Docosahexaenoic acid (DHA) has been reported to induce tumor cell death by apoptosis. However, little is known about the effects of DHA on autophagy, another complex well-programmed process characterized by the sequestration of cytoplasmic material within autophagosomes. Here, we show that DHA increased both the level of microtubule-associated protein light-chain 3 and the number of autophagic vacuoles without impairing autophagic vesicle turnover, indicating that DHA induces not only apoptosis but also autophagy. We also observed that DHA-induced autophagy was accompanied by p53 loss. Inhibition of p53 increased DHA-induced autophagy and prevention of p53 degradation significantly led to the attenuation of DHA-induced autophagy, suggesting that DHA-induced autophagy is mediated by p53. Further experiments showed that the mechanism of DHA-induced autophagy associated with p53 attenuation involved an increase in the active form of AMP-activated protein kinase and a decrease in the activity of mammalian target of rapamycin. In addition, compelling evidence for the interplay between autophagy and apoptosis induced by DHA is supported by the findings that autophagy inhibition suppressed apoptosis and further autophagy induction enhanced apoptosis in response to DHA treatment. Overall, our results demonstrate that autophagy contributes to the cytotoxicity of DHA in cancer cells harboring wild-type p53.

Docosahexaenoic acid (DHA) induces autophagy-associated apoptotic cell death in wild-type p53 cancer cells via regulation of p53. The present study investigated the effects of DHA on PC3 and DU145 prostate cancer cell lines harboring mutant p53. Results show that, in addition to apoptosis, DHA increased the expression levels of lipidated form LC3B and potently stimulated the autophagic flux, suggesting that DHA induces both autophagy and apoptosis in cancer cells expressing mutant p53. DHA led to the generation of mitochondrial reactive oxygen species (ROS), as shown by the mitochondrial ROS-specific probe mitoSOX. Similarly, pretreatment with the antioxidant N-acetyl-cysteine (NAC) markedly inhibited both the autophagy and the apoptosis triggered by DHA, indicating that mitochondrial ROS mediate the cytotoxicity of DHA in mutant p53 cells. Further, DHA reduced the levels of phospho-Akt and phospho-mTOR in a concentration-dependent manner, while NAC almost completely blocked that effect. Collectively, these findings present a novel mechanism of ROS-regulated apoptosis and autophagy that involves Akt-mTOR signaling in prostate cancer cells with mutant p53 exposed to DHA.

Although altered metabolic pathway is an important diagnostic maker and therapeutic target in cancer, it is poorly understood in cancer stem cells (CSCs). Here we show that the CD133 (+) hepatocellular CSCs have distinct metabolic properties, characterized by more active glycolysis over oxidative phosphorylation, compared to the CD133 (−) cells. Inhibition of PDK4 and LDHA markedly suppresses CD133 (+) stemness characteristics and overcome resistance to sorafenib (current chemotherapeutic agent for hepatocellular cancer). Addition of glucose or lactate to CD133 (−) cells promotes CSC phenotypes, as evidenced by increased CD133 (+) cell population, elevated stemness gene expression and enhanced spheroid formation. Furthermore, the liver-specific miRNA, miR-122, inhibits CSC phenotypes by regulating glycolysis through targeting PDK4. Our findings suggest that enhanced glycolysis is associated with CD133 (+) stem-like characteristics and that metabolic reprogramming through miR-122 or PDK4 may represent a novel therapeutic approach for the treatment of hepatocellular cancer.

MiR-122, a pivotal liver specific miRNA, has been implicated in several liver diseases including hepatocellular carcinoma (HCC) and hepatitis C and B viral infection. This study aimed to explore epigenetic regulation of miR-122 in human hepatocellular carcinoma (HCC) cells and to examine the effect of hepatitis C virus (HCV) and hepatitis B virus (HBV). We performed microRNA microarray analysis and identified miR-122 as the most up-regulated miRNA (6-fold) in human hepatocellular cancer cells treated with 5′aza-2′deoxycytidine (5-Aza-CdR, DNA methylation inhibitor) and 4-phenylbutyric acid (PBA, histone deacetylation inhibitor). Real-time PCR analysis verified significant upregulation of miR-122 by 5′aza and PBA in HCC cells, and to a lesser extent in primary hepatocytes. Peroxisome proliferator activated receptor-gamma (PPARγ) and retinoid X receptor alpha (RXRα) complex was found to be associated with the DR1 and DR2 consensus site in the miR-122 gene promoter which enhanced miR-122 gene transcription. 5-Aza-CdR and PBA treatment increased the association of PPARγ/RXRα, but decreased the association of its co-repressors (N-CoR and SMRT), with the miR-122 DR1 and DR2 motifs. The aforementioned DNA-protein complex also contains SUV39H1, a H3K9 histone methyl transferase, which downregulates miR-122 expression. Our findings establish a novel role of the PPARγ binding complex for epigenetic regulation of miR-122 in human HCC cells. Moreover, we show that hepatitis B virus X protein (HBX) binds PPARγ and inhibits the transcription of miR-122, whereas hepatitis C viral particles exhibited no significant effect; these findings provide mechanistic insight into reduction of miR-122 in patients with HBV but not with HCV infection.

A stable and persistent Hepatitis C virus (HCV) replication cell culture model was developed to examine clearance of viral replication during long-term treatment using interferon-α (IFN-α), IFN-λ, and ribavirin (RBV). Persistently HCV-infected cell culture exhibited an impaired antiviral response to IFN-α+RBV combination treatment, whereas IFN-λ treatment produced a strong and sustained antiviral response that cleared HCV replication. HCV replication in persistently infected cells induced chronic endoplasmic reticulum (ER) stress and an autophagy response that selectively down-regulated the functional IFN-α receptor-1 chain of type I, but not type II (IFN-γ) or type III (IFN-λ) IFN receptors. Down-regulation of IFN-α receptor-1 resulted in defective JAK-STAT signaling, impaired STAT phosphorylation, and impaired nuclear translocation of STAT. Furthermore, HCV replication impaired RBV uptake, because of reduced expression of the nucleoside transporters ENT1 and CNT1. Silencing ER stress and the autophagy response using chemical inhibitors or siRNA additively inhibited HCV replication and induced viral clearance by the IFN-α+RBV combination treatment. These results indicate that HCV induces ER stress and that the autophagy response selectively impairs type I (but not type III) IFN signaling, which explains why IFN-λ (but not IFN-α) produced a sustained antiviral response against HCV. The results also indicate that inhibition of ER stress and of the autophagy response overcomes IFN-α+RBV resistance mechanisms associated with HCV infection.

Hedgehog (Hh)-signaling plays a critical role in liver development, regeneration, injury repair and carcinogenesis. Activation of Hh signaling has been observed in patients with nonalcoholic fatty liver diseases (NAFLD); however, the pathobiological function and regulatory mechanism of hepatic Hh signaling in the pathogenesis of NAFLD remain to be further defined. This study was designed to examine the effect and mechanism of hepatic Hh signaling in high fat diet (HFD)-induced NAFLD by using pharmacological Smoothened inhibitors (GDC-0449 and LED225) and by using liver specific Smo knockout (Smo LKO) mice. Administration of Smo inhibitors to HFD-fed wild type mice significantly reduced the numbers of activated macrophages and decreased the expression of pro-inflammatory cytokines (TNFα, IL-1β, MCP1 and IL-6) as assessed by F4/80 immunohistochemistry and qRT-PCR, respectively. The Smo inhibitors were noted to have variable effects on hepatic fat accumulation. We observed that liver specific deletion of Smo also reduced macrophage activation and inhibited pro-inflammatory cytokine expression, while it did not significantly alter fat accumulation in the liver. Mechanistically, we found that activation of Gli1 by Hh signaling in primary hepatocytes increased the production of osteopontin (OPN) which subsequently enhanced macrophage-mediated pro-inflammatory response via a paracrine signaling. Conclusions: Hepatocyte Hh signaling can promote liver inflammation through OPN-mediated macrophage activation; this mechanism importantly contributes to the progression of NAFLD.

Our findings disclose a novel TGF-β and H19 signaling axis via Sox2 in TICs that importantly regulates hepatocarcinogenesis. This article is protected by copyright. All rights reserved.

Background: Matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and urokinase-type plasminogen activator (uPA) are involved in colorectal cancer invasion and metastasis. There is still debate whether the activity of MMP-2 and MMP-9 differs between tumors located in the colon and rectum. We designed this study to determine any differences in the expression of MMP-2, MMP-9 and uPA system between colon and rectal cancer tissues.