Knockdown of the inducible nitric oxide synthase (NOS2) splicing variant S3 promotes autophagic cell death from nitrosative stress in SW480 human colon cancer cells

Autor(es)

Castro, Eloisa D. ^[1] ; Mathias, Pedro Paulo M. ^[1] ; Batista, Wagner L. ^[2] ; Sato, Alex Yuri S. ^[3] ; Toledo, Mayte S. ^[1] ; Almeida, Victor T. ^[1] ; Curcio, Marli F. ^[1] ; Costa, Paulo E. ^[1] ; Stern, Arnold ^[4] ; Monteiro, Hugo P. ^[1]

Afiliação do(s) autor(es)

^[1] Univ Fed Sao Paulo, Ctr Cellular & Mol Therapy CTCMol, Dept Biochem, Rua Mirassol 207, Sao Paulo, SP - Brazil

^[2] Univ Fed Sao Paulo, Dept Pharmaceut Sci, Sao Paulo - Brazil

^[3] Univ Fed Sao Paulo, Dept Morphol & Genet, Sao Paulo - Brazil

^[4] NYU, Grossman Sch Med, New York, NY - USA

• Tipo de documento: Artigo Científico
• Fonte: Cell Biology International; v. 46, n. 1 NOV 2021.
• Citações Web of Science: 0

Resumo

Low levels of nitric oxide (NO) produced by constitutively expressed inducible NO synthase (NOS2) in tumor cells may be an important factor in their development. NOS2 expression is associated with high mortality rates for various cancers. Alternative splicing of NOS2 down-regulates its enzymatic activity, resulting in decreased intracellular NO concentrations. Specific probes to detect alternative splicing of NOS2 were used in two isogenic human colon cancer cell lines derived either from the primary tumor (SW480) or from a lymph node metastasis (SW620). Splicing variant of NOS2 S3, lacking exons 9, 10, and 11, was overexpressed in SW480 cells. NOS2 S3 was silenced in SW480 cells. Flow-cytometry analysis was used to estimate the intracellular NO levels and to analyze the cell cycle of the studied cell lines. Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine apoptosis and autophagy markers. SW480 and SW620 cells expressed NOS2 S3. Overexpression of the NOS2 S3 in SW480 cells downregulated intracellular NO levels. SW480 cells with knocked down NOS2 S3 (referred to as S3C9 cells) had higher intracellular levels of NO compared to the wild-type SW480 cells under serum restriction. Higher NO levels resulted in the loss of viability of S3C9 cells, which was associated with autophagy. Induction of autophagy by elevated intracellular NO levels in S3C9 cells under serum restriction, suggests that autophagy operates as a cytotoxic response to nitrosative stress. The expression of NOS2 S3 plays an important role in regulating intracellular NO production and maintaining viability in SW480 cells under serum restriction. These findings may prove significant in the design of NOS2/NO-based therapies for colon cancer. (AU)

• Processo FAPESP: 18/15038-7 - O desenvolvimento tumoral sob a ótica da sinalização celular redox: modulação temporal da produção de óxido nítrico e espécies reativas de oxigênio
• Beneficiário: Hugo Pequeno Monteiro
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 12/10470-1 - O papel do óxido nítrico e da enzima óxido nítrico sintase induzível na progressão de tumores humanos de cólon
• Beneficiário: Hugo Pequeno Monteiro
• Modalidade de apoio: Auxílio à Pesquisa - Regular