Effect of Modified Lichongtang Combined with 5-Fluorouracil on Epithelial Mesenchymal Transition of Human Hepatoma Cell Line HepG2
10.13422/j.cnki.syfjx.20190621
- VernacularTitle: 理冲汤加减联合5-氟尿嘧啶对人肝癌细胞HepG2上皮间质转化的影响
- Author:
Hui WANG
1
;
Lu-zhou XU
2
;
Jian WU
2
;
Qing-min SUN
2
;
Min CHEN
2
;
Shen-lin LIU
2
Author Information
1. Yangzhong Hospital of Traditional Chinese Medicine, Yangzhong 212200, China
2. Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, China
- Publication Type:Research Article
- Keywords:
modified Lichongtang;
5-fluorouracil;
HepG2 cells;
synergistic effect;
epithelial mesenchymal transition
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2019;25(7):14-21
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To investigate the effect of modified Lichongtang combined with 5-fluorouracil (5-FU) on epithelial-mesenchymal transition (EMT) of human hepatoma HepG2 cells. Method: The growth of HepG2 cells was detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, and the effect of Chinese medicine and 5-FU alone or combined use on the growth of HepG2 cells was analyzed by the principle of efficacy. The growth curves of HepG2 cells were plotted to determine the relationship between drug effect and combination index as well as the interaction between drugs. Scratch test was used to detect the effect of modified Lichongtang combined with 5-FU on the migration of HepG2 cells. Cell invasion assay (transwell chamber) was used to detect the effect of modified Lichongtang combined with 5-FU on the invasion ability of HepG2 cells. Real-time quantitative polymerase chain reaction (PCR) was used to detect the effect of modified Lichongtang combined with 5-FU on EMT-related genes E-cadherin, N-cadherin and Zinc finger transcription factors (snail, twist) mRNA expression after 24 hours of treatment on HepG2 cells. The expression levels of E-cadherin, N-cadherin, Snail and Vimentin in HepG2 cells were detected by Western blot after treatment by modified Lichongtang combined with 5-FU for 24 hours. Result: MTT assay showed that with the increase of drug concentration, the inhibitory effect of modified Lichongtang, 5-FU alone or combined use on HepG2 cell growth was also increased. Statistical analysis showed that the combined use of these two drugs at a low dosage could produce better synergistic effect on HepG2 cells after 24 hours of treatment. Therefore, modified Lichongtang and 5-FU were selected to treat HepG2 cells for 24 hours. 25%inhibitory concentration (IC25) was 800 mg·L-1 modified Lichongtang, 3.125 mg·L-15-FU. Blank group, 5-FU group, Lichongtang+5-FU group, and modified Lichongtang group were set for follow-up experiments. Scratch and invasion experiments showed that modified Lichongtang, 5-FU alone and combined use can inhibit HepG2 cell migration and invasion ability (P<0.05, P<0.01). As compared with 5-FU group, the inhibitory effect was more obvious in modified Lichongtang+5-FU group. As compared with the blank group, the mRNA expression of E-cadherin were up-regulated, while the mRNA expression levels of N-cadherin, Snail and Twist were and down-regulated in the 5-FU group and the 5-FU+modified Lichongtang group (P<0.05, P<0.01). As compared with 5-FU group, the mRNA expression of E-cadherin was up-regulated while the mRNA expression levels N-cadherin, Snail and Twist were down-regulated in the 5-FU+modified Lichongtang group (P<0.05, P<0.01). Western blot showed that as compared with the blank group, E-cadherin protein expression was up-regulated while N-cadherin, Snail and Vimentin protein expression levels were down-regulated in 5-FU group and 5-FU+modified Lichongtang group (P<0.05, P<0.01). As compared with the 5-FU group, E-cadherin protein expression was up-regulated while N-cadherin, Snail, and Vimentin protein expression levels were down-regulated in 5-FU+modified Lichongtang group (P<0.05, P<0.01). Conclusion: Modified Lichongtang combined with 5-FU can produce a better synergistic effect on HepG2 cells at a low dosage for 24 hours, and can significantly inhibit the migration and invasion of hepatocellular carcinoma cells, up-regulate the expression of E-cadherin, down-regulate the expression of N-cadherin, Snail, Vimentin and Twist in hepatocellular carcinoma cells. Inhibition of tumor cell proliferation, migration, invasion and EMT-related gene expression may be associated with enhancing the efficacy of chemotherapy drugs, and may act as one of the mechanisms for synergistic effect of modified Lichongtang combined with 5-FU.
