|dc.description.abstract||Consensus opinion suggests that apoptosis occurs when the intracellular redox potential
reaches its oxidative range, i.e. when the balance between oxidants and reductants is
disturbed. An understanding of the links between redox potential and the induction of
apoptosis in cells could improve our understanding of the process and help to predict
This study investigates the changes in redox potential at distinct stages of apoptosis
induced in the human cervical cancer cell line, HeLa. Stages of the apoptotic process
were defined by loss of mitochondrial membrane polarisation (ΔΨm), membrane
phosphatidyl serine exposure, caspase-3 activation, and nuclear fragmentation. To
measure real-time redox potential change in apoptotic cells two independent methods
were used: (1) expression of redox-responsive green fluorescent protein (roGFP2)
measured by flow cytometry and (2) redox-responsive nanosensors detected by surface
enhanced Raman spectroscopy (SERS).
roGFP2 measurements showed that HeLa cells demonstrate a shift towards an oxidative
redox state during the later stages of apoptosis and this was preceded by loss of ΔΨm.
The relationship between these two events was investigated by transient inhibition of
mitochondrial permeability transition pore opening using the inhibitor bongkrekic acid
At the cell population level, transient exclusion of the mitochondrial contribution
delayed two key events of apoptosis in the first two hours measured by nuclear
fragmentation and loss of ΔΨm. However, BKA treatment did not affect redox
potential, reported by roGFP2, when compared with controls. Therefore, this suggests
that mitochondria do not contribute towards the overall redox potential change in
To gain insight into the significance of redox change at the earliest stages of apoptosis,
single cell studies were performed. SERS, employing simultaneous redox potential and
intracellular pH measurements using two synthetic nanosensors AQ-NS and MBA-NS,
showed that BKA pre-treatment resulted in increased alkalinity and the cells were
consequently protected from induction of apoptosis in the first thirty minutes of the
kinase inhibitor staurosporine treatment.
Measurements with SERS nanosensors allowed for adjustment for pH, which provides a
clearer insight into redox potential dynamics, with consideration of the environment,
and accurate quantitative assessment of redox at early stages of apoptosis.
Together these data suggest that while roGFP2 is a valid method to use at a population
level, SERS is a more sensitive method for measuring the redox potential of the cell at
the early stages of apoptosis.||en