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

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.

Nitric oxide stimulates a PKC-Src-Akt signaling axis which increases human immunodeficiency virus type 1 replication in human T lymphocytes.

Human immunodeficiency virus (HIV) infections are typically accompanied by high levels of secreted inflammatory cytokines and generation of high levels of reactive oxygen species (ROS). To elucidate how HIV-1 alters the cellular redox environment during viral replication, we used human HIV-1 infected CD4+T lymphocytes and uninfected cells as controls. ROS and nitric oxide (NO) generation, antioxidant enzyme activity, protein phosphorylation, and viral and proviral loads were measured at different times (2-36 h post-infection) in the presence and absence of the NO donor S-nitroso-N-acetylpenicillamine (SNAP). HIV-1 infection increased ROS generation and decreased intracellular NO content. Upon infection, we observed increases in copper/zinc superoxide dismutase (SOD1) and glutathione peroxidase (GPx) activities, and a marked decrease in glutathione (GSH) concentration. Exposure of HIV-1 infected CD4+T lymphocytes to SNAP resulted in an increasingly oxidizing intracellular environment, associated with tyrosine nitration and SOD1 inhibition. In addition, SNAP treatment promoted phosphorylation and activation of the host's signaling proteins, PKC, Src kinase and Akt. Inhibition of PKC leads to inhibition of Src kinase strongly suggesting that PKC is the upstream element in this signaling cascade. Changes in the intracellular redox environment after SNAP treatment had an effect on HIV-1 replication as reflected by increases in proviral and viral loads. In the absence or presence of SNAP, we observed a decrease in viral load in infected CD4+T lymphocytes pre-incubated with the PKC inhibitor GF109203X. In conclusion, oxidative/nitrosative stress conditions derived from exposure of HIV-1-infected CD4+T lymphocytes to an exogenous NO source trigger a signaling cascade involving PKC, Src kinase and Akt. Activation of this signaling cascade appears to be critical to the establishment of HIV-1 infection.