Aflatoxin B1 enhances pyroptosis of hepatocytes and activation of Kupffer cells to promote liver inflammatory injury via dephosphorylation of cyclooxygenase-2: an in vitro, ex vivo and in vivo study.

Aflatoxin B1 (AFB1), a food contaminant derived from Aspergillus fungi, has been reported to cause hepatic immunotoxicity via inflammatory infiltration and cytokines release. As a pro-inflammatory factor, cyclooxygenase-2 (COX-2) is widely involved in liver inflammation induced by xenobiotics. However, the mechanism by which AFB1-induced COX-2 regulates liver inflammatory injury via hepatocytes-Kupffer cells (KCs) crosstalk remains unclear and requires further elucidation. Here, we established a COX-2 upregulated model with AFB1 treatment in vivo (C57BL/6 mice, 1 mg/kg body weight, i.g, 4 weeks) and in vitro (human liver HepaRG cells, 1 µM for 24 h). In vivo, AFB1-treated mice exhibited NLRP3 inflammasome activation, inflammatory infiltration, and increased recruitment of KCs. In vitro, dephosphorylated COX-2 by protein phosphatase 2A (PP2A)-B55δ promoted NLRP3 inflammasome activation, including mitochondrial translocation of NLRP3, caspase 1 cleavage, and IL-1ß release. Moreover, phosphorylated COX-2 at serine 601 (p-COX-2Ser601) underwent endoplasmic reticulum (ER) retention for proteasome degradation. Furthermore, pyroptosis and inflammatory response induced by AFB1 were relieved with COX-2 genetic (siPTGS2) intervention or pharmaceutic (celecoxib, 30 mg/kg body weight, i.g, 4 weeks) inhibition of COX-2 via NLRP3 inflammasome suppression in vivo and in vitro. Ex vivo, in a co-culture system with murine primary hepatocytes and KCs, activated KCs induced by damaged signals from pyroptotic hepatocytes, formed a feedback loop to amplify NLRP3-dependent pyroptosis of hepatocytes via pro-inflammatory signaling, leading to liver inflammatory injury. Taken together, our data suggest a novel mechanism that protein quality control of COX-2 determines the intracellular distribution and activation of NLRP3 inflammasome, which promotes liver inflammatory injury via hepatocytes-KCs crosstalk.

HBx combined with AFB1 triggers hepatic steatosis via COX-2-mediated necrosome formation and mitochondrial dynamics disorder.

Hepatitis B virus (HBV) infection and aflatoxin B1 (AFB1) exposure have been recognized as independent risk factors for the occurrence and exacerbation of hepatic steatosis but their combined impacts and the potential mechanisms remain to be further elucidated. Here, we showed that exposure to AFB1 impaired mitochondrial dynamics and increased intracellular lipid droplets (LDs) in the liver of HBV-transgenic mice in vivo and the hepatitis B virus X protein (HBx)-expressing human hepatocytes both ex vivo and in vitro. HBx combined with AFB1 exposure also up-regulated receptor interaction protein 1 (RIP1), receptor interaction protein 3 (RIP3) and activated mixed lineage kinase domain like protein (MLKL), providing evidence of necrosome formation in the hepatocytes. The shift of the mitochondrial dynamics towards imbalance of fission and fusion was rescued when MLKL was inhibited in the HBx and AFB1 co-treated hepatocytes. Most importantly, based on siRNA or CRISPR/Cas9 system, we found that the combination of HBx and AFB1 exposure increased cyclooxygenase-2 (COX-2) to mediate up-regulation of RIP3 and dynamin-related protein 1 (Drp1), which in turn promoted location of RIP3-MLKL necrosome on mitochondria, subsequently exacerbated steatosis in hepatocytes. Taken together, these findings advance the understanding of mechanism associated with HBx and AFB1-induced hepatic necrosome formation, mitochondrial dysfunction and steatosis and make COX-2 a good candidate for treatment.

Downregulation of mitochondrial cyclooxygenase-2 inhibits the stemness of nasopharyngeal carcinoma by decreasing the activity of dynamin-related protein 1.

Cancer stem cells (CSCs) are a small subset of malignant cells, possessing stemness, with strong tumorigenic capability, conferring resistance to therapy and leading to the relapse of nasopharyngeal carcinoma (NPC). Our previous study suggested that cyclooxygenase-2 (COX-2) would be a novel target for the CSCs-like side population (SP) cells in NPC. In the present study, we further found that COX-2 maintained the stemness of NPC by enhancing the activity of mitochondrial dynamin-related protein 1 (Drp1), a mitochondrial fission mediator, by studying both sorted SP cells from NPC cell lines and gene expression analyses in NPC tissues. Using both overexpression and knockdown of COX-2, we demonstrated that the localization of COX-2 at mitochondria promotes the stemness of NPC by recruiting the mitochondrial translocation of p53, increasing the activity of Drp1 and inducing mitochondrial fisson. Inhibition of the expression or the activity of Drp1 by siRNA or Mdivi-1 downregulates the stemness of NPC. The present study also found that inhibition of mitochondrial COX-2 with resveratrol (RSV), a natural phytochemical, increased the sensitivity of NPC to 5-fluorouracil (5-FU), a classical chemotherapy drug for NPC. The underlying mechanism is that RSV suppresses mitochondrial COX-2, thereby reducing NPC stemness by inhibiting Drp1 activity as demonstrated in both the in vitro and the in vivo studies. Taken together, the results of this study suggest that mitochondrial COX-2 is a potential theranostic target for the CSCs in NPC. Inhibition of mitochondrial COX-2 could be an attractive therapeutic option for the effective clinical treatment of therapy-resistant NPC.

Parthenolide inhibits cancer stem-like side population of nasopharyngeal carcinoma cells via suppression of the NF-κB/COX-2 pathway.

Cancer stem cells play a central role in the pathogenesis of nasopharyngeal carcinoma and contribute to both disease initiation and relapse. In this study, cyclooxygenase-2 (COX-2) was found to regulate cancer stem-like side population cells of nasopharyngeal carcinoma cells and enhance cancer stem-like cells' characteristics such as higher colony formation efficiency and overexpression of stemness-associated genes. The regulatory effect of COX-2 on cancer stem-like characteristics may be mediated by ABCG2. COX-2 overexpression by a gain-of-function experiment increased the proportion of side population cells and their cancer stemness properties. The present study also demonstrated that in contrast to the classical chemotherapy drug 5-fluorouracil, which increased the proportion of side population cells and upregulated the expression of COX-2, parthenolide, a naturally occurring small molecule, preferentially targeted the side population cells of nasopharyngeal carcinoma cells and downregulated COX-2. Moreover, we found that the cancer stem-like cells' phenotype was suppressed by using COX-2 inhibitors NS-398 and CAY10404 or knocking down COX-2 with siRNA and shRNA. These findings suggest that COX-2 inhibition is the mechanism by which parthenolide induces cell death in the cancer stem-like cells of nasopharyngeal carcinoma. In addition, parthenolide exhibited an inhibitory effect on nuclear factor-kappa B (NF-κB) nucler translocation by suppressing both the phosphorylation of IκB kinase complex and IκBα degradation. Taken together, these results suggest that parthenolide may exert its cancer stem cell-targeted chemotherapy through the NF-κB/COX-2 pathway.