Protein phosphatase 2A activation mechanism contributes to JS-K induced caspase-dependent apoptosis in human hepatocellular carcinoma cells

Abstract

Background: JS-K is a nitric oxide (NO) donor and could generate intracellularly high levels of NO. The study explores PP2A as a tumor suppressor is a major determinant mediating JS-K-caused apoptosis in human hepatocellular carcinoma (HCC) cells.

Methods: The human HCC cell lines (PLC5, Huh-7, Bel-7402, SMMC-7721 and HepG2) were used to assess effects of JS-K on cell viability, apoptosis induction and PP2A activation. Effects of JS-K on cell morphology, mitochondrial membrane potential, apoptosis and NO levels were determined in HCC cells expressing PP2A. Simultaneously, the expression of PP2A family including PP2A-A(α/β), PP2A-B55, PP2A-C(α/β) and the substrates of PP2A, such as β-catenin, c-Myc and p-Bcl-2 (Ser70) were detected in sensitive HCC cells. Furthermore, the role of NO in mediating the expression of PP2A was further validated with Z-VAD-FMK (a caspase inhibitor), Carboxy-PTIO (a NO scavenger), okadaic acid (OA, a PP2A inhibitor) and FTY720 (a PP2A agonist) in JS-K treated cells. In addition, the genetic manuplation of PP2A including overexpression and knockdown have been also performed in JS-K treated cells. Moreover, the rat model of primary hepatic carcinoma was established with diethylnitrosamine for 16 weeks to verify the anti-tumor effects of JS-K in vivo. Immunohistochemical and Western blot analysis were used to determine the expression of proteins in rat primary hepatic carcinoma tissues.

Results: JS-K significantly inhibited cell proliferation, increased apoptosis rate and activated PP2A activity in five HCC cells viability, especially SMMC7721 and HepG2 cells. It was characterized by loss of mitochondrial membrane potential, significant externalization of phosphatidylserine, nuclear morphological changes. Moreover, JS-K enhanced Bax-to-Bcl-2 ratio, released cytochrome c (Cyt c) from mitochondria, activated cleaved-caspase-9/3 and the cleavage of PARP, and decreased the expression of X-linked inhibitor of apoptosis protein (XIAP). Both Z-VAD-FMK and Carboxy-PTIO suppressed the activation of cleaved-caspase-9/3 and of cleaved-PARP in JS-K-treated sensitive HCC cells. Simultaneously, JS-K treatment could lead to the activation of protein phosphatase 2A-C (PP2A-C) but not PP2A-A and PP2A-B55, which subsequently inactivated and dephosphorylated the PP2A substrates including β-catenin, c-Myc, and p-Bcl-2 (Ser70). However, silencing PP2A-C could abolish both the activation of PP2A-C and down-regulation of β-catenin, c-Myc and p-Bcl-2 (Ser70) in sensitive HCC cells. Conversely, PP2A overexpression could enhance the effects of JS-K on activation of PP2A and down-regulation of β-catenin, c-Myc and p-Bcl-2 (Ser70). In addition, adding okadaic acid (OA), a PP2A inhibitor, abolished the effects of JS-K on apoptosis induction, PP2A activation and the substrates of PP2A dephosphorylation; FTY720, a PP2A agonist, enhanced the effects of JS-K including apoptosis induction, PP2A activation and the substrates of PP2A dephosphorylation. The mice exhibited a lower number and smaller tumor nodules in response to JS-K-treated group. A marked increase in the number of hepatocytes with PCNA-positive nuclei (proliferating cells) was evident in DEN group and tended to decrease with JS-K treatment. Furthermore, JS-K treatment could induce PP2A activation and the substrates of PP2A inactivation such as β-catenin, c-Myc and p-Bcl-2(Ser70) in DEN-induced hepatocarcinogenesis.

Conclusions: High levels of NO released from JS-K induces a caspase-dependent apoptosis through PP2A activation.

Keywords: Apoptosis; Hepatocellular carcinoma; JS-K; Nitric oxide; Protein phosphatase 2A.

Fig. 1

Differential effects of JS-K on cell death, apoptosis and PP2A activation in five HHC cells and L02 cells.a Dose-dependent effects of JS-K on cell death in the five HCC cells and L02 cells. The cells were exposed to JS-K at the indicated concentrations for 24, 48, 72 h. b Dose-dependent effects of JS-K on cell apoptosis in the five HCC cells and L02 cells. c-d Effects of JS-K on the expression of PP2A. The cells were treated with JS-K at the indicated concentration for 24 h. Cell lysates were prepared and assayed for PP2A by Western blotting. Data are mean ± SD. n = 3 for each concentration. *P < 0.05,**P < 0.01, vs. control group

Fig. 2

Effects of JS-K on cell morphology and MMP in SMMC7721 and HepG2 cells. The cells were treated with different concentrations of JS-K for 24 h. a The morphological observation were observed under a phase contrast microscopy (400 × magnification). b The morphological observation was determined by crystal violet staining assay (200 × magnification). c Morphological observation under fluorescence microscopy after Hoechst33342 staining. The arrows indicated the typical features of apoptotic cells (400 × magnification). d The change of MMP was detected by using JC-1 staining and analyzed by flow cytometric. The percentage in histogram of each profile represents the percentage of total cells with low fluorescence intensity. Data are mean ± SD. n = 3 for each concentration. *P < 0.05,**P < 0.01, vs. control group

Fig. 3

Effects of JS-K on apoptosis in SMMC7721 and HepG2 cells. a Western blotting analysis of Bcl-2, Bax, and Cyt c in mitochondria and cytosol of cells. b Western blotting analysis of caspase-9/− 3, cleaved caspase-9/− 3, PARP, cleaved PARP and XIAP of cells. c Effect of caspase inhibitors on JS-K-induced apoptosis. The cells were pretreated with caspase inhibitor Z-VAD-FMK (50 μM) for 1 h before treatment with 10 μM JS-K for 24 h and protein expression was assessed by Western blotting analysis. Data are mean ± SD. n = 3 for each concentration. *P < 0.05,**P < 0.01, vs. control group, ^#P < 0.05, ^##P < 0.01 vs. cells treated with JS-K alone

Fig. 4

NO was involved in the apoptosis induced by JS-K. a The levels of NO were determined using DAF-FM DA staining by fluorescence microscope. b The levels of NO were determined using DAF-FM DA staining by flow cytometry. c Effects of Carboxy-PTIO on the apoptosis in JS-K-treated cells. The cells were treated with the NO scavenger Carboxy-PTIO (50 μM) before treatment with 10 μM JS-K for 24 h and the apoptosis was assessed by flow cytometry. d Effects of Carboxy-PTIO on the apoptotic-related protein in JS-K-treated cells. The cells were treated with the NO scavenger Carboxy-PTIO (50 μM) before treatment with 10 μM JS-K for 24 h and the protein expressions of Bcl-2, Bax, cleaved-caspase-9/3 and cleaved-PARP were assessed by Western blotting analysis. Data are mean ± SD. n = 3 for each concentration. *P < 0.05,**P < 0.01, vs. control group, ^#P < 0.05, ^##P < 0.01 vs. cells treated with JS-K alone

Fig. 5

JS-K increased the activity of PP2A in SMMC7721 and HepG2 cells. a The levels of PP2A were measured by ELISA kit. The cells were treated with different concentrations of JS-K for 24 h and the cell lysates were prepared and assayed by enzyme-linked immunosorbent assay. b The effects of JS-K on protein levels of PP2A complexs. c Effects of JS-K on protein levels of PP2A substrates. d Effects of Carboxy-PTIO on the expression of PP2A-C and PP2A substrates. e Effects of JS-K on protein levels of PP2A substrates through silencing PP2A-C. f Effects of JS-K on protein levels of PP2A substrates through overexpression of PP2A-C. The cells were transfected for 48 h before treatment with JS-K for 24 h. Data are mean ± SD. n = 3 for each concentration. *P < 0.05, **P < 0.01, vs. cells untreated with JS-K of control siRNA /negative plasmid group, ^#P < 0.05, ^##P < 0.01 vs cells treated with JS-K of control siRNA /negative plasmid group, ^△P < 0.05, ^△△P < 0.01, vs cells untreated with JS-K of PP2A siRNA/PP2A cDNA group

Fig. 6

Effects of okadaic acid (OA) on JS-K-induced cell apoptosis and PP2A activation in SMMC7721 and HepG2 cells. a Effect of OA treatment as PP2A inhibitor on cell apoptosis. The cells were pretreated with OA (1 nM) for 1 h, and then stimulated with JS-K (10 μM) for 24 h. The apoptosis was assessed by flow cytometry. b Effect of OA treatment as PP2A inhibitor on the expression of apoptotic-related protein. c Effect of OA treatment as PP2A inhibitor on the expression of PP2A-C and its substrates. The cells were pretreated with OA (1 nM) for 1 h, and then stimulated with JS-K (10 μM) for 24 h. The expressions of protein were assessed by Western blotting analysis. Data are mean ± SD. n = 3 for each concentration. *P < 0.05,**P < 0.01, vs. control group, ^#P < 0.05, ^##P < 0.01 vs. cells treated with JS-K alone

Fig. 7

Effects of FTY720 on JS-K-induced cell apoptosis and PP2A activation in SMMC7721 and HepG2 cells. a Effect of FTY720 treatment as PP2A agonist on cell apoptosis. The cells were pre-treated with FTY720 (2 μM) for 1 h, and then stimulated with JS-K (10 μM) for 24 h. The apoptosis was assessed by flow cytometry. b Effect of FTY720 treatment as PP2A agonist on the expression of apoptotic-related protein. c Effect of FTY720 treatment as PP2A agonist on the expression of PP2A-C and its substrates. The cells were pretreated with FTY720 (2 μM) for 1 h, and then stimulated with JS-K (10 μM) for 24 h. The expressions of protein were assessed by Western blotting analysis. Data are mean ± SD. n = 3 for each concentration. *P < 0.05,**P < 0.01, vs. control group, ^#P < 0.05, ^##P < 0.01 vs. cells treated with JS-K alone

Fig. 8

JS-K inhibits the growth of rat primary hepatic carcinoma in vivo (n = 8). a The photographs of livers in control, DEN-treated and JS-K-treated groups. b-c IHC staining of PCNA and PP2A (200 × magnifcation) in control group, DEN group, and DEN+JS-K groups (0.25 mg/kg and 0.5 mg/kg). d The expression levels of PP2A-C, β-catenin, c-Myc and p-Bcl-2 in rat model of primary hepatic carcinoma were analyzed by western blot