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


RESULTS
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.


CONCLUSIONS
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.
Lluis
Josep M
JM
Department of Cell Death and Proliferation, IIBB-CSIC, and Liver Unit, Hospital Clinic, IDIBAPS-CIBEK, CIBEREHD, Barcelona, Spain.
Llacuna
Laura
L
von Montfort
Claudia
C
Bárcena
Cristina
C
Enrich
Carlos
C
Morales
Albert
A
Fernandez-Checa
José C
JC
D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D016923:Cell Death; D006801:Humans; D000860:Hypoxia; D008055:Lipids / Q000737:chemistry; D008113:Liver Neoplasms / Q000378:metabolism*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D009368:Neoplasm Transplantation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D017382:Reactive Oxygen Species; D012799:Sialyltransferases / Q000096:biosynthesis*; D015398:Signal Transduction; D015533:Transcriptional Activation; D019061:src-Family Kinases
D008055:Lipids; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D012799:Sialyltransferases; C043662:alpha-N-acetylneuraminate alpha-2,8-sialyltransferase; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human