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

Abstract

BackgroundJS-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.MethodsThe 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.ResultsJS-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-tr...

Synergistic Effect of Benzethonium Chloride Combined with Endoxan against Hepatocellular Carcinoma in Rats through Targeting Apoptosis Signaling Pathway

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Purification of Gekko Small Peptide Fraction and Its Effect of Inducing Apoptosis of EC 9706 Esophageal Cancer Cells by Inhibiting PI3K/Akt/GLUT1 Signaling Pathway

“…The SMMC 7721 and EC 9706 cell lines were cultured in RPMI 1640 while the SiHa and DU 145 cell lines were cultured in MEM. The culture mediums were replaced with freshly completed mediums supplemented with 2 m m l ‐glutamine, 10 % FBS, and 100 units/mL of penicillin/streptomycin every two days [24] …”

“…The EC 9706 cells were seeded onto 24‐well plates at a density of 5×10 5 cells/well and incubated in RPMI 1640 containing 10 % FBS for 24 h. Once the cells grew to occupy 80 % of the wells and then incubated with LH‐20‐15 (15, 30 and 60 mg L −1 ) for 48 h. The cells were subsequently washed twice in cold PBS for 3 min each time, fixed in 4 % paraformaldehyde (PFA) at 4 °C for 30 min, and stained with Hoechst 33258 solution ( MCE , Monmouth Junction, NJ, USA) for a further 5 min. Morphological changes of the cellular cytoskeleton were observed using a fluorescence microscope [24] …”

“…The HRP‐conjugated secondary antibody was incubated for 1 h at room temperature. The bands were visualized using an ECL system, and the band intensity was evaluated using Gel‐Pro analyzer software [24,31] …”