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SASP as an xCT pharmacological inhibitor is a classof sulfa drugs that is approved by the Food and Drug Administration(FDA). SASP has been applied to Chron disease therapy in the clinicfor a long time. ERA as small molecular compounds is another xCTpharmacological inhibitor that has been recently identified(). Although the two inhibitorscan effectively block cysteine uptake as well as GSH synthesis,there are other synthesis pathways besides xCT, the GSH synthesishas no absolute reliability on xCT. ERA significantly inhibits CTHactivity and affects synthesis resulting from the xCT andtranssulfuration pathways, thus the effect of TMZ is significantlyelevated. However, ERA is limited with regard to the ameliorationof TMZ cytotoxicity by applied SASP, because a low cysteine levelin cells enables CTH activity with a co-processing treatment bySASP and TMZ. This may be one reason for the controversy regardingwhether SASP has a therapeutic effect for GBM. Of note, the celldeath caused by applying TMZ and ERA together may be partiallyrescued by DFO. This type of cell death is to some degree an Fe(III)-dependent process, described as ferroptosis in a recent study(). ERA highly preferentiallyselected to injure RAS-mutant tumors (). In the present study, the results didnot show that there is a RAS-mutant in GBM cells. However, thecontinuously activated RAS was involved in the dialog between tumorand immune system (). ERA wasinvolved in the regulation of ferroptotic cancer cell death byinhibiting the activity of GPx4 (glutathione peroxidase 4)(). Moreover, GPx4 is highlyassociated with tumor growth, and is a significant risk factor forbreast cancer when GPx4 is continuously activated (,).However, whether ERA ameliorated GBM cell resistance to TMZ byinhibiting GPx4 activity and increasing the injury of oxidativestress for cells requires further investigation.
Additionally, cystathionine almost completelyreplaced cysteine and methionine. These results suggest thatanother GSH synthesis pathway besides that of xCT exists, such asthe transsulfuration pathway. TMZ strongly induces the expressionof CTH mRNA and enhances the related enzyme activity, which iscritical in the transsulfuration pathway, especially when xCT isinhibited. TMZ has been found to significantly induce theexpression of Nrf2 and ATF4. Nrf2 and ATF4 have been found toincrease GSH production via multiple mechanisms (). However, loss of ATF4 impairs GSHproduction by inhibiting the expression of CTH (). However, the relevance betweenTMZ-induced increase of Nrf2 and ATF4 expression and the CTH enzymeregulation, and whether TMZ induced other related enzymes such asCBS (cystathionine β-synthase) in the transsulfuration pathway forGSH synthesis remain to be clarified and the experimentsconfirmed.
Bocangel DB, Finkelstein S, Schold SC,Bhakat KK, Mitra S and Kokkinakis DM: Multifaceted resistance ofgliomas to temozolomide. Clin Cancer Res. 8:2725–2734.2002.
In many types of cells cysteine is derived from animported xCT process, however, there is also a transsulfurationpathway in which methionine is transferred to cysteine viacatalysis by cystathionine β-synthase (CSE) andcystathionine-γ-lyase (CTH) (,).In brain carcinoma cells such as glioma cells and astrocytes, mostcysteine originates from the reduction process of cystine importedby xCT under normal conditions. However, when xCT is blocked or GSHis decreased, the transsulfuration pathway is activated, whichinsures the cysteine supply for GSH synthesis in a compensatorymanner (,). Furthermore, CTH activity may limitcysteine synthesis via the methionine transsulfuration pathway(,). However, whether transsulfuration andCTH are involved in TMZ resistance remains to be elucidated.
The glutamate/cystine antiporter systemx is an obligate sodium-independent aminoacid antiporter, comprising 12-pass transmembrane transporterprotein xCT (SLC7A11) which is connected to the 4F2 cell surfaceantigen 4F2hc (CD98/SLC3A2) by a disulfide bridge (,).System x transports extracellular cystineinto cells in exchange for intracellular glutamate at a ratio of1:1, and maintaining intracellular cysteine pools is important(). Cysteine is a crucialmaterial in glutathione (GSH) synthesis, which is indispensable formaintaining intracellular redox balance and drug metabolism(,). xCT expression is mediated by theoxidative stress-response transcription factor NF-E2 related tofactor 2 (Nrf2) and activation of transcription factor 4 (ATF4)(). xCT is expressed in manytypes of malignancies and is associated with tumor growth andmetastasis. It is also associated with resistance to chemotherapyand poor survival (–). Accordingly, xCT has been consideredas a potential therapeutic target (,,,).Sulfasalazine (SASP) is a sulfa drug used for inflammatory boweldiseases and rheumatoid arthritis treatment and is a widelyrecognized xCT-specific inhibitor (). Although it has been suggested thatthe inhibitory effect of SASP on xCT can suppress GBM cell growth,the combination of SASP combined with TMZ for GBM treatment in theclinic has yielded controversial results (,).Erastin (ERA) is a voltage-dependent anion channels (VDAC)-bindingsmall molecule that is selectively lethal to some cancer cells(). Compared to SASP, ERA exertsa stronger inhibitory effect on xCT (). In addition, ERA is able to inhibitthe activity of certain GSH-related enzymes, such as glutathioneperoxidase 4 (GPx4), resulting in more lethal oxidative damage tocells (). ERA can cause a uniqueform of cell death on iron-dependent tumor cells as compared toconventional apoptosis, necrosis and autophagy ().
Temozolomide (TMZ, 3,4-dihydro-3-methyl-4-oxoimidazo [5, 1-d]-1, 2, 3,5-tetrazine-8-carboxamide), deferoxamine mesylate (DFO), erastin(ERA), -Acetyl-L-cysteine (NAC), sulfasalazine (SASP),propargylglycine (PPG), as well as Dulbecco’s modified Eagle’smedium (DMEM) and bovine serum albumin (BSA) were obtained fromSigma-Aldrich (St. Louis, MO, USA). Dimethylsulfoxide (DMSO) wasobtained from Wako Pure Chemical (Osaka, Japan). CellROX Orangereagent was purchased from Life Technologies (Tokyo, Japan).Anti-xCT antibody (ab37185) was obtained from Abcam (Cambridge, MA,USA). Anti-Nrf2 antibody (sc-722), anti-ATF4 antibody (sc-200) andanti-LaminB antibody (sc-56144) were purchased from Santa CruzBiotechnology, Inc. (Dallas, TX, USA).
Temozolomide (TMZ) is a class of alkylating agentapproved by the Food and Drug Administration (FDA). TMZ is widelyused as a standard-of-care during clinical treatment. However, itonly results in a slight increase of overall survival of GBMpatients. Furthermore, most patients are resistant to TMZ in theclinic ().O-methylguanine produced by DNA methyl transferasemainly mediates the cytotoxicity of TMZ and triggers cellcycle-dependent DNA damage, ensuring cell death. Thus,O-methylguanine-DNA methyl transferase (MGMT) limitsthe therapeutic effect of TMZ by removingO-methylguanine (,).However, findings of previous GBM cell line studies showed that theactivity of MGMT was not entirely consistent with the resistance ofGBM to TMZ, i.e., even in MGMT-silenced GBM cells, the effect ofTMZ was limited (). The exact celldeath pathway induced by TMZ and the molecular mechanisms affectingthe efficacy of TMZ in GBM cells remain to be determined.
In the handling of brain tumorsTMZ has been discovered as a recent and efficient second generation drugemployed in the control of advanced brain gliomas, and it is a welcomeaddition.