Inhibition of invasion and metastasis of human liver cancer HCCLM3 cells by portulacerebroside A

Figures & data

Figure 1. The chemical structure of portulacerebroside A (PCA).

Table 1. Primers used in FQ-RT-PCR analysis.

Gene	Primer sequence	Species	Amplicon size (bp)
Nm23-H1	Forward: 5′-GTGAGCGTACCTTCATTGC-3′	Human	249
	Reverse: 5′-CCGTCTTCACCACATTCAG-3′
MTA1	Forward: 5′-CTTGAAGCTGATGGTGAATG-3′	Human	134
	Reverse: 5′-AGAGTTGTACCCTGAAGTTG-3′
MMP-2	Forward: 5′-TTGACGGTAAGGACGGACTC-3′	Human	134
	Reverse: 5′-GGCGTTCCCATACTTCACAC-3′
MMP-9	Forward: 5′-AAGGGCGTCGTGGTTCCAACTC-3′	Human	210
	Reverse: 5′-AGCATTGCCGTCCTGGGTGTAG-3′
TIMP-2	Forward: 5′-TTCGTCTCCCGTCTTTGG-3′	Human	185
	Reverse: 5′-TTTGAGCGGCTTCCTCTG-3′
GAPDH	Reverse: 5′-CACCCACTCCTCCACCTTTG-3′	Human	110
	Reverse: 5′-CCACCACCCTGTTGCTGTAG-3′

Figure 2. Effects of portulacerebroside A on adhesive ability of HCCLM3 cells. The cells were pre-treated with various concentrations of portulacerebroside A (PCA) for 12 h in a 96-well plate and the adhesion rate of the cells was determined at 0.5 h, 1 h, and 1.5 h. **,ΔΔ, ††p < 0.01 compared with the control group. Data are expressed as the mean ± SD, n = 6.

Figure 3. Effects of portulacerebroside A on invasive ability of HCCLM3 cells. After 6 h of pretreatment with designated concentrations of portulacerebroside A (PCA), the cells were incubated in transwell cabinets for a further 24 h, and the invasive cell number (A) was counted. (B) Representative images (×20) for each group. **p < 0.01 compared with the control group. Data are expressed as the mean ± SD, n = 6.

Figure 4. Effects of portulacerebroside A on migration ability of HCCLM3 cells. The cells were subjected to a cross line wound by a sterile pipette tip in a 6-well plate and were then incubated with designated concentrations of portulacerebroside A (PCA) for 24 h and 48 h. The migration cells were then counted (A). (B) and (C) display typical images (×10) of cells treated with PCA. **ΔΔp < 0.01 compared with the control group. Data are expressed as the mean ± SD. n = 6.

Table 2. Effects of PCA on the mRNA expression of metastasis-related parameters.

Group	Dose (μg/ml)	nm23-H1 (%)	MTA1 (%)	MMP-2 (%)	MMP-9 (%)	TIMP-2 (%)
Control	–	4.85 ± 0.47	57.44 ± 3.93	13.21 ± 1.46	7.72 ± 0.38	1.59 ± 0.26
PCA	2.5	9.23 ± 0.86**	23.28 ± 2.02**	6.74 ± 0.64**	3.67 ± 0.05**	5.14 ± 0.35**
PCA	5	13.34 ± 1.67**	11.75 ± 1.85**	3.95 ± 0.37**	2.70 ± 0.18**	9.81 ± 0.45**
PCA	10	19.50 ± 1.08**	6.33 ± 0.77**	1.37 ± 0.17**	0.91 ± 0.09**	13.96 ± 1.24**

After HCCLM3 cells were treated with various concentrations of PCA for 6 h, the mRNA expression of metastasis-related parameters was detected by FQ-RT-PCR. **p < 0.01 compared with the control group; data are expressed as the mean ± SD, n = 6.

Figure 5. Effects of portulacerebroside A on the protein expression of metastasis-related parameters. After 24 h of treatment with portulacerebroside A (PCA), the protein expression of MTA1 (A), nm23-H1 (A), RhoC (B), RhoA (B), Rac1/Cdc42 (B), MMP-2 (C), MMP-9 (C), and TIMP-2 (C) was detected by western blot. (D) Representative western blot images of HCCLM3 cells treated with PCA. *, Δ p < 0.05; **, ΔΔ p < 0.01 compared with the control group. Data are expressed as the mean ± SD, n = 3.