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.
ABSTRACT
Objective:To investigate the anti-tumor effect mechanism of atractylenolide Ⅱ by studying its effect on macrophage polarization. Method:Phorbol myristate acetate （PMA） was used to induce THP-1 cells differentiation into macrophages， and methylthiazolyldiphenyl-tetrazolium bromide（MTT）colorimetric assay was used to detect the effect of different concentrations of atractylenolide Ⅱ on macrophage growth at different time points to screen out the safe concentration of atractylenolide Ⅱ. The macrophages were treated with different concentrations of atractylenolide Ⅱ for 24 hours and then were co-cultured with gastric cancer cells. The survival of the two types of cells was observed under light microscope. The proliferation of gastric cancer cells was detected by MTT assay to determine the effective administration concentrations of atractylenolide Ⅱ. Cells were divided into blank group， model group， atractylenolide Ⅱ high dose group （200 mg·L-1）， atractylenolide Ⅱ medium dose group （100 mg·L-1）， and atractylenolide Ⅱ low dose group（50 mg·L-1）. Wound healing assay was carried out to observe the effects of different concentrations of atractylenolide Ⅱ on the migration and morphology of gastric cancer cells. The expression levels of M1 and M2 macrophage surface markers CD86 and CD206 were detected by flow cytometry analysis（FCM）. Quantitative polymerase chain reaction（Real-time PCR）and Western blot were used to detect M1， M2 macrophage-associated tumor necrosis factor （TNF） -α， human leukocyte antigen 2 （HLA-DRA）， CD80， transforming growth factor （TGF）-β， interleukin （IL） -10 and IL-6 genes and protein expression. Western blot was used to detect intracellular phosphatidyl inositol kinase （PI3K） and p-PI3K protein expression in macrophages. Result:When the concentration of atractylenolide Ⅱ was 1， 10， 50， 100， 200 mg·L-1， it showed no inhibition on macrophage growth. As compared with the model group， macrophages treated with 50， 100， 200 mg·L-1 atractylenolide Ⅱ significantly inhibited tumor cell proliferation （P<0.01）. As compared with the model group， the migration rate of tumor cells in the atractylenolide Ⅱ （200，100 mg·L-1） groups decreased （P<0.05）. The expression levels of CD86 on M1 macrophage surfacen in the atractylenolide Ⅱ （200，100，50 mg·L-1） groups were increased（P<0.05，P<0.01）， and the expression levels of CD206 on M2 macrophagen in the atractylenolide Ⅱ （200 mg·L-1） group were decreased （P<0.05）. The expression levels of M1 macrophage-associated cytokines TNF-α， HLA-DRA， CD80 mRNA in the atractylenolide Ⅱ （200，100 mg·L-1） groups were increased（P<0.05，P<0.01）， and TNF-α protein expression in the atractylenolide Ⅱ （200 mg·L-1） group was increased （P<0.05）， M2 type macrophage-associated cytokine TGF-β mRNA expression levels in the atractylenolide Ⅱ （50 mg·L-1） group were decreased， and IL-10， IL-6 protein expression levels in the atractylenolide Ⅱ （200 mg·L-1） group were decreased （P<0.05，P<0.01）. The expression levels of p-PI3K protein in the atractylenolide Ⅱ （200，100 mg·L-1） groups were also decreased（P<0.05，P<0.01）. Conclusion:Atractylenolide Ⅱ could induce the polarization of macrophages to M1 type by reducing the expression of p-PI3K in macrophages and inhibiting the proliferation and migration of gastric cancer cells.