Abstract
BackgroundDoxycycline (DC) has been shown to possess non-antibiotic properties including Fas/Fas Ligand (FasL)-mediated apoptosis against several tumor types in the concentration range of 10–40 µg/mL. However, the effect of DC in apoptotic signaling at much low concentrations was not studied.MethodsThe present study investigated the attenuation effect of low dose of DC on FasL-induced apoptosis in HeLa cell by the methods of MTT assay, fluorescence microscopy, DNA fragmentation, flow cytometry analysis, and western blotting.Results and conclusionIn the present findings we showed that low concentration of DC (<2.0 µg/mL) exhibited protective effects against FasL-induced apoptosis in HeLa cells. FasL treatment to HeLa cells resulted in a concentration-dependent induction of cell death, and treatment with low concentrations of DC (0.1–2 µg/mL) significantly (p < 0.001) attenuated the FasL-induced cell death as measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Further, the FasL-induced apoptotic features in HeLa cells, such as morphological changes, DNA fragmentation and cell cycle arrest was also inhibited by DC (0.5 µg/mL). Tetracycline and minocycline also showed similar anti-apoptotic effects but were not significant when compared to DC, tested at same concentrations. Further, DC (0.01–16 µg/mL) did not influence the hydrogen peroxide- or cisplatin-induced intrinsic apoptotic pathway in HeLa cells. Protein analysis using Western blotting confirmed that FasL-induced cleavage/activation of caspase-8 and caspase-3, were inhibited by DC treatment at low concentration (0.5 µg/mL). Considering the overall data, we report for the first time that DC exhibited anti-apoptotic effects at low concentrations in HeLa cells by inhibition of caspase activation via FasL-induced extrinsic pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s40659-015-0025-8) contains supplementary material, which is available to authorized users.
Highlights
Doxycycline (DC) has been shown to possess non-antibiotic properties including Fas/Fas Ligand (FasL)mediated apoptosis against several tumor types in the concentration range of 10–40 μg/mL
Treatment with DC at low concentrations up to 0.5 μg/mL significantly and concentration dependently inhibited FasL-induced cell death in HeLa cells (Fig. 1a) and these effects were confirmed by crystal violet assay (Additional file 1: Figure S1)
FasL-induced cell death was observed in other cancer cells lines, and DC attenuated this cell death the results were not significant at tested doses (Additional file 2: Figure S2)
Summary
Doxycycline (DC) has been shown to possess non-antibiotic properties including Fas/Fas Ligand (FasL)mediated apoptosis against several tumor types in the concentration range of 10–40 μg/mL. The signaling events leading to apoptosis can be divided into two major pathways, the intrinsic mitochondrial and extrinsic death receptors [3, 4]. A variety of extracellular and intracellular stress stimuli converge at the mitochondrial level, resulting in the translocation of Apoptosis mediated by extrinsic death receptors, such as Fas and FasL has been shown to play an important role in regulating apoptosis. Ligands bind to their cell surface receptors and through signaling transduction cascades, lead to the activation of an initiator caspase, caspase-8.
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