Transforming growth factor-β1 (TGF-β1) is a cytokine that plays various functions in tissues. Known as a member of the family of growth and differentiation factors, TGF-β1 regulates diverse cellular functions, such as proliferation, differentiation, and apoptosis [1
]. TGF-β1 binds to TGF-β receptor II at the cell membrane, and then TGF-β receptor II is complexed with TGF-β receptor I and phosphorylated [3
]. The major signal transduction pathway in cells of the TGF-β family is driven by SMAD family proteins. The phosphorylated TGF-β receptor complex phosphorylates SMAD2 and SMAD3, which subsequently form a complex with SMAD4. This complex translocates to the nucleus and acts as a transcription factor. TGF-β1 inhibits proliferation in normal epithelial cells, but this regulation is not observed in cancer cells. In addition, TGF-β1 induces epithelial–mesenchymal transition (EMT) in the epithelium in cancer cells, leading to increased cell motility and invasiveness [4
EMT is associated with morphological changes and is an important process in cancer cells. The epithelial phenotype is transformed into a mesenchymal fibroblast phenotype that is facilitated by mesenchymal characteristics, including increased migration and invasive abilities [8
]. Since cancer cells undergo numerous extracellular stimulations in vivo, inhibiting TGF-β1-induced EMT-related factors is important in the development of cancer therapy.
EMT plays an important role and is usually associated with cancer tissue metastasis. Epithelial cells that acquire mesenchymal properties exhibit increased mobility and invasiveness, and they promote tumor formation and cancer metastasis [11
]. It has been found that each phenotype of EMT induced by TGF-β1 is regulated by distinct regulatory factors. The downregulation of E-cadherin promotes mobility and invasion by regulating various transcription inhibitors, such as SNAI1, SNAI2, and ZEB1. During EMT progression, SNAI1 and SNAI2 are overexpressed and inhibit E-cadherin expression [13
]. In contrast, mesenchymal markers N-cadherin and fibronectin are not well known and generally not regulated by the above factors [14
]. In addition, it has been reported that the changes in EMT enhance the secretion of gelatinase matrix metalloproteinase-2 (MMP2) and MMP9 [16
EMT is regulated through various pathways. In particular, the EMT pathway mediated by TGF-β is well known. TGF-β1, which induces EMT, is expressed in most human cells and plays an important role in inhibiting differentiation and growth [18
]. Three TGF-β isoforms (TGF-β1, 2, and 3) have been identified that are involved in several important biological phenomena during the processes from onset to progression [19
]. Since TGF-β1 phosphorylates SMAD2/3/4 complex and induces EMT, TGF-β1 and EMT are highly correlated, and the nuclear translocation of the SMAD complex affects the expression of target genes. Thus, inhibition of SMAD2/3 phosphorylation blocks intracellular signal transduction by TGF-β1, resulting in inhibition of EMT [21
Reducing the mobility and invasiveness of cells by inhibiting the progression of EMT mediated by TGF-β1 provides a promising strategy to improve the survival rate of patients. There is increasing research to develop TGF-β1 inhibitors using non-toxic natural compounds to suppress TGF-β-induced EMT. Therefore, it is important to develop new inhibitors that can effectively prevent EMT, an essential characteristic of cancer cells.
Chalcone is a plant metabolite commonly found in vegetables, spices, tea, and fruits. It exhibits various biological and pharmacological activities, such as anticancer, anti-inflammatory, anti-infective, immunomodulatory, antibacterial, and antioxidant activates [25
]. Natural chalcone compounds have been reported to inhibit cytotoxicity and in vitro cytotoxic activity in prostate cancer [26
]. Furthermore, chalcone compounds are potential anticancer agents and inhibitors of cathepsin B and L [27
]. Cathepsin represents a group of proteases and is involved in determining the metastasis of cancer cells [28
]. In particular, it has been reported that suppression of cathepsin L inhibits EMT induced by TGF-β1 and suppresses cancer mobility and invasiveness [26
]. We studied the TGF-β1 signaling pathway by screening 82 compounds by further designing and synthesizing chalcone compounds. We aimed to develop inhibitors that inhibit EMT in lung cancer cells through chalcone analogs. As a result, we demonstrated the inhibitory role of CTI-82 during TGF-β1-mediated EMT process in A549 lung cancer cells.
2. Materials and Methods
2.1. Cells and Reagents
HaCaT cells were cultured in DMEM (Corning, Buffalo, NY, USA) containing 10% fetal bovine serum (FBS; Corning) and 1% antibiotics (Hyclone, Waltham, MA, USA). Human lung cancer A549 cells were cultured in RPMI (Corning) containing 10% FBS and 1% antibiotics, and all cells were cultured in a 37 °C incubator with 5% CO2. TGF-β1 was obtained from PeproTech (Rocky Hill, NJ, USA). SB431542 was purchased from Selleckchem (Houston, TX, USA). The following antibodies were used: anti-SMAD2 (5339S), anti-SMAD3 (9523S), anti-p-SMAD2 (S465/467; 3108S), p-SMAD3 (S423/425; 9520S), anti-ERK1/2 (9102S), anti-p-ERK1/2 (T202/Y204; 9101S), anti-p-p38 (T180/Y182; 4631S) (Cell Signaling, Danvers, MA, USA), anti-JNK1/2/3 (ab208035), anti-p-JNK1/2/3 (Y185/Y185/Y223; ab76572), anti-p38 (ab32142) (abcam, Cambridge, MA, USA), anti-N-cadherin (sc-59987), anti-E-cadherin (sc-71009), anti-SNAI2 (sc-166476), anti-MMP9 (sc-13520), anti-COL1A2 (sc-393573), anti-PAI-1 (sc-5297) (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA). All secondary antibodies were purchased from Thermo Scientific (Waltham, MA, USA). TransIT-LT1 transfection reagent was purchased from Mirus Bio (Madison, WI, USA).
2.2. Cell Viability Assays
To determine the cytotoxicity of CTI-82 in A549 cells, cell viability was measured by one of the conventional measurement methods, the MTT reduction assay. Briefly, A549 cells were seeded at a density of 1 × 103 cells/well in a 96-well plate. After 24 h of incubation, the cells were co-treated with TGF-β1 and CTI-82 (0, 10, 20, 30, 40, and 50 μM) for 48 h. Cells were treated with 15 mL of MTT solution (2 mg/mL) for 60 min at 37 °C, and the absorbance at 570 nm was measured and recorded on a microplate reader (Model 550, BIO-RAD Laboratories, Hercules, CA, USA). All MTT assay results were expressed as the mean (±SD) of three independent experiments.
2.3. Reporter Gene Assay
A549 cells were transiently transfected with the reporter plasmid pSBE4-Luc to determine the transcriptional activity of SMAD. The β-gal luciferase reporter plasmid was co-transfected as an internal control. Then, the cells were harvested, and luciferase activity was measured according to the manufacturer’s instructions (Promega, Madison, WI, USA). All reporter activity was normalized to β-gal luciferase activity and presented as the mean (±SD) of three independent experiments.
2.4. RNA Extraction and Quantitative Real-Time PCR
Total RNA was isolated using an RNA EasySpin kit (Intron Biotechnology, Seongnam-si, Gyeonggi-do, Republic of Korea) according to the manufacturer’s instructions. cDNA synthesis was performed using a cDNA synthesis kit (primescript® RTreagent kit, Takara, Kusatsu, Shiga, Japan) to convert total RNA into cDNA. For real-time PCR, the primers for various genes were designed in Primer-BLAST (NCBI, Bethesda, MD, USA). The primers used were as follows: E-cadherin (5′-TGCCCAGAAAATGAAAAAGG-3′, 5′-GTGTATGTGGCAATGCGTTC-3′), N-cadherin (5′-CCATCACTCGGCTTAATGGT-3′, 5′-GATGATGATGCAGAGCAGGA-3′), MMP2 (5′-ACATCAAGGGCATTCAGGAG-3′, 5′-GCCTCGTATACCGCATCAAT-3′), MMP9 (5′-CATCGTCATCCAGTTTGGTG-3′, 5′-TCGAAGATGAAGGGGAAGTG-3′), COL1A1 (5′-AGCCAGCAGATCGAGAACAT-3′, 5′-TCTTGTCCTTGGGGTTCTTG-3′), ZEB1 (5′-TGCACTGAGTGTGGAAAAGC-3′, 5′-TGGTGATGCTGAAAGAGACG-3′), and GAPDH (5′-CGCGGGGCTCTCCAGAACATCATCC-3′, 5′-CTCCGACGCCTGCTTCACCACCTTCTT-3′). Real-time PCR was performed using a real-time PCR kit (EBT-1801; HiPi Real-Time PCR 2x Master Mix, ELPIS Bio, Daedeok-gu, Daejeon, Republic of Korea), and all samples were normalized using the ΔΔCt method. In addition, numerical values for all expression levels were expressed as fold changes. All reactions were repeated three times, and relative expression levels and SDs were calculated using Microsoft Excel (Office 365).
2.5. Western Blot Analysis
Briefly, after 48 h of treatment with TGF-β1 and CTI-82, A549 and HaCaT cells were washed with PBS, scraped, and harvested. Cell extracts were lysed on ice for 30 min with lysis buffer (10 mmol/L NaF, protease inhibitors, 10 mmol/L sodium pyrophosphate, 150 mmol/L NaCl, 1% NP-40, and 50 mmol/L Tris-HCl (pH 7.5)). After cell lysis, cells were centrifuged at 13,000 rpm for 20 min at 4 °C. The supernatant was transferred to a new tube, and the protein concentration of the whole cell lysate was measured with a Pierce 660 nm protein assay reagent (Thermo Scientific). The samples were separated on 8–12% SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were blocked by incubation for 2 h with w/v non-fat DifcoTM skim milk (BD Biosciences, Franklin Lakes, NJ, USA) in blocking buffer with 1X PBST. The blocked membrane was then incubated with the indicated primary antibodies for overnight at 4 °C. After washing three times with 1× PBST for 10 min each, the secondary antibody was incubated for 1.5 h. After washing three times in the same 1X PBST for 10 min each, protein bands were visualized by the developer. The signal was quantified by ImageJ (Java 1.8.0_112, NIH, Bethesda, MD, USA), and the level of protein expression was normalized to β-actin.
2.6. Matrigel Invasion Assay and Wound-Healing Assay
In the Biocoat Martigel invasion chamber (SPL Life Science, Pocheon-si, Gyeonggi-do, Republic of Korea), a Matrigel invasion assay was used to confirm the ability of cells to migrate through the extracellular matrix. A549 cells (2 × 104) were seeded in each well. The cells were then cultured for 12 h prior to co-treatment with CTI-82 and TGF-β1. After incubation for 48 h, non-invaded cells were removed with a cotton swab. The invaded cells were fixed with 100% methanol and stained with 1% crystal violet (Sigma-Aldrich). After staining, the number of invaded cells was counted with a microscope (40×, three random fields per well). Data were expressed as the mean (±SD) of at least three independent experiments.
In the wound-healing assay, A549 cells were cultured to 80% confluency, and then cells were scratched using a 20 µL pipette tip. The scraped cells were removed with DPBS, the media was changed, TGF-β1 and CTI-82 were added, and cells were cultured for 48 h. Wound healing was observed within 48 h of the scratched wounds. The TScratch program (TScratch 1.0) was used to quantify migration by measuring cell surface area. The data were expressed as the mean (±SD) of at least three independent experiments.
2.7. Confocal Microscopy
A549 cells were seeded 3 × 104 in a confocal dish (SPL, 101350). The grown A549 cells were fixed in 4% paraformaldehyde for 10 min, permeabilized in 0.3% Triton X-100 for 5 min and blocked with 10% goat serum albumin for 1 h at room temperature. Cells were incubated with the indicated primary antibody at room temperature for 3 h or overnight at 4 °C, washed three times with PBS, and then the secondary antibody was incubated at room temperature for 1 h. Antibodies for immunofluorescence were anti-N-cadherin, anti-E-cadherin, and goat anti-rabbit IgG-FITC (sc-2012, Santa Cruz). DAPI (D5942; Sigma-Aldrich) was used to stain cell nuclei. Imaging of stationary samples was performed in the ZOE Fluorescent Cell Imager (Bio-Rad).
2.8. Statistical Analysis
Statistical significance was determined via two-tailed unpaired Student’s t-test or one-way ANOVA using Microsoft Excel (Office 365) or Prism (Prism 8.0.1, GraphPad, San Diego, CA, USA) software. A two-tailed t-test was used for comparisons between two groups. All data are presented as mean ± standard error. Values were reported as the mean ± SD. p-values < 0.05 were considered significant. Detail values are shown in the figure legends.
Natural anticancer compounds inhibit tumor growth and progression by reducing cell migration, invasion, and metastasis [35
]. Interestingly, chalcone-like chain CTI-82 exhibits several pharmacological effects. It is also used as an anticancer and anti-inflammatory agent and is known as a promising new drug candidate compound with biological activity and stability [27
]. Our study showed that CTI-82, a chalcone analog, inhibited the mobility and invasiveness of A549 cells stimulated by TGF-β1.
We first performed a primary screening of a library of 84 potential TGF-β1 inhibitors to determine the concentration range of CTI-82, a chalcone analog. The secondary screening was confirmed by whether the transcriptional activity of SMAD at the selected concentration was inhibited. CTI-82 did not cause the cell cytotoxicity at 30 μM but significantly decreased the transcriptional activity of SMAD in a dose-dependent manner. In the canonical TGF-β signaling pathway, TGF-β1-induced EMT is generally initiated by phosphorylated SMAD2 and SMAD3, which translocate to the nucleus to induce the expression of EMT-related genes. We found that CTI-82, a chalcone analog, inhibited the phosphorylation of SMAD2/3 induced by TGF-β1. The expression of SMAD2/3 phosphorylation was reduced in a dose-dependent manner in A549 cells treated with CTI-82. In addition, both SMAD2/3 expression and transcriptional activity were reduced to levels similar to those in cells treated with the TGF-β1 inhibitor SB431542. Thus, these results indicate that CTI-82 antagonizes the phosphorylation of SMAD2/3 induced by TGF-β1 in the canonical TGF-β pathway.
Additionally, in the canonical signaling pathway, TGF-β1 activates the p38/JNK MAPK signaling, which proceeds through activation of TAK1 by TRAF6. Shr is mobilized by TGF-β receptor I (TGFβR1) to activate the ERK MAPK pathway. Furthermore, TGF-β1 activates phosphatidylinositol-3-kinase (PI3K)/AKT axis [44
]. Thus, to confirm CTI-82 suppresses the non-canonical signaling pathway, we confirmed that CTI-82 inhibits phosphorylation of non-canonical signaling in addition to canonical signaling pathway. As shown in Figure 4
, CTI-82 can partially inhibit not only canonical signaling but also non-canonical signaling by significantly inhibiting phosphorylation of ERK caused by TGF-β1. Thus, we strongly suggest that CTI-82 may effectively inhibit the cancer metastasis and tumorigenesis to EMT process of the growth factor-induced cancer cells.
SMAD2/3 is complexed with SMAD4 and translocated into the nucleus. This complex increases the extracellular matrix (ECM), EMT, mobility, and invasiveness by directly binding to the target gene or inducing associated gene markers such as collagenase, matrilysin, urokinase, and MMPs along with other transcription factors [14
]. Transcription factors such as SNAI2 and ZEB1 play a very important role in the process of EMT and tumor formation through inhibition of E-cadherin expression [53
]. In addition to these transcription factors, since TGF-β1 upregulates collagen type I and MMPs in ECM synthesis, this study confirmed that genes involved in the increase in TGF-β1-induced EMT are inhibited [55
]. As shown in Figure 5
, CTI-82 significantly inhibited the mRNA and protein levels of the transcription factor, collagen, and MMP genes that increase EMT. CTI-82 may be a novel inhibitor of EMT induced by TGF-β1 because it inhibits the mRNA and protein levels of various EMT markers. However, the detailed mechanism by which CTI-82 modulates the TGF-β receptor is unclear. In this study, SB431542, an inhibitor that specifically inhibits TGFβR1 phosphorylation, showed similar effects to CTI-82. Therefore, to inhibit the TGF-β signaling pathway, the TGF-β receptor can be a target of CTI-82.