Supervisors info:
Γαϊτανάκη Αικατερίνη Καθηγ. (Επιβλέπουσα), Παπαζαφείρη Παναγιώτα Αναπλ. Καθηγ., Βασιλακοπούλου Διδώ Αναπλ. Καθηγ.
Summary:
Excessive exposure of skeletal muscle to oxidative stress conditions
contributes to muscle degeneration disorders such as dystrophies and atrophies.
H2O2 is a major endogenously produced type of ROS and a very significant second
messenger. Curcumin is a chemical compound produced in the plant Curcuma longa
and can exhibit either prooxidant or antioxidant anction. The novel molecules
benzoxazine AK-AM1 & benzothiazine AK-AM2 were designed in order to combine
antioxidant, antihyperlipidemic and anti-inflammatory actions. Firstly, we
wanted to compare the prooxidant action of H2O2 1mM with that of curcumin. MTT
cytotoxicity assay unveiled that specific concentrations of curcumin (50 &
100μΜ) and H2O2 1mM decreased significantly the cellular viability. As for
curcumin, that decline presented a dose-dependent pattern and was also enhanced
by the concominant presence of H2O2 1mM . We next focused on the possible
antioxidant action of AK-AM1, AK-AM2 and other compounds against H2O2. Lower
concentrations of curcumin (30-15μΜ) presented a very effective antioxidant
action. Moreover, AK-AM1 (15-5μΜ) and AK-AM2 (100-10μΜ) showed an excellent
antioxidant action, comparable with that of melatonin (15μΜ) and catalase
(50U/ml). However, well-known antioxidant molecules such as resveratrol
(50-1μΜ), ascorbic acid (50μΜ) and uric acid (10μΜ) failed to improve the cell
viability levels. Furthermore, Western Blot analysis revealed that the
antioxidants melatonin, curcumin, resveratrol, uric acid, AK-AM1 & AK-AM2 were
able to inhibit the H2O2 induced activation of ERKs and JNKs kinases. Wanting
to clarify the way of action of the above mentioned compounds, we next decided
to use prooxidant concentrations of curcumin (50μΜ), as it produces more types
of ROS than H2O2. MTT assay revealed that only catalase (50U/ml), melatonin
(15μΜ) and AK-AM2 (30μΜ) had antioxidant action. On the contrary, ascorbic acid
(50μΜ) and AK-AM1 (15μΜ) caused a significant fall at the cellular viability by
becoming prooxidant molecules. Finally, none of these compounds was able to
reverse the extremely severe conditions of oxidative stress, induced by the
simultaneous presence of H2O2 1mM and curcumin (50μΜ). We next focused on the
signaling pathways that are induced by curcumin (50μΜ). Western blot analysis
revealed that curcumin (50μΜ) promotes maximum activation of kinases ERKs at 15
minutes, while the kinases JNKs started to be activated 1 hour after the
treatment, reaching maximul levels between 2-4 hours. Moreover, curcumin (50μΜ)
induces apoptotic cell death as a significant proteolysis of PARP-1 was
observed even at 2 hours and was highly elevated 4 hours after treatment. In
addition to that, we noticed that preatreatment with the antioxidant molecule
AK-AM2 (30μΜ) and the prooxidant factors ascorbic acid (50μΜ) and AK-AM1 (15μΜ)
partially inhbited the curcumin induced activation of ERKs. However,
preatreatment with those prooxidant factors enhanced remarkably the activation
level of kinases JNKs and their substrate c-Jun. On the contrary, the presence
of the antioxidant factors catalase (50U/ml), melatonin (15μΜ) and AK-AM2
(30μΜ) inhibited significantly the activation of both JNKs and c-Jun. In
conclusion, we believe that the novel benzothiazine derivative AK-AM2 it is
safer and more effective, as it presents antioxidant/cytoprotective action
against H2O2 in a wide concentration range (100-10μΜ) and it also exhibits an
excellent antioxidant action against curcumin (50μΜ).
Keywords:
Myoblasts, Curcumin, Antioxidants, Benzothiazines, Apoptosis
