The role of protein phosphatase 2A B56β holoenzyme in the regulation of heavy metal CdCl2 induced cytotoxicity
10.3760/cma.j.issn.0253-9624.2015.05.010
- VernacularTitle:蛋白磷酸酶2A B56β全酶调控氯化镉诱导肝细胞毒性的作用研究
- Author:
Jinmiao ZHANG
1
;
Lu MA
;
San WANG
;
Wen CHEN
;
Liping CHEN
Author Information
1. 中山大学公共卫生学院预防医学系
- Keywords:
Phosphoprotein phosphatases;
Metallothionein;
Cytotoxicity
- From:
Chinese Journal of Preventive Medicine
2015;(5):429-435
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the role of holoenzyme containing Protein Phosphatase 2A B56βin regulating CdCl2 induced cytotoxicity. Method CdCl2-induced cytotoxicity in normal human cell line L-02,AFB1-transformed hepatic cell line L-02 RT-AFB1 and tumor cell line Bel7402 was measured by modified MTT assay. Stable cell lines L-02 SHAKT, L-02 SHB56β, L-02 RT-AFB1-B56β and Bel7402-B56β were generated by infecting L-02 cells or Bel7402 cells with retroviral vectors encoding lentiviral AKT shRNA, lentiviral B56βshRNA and B56β. The relative cell viability was measured in normal human cell line AFB1-transformed hepatic cell line and tumor cell line when treated by CdCl2(0,20,40,80, 160 μmol/L). After treated by wortmannin (2.5,5.0 μmol/L) combined with 40 μmol/L CdCl2,Western blot was applied to measure the expression of associated protein in L-02.Western blot was applied to measure the expression of B56β, MT (metallothionein), AKT, and p-AKT in these cell lines treated by CdCl2. Results The levels of MT were 0.12 ± 0.02, 0.06 ± 0.06 in L-02 RT-AFB1 and Bel7402,which were lower than L02 (0.92 ± 0.14) (F=1 148.16,P<0.001)when treated by 40μmol/L CdCl2.When treated by 40μmol/L CdCl2,the expression of p-AKT in L-02 SHAKT-1 and L-02 SHAKT-2 were 0.08 ± 0.02, 0.08 ± 0.05,which levels were lower than L-02 SHGFP(0.18 ± 0.15) (F=724.70,P<0.001);and the expression of MT were both 0.62 ± 0.16 in L-02 SHAKT-1 and L-02 SHAKT-2,which levels were higher than L-02 SHGFP (0.22 ± 0.14) (F=94.73,P<0.001).After treated by wortmannin (2.5,5.0 μmol/L) combined with 40 μmol/L CdCl2, the expression of p-AKT in L-02 were 0.28±0.07, 0.15±0.11,which levels were lower than wortmannin untreated cells (0.52± 0.11) (F=578.57,P<0.001);and the expreesion of MT were 1.62 ± 0.80, 1.08 ± 0.15,which levels were higher than wortmannin untreated cells (0.69 ± 0.18) (F=12.34,P<0.001).When treated by 40 μmol/L CdCl2,the levels of p-AKT in L-02 SHB56β-1 and L-02 SHB56β-2 were 0.57±0.13, 0.59±0.02,which were higher than L-02 SHGFP(0.32 ± 0.02) (F=87.16,P<0.001); and the levels of MT were 0.35 ± 0.07, 0.20 ± 0.03 in L-02 SHB56β-1 and L-02 SHB56β-2,which were lower than L-02 SHGFP (1.51 ± 0.13) (F=2 457.10,P<0.001). After treated by 40μmol/L CdCl2, the expression of p-AKT in L-02 RT-AFB1-B56βand Bel7402-B56βwere 0.10 ± 0.11, 0.09 ± 0.01,which were lower than L-02 RT-AFB1 (0.36 ± 0.01) and Bel7402 (0.43 ± 0.11) (F=877.62,P<0.001); and the levels of MT were 0.92 ± 0.13, 0.95 ± 0.08 in L-02 RT-AFB1-B56β and Bel7402-B56β,which were higher than L-02 RT-AFB1 (0.44 ± 0.12) and Bel7402 (0.77 ± 0.06) (F=51.97,P<0.001). Conclusion Protein phosphatase 2A complexes containing B56βparticipated in the regulation of MT expression through direct dephosphorylation of AKT, finally affected the cytotoxicity responding to CdCl2. Our study revealed a key signaling pathways of PP2A involved in heavy metals induced cytotoxicity.
