Abstract
BackgroundHypoxia-mediated HIF-1α stabilization and NF-κB activation play a key role in carcinogenesis by fostering cancer cell survival, angiogenesis and tumor invasion. Gangliosides are integral components of biological membranes with an increasingly recognized role as signaling intermediates. In particular, ganglioside GD3 has been characterized as a proapoptotic lipid effector by promoting cell death signaling and suppression of survival pathways. Thus, our aim was to analyze the role of GD3 in hypoxia susceptibility of hepatocarcinoma cells and in vivo tumor growth.Methodology/Principal FindingsWe generated and characterized a human hepatocarcinoma cell line stably expressing GD3 synthase (Hep3B-GD3), which catalyzes the synthesis of GD3 from GM3. Despite increased GD3 levels (2–3 fold), no significant changes in cell morphology or growth were observed in Hep3B-GD3 cells compared to wild type Hep3B cells under normoxia. However, exposure of Hep3B-GD3 cells to hypoxia (2% O2) enhanced reactive oxygen species (ROS) generation, resulting in decreased cell survival, with similar findings observed in Hep3B cells exposed to increasing doses of exogenous GD3. In addition, hypoxia-induced c-Src phosphorylation at tyrosine residues, NF-κB activation and subsequent expression of Mn-SOD were observed in Hep3B cells but not in Hep3B-GD3 cells. Moreover, MnTBAP, an antioxidant with predominant SOD mimetic activity, reduced ROS generation, protecting Hep3B-GD3 cells from hypoxia-induced death. Finally, lower tumor growth, higher cell death and reduced Mn-SOD expression were observed in Hep3B-GD3 compared to Hep3B tumor xenografts.ConclusionThese findings underscore a role for GD3 in hypoxia susceptibility by disabling the c-Src/NF-κB survival pathway resulting in lower Mn-SOD expression, which may be of relevance in hepatocellular carcinoma therapy.
Highlights
Hypoxia is a prominent characteristic of advanced solid tumors and a major determinant of malignant progression and therapy responsiveness [1,2]
These findings underscore a role for GD3 in hypoxia susceptibility by disabling the c-Src/NF-kB survival pathway resulting in lower Mn-SOD expression, which may be of relevance in hepatocellular carcinoma therapy
Our results indicate that overexpression of GD3 synthase increases the levels of GD3, which is synthesized from endogenous GM3, rendering Hep3B cells susceptible to hypoxia-induced reactive oxygen species (ROS) generation by suppressing the hypoxia-mediated NF-kB activation via c-Src, which results in lower expression of the kB-dependent antioxidant Mn-SOD
Summary
Hypoxia is a prominent characteristic of advanced solid tumors and a major determinant of malignant progression and therapy responsiveness [1,2]. The molecular mechanisms responsible for the cellular adaptation to hypoxia is still under debate, with prolylhydroxylases playing a fundamental role [3], mitochondrial reactive oxygen species (ROS) generation is believed to contribute to this process, as hypoxia-induced mitochondrial ROS has been shown to determine HIF-1a stabilization and NF-kB activation [4,5]. Despite a key structural role in biological membranes, glycosphingolipids (GSLs) are increasingly recognized as secondary intermediates that participate in various cellular processes, including cell adhesion, differentiation, signal transduction and cell death [6,7]. Ganglioside GD3 (GD3) has been identified as a lipid death effector [8], with a dual mechanism involving its interaction with mitochondria leading to activation of apoptotic pathways [9,10] and the suppression of survival programs mediated by NF-kB activation [11,12]. Our aim was to analyze the role of GD3 in hypoxia susceptibility of hepatocarcinoma cells and in vivo tumor growth
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