title: Low concentrations of doxycycline attenuates FasL-induced apoptosis in HeLa cells.
abstract: BACKGROUND
  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. However, the effect of DC in apoptotic signaling at much low concentrations was not studied.
  
  
  METHODS
  The 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.
  
  
  CONCLUSIONS
  In 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.
authors.affiliation: Department of Biotechnology, Konkuk University, Chungju, 380-701, Republic of Korea. soul1330@naver.com.
authors.forename: Jung Mi
authors.initials: JM
authors.lastname: Yoon
authors.affiliation: Department of Biotechnology, Konkuk University, Chungju, 380-701, Republic of Korea. koppula@kku.ac.kr.
authors.forename: Sushruta
authors.initials: S
authors.lastname: Koppula
authors.affiliation: Department of Biotechnology, Konkuk University, Chungju, 380-701, Republic of Korea. yobgi104@naver.com.
authors.forename: Se Jong
authors.initials: SJ
authors.lastname: Huh
authors.affiliation: Department of Animal Science and Technology, Chung-Ang University, Anseong, Gyeonggi, 456-756, South Korea. hursj@cau.ac.kr.
authors.forename: Sun Jin
authors.initials: SJ
authors.lastname: Hur
authors.affiliation: Department of Biotechnology, Konkuk University, Chungju, 380-701, Republic of Korea. changil.kim@kku.ac.kr.
authors.forename: Chan Gil
authors.initials: CG
authors.lastname: Kim
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D020169:Caspases / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000008:administration & dosage* / Q000494:pharmacology; D004789:Enzyme Activation; D053222:Fas Ligand Protein / Q000187:drug effects*; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells
chemical_list: D053222:Fas Ligand Protein; D020169:Caspases; D004318:Doxycycline