Expression of gamma-glutamyltransferase 1 in glioblastoma cells confers resistance to cystine deprivation–induced ferroptosis

Abstract

Ferroptosis is an iron-dependent mode of cell death caused by excessive oxidative damage to lipids. Lipid peroxidation is normally suppressed by glutathione peroxidase 4, which requires reduced glutathione. Cystine is a major resource for glutathione synthesis, especially in cancer cells. Therefore, cystine deprivation or inhibition of cystine uptake promotes ferroptosis in cancer cells. However, the roles of other molecules involved in cysteine deprivation–induced ferroptosis are unexplored. We report here that the expression of gamma-glutamyltransferase 1 (GGT1), an enzyme that cleaves extracellular glutathione, determines the sensitivity of glioblastoma cells to cystine deprivation–induced ferroptosis at high cell density (HD). In glioblastoma cells expressing GGT1, pharmacological inhibition or deletion of GGT1 suppressed the cell density–induced increase in intracellular glutathione levels and cell viability under cystine deprivation, which were restored by the addition of cysteinylglycine, the GGT product of glutathione cleavage. On the other hand, cystine deprivation induced glutathione depletion and ferroptosis in GGT1-deficient glioblastoma cells even at an HD. Exogenous expression of GGT1 in GGT1-deficient glioblastoma cells inhibited cystine deprivation–induced glutathione depletion and ferroptosis at an HD. This suggests that GGT1 plays an important role in glioblastoma cell survival under cystine-limited and HD conditions. We conclude that combining GGT inhibitors with ferroptosis inducers may provide an effective therapeutic approach for treating glioblastoma. Ferroptosis is an iron-dependent mode of cell death caused by excessive oxidative damage to lipids. Lipid peroxidation is normally suppressed by glutathione peroxidase 4, which requires reduced glutathione. Cystine is a major resource for glutathione synthesis, especially in cancer cells. Therefore, cystine deprivation or inhibition of cystine uptake promotes ferroptosis in cancer cells. However, the roles of other molecules involved in cysteine deprivation–induced ferroptosis are unexplored. We report here that the expression of gamma-glutamyltransferase 1 (GGT1), an enzyme that cleaves extracellular glutathione, determines the sensitivity of glioblastoma cells to cystine deprivation–induced ferroptosis at high cell density (HD). In glioblastoma cells expressing GGT1, pharmacological inhibition or deletion of GGT1 suppressed the cell density–induced increase in intracellular glutathione levels and cell viability under cystine deprivation, which were restored by the addition of cysteinylglycine, the GGT product of glutathione cleavage. On the other hand, cystine deprivation induced glutathione depletion and ferroptosis in GGT1-deficient glioblastoma cells even at an HD. Exogenous expression of GGT1 in GGT1-deficient glioblastoma cells inhibited cystine deprivation–induced glutathione depletion and ferroptosis at an HD. This suggests that GGT1 plays an important role in glioblastoma cell survival under cystine-limited and HD conditions. We conclude that combining GGT inhibitors with ferroptosis inducers may provide an effective therapeutic approach for treating glioblastoma. Glioblastoma is the most common malignant brain tumor and has a poor prognosis (1Dolecek T.A. Propp J.M. Stroup N.E. Kruchko C. CBTRUS statistical report: Primary brain and central nervous system tumors diagnosed in the United States in 2005-2009.Neuro Oncol. 2012; 14 Suppl 5: v1-v49Google Scholar, 2Tan S.K. Jermakowicz A. Mookhtiar A.K. Nemeroff C.B. Schürer S.C. Ayad N.G. Drug repositioning in glioblastoma: A pathway perspective.Front. Pharmacol. 2018; 9: 218Google Scholar). Currently, surgical resection, radiation therapy, and chemotherapy are usually used to treat patients with glioblastoma. However, no effective treatment has been established, and the 5-year survival rate is only 5%. Therefore, it is important to find new effective therapeutic strategies for glioblastoma. Ferroptosis is a form of cell death caused by excessive oxidative damage to lipids in an iron-dependent manner (3Dixon S.J. Lemberg K.M. Lamprecht M.R. Skouta R. Zaitsev E.M. Gleason C.E. Patel D.N. Bauer A.J. Cantley A.M. Yang W.S. Morrison 3rd, B. Stockwell B.R. Ferroptosis: An iron-dependent form of nonapoptotic cell death.Cell. 2012; 149: 1060-1072Google Scholar). It is well known that inhibition of glutathione peroxidase 4, an enzyme that directly reduces lipid peroxides, induces ferroptosis. The cystine/glutamate antiport system xc− is composed of two subunits, the light chain xCT (SLC7A11) with catalytic activity and the heavy chain CD98 (4F2hc or SLC3A2) with regulatory activity, and it exchanges extracellular cystine and intracellular glutamate at the plasma membrane (4Lewerenz J. Hewett S.J. Huang Y. Lambros M. Gout P.W. Kalivas P.W. Massie A. Smolders I. Methner A. Pergande M. Smith S.B. Ganapathy V. Maher P. The cystine/glutamate antiporter system x(c)(-) in health and disease: From molecular mechanisms to novel therapeutic opportunities.Antioxid. Redox Signal. 2013; 18: 522-555Google Scholar, 5Koppula P. Zhuang L. Gan B. Cystine transporter SLC7A11/xCT in cancer: Ferroptosis, nutrient dependency, and cancer therapy.Protein Cell. 2021; 12: 599-620Google Scholar). In many cancer cells, cystine taken up by xCT is the major source of cysteine, which is required for the synthesis of reduced glutathione (GSH). As the activity of glutathione peroxidase 4 requires GSH, depletion of cystine or treatment with pharmacological inhibitors of xCT, such as erastin, induces ferroptosis (6Dixon S.J. Stockwell B.R. The hallmarks of ferroptosis.Annu. Rev. Cell Biol. 2019; 3: 35-54Google Scholar). Therefore, the synthesis of GSH is important for the survival of cancer cells to avoid excessive oxidative stress and ferroptosis. Gamma-glutamyltransferase (GGT) is a membrane-bound enzyme that is important for the metabolism of extracellular glutathione (7Meister A. Anderson M.E. Glutathione.Annu. Rev. Biochem. 1983; 52: 711-760Google Scholar, 8Hanes C.S. Hird F.J. Synthesis of peptides in enzymic reactions involving glutathione.Nature. 1950; 166: 288-292Google Scholar, 9Griffith O.W. Bridges R.J. Meister A. Evidence that the gamma-glutamyl cycle functions in vivo using intracellular glutathione: Effects of amino acids and selective inhibition of enzymes.Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 5405-5408Google Scholar). GGT can cleave the gamma-glutamyl bond of glutathione to l-cysteinylglycine (Cys-Gly) and glutamate extracellularly, and Cys-Gly is further degraded into cysteine and glycine by dipeptidase and reused by being taken up into the cell. About 13 genes of the GGT family were identified using genomic and complementary DNA database searches (10Heisterkamp N. Groffen J. Warburton D. Sneddon T.P. The human gamma-glutamyltransferase gene family.Hum. Genet. 2008; 123: 321-332Google Scholar). Among them, GGT1 is the most studied, and its expression increases in many human cancers, including glioblastoma (10Heisterkamp N. Groffen J. Warburton D. Sneddon T.P. The human gamma-glutamyltransferase gene family.Hum. Genet. 2008; 123: 321-332Google Scholar, 11Batsios G. Najac C. Cao P. Viswanath P. Subramani E. Saito Y. Gillespie A.M. Yoshihara H.A.I. Larson P. Sando S. Ronen S.M. In vivo detection of γ-glutamyl-transferase up-regulation in glioma using hyperpolarized γ-glutamyl-[1-13C]glycine.Sci. Rep. 2020; 10: 6244Google Scholar). Human GGT1 is autocatalytically cleaved into large and small subunits to generate the mature active enzyme (12Suzuki H. Kumagai H. Autocatalytic processing of gamma-glutamyltranspeptidase.J. Biol. Chem. 2002; 277: 43536-43543Google Scholar, 13West M.B. Wickham S. Quinalty L.M. Pavlovicz R.E. Li C. Hanigan M.H. Autocatalytic cleavage of human gamma-glutamyl transpeptidase is highly dependent on N-glycosylation at asparagine 95.J. Biol. Chem. 2011; 286: 28876-28888Google Scholar). Although inhibition of GGT1 was reported to inhibit the proliferation and migration of clear cell renal cell carcinoma cells (14Bansal A. Sanchez D.J. Nimgaonkar V. Sanchez D. Riscal R. Skuli N. Simon M.C. Gamma-glutamyltransferase 1 promotes clear cell renal cell carcinoma initiation and progression.Mol. Cancer Res. 2019; 17: 1881-1892Google Scholar), the role of GGT1 in cancer progression is still largely unknown. In this study, we demonstrated a role of GGT1 in ferroptosis resistance in glioblastoma cells cultured at a high density. We recently reported that cystine deprivation induces ferroptosis in glioblastoma cells (15Hayashima K. Kimura I. Katoh H. Role of ferritinophagy in cystine deprivation-induced cell death in glioblastoma cells.Biochem. Biophys. Res. Commun. 2021; 539: 56-63Google Scholar). We found that this depends on cell density. Three types of glioblastoma cell lines, A172, T98G, and LN229, were plated at a low cell density (LD; 1 × 104 cells/cm2) and high cell density (HD; 1 × 105 cells/cm2) and cultured for 24 h. Then, the culture medium was collected at 24 h after cystine depletion, and the amount of lactate dehydrogenase (LDH) released into the medium was measured to quantify cell death. Depletion of cystine significantly increased cell death in all glioblastoma cell lines at LD. In LN229 cells, cystine deprivation also induced cell death at HD. However, it had little effect on cell viability in A172 and T98G cells at HD (Fig. 1A). The addition of ferroptosis inhibitors ferrostatin-1 (Fer-1) and deferoxamine (DFO) inhibited cell death under cystine deprivation in LN229 cells at HD (Fig. 1B). Our previous study revealed that cystine deprivation–induced ferroptosis involves two processes: depletion of intracellular GSH and degradation of ferritin in glioblastoma cells (15Hayashima K. Kimura I. Katoh H. Role of ferritinophagy in cystine deprivation-induced cell death in glioblastoma cells.Biochem. Biophys. Res. Commun. 2021; 539: 56-63Google Scholar). Next, we examined whether the degradation of FTH1, the heavy chain subunit of ferritin, is affected by cell density. Western blotting of cell lysates after depletion of cystine confirmed that the protein levels of FTH1 decreased in both A172 and LN229 cells at LD and HD (Fig. 1C). On the other hand, we observed a difference in the levels of intracellular GSH between LD and HD. The intracellular GSH was depleted 15 h after cystine deprivation in three glioblastoma cell lines cultured at LD and in LN229 cells at HD. In contrast, the GSH levels decreased, but were not depleted, after cystine deprivation in A172 and T98G cells cultured at HD (Fig. 1D). l-buthionine sulfoximine (BSO), an of the enzyme of GSH synthesis, depleted GSH and cell death under cystine deprivation in A172 and T98G cells cultured at HD (Fig. and This suggests that cell density glutathione which plays a role in the resistance to ferroptosis in A172 and T98G glioblastoma cells. On the other hand, treatment depleted GSH but not cell death in the of Our previous study suggests that not ferritinophagy required for the of ferroptosis in the of cystine in glioblastoma cells (15Hayashima K. Kimura I. Katoh H. Role of ferritinophagy in cystine deprivation-induced cell death in glioblastoma cells.Biochem. Biophys. Res. Commun. 2021; 539: 56-63Google Scholar). Therefore, cell death in A172 and T98G cells at HD only in the of We examined whether the medium HD culture can inhibit cystine deprivation–induced cell death at LD. The medium A172 or T98G culture at HD suppressed cystine deprivation–induced cell death in A172 or T98G cells cultured at HD medium LN229 culture had little effect on cystine deprivation–induced cell death in LN229 cells cultured at LD (Fig. Therefore, A172 or T98G cells at HD can cell viability under cystine is in the but it is also of the cell A. Simon M.C. metabolism in cancer progression and treatment Cell Biol. 2018; Scholar). We examined whether an enzyme that glutathione to Cys-Gly and glutamate extracellularly, is involved in the cystine deprivation–induced cell death at HD. an of significantly increased the of cell death under cystine deprivation in A172 and T98G cells at HD (Fig. In LN229 cells, cystine deprivation induced cell death at HD in the or the of The ferroptosis inhibitors and inhibited cell death under cystine deprivation in A172 cells at HD (Fig. that induces cell death under cystine deprivation at HD. In depleted GSH in A172 and T98G cells cultured at HD (Fig. In LN229 cells, cystine deprivation induced GSH depletion at HD in the or the of We that the of GGT caused a difference between cells and LN229 cells in ferroptosis Human GGT1, the most of the GGT is autocatalytically cleaved into large and small subunits to generate the mature active enzyme (12Suzuki H. Kumagai H. Autocatalytic processing of gamma-glutamyltranspeptidase.J. Biol. Chem. 2002; 277: 43536-43543Google Scholar, 13West M.B. Wickham S. Quinalty L.M. Pavlovicz R.E. Li C. Hanigan M.H. Autocatalytic cleavage of human gamma-glutamyl transpeptidase is highly dependent on N-glycosylation at asparagine 95.J. Biol. Chem. 2011; 286: 28876-28888Google Scholar). We the expression levels of GGT1 by Western blotting with an the small subunit and found that GGT1 was in A172 and T98G cells. However, expression of GGT1 was in LN229 cells (Fig. The protein levels of GGT1 in A172 and T98G cells were were cultured at HD (Fig. We GGT1-deficient A172 cells using deletion of GGT1 GGT1 1 and In A172 cells, cystine deprivation not cell death cultured at HD. However, cystine deprivation significantly increased cell death in A172 GGT1 1 and cells at HD (Fig. In cystine deprivation reduced the GSH to in GGT1 1 and cells at it reduced the GSH to in cells, which were depleted by (Fig. This suggests that GGT1 plays an important role in GSH metabolism and resistance to ferroptosis in glioblastoma cells at HD. GGT1 cleaves extracellular glutathione to Cys-Gly and glutamate A. Simon M.C. metabolism in cancer progression and treatment Cell Biol. 2018; Scholar, A. M. A. A. Gamma-glutamyltransferase of cancer cells at the of tumor resistance and Res. Scholar). Therefore, we examined whether Cys-Gly restored cell viability in A172 cells with The addition of Cys-Gly to the medium suppressed cell death in A172 cells under cystine deprivation at HD. Cys-Gly also suppressed cystine deprivation–induced cell death in LN229 cells at HD in the or the of (Fig. of glutamate cystine uptake (4Lewerenz J. Hewett S.J. Huang Y. Lambros M. Gout P.W. Kalivas P.W. Massie A. Smolders I. Methner A. Pergande M. Smith S.B. Ganapathy V. Maher P. The cystine/glutamate antiporter system x(c)(-) in health and disease: From molecular mechanisms to novel therapeutic opportunities.Antioxid. Redox Signal. 2013; 18: 522-555Google Scholar, P. Cao S. K.M. Patel H. S. A.K. J. of by glutamate in cancer: 166: Scholar, K. M. Katoh H. Cystine uptake the cystine/glutamate antiporter xCT glioblastoma cell death under Biol. Chem. Scholar). However, addition of glutamate at the of Cys-Gly had little effect on the cell viability (Fig. We confirmed the effect of extracellular glutamate on cystine uptake in A172 cells. The xCT suppressed cystine uptake in A172 cells, but glutamate treatment at not (Fig. that a of extracellular glutamate is required for inhibition of cystine On the other hand, the addition of GSH had little effect on cell death in A172 cells under cystine deprivation at HD or on cystine deprivation–induced cell death in LN229 cells at HD in the or the of (Fig. We used A172 GGT1 1 cells to the effect of Cys-Gly on cell In A172 GGT1 1 cells cultured at cystine deprivation induced cell death in the and of which was inhibited by Cys-Gly (Fig. cells, increased cell death in A172 cells at which was suppressed by The addition of GSH not cell viability in A172 GGT1 1 and cells at HD (Fig. We examined the of extracellular Cys-Gly on the intracellular GSH levels in A172 and LN229 cells. Cystine deprivation with depleted intracellular GSH in A172 cells at HD. The addition of Cys-Gly significantly increased the intracellular GSH the addition of GSH not (Fig. Cys-Gly but not GSH increased the intracellular GSH in LN229 cells and A172 GGT1 1 cells at HD (Fig. and In A172 cells, the addition of GSH also increased the intracellular GSH in the of This suggests that of Cys-Gly cystine deprivation–induced cell death in glioblastoma cells at HD. is a major of metabolism and used for an pathway of glutathione synthesis M. and metabolism in Rev. Scholar). whether synthesis is involved in the of GSH levels under cystine deprivation at we used a of dehydrogenase is the enzyme that the of synthesis the with decreased the GSH levels in A172 and T98G cells at HD in the and of and were depleted by (Fig. This suggests that synthesis the plays a role in an pathway for GSH synthesis in glioblastoma cells. whether GGT1 extracellular glutathione levels in glioblastoma cells, we measured the of the glutathione and GSH of glutathione in the The levels of GSH in the medium were low in A172 and LN229 cells at both LD and HD with or cystine (Fig. most glutathione in the medium was in the and it was not affected by cystine deprivation or treatment (Fig. The extracellular of were high at and were reduced by cystine deprivation (Fig. restored the extracellular glutathione to the in the of cystine in A172 cells at but it had no effect on the extracellular glutathione levels in A172 cells at LD or in LN229 cells at LD and HD (Fig. The of extracellular glutathione was also high in A172 GGT1 1 cells (Fig. This suggests that GGT1 promotes the degradation of extracellular glutathione in glioblastoma cells cultured at HD. The intracellular cysteine levels were not significantly between LD and HD in A172 and LN229 cells (Fig. In the of glutathione cell is between LD and HD in A172 cells × × As the medium A172 HD inhibited cystine deprivation–induced cell death at LD. A172 cells were cultured at HD in the of the medium not inhibit cystine deprivation–induced cell death at LD. However, the addition of Cys-Gly to the medium HD restored cell viability of A172 cells at LD (Fig. In the of Cys-Gly on cell viability were dependent in both A172 and LN229 cells at HD (Fig. glutathione and its Cys-Gly are in the medium A172 cells at HD that cell viability under cystine The expression of GGT1 was in LN229 cells. Therefore, we LN229 cells that GGT1 and examined whether resistance to ferroptosis at HD. In LN229 cells expressing the cystine deprivation induced cell death at LD and HD. On the other hand, cystine deprivation not cell death in LN229 GGT1 cells at it induced cell death at LD (Fig. with the GGT restored cell death in LN229 GGT1 cells at HD (Fig. GSH was depleted under cystine deprivation in LN229 cells cultured at the GSH decreased, but was not depleted, after cystine deprivation in LN229 GGT1 cells cultured at HD (Fig. depleted GSH in LN229 GGT1 cells cultured at HD (Fig. that expression of GGT1 increases glioblastoma cell viability under cystine deprivation at HD. The survival of cancer cells depends on to to conditions. In this study, we found that GGT1, an enzyme for the extracellular degradation of glutathione, plays a major role in the survival of glioblastoma cells under conditions. Glioblastoma cells with high expression of GGT1 can under cystine deprivation are cultured at HD. This is in to intracellular GSH levels even cystine in the inhibition of GGT1 activity by the GGT or deletion or inhibition of GSH synthesis induced the depletion of intracellular GSH and increased cell death in glioblastoma cells at HD. The medium HD of glioblastoma cells a high of glutathione, which is to Cys-Gly and glutamate by The addition of Cys-Gly to the medium restored GSH levels and cell viability in the of or in GGT1-deficient cells. expression of GGT1 in GGT1-deficient cells increased cell viability under cystine deprivation at HD. study suggests that the expression of GGT1 is for resistance to cystine deprivation–induced ferroptosis in glioblastoma cells at HD. is to the extracellular and to Cys-Gly and Cys-Gly is to cysteine cysteine is to which is taken up into cells xCT and reused for synthesis of GSH Y. Zhuang L. K. Gan B. cystine uptake as a in 2021; Scholar). glutathione which depends on glioblastoma cells to intracellular GSH for ferroptosis cystine (Fig. Therefore, cystine depletion with the inhibition of GGT activity may an therapeutic for glioblastoma. On the other hand, Cys-Gly cystine deprivation–induced cell death in a that of glutathione and its Cys-Gly in the extracellular are required for of ferroptosis under cystine deprivation conditions. The cells in the the glutathione cells into in extracellular glutathione in Therefore, we to study this in the of HD. Cancer cells high levels of GSH, which cell and stress In addition to its role as an GSH is involved in the of cysteine an amount of intracellular cysteine is to cells Y. K. G. induces cell death by stress and protein in Scholar, C.E. by 2020; Scholar, S.J. the role of cysteine in J. 2021; Scholar). GGT functions in the of cysteine GSH, and reported that cancer cells GGT extracellular GSH as a source of intracellular cysteine and GSH A. M. A. A. Gamma-glutamyltransferase of cancer cells at the of tumor resistance and Res. Scholar, R. P. Y. Huang and cysteine in gamma-glutamyl Natl. Acad. Sci. U. S. A. Scholar, E. M. M. Groffen J. N. with transpeptidase glutathione depletion using extracellular Pharmacol. Scholar, M.H. of gamma-glutamyl transpeptidase tumor cells with a selective at of Scholar). deletion of GGT1 expression in glioblastoma cells to the depletion of intracellular GSH under cystine deprivation, that glioblastoma cells extracellular glutathione for the of intracellular GSH are to extracellular It was also reported that GGT1 is highly in cancers, including glioblastoma (10Heisterkamp N. Groffen J. Warburton D. Sneddon T.P. The human gamma-glutamyltransferase gene family.Hum. Genet. 2008; 123: 321-332Google Scholar, 11Batsios G. Najac C. Cao P. Viswanath P. Subramani E. Saito Y. Gillespie A.M. Yoshihara H.A.I. Larson P. Sando S. Ronen S.M. In vivo detection of γ-glutamyl-transferase up-regulation in glioma using hyperpolarized γ-glutamyl-[1-13C]glycine.Sci. Rep. 2020; 10: 6244Google Scholar). In GGT1 is in cancer cells, and GGT activity was reported to a for types of cancer K. Y. Y. K. K. K. Y. M. K. M. Gamma-glutamyltransferase activity in as a for 17: Scholar, K. K. Y. Y. N. K. M. gamma-glutamyltransferase activity increased in patients with renal cell carcinoma with 2020; Scholar). Therefore, it is important for to whether GGT1 in HD glioblastoma cells plays a role in the resistance to ferroptosis. It is well known that cell In inhibition and HD culture cell survival and of the mechanisms inhibition is the cells are at the pathway is the of protein with the to the and and cell proliferation is B. Li Yang J. J. J. Li L. P. K. A. G. of by the pathway is involved in cell inhibition and Scholar, N. J. V. Y. The pathway DNA inhibition of the 2013; Scholar, M. M. S.J. J. A. K. inhibition cell survival and proliferation Commun. 2018; 9: Scholar). Cancer cells inhibition and However, cell density has also been reported to cancer cell HD resistance and increases cell viability of many cancer cells, including glioblastoma H. Y. between tumor cell density and and the of or of Pharmacol. Scholar, S. K.M. S. of a novel in the of cell resistance in glioblastoma 2008; Scholar, pathway resistance to Natl. Acad. Sci. U. S. A. Scholar). HD culture also cancer cells deprivation–induced cell death I. Katoh H. cell density increases glioblastoma cell viability under deprivation degradation of the cystine/glutamate transporter xCT Biol. Chem. 2020; Scholar). On the other hand, reported that the of and increases the expression of genes involved in such as and increases cancer cell to ferroptosis. of the pathway to resistance to ferroptosis J. A.M. M. H. Li Y. Stockwell B.R. cancer cell ferroptosis 2019; Scholar, G. D. The pathway ferroptosis in renal cell Rep. 2019; Scholar, Huang J. S.K. A cell death and in Cancer Res. 2019; 18: Scholar). The expression of GGT1 in glioblastoma cells was cultured at that resistance to ferroptosis at HD is of the are to the between glutathione metabolism and the pathway in ferroptosis resistance in cancer cells. The expression was a J. The of human GGT1 was cells by and into The human GGT1 were into the expression medium was as (15Hayashima K. Kimura I. Katoh H. Role of ferritinophagy in cystine deprivation-induced cell death in glioblastoma cells.Biochem. Biophys. Res. Commun. 2021; 539: 56-63Google Scholar). and amino were used at the 1 and GSH A172 and T98G cell lines were by the the of the of and of A172, LN229 cell was were cultured in medium 4 and under with at was using generate A172 GGT1 cells, we used the system Y. S. S. R. K. for of genes by using Biol. Cell. Scholar). A172 cells in of were with human GGT1 and generate LN229 GGT1 cells, cells in of were with after cells were collected and of with medium of to cells. About after cells were were and by using Cystine deprivation was as (15Hayashima K. Kimura I. Katoh H. Role of ferritinophagy in cystine deprivation-induced cell death in glioblastoma cells.Biochem. Biophys. Res. Commun. 2021; 539: 56-63Google Scholar). Cell death was measured using the to the The was measured at using a with cell death. The amount of intracellular GSH was measured using the to the The was measured using a A was used to light into the The of extracellular and were measured using the cultured in at LD or HD were collected with the cells were in and by on with at the of for 1 The cysteine was measured using the cysteine to the The was measured using the A was used to light into the was measured using the Cystine uptake was measured using the Cystine uptake to the N. Y. M. M. Y. for cystine uptake xCT in cells using and a 2021; Scholar). The was measured using the A was used to light into the were with and by as (15Hayashima K. Kimura I. Katoh H. Role of ferritinophagy in cystine deprivation-induced cell death in glioblastoma cells.Biochem. Biophys. Res. Commun. 2021; 539: 56-63Google Scholar). were by The were used in this FTH1 Cell to peroxidase and were using and difference was was using are the The that no of with the of this We for the We also Katoh and for and This study was in by for the for the of and K. H. and H. K. K. H. and H. K. K. H. and H. K. K. H. and H. K. K. H. and H. K. K. H. and H. K. K. H. and H. K. K. H. and H. K. K. H. and H. K. H. K. H. K.

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