| Colon Cancer and Primary Liver Cancer are the most commmon malignancies,and its incidence is rising annually.It was reported that soybean and fermented soybean inhibited the proliferation of tumor.Moreover,the antiproliferative effect of soybean was significantly increased after fermented by microorganism.In this study,human colon cancer cell line HT-29,Caco-2 and human liver cancer HepG2 was used as a cell model to study the antiproliferative effects of soymilk and feremented soymilk by Lactobacillus casei 16.The change of components and the expression of apoptosis-related proteins in HT-29 cells by soymilk and feremented soymilk were also study.Firstly,soymilk was fermented by Lactobacillus casei 16.The antiproliferative effect of water and 80% ethanol extracts was determined by methylene blue method.The results showed that soymilk fermented by L.casei 16 and soymilk extracts inhibited the proliferation of HT-29 cells,Caco-2 cells and HepG2 cells in time and dose dependent manner.Compared to the 80% ethanol and water extracts of unfermented soymilk,the antiproliferative effect of the L.casei 16 fermented soymilk on HT-29 cells,Caco-2 cells and HepG2 cells were 28.08±0.07%,34.31±1.47%,34.35±0.43%,31.23±1.08%,32.40±0.99% and 53.79±0.82% for incubation 72 h at 500 ?g/mL,significantly enhanced(p<0.05)at the same dose and time level.The extracts showed the highest inhibition rate on HepG2 cells.The antiproliferative effect of water extracts on HT-29 cells and HepG2 cells showed significantly higher than ethanol extracts(p<0.05),but there was no significant difference in Caco-2 cells.Secondly,the soy isoflavone contents in the soymilk and fermented soymilk ethanol extracts was determined by High performance liquid chromatography(HPLC).The results showed that the glucoside isoflavones contents such as daidzin,daidzin and genistin were decreased 91.21%,94.67% and 89.42%(p<0.05),while the contents of glucoside isoflavone such as daidzein,daidzein and genistein was significantly increased 11.67,4.04 and 17.0 times(p<0.05).The enhance of antiproliferation effect of 80% ethanol extracts might be due to the increase of the aglycones isoflavones.Then,the changes of protein content and amino acid in soymilk and fermented soymilk water extracts was determined by Tricine-SDS-PAGE and HPLC.The results showed that the protein was converted into amino acids during fermented due to proteases producted by L.casei 16.Fermentation only changes the composition of the amino acids without affecting the type of amino acids.The amino acid apart from Aspartic was increased by L.casei 16,while phenylalanine increased 5.52 times.The enhance of antiproliferation effect of water extracts might be due to the increase of the amino acids.Finally,the expression levels of apoptosis related proteins(Bax,Bcl-2 and PCNA)in HT-29 cells was determined by Western-blot to explore the mechanism of inhibition of cancer proliferation.The results showed that soymilk and feremented soymilk could significantly increase the expression of Bax and decrease the expression of Bcl-2 in HT-29 cells.Compared with HT-29 cells treated by soymilk,the expression of Bax/Bcl-2 in the HT-29 cells treated by L.casei 16 fermented soymilk extracts was significantly changed.In addition,the expression of PCNA in the HT-29 cells treated by soymilk and L.casei 16 fermented soymilk extracts decreased compared with the control HT-29 cells.Based on the results of this study,it was found that soymilk feremented by L.casei 16 increased significantly antiproliferation effect on HT-29 cells,Caco-2 cells and HepG2 cells due to the increase of aglycone isoflavone and amino acids. |
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