Mechanism of Weichang' an on Autophagy of MKN45 Cells / 中国实验方剂学杂志

ABSTRACT

<b>Objective:To explore the effect of Weichang' an (WCA) on the autophagy of human gastric cancer MKN45 cells and its possible anti-cancer mechanism. <b>Method:MKN45 cells were cultured <italic>in vitro</italic> and incubated with different concentrations (250, 500, 1 000, 2 000 mg·L^-1) of WCA for 24, 48, 72 h. Cell counting kit-8 (CCK-8) assay was used to detect the cell proliferation. AO/EB Dyeing (AO) staining and monodansylcadaverin (MDC) staining were used to observe the changes of the effect of WCA on autophagosome and autophagic vesicles in gastric cancer MKN45 cells at 48 h. Real-time polymerase chain reaction (Real-time PCR) and Western blot were used to detect microtubule-associated protein 1 light chain 3 (LC3), Beclin1, sequestosome 1 (p62), human autophagy-related gene 5 (ATG5), human autophagy-related gene 7 (ATG7) mRNA and protein expression levels. <b>Result:WCA showed a dose-and-time-dependent growth inhibition at the concentration above 1 000 mg·L^-1. Compared with the blank group, WCA (500, 1 000, 2 000 mg·L^-1) significantly inhibited the proliferation of MKN45 cells (<italic>P</italic><0.05, <italic>P</italic><0.01). AO staining and MDC staining showed that autophagosomes and autophagic sacs increased with the rise of WCA concentration compared with the blank group. Real-time PCR and Western blot showed that the expressions of autophagy-related proteins LC3-Ⅱ, Beclin1, ATG5, ATG7, mRNA and protein increased gradually after WCA (1 000 mg·L^-1) intervention, while p62 expression decreased (<italic>P</italic><0.05, <italic>P</italic><0.01). <b>Conclusion:WCA induces the autophagy of human gastric cancer MKN45 cells in a time-and dose-dependent manner <italic>in vitro</italic>, which may be related to the up-regulation of LC3-Ⅱ, Beclin1, ATG5 and ATG7 as well as the down-regulation of p62 and LC3-Ⅰ.