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

Figures & data

Table 1. Primer sequences used for quantitative reverse transcription polymerase chain reaction (qRT-PCR) of human genes.

Genes	Primer sequences (5′–3′)
ADRB2
Forward	TTGCTGGCACCCAATAGAAGC
Reverse	CAGACGCTCGAACTTGGCA
MMP-2
Forward	TACAGGATCATTGGCTACACACC
Reverse	GGTCACATCGCTCCAGACT
MMP-9
Forward	TGTACCGCTATGGTTACACTCG
Reverse	GGCAGGGACAGTTGCTTCT
E-cadherin
Forward	GTT ATT CCT CTC CCA TCA GCT G
Reverse	CTT GGC TGA GAG GAT GGT GTA A
N-cadherin
Forward	TCAGGCGTCTGTAGAGGCTT
Reverse	ATGCACATCCTTCGATAAGACTG
GAPDH
Forward	AGCCACATCGCTCAGACAC
Reverse	GCCCAATACGACCAAATCC

Table 2. List of primary antibodies used for Western blots.

Protein	Antibody	Catalogue number	Company	Antibody dilution
MMP-2	Rabbit anti-MMP2 antibody	ab37150	Abcam	1:200
MMP-9	Rabbit anti-MMP9 antibody	ab38898	Abcam	1:1000
E-cadherin	Mouse anti-E cadherin	ab1416	Abcam	1:50
N-cadherin	Rabbit anti-N cadherin	ab18203	Abcam	1:1000
GAPDH	Rabbit anti-GAPDH	ab181602	Abcam	1:10,000
Secondary anti-body	Goat anti-rabbit IgG H&L (HRP)	ab205718	Abcam	1:2000
Secondary anti-body	Goat anti-mouse IgG H&L (HRP)	ab205719	Abcam	1:2000

Figure 1. CDN suppressed viability and ADRB2 expression in CRC cells. (A) The mRNA level of ADRB2 in human CRC tissues and matched normal tissues was determined by RT-qPCR. (B–D) The viability of HT29, HCT116 and CCD-18Co cells treated with CDN at various concentrations (5, 10 and 20 μmol/L) for 24 or 48 h was examined by CCK-8. (E, F) The ADRB2 mRNA level in HT29 and HCT116 cells treated with CDN at 10 μmol/L was examined by RT-qPCR. *p < 0.5 or ***p < 0.001 vs. control. GAPDH acted as the internal control. Control: cells only exposed to culture media containing 0.5% (v/v) DMSO; CDN: cardamonin; CRC: colorectal cancer; ADRB2: β₂ adrenergic receptor; Prop-50: propranolol (50 μmol/L); CCK-8: cell counting kit-8; DMSO: dimethyl sulphoxide; RT-qPCR: reverse transcription-quantitative polymerase chain reaction.

Figure 2. CDN inhibited migration and invasion of CRC cells. The migration and invasion of HT29 (A–D) and HCT116 cells (E–H) treated with CDN at 10 μmol/L were examined using wound healing and transwell assays, respectively. **p < 0.01 or ***p < 0.001 vs. control. Control: cells only exposed to culture media containing 0.5% (v/v) DMSO; DMSO: dimethyl sulphoxide; CDN: cardamonin; CRC: colorectal cancer; Prop-50: propranolol (50 μmol/L).

Figure 3. CDN decreased the expression of MMP-2, MMP-9 and N-cadherin, but increased E-cadherin expression in CRC cells. (A–F) The mRNA and protein levels of MMP-2, MMP-9, E-cadherin and N-cadherin in HT29 (A–C) and HCT116 (D–F) cells treated with CDN at 10 μmol/L were examined using RT-qPCR and Western blotting. **p < 0.01 or ***p < 0.001 vs. control. GAPDH acted as the internal control. Control: cells only exposed to culture media containing 0.5% (v/v) DMSO; DMSO: dimethyl sulphoxide; CDN: cardamonin; Prop-50: propranolol (50 μmol/L); CRC: colorectal cancer; RT-qPCR: reverse transcription-quantitative polymerase chain reaction; MMP: matrix metalloprotease.

Figure 4. CDN suppressed ADRB2 expression and viability of CRC cells through inhibiting ADRB2 expression. Cells transfected with siADRB2 or ADRB2 overexpression plasmid were exposed to CDN and then examined for the mRNA level of ADRB2 and the viability using RT-qPCR (A, B) and CCK-8 (C, D), respectively. GAPDH acted as the internal control. **p < 0.01 or ***p < 0.001 vs. NC; ^##p < 0.01 or ^###p < 0.001 vs. NC + CDN; ^{^^^}p < 0.001 vs. ADRB2; ⁺⁺p < 0.01 or ⁺⁺⁺p < 0.001 vs. siNC; ^&&p < 0.01 vs. NC + CDN-10; ^△△△p < 0.001 vs. siADRB2. NC: negative control (empty vector); siNC: negative control of small interfering RNA (siRNA); CDN: cardamonin; CRC: colorectal cancer; ADRB2: β₂ adrenergic receptor; CCK-8: cell counting kit-8; RT-qPCR: reverse transcription-quantitative polymerase chain reaction; siADRB2: siRNA for ADRB2.

Figure 5. CDN blocked migration and invasion of CRC cells through inhibiting ADRB2 expression. Cells transfected with siADRB2 or ADRB2 overexpression plasmid were exposed to CDN and then examined for the migration and the invasion by wound healing assay (A, B, E and F) and transwell assay (C, D, G and H), respectively. ***p < 0.001 vs. NC; ^##p < 0.01 or ^###p < 0.001 vs. NC + CDN; ^{^^}p < 0.05 or ^{^^^}p < 0.001 vs. ADRB2; ⁺p < 0.05 or ⁺⁺p < 0.01 or ⁺⁺⁺p < 0.001 vs. siNC; ^&&&p < 0.001 vs. NC + CDN-10; ^△△△p < 0.001 vs. siADRB2. NC: negative control (empty vector); siNC: negative control of small interfering RNA (siRNA); CDN: cardamonin; CRC: colorectal cancer; ADRB2: β₂ adrenergic receptor; siADRB2: small interfering RNA for ADRB2.

Figure 6. CDN lessened MMP-2, MMP-9 and N-cadherin expression but augmented E-cadherin expression in CRC cells through inhibiting ADRB2 expression. (A–F) ADRB2 overexpression or knockdown was achieved by transfection of ADRB2 overexpression plasmid or siADRB2 into CRC cells, respectively. Transfected cells were exposed to CDN and then examined for the mRNA and protein levels of MMP-2, MMP-9, E-cadherin and N-cadherin using RT-qPCR (A, D) and Western blotting (B, C, E and F). GAPDH acted as the internal control. ***p < 0.001 vs. NC; ^##p < 0.01 or ^###p < 0.001 vs. NC + CDN; ^{^^}p < 0.05 or ^{^^^}p < 0.001 vs. ADRB2; ⁺p < 0.05 or ⁺⁺p < 0.01 or ⁺⁺⁺p < 0.001 vs. siNC; ^&&p < 0.05 or ^&&&p < 0.001 vs. NC + CDN-10; ^△△△p < 0.001 vs. siADRB2. NC: negative control (empty vector); siNC: negative control of small interfering RNA (siRNA); CDN: cardamonin; CRC: colorectal cancer; ADRB2: β₂ adrenergic receptor; RT-qPCR: reverse transcription-quantitative polymerase chain reaction; MMP: matrix metalloprotease; siADRB2: small interfering RNA for ADRB2.

Figure 7. ADRB2 overexpression reversed the inhibitory effect of CDN on metastatic lung nodules in CRC metastasis model. (A) In the presence of pulmonary metastatic nodules in nude mice 5 weeks after tail vein injection of the tumour cells, the number of metastatic nodules in the lung was counted. ***p < 0.001 vs. control; ^#p < 0.05 vs. NC + CDN. CDN: cardamonin; CRC: colorectal cancer.