GD3 synthase overexpression sensitizes hepatocarcinoma cells to hypoxia and reduces tumor growth by suppressing the cSrc/NF-kappaB survival pathway

Abstract

Background: Hypoxia-mediated HIF-1alpha stabilization and NF-kappaB 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 findings: We 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% O(2)) 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-kappaB 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.

Conclusion: These findings underscore a role for GD3 in hypoxia susceptibility by disabling the c-Src/NF-kappaB survival pathway resulting in lower Mn-SOD expression, which may be of relevance in hepatocellular carcinoma therapy.

Figure 1. Characterization of GD3 synthase-overexpressing Hep3B cells.

A. Representative image of lipids extracts resolved by HPTLC from Hep3B cells transfected with and empty vector (3B) or overexpressing GD3 Synthase (3B-GD3) isotopically labeled with ³H-galactose, and quantification of three independent experiments. Commercial non-labeled standards were run in parallel, being TLC plates stained with 5% orcinol solution. *p<0.05 vs. Hep3B control cells. B. GD3 synthase activity was measured in control Hep3B (3B) and Hep3B GD3-expressing cells (3B-GD3). GD3 Synthase protein levels were analyzed by western blot and compared with β-actin levels, used as a control. C. Confocal immunofluorescence was visualized in empty-vector containing and GD3 Synthase overexpressing Hep3B cells using antibodies anti-GD3 and anti-Mn-SOD. The graphs on the right panel represent the fluorescence intensity profile plotted from a to b direction in the merge images for the different cell lines (Mn-SOD in green and GD3 in red). D. Cell growth under normoxic conditions (n = 3). *p<0.05 vs. Hep3B control cells.

Figure 2. GD3 ganglioside sensitizes Hep3B cells to hypoxia and potentiates ROS generation.

A. Cell death in Hep3B cells growing under normoxia or hypoxia for 48 h, and treated with increasing doses of GD3. Cell viability measured 24 h later (n = 3). B. Time-dependent viability of Hep3B and Hep3B-GD3 cells exposed to 2% O₂ (n = 3). C, D. ROS generation evaluated with fluorescent probes HE (5 µM) and DCF (1 µM) during 30 min at 37°C (n = 4). *p<0.05 vs. Hep3B cells. #p<0.05 vs. hypoxic Hep3B cells.

Figure 3. Overexpression of GD3 blocks NF-κB transcriptional activity and downregulates Mn-SOD expression in hypoxic cells.

A. Representative western blots of p65 and HIF-1α from nuclear extracts of control (3B) and GD3-overexpressing (3B-GD3) cells using c-jun and β-actin as representative nuclear protein and cytosolic marker, respectively (n = 3). B, Luciferase activity in 3B and 3B-GD3 cells transfected with NF-κB luciferase reporter construct (n = 4). C. Representative immunoblots with c-Src, phospho-Tyr416Src and Mn-SOD antibodies and normalized by β-actin levels. D, Mn-SOD mRNA levels were analyzed by real-time PCR after 72 hours (n = 3). E, ROS production, and F, survival of Hep3B-GD3 in the presence or absence of Mn-SOD mimetic, MnTBAP (50 µM) after 72 h hypoxia (n = 4). *p<0.05 vs. Hep3B cells. #p<0.05 vs. hypoxic control.

Figure 4. GD3 synthase overexpression decreases subcutaneous tumor growth of Hep3B cells.

mRNA levels of PDK1 (A) and VEGF (B) in Hep3B cells from subcutaneous tumors in nude mice or from cells growing under normal culture conditions with 21% O₂ (n = 3–4). *p<0.05 vs. normoxic Hep3B cells. C, Tumor volume was measured in nude mice injected with Hep3B (empty vector) and Hep3B-GD3 cells (n = 5–6 animals per group). D, E. Representative TUNEL-staining in tumor samples from injected mice, and quantification of TUNEL-positive cells in each group as indicated in methods (n = 3-4). *p<0.05 vs. Hep3B-injected mice. F. mRNA levels of Mn-SOD from Hep3B and Hep3B-GD3 subcutaneous tumors (n = 4). *p<0.05 vs. Hep3B-injected mice.

Figure 5. Schematic diagram depicting the mechanism proposed to be involved in the susceptibility of Hep3B cells to hypoxia upon GD3 synthase overexpression.

In Hep3B cells, NF-κB activation via cSrc plays a key role in the resistance to hypoxia, in part, by inducing Mn-SOD expression. However, in Hep3B-GD3 cells the overgeneration of GD3 blocks the cSrc/NF-κB pathway potentiating the mitochondrial ROS production during hypoxia and contributing to hypoxia-mediated cell death.