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.