The Effect Of Oxidative Stress On Cell Proliferation Regulated By Glutamate/Cystine Antiporter

Glutamate/cystine antiporter (xc-) is mainly located on astrocyte and neurone in the brain. xc- located in the cellular membrane is very important. In normal condition , the anionic form of extracellular cystine is transported into cells in exchange for intracellular glutamate with a stoichiometry of 1:1. Once inside a cell, cystine is rapidly reduced to cysteine, which can then enter glutathione and protein biosynthetic pathways. It is rate-limiting step of GSH synthesis. So, whether the function of xc- is normal is directly related to GSH synthesis.Malignant growth of glia cell will lead to glioma, which is a kind of malignant tumor threatening people's health. Its main bionomics are growing fast and invasion to normal brain tissue. Because there is no visible boundary between glioma and surrounding normal brain tissue resulting from its infiltrative growth, it is very difficult to be resected totally in the operation and is very easy to recur. At present, chemical therapy is also an important therapeutic tool. Although chemotherapeutics have some curative effects on glioma, their dose and side effects are evident and drug resistance is very easy to happen. So, it is very important to find some new antitumor drugs.Glioma cells uptake cystine exclusively depending on xc- system to maintain cellular anti-oxiadation ability. Sulfasalazine (SAS) is a kind of anti-inflammatory drugs frequently used clinically. It is used to treat inflammatory bowel disease and rheumatic arthritis. In recent researches, it is shown that SAS can inhibit xc-, then decrease cystine uptake by glioma cell, impact the synthesis of GSH which is a kind of antioxidant and inhibit proliferation of glioma cell. But when SAS is used singly and the effect is evident, its dose is large, so its side effect on normal cell can not be ignored.Oxidative stess plays a very key role in the process of glioma cell damage. When the oxidative metabolites increase or the ability of antioxidation and protection is not enough, ROS will accumulate and generate toxicity to cell through mitochondria damage. Nowadays, in clinically treating glioma, it is imporsibe to use oxidant to patients singlely. In this study, glioma C6 cells, primary astrocytes and pheochromobalstoma PC-12 cells were cultured to investigate the effect of inhibitor of xc---SAS on these three kinds of cells proliferation and the influence of different kinds of oxidants (VK₃ and H₂O₂) on this effect. Through analyzing the role of glutamate/cystine antiporter in tumor generation, rationale for clinic treatment of glioma will be provided.Materials and methods:1 Materials:(1) Experimental animal and cell line: Wistar neonate rat of 2nd～3rd day after newly born(red-skinned rat ), glioma C6 cell and pheochromobalstoma PC-12 cell .(2) Drugs: SAS, VK₃, H₂O₂2 Methods:(1) Culture of primary astrocyte, C6 and PC-12 cell.(2) Cell vitality rate of primary astrocyte, C6 and PC-12 cell treated with single use or combined use of SAS, VK₃ and H₂O₂ was determined through the methods of cell morphology and MTT assay.(3) Cell apoptosis was determined through TUNEL and LDH methods after C6 cells was treated with drugs.(4) The level of ROS in C6 cell was determined though DCFH-DA method using confocal microscopy.(5) GSH contents in C6 cell were determined.(6) The distribution of NF-κB (p65) protein was observed through immunocytochemical stain.(7) The mRNA levels of xCT, GCLC, GCLM, EAAT3, IκB-α,β-trcp, CyclinD1, Bcl-2, Bax and CLIC4 were investigated through RT-PCR method.(8) The protein levels of NF-κB (p65), Bcl-2, Bax, CLIC4 and 14-3-3γwere investigated through Western Blotting method.(9) Seperated the protein of cell nucleus and plasmosin and observed the shift of NF-κB (p65) protein.Results:1 The inhibitory effect of SAS on C6 cell was more significant than that on primary astrocyte and PC-12 cell.2 When SAS was used with VK₃ and H₂O₂ respectively, the synergistic inhibition of C6 cell proliferation occurred. 3 When SAS was used with VK₃ and H₂O₂ respectively, C6 cell death was induced. However, the C6 cell apoptosis rate in combined use of SAS and H₂O₂ group was much higher than that in combined used of SAS and VK₃ group; the necrosis rate in combined use of SAS and H₂O₂ group was lower than that in combined used of SAS and VK₃ group.4 SAS caused the increase of ROS in C6 cells. When SAS was used with VK₃ and H₂O₂ respectively, the increase of ROS was more significant. Moreover, the GSH contents in C6 cell gradually decreased with time.5 When SAS was used with VK₃ and H₂O₂ respectively, the xCT mRNA level of C6 cell decreased significantly.6 When SAS was used with VK₃ and H₂O₂ respectively, the GCLC, GCLM mRNA level of C6 cell decreased with time significantly.7 When SAS was used with VK₃, compared with control group, the EAAT3 mRNA level of C6 cells decreased with time. Whereas, on all the timepoints, the EAAT3 mRNA level of C6 cell had no difference from that of control group when SAS was used with H₂O₂.8 When SAS was used with VK₃ and H₂O₂ respectively, there were both identical and different aspects in the change of NF-κB related genes expression. When SAS was used with VK₃ and H₂O₂ respectively, although the p65 protein in cell nucleus increased, the p65 protein level in cell total protein decrease and the mRNA level of its target gene--CyclinD1 gradually decreased. When SAS was used with VK₃, the mRNA leveles of IκB-αandβ-trcp gradually decreased. When SAS was used with H₂O₂, the mRNA leveles of IκB-αandβ-trcp had no change on 4h and 8h and significantly increased on 12h.9 The determination results of mitochondrial apoptosis related proteins of C6 cell indicated that: when SAS was used with VK₃, the mRNA and protein levels of CLIC4 and the 14-3-3γprotein level of C6 cell gradually decreased with time; when SAS was used with H₂O₂, the Bcl-2 protein level of C6 cell decreased and Bax/Bcl-2 value increased significantly on 12h.Conclusions:1 SAS has more powerful depressant effect on C6 cell proliferation compared with primary astrocyte and neuron and this effect is dose dependent. This indicats that SAS has specific depressant effect on C6 cell proliferation. The reason may be that glioma cells uptake cystine exclusively depending on xc- system to maintain cellular anti-oxiadation ability.2 When SAS is used with different kinds of oxidant, VK₃ and H₂O₂, the depressant effect on cell proliferation is more powerful compared with single use. Here are the mechanisms as follow. (1) The inhibition of xCT expression endogeneticly leads to the lack of raw material of GSH, so the ability of cellular GSH de novo synthesis decreases. (2) The increase of ROS in C6 cell ectogenicly causes the increase of consumption of cellular GSH. (3) GCLC and GCLM expression degrades, then cellular GSH synthesis is limited endogeneticly, at last cell death is induced. (4) When SAS is used with VK₃ and H₂O₂ respectively, the changes of IκB-αandβ-trcp expression are coincident. So the change of IκB-αmay be independent of ubiquityn- proteasome pathway. (5) When SAS is used with VK₃ and H₂O₂ respectively, although NF-κB (p65) expression in cell nucleus increases, total p65 protein level and its target gene-- Cyclin D1 mRNA level decreases, then cell proliferation is inhibited. This indicates that when total p65 protein level decreases, the increase of p65 expression in cell nucleus may be insufficient to enhance the activity of p65. The balance between total and nuclear protein level of NF-κB (p65) may influence its activity. It is thus clear that GSH/NF-κB pathway is one of the mechanisms through which oxidative stress enhances the depressant effect of SAS on cell proliferation.3 Different oxidants have different influences on the effect of SAS. (1) When SAS is used with VK₃, the inhibition of EAAT3 expression causes the increase of extracellular glutamate concentration, then inhibits the function of xc-, at last the uptake of cystine decreases. (2) When SAS is used with VK₃, mRNA levels of IκB-αandβ-trcp decrease gradually. Howere, when SAS is used with H₂O₂, mRNA levels of IκB-αandβ-trcp increase significantly at last. So it is presumed that IκB-αand ubiquityn-proteasome pathway may play different roles in the influences of VK₃ and H₂O₂ of on the effect of SAS. (3) When SAS is used with VK₃, mitochondrial CLIC4 and 14-3-3γexpression can be influenced. So it is presumed that cell death may be induced by influenceing the combination of CLIC4 and 14-3-3. Whereas, when SAS is used with H₂O₂, cell death may be induced through Bax/Bcl-2 way in the mitochondrium pathway. These differences may be one of the mechanisms throuth which the combined use of SAS and VK₃ causes cell necrosis, however, the combined use of SAS and H₂O₂ leads to cell apoptosis mainly.To sum up, through observing the effect of oxidative stress on cell proliferation regulated by glutamate/cystine antiporter, the results indicated that glutamate/cystine antiporter plays a vital role in regulating cell proliferation through GSH/NF-κB/mitochondrium pathways. So this study revealed the probable mechanism through which glutamate/cystine antiporter is regarded as a target for clinic treatment of glioma more deeply. Oxidant can enhance the therapeutic efficacy of glutamate/cystine antiporter and reduce their respective side effects. But the influence of VK₃ on the effect of SAS is different from that of H₂O₂ on the effect of SAS. This indicated that VK₃ is a kind of compounds. So it has other pharmacological actions apart from oxidative stress. If its mechanism can be researched more deeply, a new target for clinic treatment of glioma will be found.