well plates, the cells were starved in MEM without FBS for an additional 18 h. And then the cells were treated with EGF (10 ng/mL) at various concentrations of LicoD (2.5, 5, 10 μM) or MK2206 (1, 3, 10 μM) in serum-free MEM. After 48 h, morphological changes in the cells treated with LicoD or MK2206 were confirmed and pictured under an inverted microscope (Leica Microsystems, Wetzlar, Hesse, Germany).
To evaluate JB6 cell transformation, we conducted a cellular anchorage-independent transformation assay according to previous study (Fu et al., 2021). BME supplemented with 10% FBS, 2 mM L-glutamine, and 25 μg/mL gentamicin (Lonza Group Ltd., Basel, Switzerland) was mixed with 0.6% agar containing DMSO, EGF (10 ng/mL) or TPA (10 ng/mL) and/or LicoD (2.5, 5, 10 μM) or MK2206 (1, 3, 10 μM). It was then solidified as the bottom agar layer in 6-well plates. JB6 cells (8,000 cells/well) were suspended in 1 mL of BME medium supplemented with 0.3% agar containing DMSO, EGF (10 ng/mL) or TPA (10 ng/mL) and/or LicoD (2.5, 5, 10 μM) or MK2206 (1, 3, 10 μM). And then it was added to the bottom agar layer. The plates were incubated at 37°C in a 5% CO2 incubator for 14 days. Colonies were visualized using a microscope (Leica Microsystems) and the colony number were analyzed using an Image-Pro Plus software ver.6.1 (Media Cybernetics, Rockville, MD, USA).
To investigate the effects of the LicoD on the cell cycle distribution of EGF- or TPA-induced transformed cells, JB6 cells were seeded at a density of 2×105 cells in a 6-well plate and incubated for 24 h. The cells were starved in serum-free MEM for an additional 18 h to eliminate the influence of growth factor. After treatment of EGF (10 ng/mL), TPA (10 ng/mL) and/or various concentrations of the LicoD (2.5, 5, 10 μM) in serum-free MEM, they were further incubated for 18 h or 24 h, respectively. The culture supernatant and trypsinized cells from each well were collected and the cell pellets were washed once with 1X cold-PBS, fixed with 70% ethanol at –20°C for more than 24 h. The cells were then stained with FxCycle™ PI/RNase Staining Solution (ThermoFisher Scientific, Rockford, IL, USA) in the dark for 15 min at room temperature (RT). Flow cytometry analysis was performed using a CytoFLEX flow cytometer and CytExpert software version 2.2 (Beckman Coulter, CA, USA).
JB6 cells were seeded in a 6-well plate (2×105 cells per well) and incubated for 24 h. After remove the media, the cells were washed with 1X cold-PBS and starved in serum-free MEM for an additional 18 h. After treatment of EGF (10 ng/mL), TPA (10 ng/mL) and/or various concentrations of the LicoD (2.5, 5, 10 μM) in serum-free MEM, they were further incubated for 48 h. The culture supernatant and cells from each well were collected and the cell pellets were washed once with 1X cold-PBS. Next, the cells were resuspended in 1X binding buffer and subsequently FITC-conjugated Annexin V and propidium iodide (ThermoFisher Scientific) were added to the suspension. After incubation in the dark for 15 min at RT, an appropriate amount of 1X binding buffer was added and flow cytometry analysis was performed using a CytoFLEX flow cytometer and CytExpert software version 2.2 (Beckman Coulter).
To confirm the molecular mechanisms underlying the growth inhibitory effect of the LicoD on the EGF- or TPA-induced transformed cells, western blot analysis was performed. After protein was extracted using PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Seongnam, Korea), and protein concentration was quantified using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific). The proteins were separated by SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 3% skim milk in 1X Tris-buffered saline with Tween-20 (TBS-T) at RT for 1 h and incubated with the primary antibodies against p-AKT with dilution ratio of 1:2,000, panAKT with dilution ratio of 1:5,000, p- GSK3β with dilution ratio of 1:1,000, GSK3β with dilution ratio of 1:3,000, p-NFκB with dilution ratio of 1:1,000, NFκB with dilution ratio of 1:1,000, p-mTOR with dilution ratio of 1:1,000, mTOR with dilution ratio of 1:1,000, cyclin D1 with dilution ratio of 1:1,000, p21 with dilution ratio of 1:1,000, Caspase-3 with dilution ratio of 1:200, Caspase-7 with dilution ratio of 1:200, Bax with dilution ratio of 1:200, and actin with dilution ration of 1:5000, overnight at 4°C. After several washes with TBS-T, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (#31430 and #31460; Invitrogen) with dilution ratio of 1:2,000~7,000 for 1 h at RT. Protein expression was detected using enhanced chemiluminescent (ECL) horseradish peroxidase (HRP) substrate (ThermoFisher Scientific) and imaged using a LAS-Amersham Imager 600 (GE Healthcare, Uppsala, Sweden). The blots were quantified using ImageJ software version 1.53 (U. S. National Institutes of Health, Bethesda, MD, USA).
The results of each experiment were expressed as the mean ± standard deviation (SD) (n=3). Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test using GraphPad Prism (version 6.01 for Windows, CA, USA, https://www.graphpad.com). A p-value less than 0.05 was considered statistically significant.
To examine the inhibitory effects of LicoD (Fig. 1A) on EGF-induced cell proliferation, JB6 cells were treated with 10 ng/mL of EGF in combination with varying concentrations of LicoD (2.5, 5, and 10 µM) for 24 and 48 h. The results demonstrated that LicoD effectively suppressed EGF-induced cell proliferation in dose-dependent and time-dependent manners (Fig. 1B). The morphological changes in EGF-treated cells induced by LicoD treatment were observed under an inverted microscope. As observed in the images of JB6 cells (Fig. 1C), it was confirmed that starved cells exhibited compromised cell survival and growth. In contrast, the cells stimulated with EGF showed enhanced cell growth. MK2206, a well-known AKT inhibitor, was evaluated for its effects on EGF-induced transformation using various assays, including cell viability, morphological changes, and anchorage-independent colony growth. MK2206 treatment significantly inhibited EGF-induced transformed cell survival in a dose-dependent manner at 24 and 48 h (Fig. 1D). Additionally, changes such as cell shrinkage induced by starvation were prevented upon EGF treatment (Fig. 1E). However, a significant anti-proliferative effect was observed with increasing concentrations of MK2206 (1, 3, and 10 μM), leading to pronounced apoptosis. These results indicate that both LicoD and MK2206 effectively inhibited cell proliferation, and inhibition of the AKT signaling pathway played a critical role in suppressing cell survival and proliferation.
Next, we conducted anchorage-independent growth assays, which is an essential tool to evaluate the effects of various compounds on tumor cell growth and colony formation, to further confirm the effect of LicoD and MK2206 on EGF and TPA-induced JB6 cell transformation (Mori et al., 2009; Chatterjee and Alfaro-Moreno, 2023). The anchorage-independent growth was performed using soft agar assays. The results demonstrated that LicoD and MK2206 effectively inhibited EGF- and TPA-induced anchorage-independent colony formation and growth in a dose-dependent manner (Fig. 2A, 2B). As shown in Fig. 2C, treatment with LicoD (2.5, 5, and 10 μM) significantly inhibited with rates of 31.9%, 63.9%, and 80.7% in EGF-induced cell transformation. LicoD exhibited inhibitory effects of 40.7%, 56.8%, and 80.2%, respectively, on TPA-induced cell transformation compared to the TPA-only treated group (Fig. 2D). Thus, LicoD exhibited potent inhibitory effects on colony formation and growth in the presence of EGF or TPA, similar to MK2206 (Fig. 2E, 2F).
Figure 2. LicoD and MK2206 suppress EGF- or TPA-induced JB6 cell transformation in soft agar. (A) Anchorage-independent cell growth was assessed by conducting soft agar assays with various concentrations of LicoD or MK2206 in combination with EGF. (B) Additionally, TPA and various concentrations of LicoD or MK2206 were co-treated to evaluate anchorage-independent cell growth. (C, D) The bar represents the relative ratio of colonies in soft agar with EGF or TPA and LicoD represented as the means ± SD of three replicates. (E, F) The bar graph indicates the relative ratio of colonies in soft agar with EGF or TPA and MK2206 represented as the means ± SD of three replicates. Significantly different at: ###p<0.001 compared to the control; ***p<0.001 compared to group treated with EGF or TPA only.
JB6 cells were treated with varying concentrations of LicoD in the presence of EGF or TPA to identify the effects of LicoD on cell cycle regulation in EGF and TPA-induced transformed JB6 cells and the underlying molecular mechanisms. Cell cycle distribution was assessed by flow cytometry after staining with FxCycle™ PI/RNase Staining Solution. Flow cytometric analysis showed that treatment with either EGF or TPA led to an increase in the S phase compared to untreated cells, consistent with the results shown in Fig. 3A. LicoD treatment induced a dose-dependent increase in the proportion of G1 phase cells, whereas the proportion of cells in the S phase decreased in both EGF and TPA-induced transformed cells. The distribution of G1 phase EGF and TPA-induced cells treated with increasing concentrations of LicoD (2.5, 5, 10 μM) increased to 71%, 87%, and 88% (for EGF; Fig. 3B) and 78%, 76%, and 74% (for TPA; Fig. 3C), respectively, compared to controls (65% and 68%). The expression levels of cell cycle-related proteins at the G1 phase were determined by Western blotting. LicoD treatment decreased the expression of cyclin D1 and increased the expression of p21 in both EGF (Fig. 3D, 3E) and TPA-treated (Fig. 3F, 3G) cells. These results suggest that LicoD induced cell cycle arrest at the G1 phase by modulating cell cycle-regulating proteins.
Figure 3. LicoD induces cell cycle arrest in EGF- or TPA-induced JB6 cells. (A) Flow cytometry analysis of EGF or TPA-induced JB6 cells treated with different concentrations of LicoD for 18 h. The representative images are shown. (B, C) The cell cycle distribution measured with a flowcytometry was represented as bar graphs. The effect of LicoD on the expression of proteins associated with cell cycle in EGF- (D, E) or TPA- (F, G) induced JB6 cells was investigated. JB6 cells were treated with EGF or TPA with 0, 2.5, 5, and 10 μM LicoD. The cell lysates were performed to western blotting using CyclinD1 and p21 antibodies. β-actin was used as a loading control. Densitometry quantification of proteins compared to β-actin is represented as the means ± SD. Significantly different at: ###p<0.001 compared to the control; **p<0.01 and ***p<0.001 compared to group treated with EGF or TPA only.
Flow cytometry of Annexin V and propidium iodide-stained cells was performed to determine whether LicoD induced apoptosis in EGF and TPA-induced transformed cells (Fig. 4A). After LicoD treatment for 48 h, the total percentage of apoptotic cells increased from 1.8% to 56.9% (for EGF; Fig. 4B) and 4.2% to 34.2% (for TPA; Fig. 4C) in a dose-dependent manner. Western blot experiments were conducted to investigate the expression of apoptosis-related proteins, such as caspase3, cleaved-caspase3, caspase7, cleaved-caspase7, and Bax. The expression of cleaved-caspase3, cleaved-caspase7, and Bax was increased in both EGF and TPA-treated cells (Fig. 4D-4G). The results confirmed that LicoD treatment induced apoptosis in transformed JB6 cells.
Figure 4. Lico D affects apoptosis in EGF- or TPA-induced JB6 cells. (A) The representative images of flow cytometry analysis in EGF- or TPA- induced JB6 cells treated with LicoD (2.5, 5, 10 μM) for 48 h. (B, C) The proportion of cells by stained with Annexin V/propidium iodide was represented as bar graphs. (D, E) Representative Western blots of apoptosis-related proteins expression of Caspase-3, Caspase-7 and Bax in EGF- or TPA-induced JB6 cells. JB6 cells were treated with EGF or TPA with 0, 2.5, 5, and 10 μM LicoD. The cell lysates were performed to western blotting using Caspase-3, Caspase-7 and Bax antibodies. β-actin was used as a loading control. (F, G) Quantitation of band intensity from blots by densitometry. The relative expression of cleaved-Caspase-3, cleaved-Caspase-7 and Bax was shown after normalization against Caspase-3, Caspase-7 and actin, respectively. Significantly different at: #p<0.05 and ##p<0.01 compared to the control; *p<0.05, **p<0.01, and ***p<0.001 compared to group treated with EGF or TPA only.
Western blotting analysis was performed using specific antibodies for p-AKT, AKT, p-GSK3β, GSK3β, p-NFκB, NFκB, p-mTOR, and mTOR to investigate whether LicoD regulated the AKT signaling pathway. JB6 cells were treated with varying concentrations of LicoD in the presence of EGF or TPA for 24 h. The results showed a decrease of p-AKT, p-GSK3β, p-NFκB, and p-mTOR expression in both transformed cells (for EGF; Fig. 5A, 5B, for TPA; Fig. 5C, 5D). The results indicate that LicoD regulated the AKT signaling pathway in EGF and TPA-induced transformed cells.
Figure 5. LicoD alters the expression of proteins associated with AKT signaling in EGF- or TPA-induced JB6 cells. (A) The effect of LicoD on the expression of AKT signaling-related proteins in EGF-induced JB6 cells was investigated. JB6 cells were treated with EGF with 0, 2.5, 5, and 10 μM LicoD and then incubated for 30 min. The cell lysates were performed to western blotting using p-AKT, panAKT, p-GSK3β, GSK3β, p-NFκB, NFκB, p-mTOR and mTOR antibodies. β-actin was used as a loading control. (B) The relative expression of p-AKT, p-mTOR, p-GSK3β and p-NFκB in EGF-induced JB6 cells was shown after normalization against panAKT, mTOR, GSK3β, and NFκB, respectively. (C) The effect of LicoD on the expression of AKT signaling-related proteins in TPA-induced JB6 cells was examined. JB6 cells were treated with TPA with 0, 2.5, 5, and 10 μM LicoD and then incubated for 60 min. (D) The relative expression of p-AKT, p-mTOR, p-GSK3β and p-NFκB in TPA-induced JB6 cells was shown after normalization against panAKT, mTOR, GSK3β, and NFκB, respectively. Significantly different at: #p<0.05, ##p<0.01, and ###p<0.001 compared to the control; *p<0.05, **p<0.01, and ***p<0.001 compared to group treated with EGF or TPA only.
Cancer is one of the leading causes of morbidity and mortality worldwide. More precise and effective treatment methods with fewer side effects are needed to complement the limitations of traditional cancer treatment approaches. Cell transformation refers to the process by which a normal cell undergoes changes that result in its conversion to a cancerous state. The process is a critical event in the initial step of malignant tumor formation. Therefore, the investigation of cell transformation and its underlying mechanisms is crucial for understanding cancer development and progression, and EGF or TPA-induced cell transformation can be an important technique for understanding molecular mechanisms. The excessive activation of cell signaling pathways by EGF or TPA can lead to cancer characteristics, such as persistent cell growth, invasion, and metastasis. In this study, we confirmed that LicoD effectively suppressed EGF-induced cell proliferation and anchorage-independent colony growth (Fig. 1, 2).
Licorice is a traditional herbal medicine widely used around the world, with various pharmacological properties. Licochalcones, the representative flavonoids in licorice, also have high potential as natural compounds for cancer treatment. An understanding of the molecular mechanisms involved in cancer development and progression may inform the use of these compounds in more comprehensive therapeutic strategies. Among them, LicoD has been reported to inhibit the growth of several types of cancer cells, but the mechanism involved has not yet been sufficiently identified. In this study, we investigated the inhibitory effects of LicoD on EGF and TPA-induced skin epithelial cell transformation for cancer prevention. LicoD effectively inhibited cell transformation and cell growth by accelerating cell cycle arrest and apoptosis (Fig. 3, 4). Flow cytometry and Western blot analysis showed that treatment with LicoD decreased cyclin D1 expression and increased p21 expression and G1 cell cycle arrest in EGF and TPA-transformed cells (Fig. 3D-3G). This indicates that LicoD regulated the expression of cyclin D1 and p21, which are important in cell cycle progression, leading to cell cycle arrest in the G1 phase. Cyclin D1 is a member of the cyclin family that is primarily activated during the G1 phase and is known to promote transition from the G1 phase to the S phase. The cyclin D1-CDK complex formed by binding to cyclin-dependent kinase (CDK) 4 or CDK6 promotes cell cycle progression (Montalto and De Amicis, 2020). However, the overexpression of cyclin D1 often leads to cell cycle dysregulation, which is associated with cancer development. In contrast, p21 is a member of the Cip/Kip family of CDK inhibitors and plays a crucial role in controlling cell cycle progression, particularly the transition from the G1 to the S phase (Karimian et al., 2016). The activation of p21 blocks cell cycle progression, which can lead to programmed cell death (Gartel and Tyner, 2002). Therefore, changes in the expression of these proteins ultimately resulted in G1 phase arrest, indicating the potential of LicoD to inhibit cell transformation (Fig. 3).
Annexin V/propidium iodide staining was performed, followed by flow cytometric analysis, to determine the occurrence of apoptosis induced by LicoD treatment (Fig. 4A). We also investigated the expression of apoptosis-related proteins and AKT signaling-related proteins by Western blot analysis to provide further insight into the molecular mechanisms underlying the anticancer activity of LicoD (Fig. 4, 5). Bax is a pro-apoptotic protein that promotes apoptosis and leads to the activation of caspase-3 and caspase-7 through a series of apoptotic processes (Schmitt et al., 1998). According to our findings, LicoD upregulated the expression of Bax, cleaved caspase-3, and cleaved caspase-7 in a concentration-dependent manner (Fig. 4D-4G). The changes in the expression of these proteins suggest that LicoD may induce apoptosis in cancer cells.
Licochalcones regulate signaling pathways, including EGFR/ERK (Gao et al., 2021), PI3K/Akt/mTOR (Xue et al., 2018), p38/JNK (Kwak et al., 2023), JAK2/STAT3 (Park et al., 2022), MEK/ERK (Park et al., 2015), and MKK4/JNK (Wu et al., 2018), and their downstream protein expression. They have been shown to exert various biological effects on cancer cells, such as inducing autophagy and apoptosis and inhibiting cell proliferation, migration, and invasion (Deng et al., 2023). In this study, decreases in p-AKT, p- GSK3β, p-NFκB, and p-mTOR protein levels shown through Western blot analysis revealed a marked inhibition of the AKT/mTOR pathway by LicoD (Fig. 5). The results were verified by treating with the pharmacological AKT inhibitor MK-2206 (Fig. 1, 2). Considering the role of the AKT signaling pathway in cancer progression, this study investigated the effects of LicoD on the AKT signaling pathway in EGF and TPA-induced cellular transformation, as well as its potential as an anticancer agent.
Taken together, our findings demonstrate that LicoD suppressed EGF and TPA-induced cell transformation by targeting the AKT signaling pathway, leading to G1 phase arrest and the modulation of cell cycle and apoptosis-related proteins. The results suggest that LicoD could be used as an alternative therapeutic agent for cancer. Further research should be conducted to understand the various anticancer effects and mechanisms of LicoD.
This work was supported by a Korea Innovation Foundation (INNIPOLIS) grant funded by the Korean government (Ministry of Science and ICT) through a science and technology project that opens the future of the region, grant number: 2021-DD-UP-0380, the National Research Foundation of Korea (NRF), funded by the Ministry of Science and ICT (No. 2022R1A5A2029546) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1I1A3070556). This research was supported by the Dongshin University research grants.
The authors have no financial conflicts of interest to declare.
SYH, JHS and MHL were involved in study concept and design, acquisition of data, analysis and interpretation of data, and drafting of the manuscript. SYH, KHW, and MHL performed experiments. GY, SIL, JGJ, HWJ, JSK, CHC and CSN supported the data analysis and materials. SYH and MHL wrote the manuscript. JHS and MHL supervised the study. All authors read and approved the final manuscript.
Funding Information
Korea Innovation Foundation (INNIPOLIS) Ministry of Science and ICT, South Korea National Research Foundation of Korea Ministry of Education Dongshin University |
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