In our lab, we often use doxycycline to induce gene expression or knockdown whose expression was regulated by Tet-regulated systems. This system is quite powerful and efficient until I found doxycycline could induce protein oxidation using oxyblot.
In a Tet-regulated gene knockdown system, I added dox to downregulate my target gene, which has been proven to induce ROS in my hands, the expected result is that, without dox, EV and gene knockdown cells should show kind of basal protein oxidation, while gene knockdown cells exhibit increase in protein oxidation and EV cells remained largely unchanged once dox added.
The result surprised me, which shows without Dox, gene knockdown induce cells exhibit dramatic increase of protein oxidation. Once dox added, both EV cells and knockdown cells all exhibit dramatic surge of protein oxidation, similar to gene knockdown cells without dox.
While gene knockdown might surpass the regulation of Dox in a Tet-regulated system is relatively easy to understand, I am puzzled by the effect of Doxycycline alone on protein oxidation. Do you guys have any clues about this effect? Besides, I also observed p21 and p16 upregulation by Dox in EV cells.
Given its structure, it is not inconceivable that doxycyclin or one of its metabolite act as a catalytic source of superoxide and other activated oxygen species through redox cycling. Another possibility would be induction of enzymes producing peroxides, for example flavin monooxygenases or cyt P450-dependent monooxygenases, or the down-regulation of key antioxidant enzymes such as SOD or Se-GPx
The truth might be complex, because it has recently been published (Eur J Pharmacol. 2010 Oct 10;644(1-3):176-87) that doxycycline extensively counteracts multiple oxidative and apoptotic actions of doxorubicin in heart in vivo.
If the answer is really important to you, you should think about monitoring SOD and Se-GPx activities in the cell lysates.
For SOD activity, use either the SOD-525 kit (commercial source today) which I developped many years ago (Anal. Biochem., 214, 442-451) or the cytochrome C based assay which is more cumbersomle and sample-consuming but works well. For Se-GPX activity use t-butylhydroperoxide as substrate [cf . Eur J Biochem. 1992 205(3):955-60] and mercaptosuccinate as a specific inhibitor of the selenoenzyme.
Some studies report that the modified tetracyclines exhibit many non-antimicrobial properties (anti-inflammatory, anti-angiogenic and particularly anti-oxidant). Although both the Tet -regulated system and oxyblot are a unknown world for me, your question has aroused my interest. Regarding the formation of radical intermediates from doxycicline: the molecule's chemical structure allows the electron shift (resonance structures) and consequently this intermediate is less reactive than common radical species, rather I'd associate both the radical formation and its catalytic role to the doxycycline concetration since many molecules switch from antioxidant to pro-oxidant with the concentration's increase.Could you modify this parameter without alterating the Tet-regulated systems efficacy?
It's possible that the protein oxidation could depend on a bias during either the sample handling or the executive phase of oxyblot protocol that in turn could produce protein oxidation. In order to be sure that the protein oxidation is linked to an pro-oxidant enviroment inside the cell , have you ever measured the ROS concetration in all your cell samples (EV, gene ko, w or wo Doxycycline) ? As Jean correctly suggested you should evaluate the expression and/or the activity of key enzymes (oxidant and antioxidant) involved in the cellular redox balance to get a clear view about what's happened..
B.
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