TNFR1/TNF-α and mitochondria interrelated signaling pathway mediates quinocetone-induced apoptosis in HepG2 cells

Abstract

Quinocetone, a new quinoxaline 1, 4-dioxide derivative, has been widely used as an animal feed additive in China. This study was conducted to explore the molecular mechanisms of apoptosis induced by quinocetone in HepG2 cells. MTT assay revealed that the viability of HepG2 cells was significantly inhibited by quinocetone in a dose- and time-dependent manner. Quinocetone-induced apoptosis in HepG2 cells was characterized by cell and nuclei morphology change, cell membrane phosphatidylserine translocation, DNA fragmentation, cleavage of poly (ADP-ribose) polymerase (PARP) and a cascade activation of caspase-8, caspase-9 and caspase-3. Simultaneously, quinocetone induced HepG2 cell cycle arrest, which was supported by overexpression of p21. Cytochrome c release was caused by the mitochondrial membrane potential dissipation, a process related to quinocetone-induced Bid cleavage and elevated Bax/Bcl-2 ratio. Moreover, quinocetone treatment caused the up-regulation of TNF-α and TNFR1 in HepG2 cells. Both soluble TNFR1 receptors and caspase inhibitors suppressed quinocetone-induced apoptosis. In addition, the protein levels of p53, p-p38 and p-JNK were increased in quinocetone-treated cells. Taken together, quinocetone induced apoptosis in HepG2 cells via activation of caspase, interaction of TNF-α and TNFR1 and modulation of the protein levels of Bid, Bax and Bcl-2, involving the participation of p53, p38 and JNK.

Keywords: 2′-(4-Ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole trihydrochloride; 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; AV; Apoptosis; Caspase; Cell cycle arrest; FCS; HepG2; Hoechst 33342; MAPKs; MEM; MMP/△ψ(m); MOMP; MTT; Mitochondria; NC; PARP; PCR; PI; PMSF; QdNOs; Quinocetone; Rh123; SDS–PAGE; TBST; TNF-α; TUNEL; Tris–HCl; annexin V-FITC; fetal calf serum; human liver carcinoma cell line; minimum essential medium; mitochondrial membrane potential; mitochondrial outer membrane permeabilization; mitogen-activated protein kinases; nitrocellulose; phenylmethyl sulfonylfluoride; poly(ADP-ribose) polymerase; polymerase chain reaction; propidium iodide; quinoxaline 1,4-dioxides; rhodamine123; sodium dodecyl sulfate polyacrylamide gel electrophoresis; terminal deoxynucleotidyl transferase mediated dUTP nick end labeling; tris (hydroxymethyl) aminomethane hydrochloride; tris buffered saline with Tween 20.