Abstract:

Objective To study the influence of betulin in the proliferation and apoptosis of human colon cancer SW480 cells, and to explore its related mechanism. Methods The SW480 cells were cultured in Dulbecco's modified Eagle's medium and used in this experiment after passages.The SW480 cells were divided into control group and different doses (1, 5, 10, 20, 50, 100 mg·L^-1) of betulin groups.MTT colorimetric assay was used to determine the inhibitory rates of proliferation of SW480 cells in various groups.The morphological changes of SW480 cells were detected by DAPI staining after treated with 15 mg·L^-1 betulin for different time(6, 12, 24 h).The apoptotic cells were quantified by flow cytometry (FCM);the activities of caspase-3, caspase-8 and caspase-9 were determined through Caspase Assay in vitro;Western blotting method was used to detect the breakage of poly (ADP-ribose) polymerase (PARP) and the protein expression of Bcl-2, Bcl-xL and cytochrome C. Results The MTT results showed that the inhibitory rates of proliferation of SW480 cells were gradually increased with the increasing of betulin dose (1, 5, 10, 20, 50, 100 mg·L^-1) in a dose-dependent manner compared with control group(P<0.05 or P<0.01).The 50% inhibitory concentration (IC₅₀) of SW480 cells were 12.875 mg·L^-1.The typical morphological changes of apoptosis were observed in SW480 cells with DAPI staining after induction in 15 mg·L^-1 betulin group such as membrane bubbles, chromatin condensation, nuclear condensation and deformation, especially at 12 or 24 h;compared with control group, the number of normal cells in the experimental group (12, 24 h)was reduced.The FCM results showed the percentage of cells at sub-G₁ arrest in 15 mg·L^-1 betulin group was increased compared with control group.Compared with control group, the activities of caspase-3 and caspase-9 were significantly increased (P<0.05 or P<0.01).The Western blotting method results showed that poly- (ADP-ribose) polymerase (PARP) was cleaved in 15 mg·L^-1 betulin group, the expression of apoptotic protein Bcl-xL was down-regulated, and the expression of cytochrome C was up-regulated, but the expression of Bcl-2 had no changes. Conclusion Betulin can inhibit the proliferation and induce the apoptosis of SW480 cells.This effect may be related to Bcl-xL degradation through caspase-9 related mitochondrial-dependent signal pathway.

Key words: betulin, colon neoplasms, SW480 cell, apoptosis, Bcl-xL