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Studies on the Mechanisms of TNFα-and Oridonin-induced Murine Fibrosarcoma L929Cell Death

Author: YeYuanChao
Tutor: ChiDaoQiao
School: Shenyang Pharmaceutical University
Course: Pharmacology
Keywords: TNFα necroptosis autophgy zVAD p38-NF-κB NO-ERK-p53 apoptosis L929cell
CLC: R285.5
Type: PhD thesis
Year: 2012
Downloads: 148
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Abstract

Necroptosis has been involved in inflammatory and cancer, while its detailed mechanisms remain unclear. It was reported that tumor necrosis factor alpha (TNFa) induced murine fibrosarcoma L929cell necroptosis. In this study, we further investigated the molecular mechanisms in TNFa-induced necroptosis and autophagy, and the relationship between these two processes.We found that TNFa-treated L929cells showed typical necrotic features. Necroptosis inhibitor necrostatin-1(Nec-1) completely blocked TNFa-induced cell death, and caspase-8signaling pathway was not activated. These suggested that TNFa-induced cell death was associated with necroptosis. The cytotoxicity of TNFa was accompanied by decreased expressions of phosphorylated p38mitogen-activated protein kinase (p-p38) and nuclear factor-kappa B (NF-кB), while ERK and JNK were not involved. Moreover, inhibition of p-p38and NF-кB by chemical inhibitors or siRNA augmented these necroptotic responses to TNFa. Inhibition of p38activation suppressed NF-kB expression. Pan-caspase inhibitor z-VAD-fink (zVAD) exacerbated TNFa-induced necroptosis. Combined treatment with TNFa and zVAD further decreased the expressions of p-p38and NF-кB compared with TNFa alone treatment. Together, these indicated that suppression of p38-NF-кB survivial signaling pathway promoted necroptotic cell death in TNFa-treated L929cells.Autophagy was accompanied with necroptosis. We also found that TNFa induced L929cell autophagy, demonstrated by extensive autophagic vacuolization, the punctuate distribution of MDC staining and GFP-LC3, as well as Beclin1activation and the conversion from LC3Ⅰ to LC3Ⅱ. Subsequently, we found that p38-NF-кB also inhibited TNFa-induced autophagy. Further, Nec-1blocked TNFa-or TNFa+zVAD-induced autophagy; however, inhibition of autophagy by3-methyladenine (3MA) or small interfering RNA (siRNA) against Beclin1augmented TNFa-induced necropotosis, while, autophagy inhibition did not influence TNFa+zVAD-induced necroptosis. These results suggested that autophagy was a downstream consequence of necroptosis, and had a negative-feedback function to necroptosis in TNFa-treated L929cells, but not in the presence of zVAD. Subsequently, TNFa administration was accompanied with caspase-6activation. Inhibition of caspase-6activity by z-V-E(OMe)-I-D(OMe)-fink (zVEID) or caspase-6(p20) siRNA had no effect on necroptosis but promoted TNFa-induced autophagy. Meanwhile, autophagy inhibition further increased caspase-6activation. Caspase-6(p20) siRNA sequestered the increased necroptotic ratio by3MA pretreatment in TNFa-treated L929cells. In addition, caspase-6activation induced by TNFa adminstration was inhibited by zVAD. Further, high concentration of zVAD-induced autophagy also did not negatively regulate necroptosis because caspase-6was not activated.The involvement of reactive oxygen species (ROS) in induction of necroptosis of various cancer cells has been widely reported, thus we investigated the role of ROS in necroptosis and autophagy. We found that TNFa induced mitochondrial dysfunction accompanied with ROS production and cytochrome c release via RIP1, leading to necroptosis and resulting autophagic cell death.Our previous study showed that ROS production played an important role in oridonin-induced L929cell necroptosis and autophagy. Here, we further investigate the role of nitric oxide (NO) in oridonin-treated L929cells. Exposure of L929cells to oridonin led to intracellular NO production. Pretreatment with the NO synthases (NOS) inhibitor N-(3-(Aminomethyl)benzyl) acetamidine dihydrochloride (1400w), NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) or NO scavenger DL-dithiothreitol (DTT) inhibited oridonin-induced apoptosis and autophagy. In additon, oridonin induced ERK and p53activation and the interruption of ERK and p53activation by PD98059, pifithrin-a, or ERK siRNA decreased the oridonin-induced apoptosis and autophagy. The inhibition of NO production reduced oridonin-induced ERK and p53activation, and NO production was down-regulated by blocking ERK and p53activation.In conclusion, these findings indicate that TNFa induced L929cell necroptosis and autophagy via inhibiting p38-NF-KB pathway. Autophagy was a downstream consequence of necroptosis, and negatively regulated necroptosis when caspase-6was activated in TNFa-treated L929cells. Further, ROS production was involved in TNFa-induced L929cell necroptosis and autophagy. Oridonin-induced apoptosis and autophagy were modulated by the NO-ERK-p53signaling molecular mechanism in L929cells.

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