TY - JOUR
T1 - S-nitrosocysteine and glutathione depletion synergize to induce cell death in human tumor cells
T2 - Insights into the redox and cytotoxic mechanisms
AU - Knany, Alaa
AU - Engelman, Rotem
AU - Hariri, Hiba Abu
AU - Biswal, Shyam
AU - Wolfenson, Haguy
AU - Benhar, Moran
N1 - Funding Information:
This work was supported by Israel Science Foundation grants no. 1574/14 and 939/19 (to M.B.), by U.S-Israel Binational Science Foundation grant no. 2013451 (to M.B. and S.B.) and by National Institutes of Health R01CA206155 (to S.B.)
Publisher Copyright:
© 2020 Elsevier Inc.
PY - 2020/11/20
Y1 - 2020/11/20
N2 - Nitric oxide (NO)-dependent signaling and cytotoxic effects are mediated in part via protein S-nitrosylation. The magnitude and duration of S-nitrosylation are governed by the two main thiol reducing systems, the glutathione (GSH) and thioredoxin (Trx) antioxidant systems. In recent years, approaches have been developed to harness the cytotoxic potential of NO/nitrosylation to inhibit tumor cell growth. However, progress in this area has been hindered by insufficient understanding of the balance and interplay between cellular nitrosylation, other oxidative processes and the GSH/Trx systems. In addition, the mechanistic relationship between thiol redox imbalance and cancer cell death is not fully understood. Herein, we explored the redox and cellular effects induced by the S-nitrosylating agent, S-nitrosocysteine (CysNO), in GSH-sufficient and -deficient human tumor cells. We used L-buthionine-sulfoximine (BSO) to induce GSH deficiency, and employed redox, biochemical and cellular assays to interrogate molecular mechanisms. We found that, under GSH-sufficient conditions, a CysNO challenge (100–500 μM) results in a marked yet reversible increase in protein S-nitrosylation in the absence of appreciable S-oxidation. In contrast, under GSH-deficient conditions, CysNO induces elevated and sustained levels of both S-nitrosylation and S-oxidation. Experiments in various cancer cell lines showed that administration of CysNO or BSO alone commonly induce minimal cytotoxicity whereas BSO/CysNO combination therapy leads to extensive cell death. Studies in HeLa cancer cells revealed that treatment with BSO/CysNO results in dual inhibition of the GSH and Trx systems, thereby amplifying redox stress and causing cellular dysfunction. In particular, BSO/CysNO induced rapid oxidation and collapse of the actin cytoskeletal network, followed by loss of mitochondrial function, leading to profound and irreversible decrease in ATP levels. Further observations indicated that BSO/CysNO-induced cell death occurs via a caspase-independent mechanism that involves multiple stress-induced pathways. The present findings provide new insights into the relationship between cellular nitrosylation/oxidation, thiol antioxidant defenses and cell death. These results may aid future efforts to develop NO/redox-based anticancer approaches.
AB - Nitric oxide (NO)-dependent signaling and cytotoxic effects are mediated in part via protein S-nitrosylation. The magnitude and duration of S-nitrosylation are governed by the two main thiol reducing systems, the glutathione (GSH) and thioredoxin (Trx) antioxidant systems. In recent years, approaches have been developed to harness the cytotoxic potential of NO/nitrosylation to inhibit tumor cell growth. However, progress in this area has been hindered by insufficient understanding of the balance and interplay between cellular nitrosylation, other oxidative processes and the GSH/Trx systems. In addition, the mechanistic relationship between thiol redox imbalance and cancer cell death is not fully understood. Herein, we explored the redox and cellular effects induced by the S-nitrosylating agent, S-nitrosocysteine (CysNO), in GSH-sufficient and -deficient human tumor cells. We used L-buthionine-sulfoximine (BSO) to induce GSH deficiency, and employed redox, biochemical and cellular assays to interrogate molecular mechanisms. We found that, under GSH-sufficient conditions, a CysNO challenge (100–500 μM) results in a marked yet reversible increase in protein S-nitrosylation in the absence of appreciable S-oxidation. In contrast, under GSH-deficient conditions, CysNO induces elevated and sustained levels of both S-nitrosylation and S-oxidation. Experiments in various cancer cell lines showed that administration of CysNO or BSO alone commonly induce minimal cytotoxicity whereas BSO/CysNO combination therapy leads to extensive cell death. Studies in HeLa cancer cells revealed that treatment with BSO/CysNO results in dual inhibition of the GSH and Trx systems, thereby amplifying redox stress and causing cellular dysfunction. In particular, BSO/CysNO induced rapid oxidation and collapse of the actin cytoskeletal network, followed by loss of mitochondrial function, leading to profound and irreversible decrease in ATP levels. Further observations indicated that BSO/CysNO-induced cell death occurs via a caspase-independent mechanism that involves multiple stress-induced pathways. The present findings provide new insights into the relationship between cellular nitrosylation/oxidation, thiol antioxidant defenses and cell death. These results may aid future efforts to develop NO/redox-based anticancer approaches.
KW - Cancer
KW - Cell death
KW - Glutathione
KW - Nitrosylation
KW - Oxidation
KW - Thiols
KW - Thioredoxin
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UR - http://www.scopus.com/inward/citedby.url?scp=85090746024&partnerID=8YFLogxK
U2 - 10.1016/j.freeradbiomed.2020.08.026
DO - 10.1016/j.freeradbiomed.2020.08.026
M3 - Article
C2 - 32898624
AN - SCOPUS:85090746024
SN - 0891-5849
VL - 160
SP - 566
EP - 574
JO - Free Radical Biology and Medicine
JF - Free Radical Biology and Medicine
ER -