COL-3 prevents ROS production and consequent LPS or αSa-evoked oxidative stress
To finally assume that the anti-inflammatory effect of COL-3 was acquired from an underlying and indirect anti-oxidant action, we observed LPS and αSa-increased ROS levels after treatment with COL-3. Coherent with the previous results, COL-3 (20 µM) strongly inhibited ROS production in both scenarios. An intrinsic antioxidant effect from COL-3 would implicate in a passive diffusion from this compound into the intracellular content. Despite being shown that from the eight chemically modified tetracyclines existent, COL-3 is the most lipophilic and consistently exhibited the greatest in vivo efficacy in animal models of tissue breakdown and tissue uptake [51], if COL-3 has an intrinsic antioxidant ability or if it is only an indirect mechanism through the reduction of glucose uptake and NADPH production remains to be determined.
It has been well described by our group and others that the inflammatory profile of microglial cells evoked by LPS and αSa is associated with a robust elevation of intracellular oxidative stress [30,32,35,52,53]. Interestingly this response was efficiently inhibited by COL-3, that reduced microglial inflammatory-type responses, and by APO and TROL, an inhibitor of the superoxide producing enzyme NADPH oxidase, and an antioxidant analog of vitamin E, respectively [54,55]. As previously hypothesized, these data point that ROS production through a NOX2-dependent manner – i.e., the NADPH oxidase isoform that is most abundant in microglial cells [56] – is a critical participant of the inflammatory response to the inflammogens LPS and αSa, and that COL-3 potentially hindered with this signaling event. The expression of the phosphorylated form of the p65 subunit of the NF-κB transcription factor is an important sequential event in the ROS signaling cascade, as ROS operate as a trigger for NF-κB activation [57]. In addition to ROS direct toxic impact on biological macromolecules, they can trigger the inflammatory response – i.e., microglial activation and cytokine release – by stimulating a number of genes which are regulating the inflammatory-signaling cascades, including NF-κB [58].
Again, we noticed COL-3 was proportionally more efficacious to inhibit oxidative stress in LPS- and αSa-activated microglial cells than DOX, as a higher concentration of the last (50 µM was necessary to mimic COL-3 (20 µM) action in avoiding ROS production. Several studies have compared the pleiotropic actions od COL-3 and DOX, with converging results. Protasoni et al. [1] performed a comparative study of COL-3 and DOX to investigate the role of the mitochondrial energy generating capacity in an anticancer mechanism and found that both drugs caused a severe decrease in the levels of mitochondrially encoded cytochrome-c oxidase subunits and cytochrome-c oxidase activity. In addition to that, COL-3 was the only drug to produce a marked drop in the level of nuclear-encoded succinate dehydrogenase subunit A and citrate synthase activity, indicating that COL-3, differently from DOX, had multiple inhibitory effects. Opposingly, Onoda et al. [59], investigating whether tetracyclines could induce apoptosis in human HT29 colon cancer cells, described that both COL-3 and DEX inhibited the proliferation of six different colorectal cancer cell lines in a dose-dependent manner; however, COL-3 had a stronger effect on cancer cells than DOX. This study is the first to suggest a more effective action of COL-3 when compared to a tetracycline with antibiotic activity such as DOX in an inflammatory context for microglial cells.
Our results point to a higher efficacy of COL-3 than DOX in their anti-inflammatory properties. That might be explained by the structural differences of these two molecules: the presence of methyl groups that play a role in the antibiotic activity of DOX might not influence or even reduce the anti-inflammatory/antioxidant response shaped by this tetracycline. Gonzalez-Lizarraga et al. [17] described that COL-3 is also more effective than DOX as an antiaggregant molecule due to these structural distinctions. In this way, COL-3 not only efficiently prevented the formation of toxic α-synuclein amyloid aggregates in vitro , but also disaggregated α-synuclein amyloid fibrils, while DOX was only efficient in the former analysis. This suggests that COL-3 and DOX structural differences can impact the efficacy of each compound concerning their anti-inflammatory responses.