Cardamonin suppressed the migration, invasion, epithelial mesenchymal transition (EMT) and lung metastasis of colorectal cancer cells by down-regulating ADRB2 expression

Abstract Context Cardamonin (CDN) can suppress cell growth in colorectal cancer (CRC), a common digestive malignancy. Objective We explored the effect and mechanism of CDN on metastatic CRC. Materials and methods Two cell lines (HT29 and HCT116) were initially treated with CDN at different concentrations (5, 10 and 20 μmol/L) or 50 μmol/L propranolol (positive control) for 24 or 48 h. Then, the two cell lines were separately transfected with siADRB2 and ADRB2 overexpression plasmids, and further treated with 10 μmol/L CDN for 24 h. The cell viability, migration and invasion were determined by cell counting kit-8 (CCK-8), wound healing and transwell assays, respectively. The levels of ADRB2, matrix metalloprotease (MMP)-2, MMP-9, E-cadherin and N-cadherin were measured by Western blotting or/and RT-qPCR. A CRC metastasis model was established to evaluate the antimetastatic potential of CDN (25 mg/kg). Results ADRB2 (3.2-fold change; p < 0.001) was highly expressed in CRC tissues. CDN at 10 μmol/L suppressed viability (69% and 70%), migration (33% and 66%), invasion (43% and 72%) and ADRB2 expression (2.2- and 2.84-fold change) in HT29 and HCT116 cells (p < 0.001). CDN at 10 μmol/L inhibited MMP-2, MMP-9 and N-cadherin expression but promoted E-cadherin expression in CRC cells (p < 0.001). Importantly, the effect of CDN on CRC cells was impaired by ADRB2 overexpression, but further enhanced by ADRB2 down-regulation (p < 0.01). Additionally, ADRB2 overexpression reversed the inhibitory effect of CDN on metastatic lung nodules (p < 0.05). Discussion and conclusions: CDN is a potential candidate for the treatment of metastatic CRC in clinical practice.


Introduction
Colorectal cancer (CRC) is one of the most common types of cancer that is responsible for numerous cancer-related deaths worldwide . Based on global cancer statistics database, in 2018, about 1.09 million people suffered from CRC, of which 551,269 CRC patients died (Bray et al. 2018). The 5-year overall survival rate of patients with metastatic (stage IV) disease is 14% (James et al. 2017;Hou S et al. 2019), and metastatic disease occurs within 5 years in half of patients who underwent surgery for curative intent (Ferlay et al. 2015). Despite recent advances in the treatment of the disease, challenges still exist in the aspect of CRC metastasis which is extremely hard to be cured (Piawah and Venook 2019). Fortunately, targeted therapies, to our excitement, are now a part of the treatment modality for metastatic CRC, and may improve therapeutic outcome (Piawah and Venook 2019). Therefore, it is of great significance to discover molecular targeted therapeutic drugs for the treatment of metastatic CRC.
As a kind of pungent and warm Chinese herbal medicine, Caodoukou [also termed as semen Alpiniae katsumadai or Alpinia katsumadai Hayata (Zingiberaceae)] has various properties (Wang S et al. 2016). Cardamonin (2,4-dihydroxy-6-methoxychalcone, CDN), a natural chalcone compound, is the major active ingredient isolated from Alpinia katsumadai with potential effects in anti-inflammation and antitumor (Jin et al. 2019). For instance, CDN restrains the proliferation and metastasis of nonsmall-cell lung cancer cells (Zhou et al. 2019), and suppresses TGF-b1-provoked epithelial mesenchymal transition (EMT) of A549 cells (Kim et al. 2015). In addition, CDN inhibits proliferation, migration and invasion of prostate cancer cells (Zhang J et al. 2017), HT-1080 sarcoma cells (Park MK et al. 2013) and gastric cancer cells (Wang Z et al. 2019). These inhibitory effects of CDN on cancer metastasis make it a promising novel drug candidate to prevent or impede metastasis. A previous report demonstrated that CDN hinders the proliferation of CRC cells through enhancing b-catenin degradation (Park S et al. 2013). Moreover, a recent result verifies that CDN reduces chemotherapy resistance of CRC cells through the TSP50/NF-jB pathway in vitro (Lu et al. 2018). However, the precise effect and molecular mechanism of CDN on metastasis of CRC remain to be elucidated.
Metastatic dissemination represents the real reason of the malignant nature of tumours, the targeting of which is far more difficult than that of cell proliferation (Robert 2013). EMT is a critical metastasis-related process, where cancer cells gain the metastatic competence of migratory and invasive capabilities (Heerboth et al. 2015). Consequently, controlling EMT is fundamental to prevent and cure metastasis. In addition, accumulative evidence has indicated that adrenergic signalling plays an essential part in chronic stress-induced progression and metastasis in tumour . b 2 Adrenergic receptor (ADRB2) specifically binds to and is also provoked by the endogenous class of ligands known as catecholamines and epinephrine (Litonjua et al. 2010). In cancer cells, ADRB2 modulates the proliferation, migration, apoptosis, angiogenesis and metastasis (Ha et al. 2019). ADRB2 is strikingly implicated with tumour grade, size, invasion and lymph node metastasis of CRC (Ciurea et al. 2017) and selective inhibition on ADRB2 can repress growth of CRC (Chin et al. 2016), indicating that ADRB2 might be a promising therapeutic target for combating the development of ADRB2-dependent CRC. The current research therefore aimed to explore the effect of CDN on biological behaviours mainly including EMT, migration and invasion of CRC cells, and further investigated whether these effects are associated with ADRB2 expression.

Ethics statement
We collected 56 pairs of colorectal carcinoma tissues and corresponding normal mucosa tissues from CRC patients undergoing operation in the Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine from March 2018 to April 2020. The tissues were snap frozen in liquid nitrogen. The present study was reviewed and approved by the Ethics Committee of the Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine (ethical batch number: NJ201801005). All patients agreed to the use of their specimens before sampling.
This study was recommended by the Committee of Experimental Animals of Nanfang Hospital (ethical batch number: NF20200603015). All animal experiments were carried out in Nanfang Hospital complying with the principles of the China Council on Animal Care and Use. Every effort was made to minimize the pain and discomfort in the animals.
Transfection and treatment ADRB2 overexpression vector was generated by inserting its fulllength sequence (NCBI accession number: NC_000005.10) into the pUC57 vector purchased from GenePharma (Shanghai, China), with the empty pUC57 vector as a negative control (NC). SiADRB2 (siG000000154A-1-5) and siNC (siN0000002-1-5) were ordered from RiboBio Co. Ltd. (Guangzhou, China). SiNC served as the control for siADRB2. HT29 or HCT116 cells were seeded in a sixwell plate at a density of 3.0 Â 10 5 cells/well. When cell confluence reached 70%, ADRB2 overexpression vector, empty pUC57, siADRB2 and siNC were separately transfected into cells using Lipofectamine 3000 reagent for 24 h. CDN was dissolved in DMSO. HT29 and HCT116 cells were treated with CDN at various concentrations (5, 10 and 20 lmol/L) for 24 or 48 h, and with 50 lmol/L Prop for 24 h (Wang F et al. 2018) as a positive control. To further explore the mechanism of CDN in CRC, the expression of ADRB2 was up-regulated or down-regulated using ADRB2 overexpression vector or siADRB2 in HT29 and HCT116 cells that were then treated with CDN. Controls were only exposed to culture media containing 0.5% (v/v) DMSO.

Cell viability
The viability of HT29 and HCT116 cells was detected by CCK-8. In short, HT29 and HCT116 cells with or without transfection were plated in a 96-well plate at a density of 1.0 Â 10 4 cells/well and incubated overnight. Subsequently, the cells were treated with CDN at different concentrations. After culture for indicated times, CCK-8 solution (10 lL) was added to each well for additional 1 h of incubation. Next, cell viability was assessed by detecting absorbance at a wavelength of 450 nm with a Microplate Reader (GENios-Pro, Tecan, Milan, Italy).

Wound healing assay
To reveal the migration capacity, the wound healing assay was performed as previously reported (Gu et al. 2019). The transfected or untransfected HT29 and HCT116 cells were separately placed into six-well plates (3 Â 10 5 cells/well). When the cells reached about 90% confluence, a 200 lL pipette tip was used to make separate wounds on cell monolayers. Afterwards, the damaged cells were washed with PBS, and the adherent cells were cultured in FBS-free medium with CDN or Prop at the indicated concentrations. The distance covered by migrated cells was quantified at 0 and 24 h utilizing a microscope (Â100 magnification, E800, Nikon, Tokyo, Japan).

Transwell assay
To uncover cell invasion capacity, the transwell assay was carried out in accordance with a previous report (Gu et al. 2019). The 24-well chambers (#3422, 8 lm pore size, Corning, Corning, NY) were pre-coated with 50 mg/L Matrigel (1:8). After transfection, HT29 and HCT116 cells were starved in FBS-free medium for 24 h. Subsequently, 200 lL cells were added into the upper chamber (3 Â 10 5 cells/well) and 500 lL medium containing 20% FBS with CDN or Prop at the indicated concentrations was transferred to the lower chamber. After incubation at 37 C for 24 h, the invading cells were fixed with 4% paraformaldehyde for 10 min and then dyed with 1% crystal violet for 30 min. The invasion ability of cells was determined with a microscope (Â100 magnification).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was isolated from cells and tissues with TRIzol and reverse-transcribed into complementary DNAs (cDNAs) with Advantage RT-for-PCR Kit. The cDNAs were amplified based on the standard qPCR protocol with TB Green V R Advantage V R qPCR Premix in an Applied Biosystems 7500 Fast Real-Time PCR System (Foster City, CA). Quantitative PCR was conducted at 95 C for 10 min, followed by 35 cycles of 95 C for 15 s and 60 C for 60 s. Primers used in this research are listed in Table 1. GAPDH acted as the internal control and the results were calculated using 2 -DDCt method (Livak and Schmittgen 2001).

Western blotting
Western blotting was performed as previously reported (Gu et al. 2019). Whole-cell lysates were isolated from cells and tissues with RIPA buffer. Protein concentration was determined with BCA Protein Assay Kit. ColorMixed Protein Marker was applied as a protein size marker. The protein lysate (25 mg) was then separated by 6-10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (3010040001, Sigma-Aldrich, St. Louis, MO). After being blocked with 5% non-fat milk at 37 C for 1 h, the membrane was incubated first with primary antibodies at 4 C overnight and then with corresponding secondary antibodies at 37 C for 1 h. The protein was visualized using an ECL detection reagent and quantified by means of Bio-Rad ChemiDoc system (version 6.0 Bio-Rad Laboratories, Inc., Hercules, CA) with software Image J v2.1.4.7 (National Institutes of Health, Bethesda, MD). Antibodies used are listed in Table 2. GAPDH served as the internal control.

Animal studies
Six-week-old female BALB/c nude mice purchased from Shanghai Animal Laboratory Centre (Shanghai, China) were used to conduct all the in vivo studies. The mice were reared in Laboratory Animal Centre of Nanfang Hospital in a specific pathogen-free atmosphere.
For the metastasis model (Zhu et al. 2020), mice were randomly divided into five groups (n ¼ 3) as follows: normal, control, CDN, NC þ CDN and ADRB2 þ CDN groups.
Normal group: Mice were injected with 200 mL PBS through the tail veins; Control group: Mice were injected with 200 mL HT29 cell suspension (1 Â 10 7 cells/mL) through the tail veins; CDN group: Mice were injected with 200 mL HT29 cell suspension (1 Â 10 7 cells/mL) through the tail veins. Seven days later, mice were treated with CDN (25 mg/kg) (Hou G et al. 2020) twice a week via intraperitoneal injection; NC þ CDN group: Mice were injected through the tail veins with 200 mL HT29 cell suspension (1 Â 10 7 cells/mL) that was infected with lentivirus control by 8 mg/mL polybrene. After seven days, mice were treated with CDN (25 mg/kg) twice a week via intraperitoneal injection; ADRB2 þ CDN group: Mice were injected through the tail veins with 200 mL HT29 cell suspension (1 Â 10 7 cells/mL) that was infected with ADRB2 sequence by 8 mg/mL polybrene. Seven days later, mice were treated with CDN (25 mg/kg) twice a week via intraperitoneal injection.
After 28 days of continuous treatment, the mice were sacrificed. The number of metastatic lung nodules was counted.

Data analysis
The experiment was repeated three times. GraphPad prism 8.0 software (GraphPad Software, San Diego, CA) was utilized for processing the data that were presented as mean ± standard deviation (SD). A paired t-test was used to analyse paired samples (Figure 1(A)). Comparison between two groups was fulfilled using Student's t-test, and comparison among multiple groups was achieved by analysis of variance (ANOVA) followed by post hoc Tamhane's test. p < 0.05 was considered as statistically significant.

CDN suppressed CRC cell viability and expression of ADRB2 which was high-expressed in CRC
As shown in Figure 1(A), the mRNA level of ADRB2 was higher in CRC tissues than in normal tissues (p < 0.001). In addition, CCK-8 results indicated that CDN concentration-dependently restrained the viability of HT29 and HCT116 cells at 24 and 48 h (Figure 1(B,C), p < 0.05). When the concentration of CDN exceeded 10 lmol/L at 24 and 48 h, the viability of HT29 and HCT116 cells was less than 50%, but that of CCD-18Co cells was greater than 50% (Figure 1(D)). Thus, CDN at a concentration of 10 lmol/L was chosen for all subsequent experiments. It can be noted from RT-qPCR results that the mRNA level of ADRB2 in HT29 and HCT116 cells was strikingly suppressed by CDN and Prop (Figure 1(E,F), p < 0.001).

CDN hindered migration and invasion of CRC cells
According to Figure 2(A), in control group, cell wound width was obviously reduced at 24 h when compared to that at 0 h. By contrast, in CDN group or Prop-50 group, the wound width at 24 h showed no obvious changes compared to that at 0 h. Figure 2(B) depicts that the migration rate of HT29 cells in CDN or Prop-50 group was evidently decreased (p < 0.001) in contrast with that in control group. Also, similar results were observed in HCT116 cells (Figure 2(E-H), p < 0.001).

Discussion
To the best of our knowledge, this is the first study to elucidate the molecular mechanism underlying the inhibitory effect of CDN on lung metastasis of CRC via regulation of ADRB2 expression. The present study revealed that CDN inhibited ADRB2 expression, viability, migration, invasion and EMT of CRC cell lines (HT29 and HCT116) via down-regulating ADRB2 expression. Furthermore, a metastasis mouse model was used to confirm the findings in vitro, which emphasized the importance of CDN as a promising therapy to lung metastasis in CRC patients.
To initiate metastasis, cancer cells undergo a phenotypic alteration from epithelial cells to mesenchymal cells, a process known as EMT (Hassan et al. 2017). EMT is a possible mechanism of migration, invasion and subsequent metastasis in cancers (Xu et al. 2020). In certain tumour types, the critical markers of EMT are loss of E-cadherin (epithelium-derived labelled protein) expression and induction of N-cadherin (the labelled protein of the mesenchymal cells) expression (Moln ar et al. 2019). In the EMT process, mesenchymal-like pro-migratory phenotypes enable cells to cross anatomical boundaries such as the extracellular matrix (ECM) or the basement membrane (Kryczka et al. 2019). Additionally, MMP-2 and MMP-9 are responsible for ECM degradation and basement membrane impairment (Angelone et al. 2017). In this study, CDN suppressed viability of CRC cells, which was consistent with Park's findings (Park S et al. 2013). Moreover, previous studies also put forward that CDN retards migration, invasion and EMT of cancer cells (Park MK et al. 2013;Shrivastava et al. 2017;Gmerek et al. 2019;Zhou et al. 2019;Zhang L et al. 2021). Our novel data extended previous observations and provided the first evidence to directly demonstrate that CDN suppressed cell migration, invasion and EMT through upregulation of E-cadherin and downregulation of Ncadherin, and facilitated ECM degradation via down-regulations of MMP-2 and MMP-9 in CRC cells. Therefore, CDN may serve as a therapeutic drug in treating metastatic CRC.
reported that ADRB2 expression is up-regulated in gastric cancer ) and tongue squamous cell carcinoma . The current data implicated that ADRB2 was overexpressed in CRC tissues but was strikingly under-expressed in CRC cells treated with CDN, indicating that ADRB2 may act as an oncogene involved in the regulation of CDN on CRC progression. To further explore whether ADRB2 mediates the function of CDN in metastasis of CRC, the expression of ADRB2 was manipulated in HT29 and HCT116 cells by transfection with siADRB2 or ADRB2 overexpression plasmid. Previous studies corroborated that ADRB2 activation inhibits tumour progression (migration, invasion, EMT or metastasis) of oral cancer cells , breast cancer cells (Massaro et al. 2020) and tongue squamous cell carcinoma cells . The present study uncovered that ADRB2 overexpression strikingly promoted viability, migration and invasion and remarkably upregulated MMP-2, MMP-9 and N-cadherin expressions, but suppressed E-cadherin expression in HT29 cells. By contrast, ADRB2 deficiency produced the opposite effects on the same aspects in HCT116 cells. Furthermore, ADRB2 overexpression reversed the inhibitory effect of CDN on metastasis nodules of lung in mouse metastasis model. The data verified that CDN inhibited migration, invasion and EMT of CRC cells as well as lung metastasis through targeting ADRB2, manifesting that CDN may suppress metastasis of CRC as a molecular targeted therapeutic drug.
Recent publications have reported that CDN attenuates CRC cell growth through signal transducers and transcription (STAT) signal activators (Hou S et al. 2019). CDN inhibits tumorigenesis in CRC through regulation of microRNAs (miRNAs) expression (James et al. 2017(James et al. , 2021. Increasing evidence has indicated that miRNAs play important roles in the development and treatment of CRC Gmerek et al. 2019;Massaro et al. 2020;Wang H 2020). Other reports have also demonstrated the importance of STAT signal in regulation of CRC development Fang et al. 2020;Dariya et al. 2021). Thus, there is a possibility for the potential crosstalk between ADRB2 and STAT3 or miRNA in CRC. This would be further explored in our future research.
Several limitations existed in this study that should be overcome in the future study. First, several experiments were performed without a positive control, which therefore should be further investigated in the future study. Second, more extensive investigations in clinical trials are required to further confirm our results.

Conclusions
The present research demonstrates that CDN could suppress the CRC cell viability, migration and invasion in vitro and the metastasis to lung in vivo. The potential mechanism attributes to CDN-induced down-regulation of ADRB2, which further increases the level of E-cadherin, decreases those of N-cadherin, MMP-2 and MMP-9, and finally prevents EMT. These findings indicate that CDN may be perceived as a molecular targeted therapeutic drug for metastatic CRC.