Nitrosative stress induces DNA strand breaks but not caspase mediated apoptosis in a lung cancer cell line

Abstract

BACKGROUND: Key steps crucial to the process of tumor progression are genomic instability and escape from apoptosis. Nitric oxide and its interrelated reactive intermediates (collectively denoted as NOX) have been implicated in DNA damage and mutational events leading to cancer development, while also being implicated in the inhibition of apoptosis through S-nitrosation of key apoptotic enzymes. The purpose of this study was to explore the interrelationship between NOX-mediated DNA strand breaks (DSBs) and apoptosis in cultured tumor cell lines. METHODS: Two well-characterized cell lines were exposed to increasing concentrations of exogenous NOX via donor compounds. Production of NOX was quantified by the Greiss reaction and spectrophotometery, and confirmed by nitrotyrosine immunostaining. DSBs were measured by the alkaline single-cell gel electrophoresis assay (the COMET assay), and correlated with cell viability by the MTT assay. Apoptosis was analyzed both by TUNEL staining and Annexin V/propidium iodine FACS. Finally, caspase enzymatic activity was measured using an in-vitro fluorogenic caspase assay. RESULTS: Increases in DNA strand breaks in our tumor cells, but not in control fibroblasts, correlated with the concentration as well as rate of release of exogenously administered NOX. This increase in DSBs did not correlate with an increase in cell death or apoptosis in our tumor cell line. Finally, this lack of apoptosis was found to correlate with inhibition of caspase activity upon exposure to thiol- but not NONOate-based NOX donor compounds. CONCLUSIONS: Genotoxicity appears to be highly interrelated with both the concentration and kinetic delivery of NOX. Moreover, alterations in cell apoptosis can be seen as a consequence of the explicit mechanisms of NOX delivery. These findings lend credence to the hypothesis that NOX may play an important role in tumor progression, and underscores potential pitfalls which should be considered when developing NOX-based chemotherapeutic agents.

Figure 1

Nitrite Production in Media and Supernatants in the Presence of Increasing Concentrations of NO_X Donors: A. Nitrite production in media without cells, B. Nitrite production in WI38 supernatants, C. Nitrite production in A549 Supernatants. Measurements of nitrite production in media alone or the supernatant of WI38 and A549 as measured by the Greiss reaction demonstrate increasing concentrations of nitrite production in the presence of increasing concentrations of NO_X donors. This confirms that our NO_X donors are liberating NO_X in a concentration-dependent fashion. Interestingly, the curves for SNAP and glycol-SNAP were shifted toward the right in the supernatants of both WI38 and A549 possibly reflecting the possibility that these thiol-based NO_X donors were preferentially donating their NO_X to intracellular thiols.

Figure 2

COMET Tail Moment Lengths After 24 hour Exposure to Various NO_X Donors: A. COMET tail moment of WI38 after 24 hours of exposure to increasing concentrations of NO_X, B. COMET tail moment of A549 after 24 hours of exposure to increasing concentrations of NO_X. Measurements of the COMET tail moments for WI38 and A549 in the presence of increasing concentrations of NO_X donors demonstrate that A549 was found to have increasing DNA strand breaks as nitrosative stress increased which was not seen in the WI38 control cell line. As seen in the graph, NO_X donors with a long half-life [i.e. glyco-SNAP with a t^1/2 = 28 hours and DETA-NONOate with a t^1/2 of 20 hours] demonstrated significant increases in COMET tail moments only at the higher concentrations of NO donor, whereas NO donors with a shorter half-life [i.e. SNAP with a t^1/2 of 10 hours and Spermine-NONOate with a t^1/2 of 39 minutes] demonstrated significant increases in DNA strand breaks at lower concentrations. This confirms that A549 demonstrates more genomic instability in the presence of increasing nitrosative stress, which is not seen in control cells.

Figure 3

Example COMET Tail Moments for A549: These are example COMET tail moments for A549 in the presence of A.) 0 μM, B.) 75 μM, and C.) 150 μM Spermine-NONOate. Note that a significant increase in tail moment can be visualized with increasing concentrations of this NO_X donor.

Figure 4

Cell Viability as Determined by the MTT Assay with Exposure to Increasing Concentrations of NO_X Donors: A. Cell viability of WI38 as measured by the MTT assay after exposure to increasing concentrations of NO_X, B. Cell viability of A549 as measured by the MTT assay after exposure to increasing concentrations of NO_X. The cell viability was significantly reduced in WI38 the presence of increasing concentrations of NO_X donors at all concentrations greater than 150 μM (p < 0.007 for all) for DETA- and spermine-NONOate, whereas the thiol-based glycol-SNAP and SNAP did not significantly decrease cell viability until 300 μM concentrations. These results are contrasted with A549 cells which did not demonstrate a significant decrease in cell viability (p < 0.03) until 600 μM concentrations of either thiol-based or NONOate-based NO_X donors. Interestingly, the lowest concentration of SNAP (75 μM) demonstrated a significant growth advantage (p = 0.01).

Figure 5

Annexin-V/Propidium Iodine FACS in A549 Cells Unexposed and Exposed to High Concentrations of NO_X Donors: A. Untreated control, B. 600 μM Spermine NONOate treated, C. 600 μM SNAP treated. Annexin-V/Propidium Iodine FACS confirms the TUNEL results that demonstrate that no significant increase in apoptosis is noted in A549 with or without exposure to high nitrosative stress.

Figure 6

Caspase 3 (DEVD-pNa) and Caspase 9 (LEDH-pNa) Activity as Measured by the in vitro Fluorogenic Caspase Assay in the Absence and Presence of High Concentrations of NO_X Donors: Both endogenous and exogenous NO_X appears to influence caspase activity in A549 cells. Caspase 3 activity appears to demonstrate endogenous NO_X inhibition since the addition of 3 mM L-NMMA or 1 mM DTT significantly increased caspase 3 activity (p = 0.001, p = 0.003 respectively). No similar endogenous inhibition was seen with caspase 9. The addition of the thiol-based NO_X donor, SNAP (600 μM), significantly inhibited both caspase 3 and caspase 9 (p < 0.001 for both) when compared with control cell caspase activity. This caspase activity was significantly reversed with the addition of DTT (p < 0.001 for both caspase 3 and 9). Treatment groups: 1) Control, 2) 3 mM L-NMMA, 3) 1 mM DTT, 4) 600 μM Spermine-NONOate, 5) 600 μM SNAP, 6) 600 μM SNAP + 1 mM DTT.