ABSTRACT
Objective:
To investigate the mechanism of Jianpi Yangzheng recipe in inhibiting aerobic
glycolysis by down-regulating the expression of
pyruvate kinase isoenzyme M2 (PKM2)
protein, in order to promote
apoptosis and inhibite
epithelial-mesenchymal transition(EMT)in
HCT116 cells of
colorectal cancer.
Method:
The effect of different concentrations of Jianpi Yangzheng recipe on
HCT116 cell proliferation was detected by methylthiazolyldiphenyl-tetrazolium
bromide(MTT)
colorimetry.
Flow cytometry was used to detect the effect of different concentrations of Jianpi Yangzheng recipe(2.0, 4.0, 8.0 g·L-1) on
HCT116 cell apoptosis. The effect of Jianpi Yangzheng recipe(2.0, 4.0, 8.0 g·L-1) on the migration and invasion
ability of
HCT116 cells was observed by
cell scratch and
cell invasion assay (Transwell). The effect of different concentrations of Jianpi Yangzheng recipe(2.0, 4.0, 8.0 g·L-1) on
glycolysis metabolism of
HCT116 cells were detected by
lactic acid (LD) test kit and
glucose assay kit, respectively.
Western blot was used to detect the expressions of
apoptosis-related
proteins, like
B lymphocyte tumor-2
gene (Bcl-2), Bcl-2 related X
protein (Bax) and EMT-related
proteins, like
epithelial cadherin (
E-cadherin),neurogenic
cadherin(
N-cadherin),
Vimentin, and PKM2, the key
protein of
glycolysis, in each group.
Result:
MTT assay showed that, compared with the blank group,
HCT116 cells were treated with Jianpi Yangzheng recipe for 48 h. With the increase of
drug concentration, the inhibitory effect of Jianpi Yangzheng recipe on the proliferation of
HCT116 cells was also increased; and when the concentration was 4.0 g·L-1, the inhibition rate of
HCT116 cells was about 53.87%. Therefore, 2.0,4.0,8.0 g·L-1 were selected as low, medium and high-
dose groups for the study. The
cell flow cytometry results indicated that, compared with the blank group, the low, medium and high-
dose groups all significantly induced the
apoptosis of
HCT116 cells (P<0.05), and the effect in inducing
apoptosis was more obvious with the increase of
drug concentration (P<0.05).
Cell scratch and Transwell showed that, compared with the blank group, all the groups had an inhibitory effect on migration and invasion of
HCT116 cells (P<0.05), and the effect was more significant with the increase of
drug concentration (P<0.05). The
determination of
lactic acid and
glucose indicated that compared with the blank group, with the increase of
drug concentration, the amount of
lactic acid produced by
cells in each group gradually decreased (P<0.05), while the
glucose dosage also gradually decreased (P<0.05).
Western blot showed that, compared with the blank group, the
protein expressions of
E-cadherin and Bax were up-regulated in groups with different concentrations, whereas the
protein expressions of
N-cadherin,
Vimentin, Bcl-2 and PKM2 were down-regulated (P<0.05).
Conclusion:
Jianpi Yangzheng recipe can effectively induce the
apoptosis of
HCT116 cells and inhibit EMT in
colorectal cancer. The possible mechanism may be related to the inhibition of aerobic
glycolysis pathway of
HCT116 cells by down-regulating PKM2
protein expression.