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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