4 | DISCUSSION
This study quantifies chemotherapeutic efficacy of drugs metformin,
sulindac, and their combination in terms of superoxide production using
time-lapse fluorescence microscopy. Superoxide levels were quantified
over an 8-hour period for each treatment through time-lapse fluorescence
microscopy. The various drug treatments exhibited distinct trends in the
generation of superoxide, as depicted in Figure 2. We report i) the
combination of sulindac and metformin mediated robust superoxide
generation out of all treatment conditions and ii) individual treatments
of metformin and sulindac do not generate an effective response when
compared to the combination treatment using the same dosages. These
findings suggest the co-treatment to cancer cells could increase
apoptosis in the presence of an oxidative agent, functioning as a more
effective cancer treatment option.
Previous experiments examined the combination of pre-treatment with
sulindac before the addition of another agent and concluded that
mitochondrial dysfunction played a role in anticancer mechanisms
[21,
23]. This present study is an expansion
of this work, as it investigates the onset of cellular effects while the
drug combination is administered together, validating cell viability
data. Additionally, this study demonstrates in further detail the
mechanism of action for superoxide formation and its potential role in
cancer cell death.
Cancer research using chemotherapeutic agents indicates evidence of
heavily fostered apoptosis of tumor cells by these agents
[43-45]. Specifically, research has
focused on agents that elevate superoxide levels in cancer tissue,
utilizing superoxide to induce apoptotic pathways
[46]. Superoxide form key agents of
mitochondrial dysfunction often associated with apoptosis-inducing
cancer drugs [45]. This coincides
with the results from this research where highest superoxide elevation
was observed with the combination drug treatment (Figure 4).
The inhibition of complex I by metformin causes superoxide accumulation
in the mitochondrial matrix, promoting a collapse of cellular
homeostasis and apoptosis [47,
48]. Previous experiments have
suggested that administering metformin increased superoxide production,
decreased mitochondrial membrane potential, activated caspase family
enzymes, which are closely related to apoptosis, increased
apoptosis-promoting BAX (BCL-2 associated X protein) levels, and
decreased anti-apoptotic BCL-2 (B-cell lymphoma-2) and MCL-1 (Myeloid
leukemia 1) protein levels [47,
49-51]. Sulindac-induced apoptosis
heavily relies on superoxide generation
[23,
45]. The oxidative stress induced by
superoxide phosphorylates p38 MAPK (p38 mitogen-activated protein
kinase) [52], a protein prominently
involved in apoptotic pathways and p53 a tumor suppressor gene
[53]. The Bcl-protein family controls
proapoptotic responses by the inhibition of BAX and BAK (Bcl-2
homologous antagonist killer) [54,
55]. Cancer cells have been found to
evade apoptosis predominantly by inhibiting BAX and BAK proteins
[56]. The oligomerization of BAX and
BAK at the mitochondrial outer membrane (MOM) opens pores mediating the
release of cytochrome c (cyt c) from the mitochondria
[56]. Cyt c activates caspases that
cleave all cellular components of the cell. Accordingly, administering
metformin and sulindac together brings forth significant superoxide
accumulation in the mitochondria supporting a chemotherapeutic mechanism
for activation of the apoptotic events.
We have demonstrated a combination of two FDA approved drugs with
anti-cancer activity can more intensely induce superoxide production in
cancer cells than when treated alone. This approach is advantageous
because it reduces dosage requirements as well as the development of
drug resistance often seen in monotherapies. When drugs with different
mechanisms of action are combined, each drug can be used at its optimal
dose, limiting adverse side effects. A two-drug treatment can reduce
cytotoxicity to peripheral non-tumor cells
[57] and provide a more efficacious
treatment.
5 | CONCLUSION
Time- lapse microscopy was used to monitor ROS generation in lung cancer
cells treated with a combinatorial treatment, metformin and sulindac.
Results from this study report significant superoxide accumulation due
to the combined effect of metformin and sulindac on A549 lung cancer
cells. However, superoxide production for individual metformin and
sulindac treatments showed only modest increases. The combinatorial
treatment has shown a notable anticancer effect, specifically targeting
the metabolic dysfunction caused by excess superoxide, which ultimately
may lead to increased cell death in lung cancer cells. This study
demonstrates elevated superoxide levels in the dual drug combination,
which can be used in chemotherapeutic treatments. We aim to expand on
our prior protocol and investigation to quantitatively measure and
compare O2•− production across
different cancer treatment conditions
[39]. Our ongoing cancer therapy
regime examines the influence of extremely low frequency electromagnetic
field radiation combined with proapoptotic agents.
ACKNOWLEDGEMENTS
This work was funded by National Institutes of Health (EY031533).
CONFLICT OF INTEREST
The authors declare no conflict of interest.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
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