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• W3088591201 abstract "Large regions in tumor tissues, particularly pancreatic cancer, are hypoxic and nutrient-deprived because of unregulated cell growth and insufficient vascular supply. Certain cancer cells, such as those inside a tumor, can tolerate these severe conditions and survive for prolonged periods. We hypothesized that small molecular agents, which can preferentially reduce cancer cell survival under nutrient-deprived conditions, could function as anticancer drugs. In this study, we constructed a high-throughput screening system to identify such small molecules and screened chemical libraries and microbial culture extracts. We were able to determine that some small molecular compounds, such as penicillic acid, papyracillic acid, and auranofin, exhibit preferential cytotoxicity to human pancreatic cancer cells under nutrient-deprived compared with nutrient-sufficient conditions. Further analysis revealed that these compounds target to redox systems such as GSH and thioredoxin and induce accumulation of reactive oxygen species in nutrient-deprived cancer cells, potentially contributing to apoptosis under nutrient-deprived conditions. Nutrient-deficient cancer cells are often deficient in GSH; thus, they are susceptible to redox system inhibitors. Targeting redox systems might be an attractive therapeutic strategy under nutrient-deprived conditions of the tumor microenvironment. Large regions in tumor tissues, particularly pancreatic cancer, are hypoxic and nutrient-deprived because of unregulated cell growth and insufficient vascular supply. Certain cancer cells, such as those inside a tumor, can tolerate these severe conditions and survive for prolonged periods. We hypothesized that small molecular agents, which can preferentially reduce cancer cell survival under nutrient-deprived conditions, could function as anticancer drugs. In this study, we constructed a high-throughput screening system to identify such small molecules and screened chemical libraries and microbial culture extracts. We were able to determine that some small molecular compounds, such as penicillic acid, papyracillic acid, and auranofin, exhibit preferential cytotoxicity to human pancreatic cancer cells under nutrient-deprived compared with nutrient-sufficient conditions. Further analysis revealed that these compounds target to redox systems such as GSH and thioredoxin and induce accumulation of reactive oxygen species in nutrient-deprived cancer cells, potentially contributing to apoptosis under nutrient-deprived conditions. Nutrient-deficient cancer cells are often deficient in GSH; thus, they are susceptible to redox system inhibitors. Targeting redox systems might be an attractive therapeutic strategy under nutrient-deprived conditions of the tumor microenvironment. Pancreatic cancer is an aggressive disease that frequently presents at an advanced stage (1Kleeff J. Korc M. Apte M. La Vecchia C. Johnson C.D. Biankin A.V. Neale R.E. Tempero M. Tuveson D.A. Hruban R.H. Neoptolemos J.P. Pancreatic cancer.Nat. Rev. Dis. Primers. 2016; 2 (27158978)16022 10.1038/nrdp.2016.22Crossref PubMed Scopus (868) Google Scholar), and the majority of the patients with pancreatic cancer have surgically unresectable disease. Pancreatic cancer is a major cause of cancer-associated mortality, and the 5-year survival rate is only 8% (2Siegel R.L. Miller K.D. Jemal A. Cancer statistics, 2018.CA Cancer J. Clin. 2018; 68 (29313949): 7-3010.3322/caac.21442Crossref PubMed Scopus (6017) Google Scholar). Effective drug therapies for patients with pancreatic cancer are desired. The tumor microenvironment plays an important role in tumor progression and metastasis; thus this should be a potential target for anticancer drugs (3Altorki N.K. Markowitz G.J. Gao D. Port J.L. Saxena A. Stiles B. McGraw T. Mittal V. The lung microenvironment: an important regulator of tumour growth and metastasis.Nat. Rev. Cancer. 2019; 19 (30532012): 9-3110.1038/s41568-018-0081-9Crossref PubMed Scopus (363) Google Scholar, 4Quail D.F. Joyce J.A. Microenvironmental regulation of tumor progression and metastasis.Nat. Med. 2013; 19 (24202395): 1423-143710.1038/nm.3394Crossref PubMed Scopus (3981) Google Scholar). Large regions of tumor tissues are often nutrient-deprived and hypoxic because of aberrant cell proliferation and abnormal blood vessels (5Vaupel P. Kallinowski F. Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review.Cancer Res. 1989; 49 (2684393): 6449-6465PubMed Google Scholar, 6Brown J.M. Giaccia A.J. The unique physiology of solid tumors: opportunities (and problems) for cancer therapy.Cancer Res. 1998; 58 (9537241): 1408-1416PubMed Google Scholar). In particular, pancreatic cancer tumors are typically hypoxic and low in nutrients because of poor vascularization and high interstitial pressure (7Kamphorst J.J. Nofal M. Commisso C. Hackett S.R. Lu W. Grabocka E. Vander Heiden M.G. Miller G. Drebin J.A. Bar-Sagi D. Thompson C.B. Rabinowitz J.D. Human pancreatic cancer tumors are nutrient poor and tumor cells actively scavenge extracellular protein.Cancer Res. 2015; 75 (25644265): 544-55310.1158/0008-5472.CAN-14-2211Crossref PubMed Scopus (448) Google Scholar, 8Koong A.C. Mehta V.K. Le Q.T. Fisher G.A. Terris D.J. Brown J.M. Bastidas A.J. Vierra M. Pancreatic tumors show high levels of hypoxia.Int. J. Radiat. Oncol. Biol. Phys. 2000; 48 (11072146): 919-92210.1016/S0360-3016(00)00803-8Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar, 9Duffy J.P. Eibl G. Reber H.A. Hines O.J. Influence of hypoxia and neoangiogenesis on the growth of pancreatic cancer.Mol. Cancer. 2003; 2 (12605718): 1210.1186/1476-4598-2-12Crossref PubMed Scopus (130) Google Scholar, 10Katsuta E. Qi Q. Peng X. Hochwald S.N. Yan L. Takabe K. Pancreatic adenocarcinomas with mature blood vessels have better overall survival.Sci. Rep. 2019; 9 (30718678)1310 10.1038/s41598-018-37909-5Crossref PubMed Scopus (48) Google Scholar). Certain cancer cells can tolerate severe conditions, such as poor nutrient availability and oxygen deprivation and survive for prolonged periods (11Izuishi K. Kato K. Ogura T. Kinoshita T. Esumi H. Remarkable tolerance of tumor cells to nutrient deprivation: possible new biochemical target for cancer therapy.Cancer Res. 2000; 60 (11085546): 6201-6207PubMed Google Scholar). To survive in a nutrient-deprived environment, cancer cells use the phosphoinositide 3-kinase/Akt pathway that is associated with nutrient acquisition, cell proliferation, and apoptosis inhibition (12Zhu J. Thompson C.B. Metabolic regulation of cell growth and proliferation.Nat. Rev. Mol. Cell Biol. 2019; 20 (30976106): 436-45010.1038/s41580-019-0123-5Crossref PubMed Scopus (274) Google Scholar, 13Manning B.D. Toker A. AKT/PKB signaling: navigating the network.Cell. 2017; 169 (28431241): 381-40510.1016/j.cell.2017.04.001Abstract Full Text Full Text PDF PubMed Scopus (1616) Google Scholar). Previously, we showed that kigamicins (polycyclic xanthone compounds produced by Amycolatopsis sp. ML630-mF1) inhibit Akt activation and demonstrate preferential cytotoxicity to human pancreatic cancer cells under nutrient-deprived conditions compared with nutrient-sufficient conditions (14Lu J. Kunimoto S. Yamazaki Y. Kaminishi M. Esumi H. Kigamicin D, a novel anticancer agent based on a new anti-austerity strategy targeting cancer cells' tolerance to nutrient starvation.Cancer Sci. 2004; 95 (15182438): 547-55210.1111/j.1349-7006.2004.tb03247.xCrossref PubMed Scopus (92) Google Scholar, 15Kunimoto S. Someno T. Yamazaki Y. Lu J. Esumi H. Naganawa H. Kigamicins, novel antitumor antibiotics: II. Structure determination.J. Antibiot. (Tokyo). 2003; 56 (15015728): 1012-101710.7164/antibiotics.56.1012Crossref PubMed Scopus (38) Google Scholar, 16Kunimoto S. Lu J. Esumi H. Yamazaki Y. Kinoshita N. Honma Y. Hamada M. Ohsono M. Ishizuka M. Takeuchi T. Kigamicins, novel antitumor antibiotics: I. Taxonomy, isolation, physico-chemical properties and biological activities.J. Antibiot. (Tokyo). 2003; 56 (15015727): 1004-101110.7164/antibiotics.56.1004Crossref PubMed Scopus (47) Google Scholar). Therefore, targeting cancer cells that have adapted to nutrient deprivation might be an effective strategy for cancer therapy. Further, the tumor microenvironment plays a major role in determining the metabolic phenotypes of cancer cells (17Cairns R.A. Harris I.S. Mak T.W. Regulation of cancer cell metabolism.Nat. Rev. Cancer. 2011; 11 (21258394): 85-9510.1038/nrc2981Crossref PubMed Scopus (3428) Google Scholar). Metabolic alterations affect reactive oxygen species (ROS) production, which modulates the cellular reduction–oxidation (redox) status (18Gorrini C. Harris I.S. Mak T.W. Modulation of oxidative stress as an anticancer strategy.Nat. Rev. Drug Discov. 2013; 12 (24287781): 931-94710.1038/nrd4002Crossref PubMed Scopus (2076) Google Scholar). Pancreatic cancer is characterized by the expression of oncogenic KRAS, and >90% of the patients with pancreatic cancer have oncogenic mutations in the KRAS (19Morris J.P.T. Wang S.C. Hebrok M. KRAS, Hedgehog, Wnt and the twisted developmental biology of pancreatic ductal adenocarcinoma.Nat. Rev. Cancer. 2010; 10 (20814421): 683-69510.1038/nrc2899Crossref PubMed Scopus (428) Google Scholar). Oncogenic KRAS induces ROS production through activation of NADPH oxidase 1 and mitochondrial dysfunction (20Park M.T. Kim M.J. Suh Y. Kim R.K. Kim H. Lim E.J. Yoo K.C. Lee G.H. Kim Y.H. Hwang S.G. Yi J.M. Lee S.J. Novel signaling axis for ROS generation during K-Ras–induced cellular transformation.Cell Death Differ. 2014; 21 (24632950): 1185-119710.1038/cdd.2014.34Crossref PubMed Scopus (77) Google Scholar, 21Liou G.Y. Döppler H. DelGiorno K.E. Zhang L. Leitges M. Crawford H.C. Murphy M.P. Storz P. Mutant KRas-induced mitochondrial oxidative stress in acinar cells upregulates EGFR signaling to drive formation of pancreatic precancerous lesions.Cell Rep. 2016; 14 (26947075): 2325-233610.1016/j.celrep.2016.02.029Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). KRAS-induced ROS causes DNA damage and genomic instability, which, in turn, facilitates the acquisition of malignant phenotypes (21Liou G.Y. Döppler H. DelGiorno K.E. Zhang L. Leitges M. Crawford H.C. Murphy M.P. Storz P. Mutant KRas-induced mitochondrial oxidative stress in acinar cells upregulates EGFR signaling to drive formation of pancreatic precancerous lesions.Cell Rep. 2016; 14 (26947075): 2325-233610.1016/j.celrep.2016.02.029Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Appropriate levels of ROS are deemed beneficial for tumor development and progression; however, an excess of ROS leads to senescence and cell death because ROS can damage DNA, RNA, lipids, and proteins. The cells use redox systems, such as GSH and thioredoxin (Trx) systems, to counteract the detrimental effects of ROS. The GSH system involves NADPH, GSH reductase, GSH, and GSH peroxidase, whereas the Trx systems involves NADPH, thioredoxin reductase (TrxR), Trx, and peroxiredoxin (22Ren X. Zou L. Zhang X. Branco V. Wang J. Carvalho C. Holmgren A. Lu J. Redox signaling mediated by thioredoxin and glutathione systems in the central nervous system.Antioxid. Redox Signal. 2017; 27 (28443683): 989-101010.1089/ars.2016.6925Crossref PubMed Scopus (150) Google Scholar). GSH peroxidase and peroxiredoxin are antioxidant enzymes that efficiently catalyze the decomposition of H2O2 and that are known to reduce excessive ROS levels and prevent cellular damage. Many genes involved in the GSH and Trx systems are regulated by the transcription factor Nrf2 (nuclear factor erythroid 2–related factor 2). Oncogenic KRAS induces Nrf2 transcription and promotes ROS detoxification that supports pancreatic tumor maintenance (23DeNicola G.M. Karreth F.A. Humpton T.J. Gopinathan A. Wei C. Frese K. Mangal D. Yu K.H. Yeo C.J. Calhoun E.S. Scrimieri F. Winter J.M. Hruban R.H. Iacobuzio-Donahue C. Kern S.E. et al.Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis.Nature. 2011; 475 (21734707): 106-10910.1038/nature10189Crossref PubMed Scopus (1452) Google Scholar, 24Chio I.I.C. Jafarnejad S.M. Ponz-Sarvise M. Park Y. Rivera K. Palm W. Wilson J. Sangar V. Hao Y. Öhlund D. Wright K. Filippini D. Lee E.J. Da Silva B. Schoepfer C. et al.NRF2 promotes tumor maintenance by modulating mRNA translation in pancreatic cancer.Cell. 2016; 166 (27477511): 963-97610.1016/j.cell.2016.06.056Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Cancer cells exhibit often high ROS levels compared with normal cells because of metabolic and signaling aberrations caused by the accumulation of multiple genetic alterations (25Durand N. Storz P. Targeting reactive oxygen species in development and progression of pancreatic cancer.Expert Rev. Anticancer Ther. 2017; 17 (27841037): 19-3110.1080/14737140.2017.1261017Crossref PubMed Scopus (42) Google Scholar, 26Trachootham D. Alexandre J. Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach?.Nat. Rev. Drug Discov. 2009; 8 (19478820): 579-59110.1038/nrd2803Crossref PubMed Scopus (3613) Google Scholar). Accordingly, these malignant cells are more dependent on antioxidants for cell survival and thus are more vulnerable to further oxidative stress induced by inhibition of redox systems (26Trachootham D. Alexandre J. Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach?.Nat. Rev. Drug Discov. 2009; 8 (19478820): 579-59110.1038/nrd2803Crossref PubMed Scopus (3613) Google Scholar, 27Toyokuni S. Okamoto K. Yodoi J. Hiai H. Persistent oxidative stress in cancer.FEBS Lett. 1995; 358 (7821417): 1-310.1016/0014-5793(94)01368-BCrossref PubMed Scopus (999) Google Scholar). In the present study, we found that inhibitors of redox system display preferential cytotoxicity to cancer cells in nutrient-deprived conditions. Targeting redox systems might be an attractive therapeutic strategy under nutrient-deprived conditions of the tumor microenvironment. We constructed a high-throughput screening system to explore small molecules that preferentially reduce the survival of nutrient-deprived cancer cells (Fig. 1A). To identify selective cytotoxic agents that act preferentially on human pancreatic cancer PANC-1 cells grown in nutrient-deprived medium (NDM), but not in nutrient-sufficient medium (Dulbecco's modified Eagle's medium (DMEM)), we screened chemical libraries and microbial culture extracts. We found two culture extracts of fungi. The first was penicillic acid (PCA), which was isolated from a culture extract of fungal strain CR44035 (Fig. 1B) (28Birkinshaw J.H. Oxford A.E. Raistrick H. Studies in the biochemistry of micro-organisms: penicillic acid, a metabolic product of Penicillium puberulum Bainier and P. cylopium Westling.Biochem. J. 1936; 30 (16746037): 394-41110.1042/bj0300394Crossref PubMed Google Scholar). PCA is a classical mycotoxin produced by various fungi, such as Penicillium and Aspergillus (29Natori S. Sakaki S. Kurata H. Udagawa S. Ichinoe M. Chemical and cytotoxicity survey on the production of ochratoxins and penicillic acid by Aspergillus ochraceus Wilhelm.Chem. Pharm. Bull. (Tokyo). 1970; 18 (5494852): 2259-226810.1248/cpb.18.2259Crossref PubMed Scopus (26) Google Scholar). The second was papyracillic acid (PPA), a PCA analog isolated from a culture extract of fungal strain CR45365 (Fig. 1C) (30Shan R.D. Anke H. Stadler M. Sterner O. Papyracillic acid, a new penicillic acid analogue from the ascomycete Lachnum papyraceum.Tetrahedron. 1996; 52: 10249-1025410.1016/0040-4020(96)00559-5Crossref Scopus (12) Google Scholar). PCA and PPA clearly showed preferential cytotoxicity to PANC-1 cells under nutrient-deprived compared with nutrient-sufficient conditions (Fig. 1, D and E). The preferential cytotoxicity of PCA and PPA under nutrient-deprived conditions was exhibited in PANC-1 cells and other human pancreatic cancer cell lines (Fig. 1F and Fig. S1, A and B). We also examined cell survival under nutrient-deprived conditions in colony formation assays (Fig. 1, G and H, and Fig. S1C). PCA and PPA inhibited colony formation of PANC-1 cells under nutrient-deprived but not under nutrient-sufficient conditions. Next, we examined apoptosis induced by PCA and PPA using annexin V and propidium iodide (PI) double staining (Fig. 1I). PCA and PPA significantly increased the number of early (annexin V–positive/PI-negative) and late (annexin V–positive/PI-positive) apoptotic cells under nutrient-deprived conditions. We also observed PCA- and PPA-triggered activation of caspase-3/7 in only nutrient-deprived cells (Fig. 1, J and K). These results indicated that PCA and PPA preferentially inhibit cell growth of human pancreatic cancer under nutrient-deprived conditions. We examined the sensitivity of a panel of human cancer cell lines (JFCR39) to PCA and PPA to predict the molecular mechanism of PCA and PPA (31Yamori T. Matsunaga A. Sato S. Yamazaki K. Komi A. Ishizu K. Mita I. Edatsugi H. Matsuba Y. Takezawa K. Nakanishi O. Kohno H. Nakajima Y. Komatsu H. Andoh T. et al.Potent antitumor activity of MS-247, a novel DNA minor groove binder, evaluated by an in vitro in vivo human cancer cell line panel.Cancer Res. 1999; 59 (10463605): 4042-4049PubMed Google Scholar). Sensitivity patterns in JFCR39 were determined to be different from existing clinical drugs, indicating that PCA and PPA have unique molecular mechanisms (Fig. S2, A and B). Next, we examined metabolites of PANC-1 cells altered by PCA using capillary electrophoresis (CE)–TOF MS. In total, 218 metabolites related to primary metabolism were detected in PCA-treated PANC-1 cells. GSH, a major cellular antioxidant, was markedly decreased by PCA (Fig. 2A, and Fig. S2, C and D, and Table S1). Detailed analysis revealed that GSH rapidly decreases after PCA and PPA treatment and that GSH levels are less than half of its initial levels at ∼1 h (Fig. 2B). Similarly, the reduced GSH/GSSG ratio also decreased after PCA and PPA treatment, which depended on the PCA and PPA concentration (Fig. 2C and Fig. S2E). Further, we also observed such decreases in GSH after PCA and PPA treatment in Capan-1, MIA Paca-2, and KP-3 cells (Fig. 2D and Fig. S2F). GSH is a thiol-containing tripeptide (γ-glutamyl-cysteinyl-glycine). We monitored the direct reaction of PCA and PPA with GSH using LC–MS. We detected PCA–GSH and PPA–GSH conjugates as new peaks (Fig. 2, E and F, and Fig. S2, G and H). These data suggest that PCA and PPA form an adduct with GSH nonenzymatically. The chemical structures of PCA–GSH and PPA–GSH conjugates were assumed as shown in Fig. 2 (E and F). We used N-Ac-cysteine methyl ester (NACM) as a simple model compound instead of GSH in determining the detailed chemical structures of PCA–GSH or PPA–GSH conjugates (Fig. S2I). A PCA–NACM conjugate was quickly formed by incubation of PCA and NACM in PBS. However, the conjugate was a mixture of four isomers, which could not be isolated by chromatography. Using derivatization by methylation, we separated four methyl derivatives of isomers by reverse-phase HPLC and chiral HPLC (Fig. S2J). Each chemical structure was determined by analyzing NMR and MS spectroscopic data (Fig. S2, K–R). Further, we examined PCA binding to GSH in cells. The PCA–GSH conjugate was detected in PANC-1 cells treated with PCA, indicating that PCA binds to GSH in cells as well as in vitro (Fig. 2G and Fig. S2S). These results suggested that the exomethylene groups of PCA and PPA covalently bind to the thiol group of GSH, decreasing free GSH (Fig. 2H). Several small thiol metabolites exist in cells. PCA and PPA might bind to other small thiol metabolites in addition to GSH. Cys was undetectable in PANC-1 cells by LC–MS, but BxPC-3 cells contained detectable levels of Cys. Treatment of BxPC-3 cells with PCA decreased Cys levels and generated a PCA-Cys conjugate (Fig. S2, T–V). Therefore, we might also consider the relationship between PCA/PPA and other small thiol metabolites. PCA and PPA decrease intracellular GSH levels, shifting intracellular ROS generation–antioxidant balance toward an increase in ROS levels. PCA and PPA treatment dose- and time-dependently increased intracellular ROS levels in PANC-1 cells, as shown by intracellular H2O2 concentrations (Fig. 3, A and B). This increase was observed not only in PANC-1 cells but also in Capan-1, MIA Paca-2, and KP-3 cells (Fig. 3C). These results indicated that PCA and PPA decrease GSH and increase ROS levels, leading to apoptosis. Investigation of the relationship between nutrient deprivation and GSH levels showed that intracellular GSH in PANC-1 cells gradually decreases under nutrient-deprived conditions, but nutrient-sufficient conditions, understandably, did not affect GSH levels (Fig. 3D). PCA and PPA decreased GSH levels more rapidly under nutrient-deprived compared with nutrient-sufficient conditions. Investigation of the relationship between nutrient deprivation and ROS levels showed that intracellular ROS levels are higher in nutrient-deprived cells than in nutrient-sufficient cells at basal levels (Fig. 3E), indicating that nutrient deprivation increases oxidative stress in cancer cells. In addition, PCA and PPA treatment induced large accumulation of ROS in nutrient-deprived compared with nutrient-sufficient cells, which might contribute to increased apoptosis when nutrients are deemed insufficient. However, preferential cytotoxicity under nutrient-deprived conditions by PCA and PPA remains to be elaborated. We investigated the effects of nutritional composition on PCA and PPA cytotoxicity under nutrient-deprived conditions. Preferential cytotoxicity of PCA and PPA was induced by deprivation of amino acids, not glucose (Fig. 3F). NDM lacks 15 amino acids available in DMEM. We investigated the effect of 15 amino acids on PCA cytotoxicity in NDM. Single amino acid supplementation could not rescue PCA cytotoxicity (Fig. S3A). However, simultaneous addition of Gln, Gly, and Cys (precursor amino acids of GSH) abrogated PCA preferential cytotoxicity under nutrient-deprived conditions (Fig. 3G). The decrease in cellular GSH by nutrient deprivation can be attributed to a limitation of the supply of precursor amino acids of GSH. Consequently, we propose the molecular mechanism of PCA and PPA (Fig. 3H). Tumor tissue shows reduced content of glucose and amino acids because of the aberrant cell proliferation and abnormal blood vessels, which decrease GSH levels. PCA and PPA nonenzymatically bind to depleted GSH, further decreasing GSH levels. The resulting small amount of GSH can no longer prevent cellular damage caused by ROS, often resulting in induction of apoptosis. In contrast, nutrient-sufficient cells can produce sufficient GSH in normal tissue. Even if PCA and PPA decrease GSH, the remaining GSH will be enough to control redox status and prevent cellular damage by ROS. Small molecular compounds that decrease antioxidant GSH, such as PCA and PPA, show selective cytotoxicity to human pancreatic cancer cells under nutrient-deprived conditions. Trx serves a partially overlapping and complementary role to GSH for protection from oxidative stress (32Watson W.H. Yang X. Choi Y.E. Jones D.P. Kehrer J.P. Thioredoxin and its role in toxicology.Toxicol. Sci. 2004; 78 (14691207): 3-1410.1093/toxsci/kfh050Crossref PubMed Scopus (199) Google Scholar). Small molecular compounds that decrease Trx levels might also show such selective cytotoxicity. Auranofin is an Au(I) complex containing an gold–sulfur bond stabilized by a triethylphosphine group. This agent inhibits cytosolic Trx reductase 1 (TrxR1) and mitochondrial TrxR2, thus decreasing the reduced form of Trx (Fig. 4A) (33Rackham O. Shearwood A.M. Thyer R. McNamara E. Davies S.M. Callus B.A. Miranda-Vizuete A. Berners-Price S.J. Cheng Q. Arnér E.S. Filipovska A. Substrate and inhibitor specificities differ between human cytosolic and mitochondrial thioredoxin reductases: implications for development of specific inhibitors.Free Radic. Biol. Med. 2011; 50 (21172426): 689-69910.1016/j.freeradbiomed.2010.12.015Crossref PubMed Scopus (86) Google Scholar, 34Gromer S. Arscott L.D. Williams Jr., C.H. Schirmer R.H. Becker K. Human placenta thioredoxin reductase: isolation of the selenoenzyme, steady state kinetics, and inhibition by therapeutic gold compounds.J. Biol. Chem. 1998; 273 (9685351): 20096-2010110.1074/jbc.273.32.20096Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). Auranofin markedly inhibited TrxR activities of human recombinant TrxR1 and TrxR2 proteins (Fig. 4B). In addition, auranofin inhibited total intracellular TrxR activity in PANC-1 and PSN-1 cells (Fig. 4C and Fig. S4A), and auranofin treatment showed preferential cytotoxicity under nutrient-deprived compared with nutrient-sufficient conditions in PANC-1 cells and other human pancreatic cancer cells (Fig. 4, D and E, and Fig. S4B). Furthermore, preferential cytotoxicity of auranofin was induced by deprivation of amino acids, but not glucose (Fig. S4C). Auranofin might cause an imbalance between reduced and oxidized Trx, because alteration of the intracellular redox state triggers tumor cell apoptosis (35Trachootham D. Lu W. Ogasawara M.A. Nilsa R.D. Huang P. Redox regulation of cell survival.Antioxid. Redox Signal. 2008; 10 (18522489): 1343-137410.1089/ars.2007.1957Crossref PubMed Scopus (1287) Google Scholar). We have examined the redox states of Trx in PANC-1 cells incubated with auranofin using modified redox Western blotting (Fig. 4F) (36Folda A. Citta A. Scalcon V. Calì T. Zonta F. Scutari G. Bindoli A. Rigobello M.P. Mitochondrial thioredoxin system as a modulator of cyclophilin D redox state.Sci. Rep. 2016; 6 (26975474)23071 10.1038/srep23071Crossref PubMed Scopus (37) Google Scholar, 37Du Y. Zhang H. Zhang X. Lu J. Holmgren A. Thioredoxin 1 is inactivated due to oxidation induced by peroxiredoxin under oxidative stress and reactivated by the glutaredoxin system.J. Biol. Chem. 2013; 288 (24062305): 32241-3224710.1074/jbc.M113.495150Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The uppermost band indicates the fully reduced form of Trx, whereas the more oxidized forms appeared in the lower part of the gel. At basal levels, we observed the oxidized forms of Trx in nutrient-deprived compared with nutrient-sufficient cells. Auranofin treatment obviously dose-dependently increased the oxidized forms of Trx under nutrient-deprived conditions. In parallel, auranofin treatment increased ROS levels under nutrient-deprived conditions in PANC-1, MIA Paca-2, and PSN-1 cells (Fig. 4G and Fig. S4D). However, the same treatment did not increase ROS levels in KP-4 cells that display only a small difference in cytotoxicity in DMEM versus NDM (Fig. 4E). Moreover, auranofin-induced ROS generation was eliminated by treatment with an antioxidant, such as N-acetyl-l-cysteine (NAC). NAC treatment also counteracted auranofin cytotoxicity (Fig. 4H and Fig. S4E). Therefore, accumulation of ROS can be attributed to auranofin-induced cytotoxicity. We also examined caspase 3/7 activity, activation of poly(ADP-ribose) polymerase (PARP), and occurrence of apoptotic cells to assess auranofin impact on apoptosis. Auranofin significantly up-regulated caspase 3/7 activity in PANC-1 cells time-dependently under nutrient deprivation (Fig. 4, I and J). Caspase 3/7 activation subsequently induced proteolytic cleavage of PARP, and finally, underwent apoptosis. Auranofin treatment in nutrient-deprived cells clearly showed activation of PARP cleavage (Fig. 4K). The percentage of apoptotic cells (annexin V and PI double-positive) were significantly increased by auranofin treatment under nutrient-deprived conditions (Fig. 4L). In addition, NAC was able to reduce the percentage of auranofin-induced apoptotic cells. Finally, we evaluated the antitumor activity of auranofin using a nude mouse xenograft model of human pancreatic cancer. Intraperitoneal administration of auranofin showed significant suppression of tumor growth in PSN-1 cancers. Auranofin suppressed tumor growth up to ∼40% of vehicle control, and its antitumor activity was comparable with that of cisplatin (Fig. 4M). We found no difference in body weight between auranofin-treated and control mice after treatment, which had no severe side effects. We also evaluated the anti-tumor activity of PCA on PSN-1 tumor-bearing mice. PCA suppressed tumor growth up to ∼30% of vehicle control (Fig. 4N). Next, we investigated the combined effect of PCA/auranofin and cisplatin. Combination therapy has been considered as an effective tool for augmenting efficacy, preventing the development of drug resistance, and reducing treatment duration. PCA/PPA is expected to increase the sensitivity of pancreatic cancer cells to cisplatin because cisplatin is inactivated by GSH (38Godwin A.K. Meister A. O'Dwyer P.J. Huang C.S. Hamilton T.C. Anderson M.E. High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis.Proc. Natl. Acad. Sci. U.S.A. 1992; 89 (1348364): 3070-307410.1073/pnas.89.7.3070Crossref PubMed Scopus (847) Google Scholar, 39Ishikawa T. Ali-Osman F. Glutathione-associated cis-diamminedichloroplatinum(II) metabolism and ATP-dependent efflux from leukemia cells: molecular characterization of glutathione-platinum complex and its biological significance.J. Biol. Chem. 1993; 268 (8376370): 20116-20125Abstract Full Text PDF PubMed Google Scholar). PCA/PPA/auranofin showed increased efficacy in colony formation assays and 3D spheroid models (Fig. S4, F–H). Unfortunately, PCA and auranofin did not show this effect with cisplatin on in vivo mouse xenografts (Fig. S4, I and J). A more detail investigation will be required to enhance a combined effect with cispla" @default.
• W3088591201 created "2020-10-01" @default.
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• W3088591201 date "2020-12-01" @default.
• W3088591201 modified "2023-09-30" @default.
• W3088591201 title "Human pancreatic cancer cells under nutrient deprivation are vulnerable to redox system inhibition" @default.
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