In our study, we showed that ciprofloxacin increased the viability of MDCK cells by inhibiting apoptosis through an autophagy mechanism. Although all cells eventually undergo apoptosis, it was noteworthy that ciprofloxacin could increase cell viability in the process of apoptotic cell death.
When energy is lacking, the organism tries to save cells by recycling dead cells through autophagy activation. At this time, it is not yet clear whether autophagy is being promoted or whether apoptosis is being suppressed. Apoptosis refers to programmed cell death, which can be observed through DNA breakage1. In our study, the TUNEL assay demonstrated a status of apoptosis. Recently, several antibiotics have been reported to control cell viability. Therefore, for the first time, we screened using an MTT assay whether cell viability increased when antibiotics representing each class were administered. We examined four antibiotics: ciprofloxacin, a quinolone, ceftizoxime, a cephalosporin, minocycline, a tetracycline, and netilmicin, an aminoglycoside. Ceftizoxime was effective only at high concentrations under normoxia conditions, but not under hypoxia conditions. Netilmicin was not effective under normoxia or hypoxia conditions. Minocycline was effective at lower concentrations but deteriorated cell viability at higher concentrations under both normoxia and hypoxia conditions. Therefore, we used the most effective and safe antibiotic, ciprofloxacin, for further studies.
The first experiment was performed to investigate whether ciprofloxacin improved cell viability by inhibiting apoptosis using the TUNEL assay. The results showed that apoptosis was reduced qualitatively and quantitatively when ciprofloxacin was added under the normoxia conditions. Therefore, ciprofloxacin saves cells by inhibiting apoptosis.
The second experiment was performed to investigate the mechanism of increased ciprofloxacin-induced cell viability. In previous studies, apoptosis has been reported to be mainly responsible for programmed cell death9, but autophagy has also been reported to play important roles2. In addition, although apoptosis and autophagy have been considered as different concepts, many studies have shown that the homeostasis of cells is maintained through the interaction between these two molecular mechanisms10,11. Firstly, we investigated apoptosis mechanism with the relationship among caspase-3, Bax, and Bcl-2. In general, when Bax/Bcl-2 decreases and caspase-3 decreases, apoptosis is suppressed. Our study showed reduction of the caspase-3 level in the ciprofloxacin-treated group, and this result means that the inhibition of apoptosis occurs inhibiting caspase-3 activity by ciprofloxacin. However, conversely, ciprofloxacin-treated cells showed an increase in Bax/Bcl-2 level in this study. This may not be explained by the fact that the whole process is caused by apoptosis because this means that an increased Bax/Bcl-2 level caused more apoptosis, which leads to conflicting results (Fig. 2 and Fig. 3B). Therefore, we investigated the influence of ciprofloxacin on autophagy in an energy-insufficient environment. Since Beclin-1 is inhibited by Bcl-2, when Bcl-2 is phosphorylated and inactivated, Beclin-1 and Bcl-2 are separated from each other and an increased level of free Beclin-1 becomes operational, resulting in an increase in pBcl-2. This means that the inhibition of autophagy induced by Bcl-2 is reduced, which can lead to a better progression of autophagy. In other words, an increase in the amount of free Beclin-1 promotes autophagy12. First, the level of Beclin-1 increases steeply. Second, when the level of unbounded pBcl-2 is increased, the level of free Beclin-1 is increased due to a decrease in the binding to Beclin-1. Third, as Bax increases, more Bcl-2 and more bonds can be obtained, and the amount of free Beclin-1 can be increased. Finally, caspase-3 cleaves and deactivates Beclin-1, and the decreasing level of caspase-3 increases the level of free Beclin-1. Finally, in our study, ciprofloxacin enhanced autophagy, increasing the level of free Beclin-1, and inhibited caspase activity and reduced cell death due to the energy produced during autophagy as summarized in Fig. 4. To survive in the absence of energy, cells die to generate energy sources for the living cells. In other words, when the energy source is insufficient, apoptosis is promoted upstream, but at the same time, autophagy is promoted to decompose the unnecessary proteins in the cell and use them as energy sources. Accordingly, the activity of caspase-3 is inhibited, thereby suppressing apoptosis.
The strength of this study is that it has shown a positive effect on cell viability using antibiotics, and this is the first study to apply the concept of autophagy and apoptosis. In previous studies, minocycline had potent anti-apoptotic and anti-inflammatory effects and protected renal function in a rat model of ischemia-reperfusion injury8,13,14. Because minocycline showed toxic effects at high concentrations in the MDCK cell line in our study, we could explain the effect of ciprofloxacin, which is safer than other antibiotics for the action of autophagy, on cell viability. Recently, these concepts of autophagy have been applied in several clinical fields, such as acute kidney injury15, chronic kidney disease (obstructive nephropathy, immunoglobulin A nephropathy, autoimmune kidney diseases)16, diabetic nephropathy17, autosomal dominant polycystic kidney disease18, cystinosis19, and chronic cyclosporine A renal toxicity20. Our findings may have a positive impact on acute kidney injury and kidney diseases associated with chronic inflammation.
However, our study has some limitations. First, the order of apoptosis and the autophagy process is still unclear. Autophagy is known to precede apoptosis21. However, our study did not show the preceding autophagy process, although the apoptosis and autophagy markers were checked at 0, 6, 12, 24, 36, and 48 h. Second, in our study, research on other markers of autophagy and apoptosis was insufficient, and further studies are needed. Third, a slightly stronger stimulus leads directly to apoptosis without the action of autophagy. Although nutrient depletion is the most powerful factor in inducing autophagy, the combined metabolic stress of hypoxia and nutrient depletion damages organs, proteins, and DNA, which ultimately leads to apoptosis. Recent studies have shown that autophagy mitigates metabolic stress to protect cells against extreme cell damage4. Autophagy is not only necessary as a tool to generate energy in a nutrient-free environment, but it also plays an important role in regulating protein and organ function and maintaining homeostasis. It is also important in situations of metabolic stress, that is, when energy is limited and intracellular damage is accelerated. However, it is important to recognize that autophagy cannot inhibit apoptosis completely in conditions in which apoptosis is induced. In other words, when cells are exposed to stresses, autophagy takes place temporally, but it cannot completely prevent the progression to apoptosis. Finally, extreme environments with no nutrient supply and no oxygen are unlikely to reveal the mechanisms by which antibiotics affect cell viability and are most likely to be biased by apoptosis processes. In the future, it will be necessary to study the difference between normoxia and hypoxia conditions in a nutrient-free environment.
In conclusion, ciprofloxacin can help to restore cell viability against renal tubular damage under nutrient-free conditions associated with autophagy. Therefore, ciprofloxacin might be helpful in increasing renal cell viability through the activation of autophagy under acute kidney injury. Further studies are needed to confirm the mechanism by which ciprofloxacin affects cell viability.