Betulinic Acid Induces Autophagy and Apoptosis of Human Colorectal Cancer SW620 Cells by Regulating PI3K/Akt/mTOR Signaling Pathway / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo investigate the effect of betulinic acid （BA） on apoptosis and autophagy of human colorectal cancer SW620 cells and the regulatory role of phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway. MethodCell viability was detected by methyl thiazolyl tetrazolium （MTT） colorimetry to determine the optimal administration time and dosage for subsequent experiments. Four groups were designed， including blank group and low-， medium-， and high-dose BA groups. Hematoxylin-eosin （HE） staining was conducted for the observation of SW620 cell morphology， and annexin-V/propidium iodide double staining for the determination of apoptosis rate in SW620 cells. Hoechst33258 staining and MDC staining were used for the observation of apoptosis and autophagy， respectively. Western blotting was employed to determine the protein levels of B-cell lymphoma/leukemia-2（Bcl-2）-associated X protein （Bax）， aspartate proteolytic enzyme-9 （Caspase-9）， activated aspartate proteolytic enzyme-3 （cleaved Caspase-3）， microtubule-associated protein 1 light chain 3 （LC3）， the mammalian homolog of yeast Atg6 （Beclin-1）， p62， phosphorylated PI3K （p-PI3K）， phosphorylated Akt （p-Akt）， and phosphorylated mTOR （p-mTOR） in SW620 cells. ResultBA inhibited the activity of SW620， HT29， and HCT116 cells in a concentration- and time-dependent manner. The cells treated with BA for 48 h had lower viability than those treated for 24 h （P<0.05， P<0.01）. The half maximal inhibitory concentration (IC50） value of BA at the time point of 48 h was also lower than that at the time point of 24 h （P<0.01）， and that for SW620 cells was the minimum. BA induced the apoptosis in a concentration-dependent manner and increased the autophagosomes. Compared with the blank group， BA increased the apoptosis rate （P<0.01）， up-regulated the protein levels of Bax， Caspase-9， cleaved Caspase-3， and LC3 Ⅱ （P<0.05， P<0.01）， and down-regulated the protein levels of p62， p-Akt， p-PI3K， and p-mTOR （P<0.01）. Additionally， medium- and high-dose BA up-regulated the protein level of beclin-1 （P<0.01）. ConclusionBA may inhibit the activity of SW620 cells by hindering the PI3K/Akt/mTOR signaling pathway to induce cell apoptosis and autophagy.