1. Introduction

According to the National Breast Cancer Foundation, breast cancer is the most common cancer diagnosed in women, and one in eight women will be diagnosed with breast cancer in their lifetime [1]. A subtype of breast cancer is basal-like breast cancer, also known as triple-negative breast cancer (TNBC). Given its lack of estrogen receptors (ER), progesterone receptors (PR), and low expression of human epidermal growth factor receptor 2 (HER2), there is no effective biological targeted therapy [2]. MDA-MB-231 is a known representative of triple-negative breast cancer, which has aggressive behavior as they go through reattachment, cell metastasis, and cell aggregation [3]. There is a need for an effective therapy that treats triple-negative breast cancer [4,5].

Cisplatin is commonly used as a chemotherapy drug to treat different cancers today [6]. Cisplatin is a DNA cross-linking agent which induces apoptosis by introducing DNA damage through the distortion of the structure of the DNA duplex by binding covalently to the N⁷ position of purines to form 1,2- or 1,3-intrastrand crosslinks and interstrand crosslinks [7]. During cisplatin’s DNA damage mechanism, chlorines in the platinum compound allow the platinum to attach to guanine’s N⁷ position, cross-linking DNA strands. At a high enough concentration of cisplatin, the cells cannot repair the damage cisplatin has done and undergo apoptosis [7]. Neoadjuvant cisplatin has been proven to be efficacious in treating testicular, ovarian, and bladder cancers [6,8,9]. However, cisplatin also damages non-cancerous cells [10]. Therefore, it is important to find a treatment that specifically targets cancer cells. Despite the short-term results, the effectiveness of cisplatin declines as the cancer cells becomes more resistant to the drug [11].

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Caffeine is a common chemical found in our daily diets, which is both a central nervous stimulant and a protein kinase inhibitor [12,13]. Caffeine affects specific protein kinases, including ATM and ATR, which play key roles in DNA damage repair that induce cell cycle arrest at the G1 phase and apoptosis signaling [14]. Studies have shown that the combination of caffeine and cisplatin is an even more effective treatment than cisplatin alone [11,15,16]. Caffeine has received considerable attention in the past decade because of its ability to inhibit carcinogenesis in the lungs, skin, and ovaries [17,18,19,20]. The aim of this paper is to characterize the enhancing effects of caffeine effects in altering energy metabolism specifically to triple-negative breast cancer cells, which are characterized by a high frequency of mutations in ATM and BRCA2, as well as RB1 loss and cyclin E1 amplification [21,22]. These mutations also alter the function of mitochondria and energy metabolism [23,24].

Cancer cells undergo the Warburg effect, a shift in metabolic behavior away from mitochondrial oxidative phosphorylation (OXPHOS), towards aerobic glycolysis (GLY), even in the presence of excess oxygen [25]. Therefore, it has been of great interest to target the mitochondrial function as well as energy-related metabolic pathways for therapeutic development [26]. Cisplatin, a well-known platinum-based chemotherapeutic drug, is widely used as one of the major therapeutic options against cancer due to its ability to activate the DNA damage response and the induction of mitochondrial apoptosis exerts. After the initial therapeutic success associated with partial responses or disease stabilization, cisplatin treatment often results in the development of chemoresistance, leading to therapeutic failure. Approaches that can reverse cancer cell resistance to cisplatin treatment need to be explored. This study utilizes a phasor fluorescence lifetime imaging microscopy (phasor-FLIM) approach to evaluate caffeine and cisplatin’s ability to degrade a triple-negative cell line’s metabolic profile in comparison to a normally proliferating, or wild-type cell. Previous studies revealed that the presence of caffeine caused a decrease of cisplatin-induced cell cycle arrests at the S and the G2 phase in lung cancer [11]. FLIM provides a state-of-art way to measure the decay curve of a fluorescent species without fitting and identify biochemical reactions like oxidation and reduction, which can be used to map the effects of the potential drug in real-time without perturbing the cell behavior [6]. The coenzyme nicotinamide adenine dinucleotide (NADH) is the principal electron acceptor in glycolysis and electron donor in oxidative phosphorylation, which has been found to have endogenous fluorescence within the cells [27]. The protein-bound form of NADH is associated with energy production through oxidative phosphorylation with a lifetime of 3.4 ns (bound with lactate dehydrogenase) and maps on the left top of the phasor plot, whereas the free form of NADH is linked to glycolysis with a lifetime of 0.38 ns which maps on the right bottom of the phasor plot [28].

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This change in signature correlates to a shift towards OXPHOS and GLY, respectively, which has been previously described as the metabolic or M-trajectory [27,28,29,30]. This trajectory has also been shown to correlate with results found in conventional biochemical assays when OXPHOS or GLY inhibitors are used to shift metabolic signatures towards one another [27,31,32,33]. To ensure the collection of the fluorescence lifetime of NADH, all of the measurements were done using a 2-photon laser excitation set to 740 nm, and the emission was collected with an emission bandpass filter (460/40 nm) to select the emission of NADH. Our study focuses on the energy metabolism of three cell lines: triple-negative breast cancer (MDA-MB-231), estrogen-receptor lacking breast cancer (MCF7), and normal breast epithelial cells (MCF10A) with caffeine, cisplatin, or a combination of the two. We have found that caffeine alters energy metabolism and significantly increases the effectiveness of the treatment of cisplatin for MDA-MB-231 and MCF7. On the other hand, MCF10A cells were not affected by any of the three treatments. These results indicate that monitoring the effects of caffeine together with DNA damaging agents on energy metabolism in different breast cancer cells can provide effective therapy, which raises the possibility of pharmacologic targeting of triple-negative breast cancers.