KEYWORDS
antimycin A, fluorescence microscopy, metformin, MitoSOX red, oxidative
stress, sulindac, superoxide, time-lapse imaging
1 | INTRODUCTION
Reactive oxygen species (ROS) contain an unpaired electron in their
outermost shell, making them extremely reactive ions. They play a
critical role in the cell, primarily in maintaining homeostasis and
facilitating cellular signaling. In mitochondria, ROS are generated
through the movement of electrons across the electron transport chain
(ETC) during cellular respiration. The ETC consists of complexes I
through IV and enzyme ATP synthase in the inner membrane of the
mitochondria. As electrons move through the ETC, a portion of these
electrons unintentionally leak and are captured by O2,
resulting in a continuous production of superoxide anions
(O2•−) on a significant scale
[1]. However, antioxidant defense
mechanisms, such as manganese superoxide dismutase (MnSOD) in the
mitochondrial matrix and copper–zinc superoxide dismutase (CuZnSOD) in
the intermembrane space and cytosol, typically quickly convert
superoxide (O2•−) to hydrogen peroxide
(H2O2)
[2]. While the production of
mitochondrial r is a natural result of typical cellular metabolic
processes, its accumulation in large quantities is often implicated in
the progression of various diseases and injuries
[3-9].
ROS have a diverse range of effects on cancer cells; ROS increase
migration, proliferation, and tumor progression, as well as induce cell
senescence and death [10]. Otto
Warburg discovered that in the presence of oxygen, cancer cells produce
excess lactate, which he deemed “aerobic glycolysis” to explain that
cancer cells exhibit a shift towards glycolysis for energy production,
rather than oxidative phosphorylation
[11,
12]. Though the Warburg effect
initially suggested that the respiration process of cancerous cells is
damaged, it is widely understood today that their regulation of
glycolysis is instead compromised [13,
14]. When ROS production exceeds
permissible levels in cancer cells, antioxidant defense mechanism
capabilities are depleted, leading to apoptosis (programmed cell death),
which highlights the anti-tumorigenic signaling feature of ROS as a
promising cancer therapy option [15].
Sulindac is an FDA-approved non-steroidal anti-inflammatory drug (NSAID)
that has demonstrated anticancer potency
[16-20]. Former studies have shown
that treating cancer cells with sulindac and subsequently exposing them
to oxidizing agents capable of generating ROS, such as hydrogen
peroxide, tert-butyl hydroperoxide (TBHP), and dichloroacetate (DCA),
leads to apoptosis [21]. Sulindac is
also an inhibitor of cyclooxygenases (COX-1 and COX-2), enzymes that
convert arachidonic acid to prostaglandins, which are lipid compounds
involved in inflammatory responses [22,
23]. However, experiments with lung
cancer cells demonstrated that sulindac’s role as a COX inhibitor is
unrelated to its function as a cancer-killing drug
[23]. Additionally, data has
supported sulindac’s protection of normal cells from oxidative damage,
another facet of interest when developing cancer treatments
[23]. While sulindac’s ability as a
cancer therapy option has been proven, there may be other combinations
involving the drug that have yet to be fully understood.
Metformin is a longtime FDA-approved drug of the biguanide class used to
treat type 2 diabetes (T2D) and has also become of interest in cancer
therapy [24]. Metformin’s pleiotropic
effects are primarily due to its interactions with the mitochondria,
specifically through inhibition of complex I of the ETC, which
interferes with oxidative metabolic activity
[25,
26]. In 2005, researchers proposed that
the administration of metformin may lead to a reduction in instances of
cancer in T2D patients [27]. Studies
have shown that metformin’s anticancer properties are attributed to its
inhibition of mitochondrial ETC complex I and of crucial signaling
pathways [28].
MitoSOX Red is a derivative of hydroethidine (HE) that functions as a
fluorescent probe designed for the selective detection of superoxide in
mitochondria of live cells [29].
MitoSOX Red has a positively charged phosphonium group that specifically
targets the cell-permeative HE derivative to the mitochondria,
accumulating in the mitochondrial matrix, where its oxidization by
O2•− produces fluorescence
proportionate to the concentration of
O2•−[29,
30]. Numerous studies incorporating
different cell lines have used MitoSOX Red for selective detection of
superoxide anion [31-36].
Sulindac and metformin are both known to have anticancer effects related
to the induction of key apoptotic pathways and mitochondrial mechanisms
through the inhibition of complexes in the ETC. Therefore, our approach
is designed to monitor superoxide dynamics over time in response to each
drug as well as the combination of both drugs.
It is hypothesized that oxidative stress, due to oxidative metabolic
mitochondrial dysfunction, may play an important role in the anticancer
activity of the combination of metformin and sulindac. The aim of this
experiment is to measure superoxide levels in lung cancer cells when
exposed to metformin, sulindac, and a combined treatment of metformin
and sulindac through time-lapse fluorescence imaging, which provides a
measurement of dynamic changes in the slopes of superoxide anion
production quantitatively over time.
2 | MATERIALS & METHODS
2.1 | Cell Preparation
A549 adenocarcinoma human alveolar basal epithelial lung carcinoma cells
were obtained from ATCC (Rockville, MD). The cells were grown in
Dulbecco’s Modified Eagles Medium (DMEM) (Gibco, Grand Island, NY) and
supplemented with 10% FBS (fetal bovine serum) (Gibco, Grand Island,
NY), 100 IU/ml penicillin (Gibco, UK), and 100 µg/ml streptomycin
(Gibco, Grand Island, NY), and maintained at 37℃ and 5%
CO2 in a temperature- and gas-controlled incubator. For
repeatability, frozen cell stocks with of the same early passage were
prepared.
2.2 | Fluorescence Microscopy
Two days prior to imaging, cells were thawed and seeded to a 24-well
plate (50,000 cells/well). Before plating, cell viability was assessed
with a Cell Countess II FL Automated Cell Counter (Invitrogen, Carlsbad,
CA). A549 cells were cultivated in phenol-free Dulbecco’s Modified
Eagles Medium (DMEM) (Gibco, Grand Island, NY) and supplemented with
10% FBS (Gibco, Grand Island, NY), 100 IU/ml penicillin (Gibco, Grand
Island, NY), and 100 µg/ml streptomycin (Gibco, Grand Island, NY) and
cultured at 37℃ and 5% CO2 in a temperature- and
gas-controlled incubator. Phenol-free media is used to avoid
interference with the red channel dye while imaging
[37].
Live cell imaging was performed with a Nikon Ti-E inverted microscope
(Nikon Instruments, Melville, NY) customized in the Biophotonics Lab
(Florida Atlantic University, Boca Raton, FL) to include a temperature-
and gas-controlled incubation chamber (Okolab, Sewickley, PA). An
overhead halogen lamp was utilized for brightfield imaging of cells at
the beginning of the experiment. Fluorescence excitation was achieved
through pairing a mercury arc lamp with a filter cube for the red
channel (510 nm) and fluorescent emission was filtered with a filter
cube (580 nm) and captured with a Rolera EM-C2 CCD
camera (Teledyne Photometrics, Tucson, AZ). The imaging protocol begins
with a 20-minute baseline imaging session, followed by the addition of 1
µM MitoSOX Red Mitochondrial Superoxide Indicator (Thermo Fisher
Scientific, Waltham, MA), where imaging takes place for a duration of 40
minutes. At the 60th minute, cells are subjected to
various drug treatments: 25 µM antimycin A (AA, positive control), 250
µM sulindac, 1.6 mM metformin, and a combination of 250 µM sulindac and
1.6 mM metformin. The 8-hour experiment involved capturing images at
20-minute intervals, using 20x magnification and an exposure time of 900
ms. Our experimental protocol is described in detail in previous
manuscripts [38-41].