Abstract

Background
and Aim:

Functional recovery in patients who have
suffered debilitating neurological events such as stroke and traumatic brain
injury critically depends on the time window within which appropriate
therapeutic interventions are initiated following the event, and delayed
treatments are associated with poorer functional recovery outcomes. One major
pathway now identified to underlie neuronal death in neurological and
neurodegenerative conditions involves over-activation of the nuclear enzyme poly
(ADP-ribose) polymerase (PARP), leading to PARP-mediated death (parthanatos).  PARP inhibitors are, therefore, being
investigated for their potential as neuroprotective agents. However, the time
course of potential protection by PARP inhibitors in the context of delayed
intervention following the initiation of PARP-mediated cell death has not been
examined in detail. We, therefore, investigated the extent of protection against
PARP-mediated cell death that could be elicited by experimental and
clinically-used PARP inhibitors when they are first added up to several hours
after the initiation of the insult.   

Methods:

PARP-dependent cell death was induced in cultured
human cervical adenocarcinoma (HeLa) cells by treatment with the alkylating
agent, N-methyl-N'-nitro-N-nitrosoguanidine
(MNNG) (50µM, 25 minutes). The cells were then treated with each inhibitor
(olaparib (10 – 1000nM), a clinically-used PARP inhibitor, and DPQ (1 – 50µM),
an experimental PARP inhibitor). Each inhibitor was then added once, immediately
after death induction or at each subsequent 4 h time point up to 16 hours
post-insult. Protection against cell death (as evidence of PARP inhibition) was
then assessed 24 h post-MNNG treatment by quantifying cell viability using the
MTT viability assay. Photomicrographs were also acquired to complement the
viability data. Viability values were reported as means ± SEM of n=3-5
independent experiments. Statistical analysis (ANOVA with Tukey’s post-hoc test)
was performed using GraphPad (Version 8.2.0), with P<0.05 considered
statistically significant.

Results:

Olaparib and DPQ each when added immediately
after the initiation of insult elicited concentration-dependent protection
against MNNG-induced reduction in cell viability, which was significant at 100
and 1000nM (P<0.0001) for olaparib and at 10 and 50µM (P<0.0001) for DPQ.
The insult MNNG reduced viability to about 18% of the negative control, and
olaparib and DPQ protected against this deleterious effect by raising viability
to nearly 80% and 70% of the negative control, at their maximal effects, respectively.
While the protection by olaparib progressively decreased as the number of hours
that had elapsed post-insult when it was added increased, it continued to
elicit concentration-dependent protection up to 12 h post-insult. Its
protection profile revealed that the highest significant decrease in its protection
occurred between 0 and 4 h time points, followed by less steep decreases in
viability between subsequent 4 h time-point pairs. A profile similar to that of
olaparib was exhibited by DPQ, although its concentration-dependent protection
was significant only up to 8 h post-insult. Olaparib was found to exhibit
significantly higher potency than DPQ.

Conclusions:

Our results demonstrate that PARP inhibitors could
elicit protection against PARP-mediated cell death when added as late as 8-12 h
after the initiation of insult. This suggests that PARP inhibitors could provide
significant therapeutic benefit and at least some functional recovery if used clinically
in managing neurological condition scenarios that have been exacerbated by
dela