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].