Role of the 26S proteasome inhibitor b-AP15 on Jurkat cells and RS4; 11 cells

Abstract

Objective To investigate the effect of 26S proteasome inhibitor b-AP15 on proliferation and apoptosis of Jurkat cells and RS4; 11 cells and to explore its possible mechanism. Methods Jurkat cells and RS4; 11 cells were cultured under asepsis condition. According to the presence of b - AP15 during culturing, Jurkat cells and RS4; 11 cells were divided into four groups: ①Jurkat cells experimental group, ②Jurkat cells control group, ③RS4; 11 cells experimental group, ④RS4; 11 cells control group. Jurkat cells experimental group and RS4; 11 cells experimental group were cultured with 0.05, 0.10, 0.50, 1.00, 5.00 μmol/L b-AP15 for 24 h and 48 h, while Jurkat cells control group and RS4; 11 cells control group were cultured synchronously using RPMI1640 medium. The cell growth curves of the four groups were analyzed by CCK-8. The cell apoptosis of the four groups were analyzed by Annexin V- allophycocyanin (APC)/7-aminoactinomycin D (7-AAD) double labeling flow cytometry. The cell cycle changes of the four groups were analyzed by flow cytometry.The mRNA expressions levels of B cell lymphoma/ leukemia-2 associated X protein (Bax) gene, B cell lymphoma/ leukemia (Bcl)-2 gene, X-linked inhibitor of apoptosis protein (XIAP) gene, cyclin-dependent kinase (CDK)1 gene and cysteine-aspartic proteases (caspase)-3 gene of the four groups were determined by real- time PCR. Results ①b-AP15 significantly inhibited the growth of Jurkat cells and RS4; 11 cells, and both the inhibitory effect were in a time-and dose-dependent manner. The IC50 values of Jurkat cells were(1.52±0.35)μmol/L and(0.54±0.01)μmol/L after culturing with b-AP15 for 24 h and 48 h. The IC50 values of RS4; 11 cells were (0.97±0.02)μmol/L and(0.08±0.03)μmol/L after culturing with b-AP15 for 24 h and 48 h. ②The apoptosis rates of Jurkat cells were significantly higher than the control groups after cultured with 1.5 μmol/L b-AP15 (P<0.001). The apoptosis rates of RS4; 11 cells significantly higher than the control groups after cultured with 1.0 μmol/L b-AP15 (P<0.001). Meanwhile, The apoptosis rates of both Jurkat cells and RS4; 11 cells cultured with b-AP15 for 48 h were significantly higher than these for 24 h (t=11.887, 7.449; P<0.001). ③ Compared with Jurkat cells control group, cell cycle of Jurkat cells experimental group cultured with 1.5 μmol/L b-AP15 for 24 h was arrested at G2/M period (t=10.672, P<0.05). At the same time, the proportion of S phase cells were reduced significantly (t=19.053, P<0.05). Compared with RS4; 11 cells control group, cell cycle of RS4; 11 cells experimental group cultured with 1.0 μmol/L b-AP15 for 24 h was also arrested at G2/M period (t=13.643, P<0.05). and the proportion of S phase cells were also reduced significantly(t=6.992, P<0.05). ④For Jurkat cells experimental group and RS4; 11 cells experimental group, the real-time PCR assay revealed that the relative mRNA expression levels of Bax gene, caspase-3 gene were increased significantly, while the relative mRNA expression levels of Bcl-2 gene, XIAP gene were reduced significantly. Meanwhile, mRNA expression level of the cell cycle related gene CDK1 was reduced significantly compared with their own control groups (P<0.05). The mRNA expression level of caspase-3 gene in RS4; 11 cells experimental group increased more significantly than that of Jurkat cells experimental group (P<0.05). Conclusion b-AP15 can inhibit proliferation and induce apoptosis of Jurkat cells and RS4; 11 cells, and the cell cycle of the two cells were both arrested at G2/M period. The mechanism of b-AP15 promoting apoptosis may be related with up-regulating the expression levels of Bax gene, caspase-3 gene and down-regulating the expression levels of Bcl-2 gene, XIAP gene. And the cell cycle arrested at G2/M period may be associated with down-regulating the expression levels of CDK1 gene. Key words: Protease inhibitor; Cell proliferation; Apoptosis; Cell cycle; Jurkat cells; RS4; 11 cells; b-AP15