Increased chemosensitivity and elevated reactive oxygen species are mediated by glutathione reduction in glutamine deprived neuroblastoma cells.
OBJECTIVE
Glutamine is an essential amino acid for the synthesis of glutathione (GSH), the major endogenous antioxidant which protects cells from oxidative injury. To evaluate the effects of glutamine concentrations, cell growth, GSH levels, oxidative stress, and chemosensitivity were evaluated in neuroblastoma cell lines.


METHODS
Three human neuroblastoma cell lines (SMS-KCNR, SMS-KANR, SMS-LHN) were cultured with different concentrations of glutamine (2, 0.2 and 0 mM) under hypoxic (5% O(2)) or normoxic (20% O(2)) condition. Cell proliferation and chemosensitivity were determined by MTT assay, and the levels of intracellular GSH were measured by DTNB-GSSG reductase method. Cellular reactive oxidative species (ROS) were quantified by flow cytometry.


RESULTS
There was a significant decrease of cell growth in low glutamine (0.2 and 0 mM) compared with control (2 mM) in all three cell lines (P < 0.01), while adding GSH partially restored the reduced cell proliferation by low glutamine. The levels of GSH in neuroblastoma cells decreased significantly in low glutamine compared with the levels of control cells cultured in 2 mM glutamine (P < 0.05), and the accumulation of cellular ROS was significantly higher in 0 mM glutamine compared to the control. Moreover, glutamine deprivation significantly enhanced cytotoxicity of L-PAM in all three cell lines, which was abolished after addition of GSH.


CONCLUSIONS
Glutamine deprivation decreased cell proliferation and enhances cell chemosensitivity in neuroblastoma, which is presumably associated with GSH depletion.
Izaki
Sakurako
S
Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama City, 236-0004, Japan. sakura-306@cronos.ocn.ne.jp
Goto
Hiroaki
H
Yokota
Shumpei
S
D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D015687:Cell Hypoxia / Q000502:physiology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D005434:Flow Cytometry; D005973:Glutamine / Q000172:deficiency* / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D008558:Melphalan / Q000494:pharmacology; D009447:Neuroblastoma / Q000378:metabolism*; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism*
D018906:Antineoplastic Agents, Alkylating; D017382:Reactive Oxygen Species; D005973:Glutamine; D005978:Glutathione; D008558:Melphalan