Abstract
Objectives
Camphorquinone (CQ) is a widely used photoinitiator in dental visible light (VL)-cured resinous materials. However, little is known about the toxicity of CQ in human cells. This study was designed to investigate CQ induced oxidative strain and apoptosis in cultured human oral keratinocytes (OKF6/TERT 2). Furthermore, the effects of visible-light (VL)-irradiation and the reducing agent N , N -dimethyl- p -toluidine (DMT) were investigated. In addition, the preventive potential of the antioxidant glutathione (GSH) against CQ induced toxicity was analyzed as well.
Methods
The fluorescent DNA-staining dye Hoechst 33342 was used to quantify total cell numbers. Intracellular levels of reactive oxygen species (ROS) were measured by the fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Apoptosis was determined by FACS analysis (Annexin V-FITC/propidium iodide), by measuring caspase-3/7 activity (ELISA) and by DNA laddering.
Results
Our data show that CQ was dose-dependent cytotoxic and caused oxidative stress by inducing reactive oxygen species (ROS). The redistribution of phosphatidylserine (PS) to the outer layer of the plasma membrane, induction of caspase-3 enzyme activity and DNA fragmentation were also observed in CQ exposed cells. Interestingly, CQ-induced ROS generation enhanced by VL irradiation or a simultaneous treatment with DMT showed no quantitative effect on apoptosis. However, co-exposure of cells with GSH significantly reduced the intracellular ROS generation as well as apoptosis caused by CQ.
Significance
This is the first report showing that ROS-induced apoptosis, which is caused by CQ, is prevented by GSH.
1
Introduction
It has been documented that high amounts of organic compounds may leach from resinous restorative materials in the first days after application. Subsequently, lower quantities of these compounds are continuously released into the aqueous environment of the oral cavity due to degradation and/or erosion over time . The vast majority of light-curing dental resins contain the initiator camphorquinone (CQ) (∼0.2% to 1.0% w/w) in combination with a reducing agent or accelerator, like dimethyl- p -toluidine (DMT) . These components are not incorporated in the polymer network and may completely leach after polymerization by and by . After polymerization, a maximum of 14 mM CQ could be potentially released into the oral cavity . However, contrary to the rapid increase in clinical use, only scarce data are available about chemical–biological interactions of CQ and cells’ metabolism.
The initiating substances CQ and DMT revealed EC 50 -concentrations (concentration that reduce the total cell number to 50% of untreated control cells) between 2.17 mM and 4.25 mM, respectively . But already lower concentrations of CQ (10 μM–1 mM) in combination with DMT (20 μM–2 mM) caused an increase of strand breaks of DNA in a dose-dependent manner after irradiation with a resin light-curing device in the range of 460–480 nm, which may significantly enhance the compounds’ cytotoxic potency. Most recently, we exposed immortalized murine cementoblasts to CQ ± DMT and found that CQ with and without the co-initiator induces initial DNA lesions (apurinic/apyrimidinic sites/AP sites) . CQ caused clear mutagenic effects in the bacterial umu test, and genotoxicity was detected for CQ in the DNA synthesis inhibition test . In a previous study, our results indicated genotoxic effects of oxidative nature induced by non-irradiated CQ using primary human gingival fibroblasts. This may be due to the activation of CQ by intracellular mechanisms . Taken together, the available data show that both irradiated and non-irradiated CQ exerts cytotoxic effects.
Although cells possess antioxidant systems to control the intracellular redox state, which is important for their survival, it was documented that irradiated CQ and CQ-related photoinitiators can generate high amounts of ROS in various human cell types . These authors identified elevated concentrations of free radicals due to irradiated CQ, which enhanced the toxicity of this initiator in a concentration dependent manner. Interestingly, CQ also caused a very pronounced increase of ROS in cells without light irradiation . Low levels of prolonged ROS generation are known to initiate cell proliferation in many cell systems, whereas the excessive production of ROS activates reactions that lead to apoptosis or necrosis in several cell types . Hence, ROS serve as critical signaling molecules, either for cell proliferation or for apoptosis . The exact mechanisms involved in cell death induced by ROS are not yet fully understood.
ROS also regulate a variety of cellular processes including inflammation, cell cycle progression, and aging . Noda et al. showed for the first time that CQ alone or in combination with DMT suppressed transactivation of NFkB, a redox-sensitive transcription factor that regulates inflammatory responses and glutathione synthetic enzyme levels in LPS-treated THP1 human monocytes. Kim et al. suggested that a CQ-mediated inhibition of mineralization in a cell culture containing primary dental pulp stem cells occurs either directly via ROS generation or indirectly via triggering the release of inflammatory cytokines. These findings point out that cellular redox responses might be significantly influenced by dental (co-)initiators.
Very few studies addressed the question, if the cytotoxic potency of dental resins is associated with apoptosis and/or necrosis. In a previous investigation we have shown that non-irradiated CQ induced a pronounced ROS generation in primary human gingival fibroblasts and damaged chromosomal DNA . Under oxidative stress ROS produced by various agents have been shown to damage DNA resulting in DNA degradation and to induce apoptosis . The mechanisms behind this cellular effect are rather complex and not yet fully understood. However, to find novel strategies to minimize or significantly prevent adverse effects of dental materials, studies should be designed (1) to understand the exact mechanisms by which these materials induce cell death and (2) to find strategies to decrease or eliminate their toxicities while preserving their beneficial effects.
Therefore, it was the purpose of this investigation to analyze, which type of cell death, necrosis or apoptosis, is triggered by CQ at low concentrations in immortalized human oral keratinocytes. Further, it was our goal to characterize the role of ROS during these events. Taken together, the hypothesis was set forth that CQ combined with or without DMT will generate ROS that increase cellular death and that a simultaneous treatment of cells with the ROS scavenger glutathione (GSH) will reduce or prevent these cytotoxic effects.
2
Materials and methods
2.1
Cell cultures
The immortalized human oral keratinocyte cell line OKF6/TERT2 was provided by Dr. J. Rheinwald (Harvard University) and cultured in accordance with the protocols described by Dickson et al. . Briefly, cells were cultured in a keratinocyte serum-free medium (ker-sfm #17005-042) containing 25 μg/mL bovine pituitary extract (BPE), 0.2 ng/mL epidermal growth factor (EGF) (all from GIBCO/Invitrogen, Carlsbad, CA, USA), 0.3 mM CaCl 2 , and penicillin (100 U/mL)/streptomycin (100 μg/mL) (all from Biochrom KG, Berlin, Germany).
For passaging, a small amount of 0.125% trypsin/0.01% EDTA (T/E) solution (Sigma, Deisenhofen, Germany) in PBS and Dulbecco’s modified Eagle medium/F-12 medium (DMEM/F-12, Biochrom KG, Berlin, Germany) including 10% fetal bovine serum (FCS, Lonza, Verviers, Belgium) was used. Exponentially growing cultures (4–8 days old) were used as source of cells for the next passage.
For both experimental and control groups, cells were grown in a medium containing higher concentrations of nutrients (HD-ker medium), mixed (1:1) of GIBCO ker-sfm and a DMEM/F-12 medium containing calcium-free, glutamine-free DMEM (GIBCO/Invitrogen, #21068-028) with Ham’s F-12 supplemented with 0.2 ng/mL EGF, 25 μg/mL BPE (all GIBCO/Invitrogen, #11765-054), 1.5 mM l -glutamine, and penicillin (100 U/mL)/streptomycin (100 mg/mL). Cell viability (95–98%) was analyzed before plating for experiments using trypan blue dye (Sigma, Taufkirchen, Germany) exclusion tests. All cultures were routinely tested for mycoplasma contamination by means of the mycoplasma detection kit Venor GeM (Minerva Biolabs, Berlin, Germany).
2.2
Preparation of materials
Stock solutions (200-fold) of CQ and DMT (VOCO, Cuxhaven, Germany) were prepared in ethanol (Baker, Griesheim, Germany) and were freshly diluted in keratinocyte medium (HD-ker) prior to each experiment. GSH (Sigma, Taufkirchen, Germany) was prepared in distilled water. The final concentration of ethanol did not exceed 0.25% (v/v). In previous experiments it was found that an ethanol concentration of 0.25% (v/v) is nontoxic for OKF6/TERT2 cells and has no effect on redox balance (data not shown). Cells incubated with fresh growth medium containing 0.25% ethanol (c1) and fresh growth medium (HD-ker) without ethanol (c2) served as solvent controls (c1) and negative controls (c2). The complete preparation of the used CQ solutions was done under dim green safe light, prepared solutions were wrapped in aluminum foil and the cells were treated under dim room light to avoid the photoactivation of CQ.
2.3
Cytotoxicity
2.3.1
H33342 assay
The chromatin staining and membrane-permeable dye H33342 (Riedel de Haen, Seelze, Germany) was used to determine the effective concentration for a 50% (EC 50 ) growth reduction due to CQ and DMT (both VOCO, Cuxhaven, Germany). For this, CQ and DMT were dissolved and pre-diluted in ethanol (EtOH, 1 M stock solutions), then finally diluted at least 1:400 in culture medium (HD-ker) and used in concentrations ranging between 0.125 and 2.5 mM. Cells were seeded in 96-well microplates at a density of 2 × 10 4 cells/well and grown for 24 h. Subsequently, cultures were treated with CQ and DMT, respectively, for 24 h and 48 h. The materials were added by medium change. Untreated cells grown in HD-ker served as growth medium control (c2). After treatment, medium was removed and H33342 (working solution: 1 mg/mL in growth medium) was added to determine the total cell number. The fluorescence intensity of the cultures at 460 nm (excitation wavelength of 360 nm) (Ex/Em = 360 nm/460 nm) was evaluated using a FLx800 microplate reader (BioTec, Neufahrn, Germany). Each assay with each 6 replicates was repeated at least three times to ensure reproducibility.
2.3.2
ROS detection
Formation of reactive oxygen species (ROS) was evaluated using the oxidation-sensitive dye 2 1 ,7 1 -dichlorofluorescin diacetate (DCFH-DA, Sigma, Deisenhofen, Germany). Cells were seeded in transparent 96-well microplates at a density of 2 × 10 4 cells/well. After growing for 48 h, cells were loaded with DCFH-DA in HBSS for 20 min in the dark and then washed with HBSS. Cells were treated with CQ (0. 5–2.5 mM) in HBSS alone, with 1.0 mM CQ in combination with DMT (1.0 mM, 2.5 mM), with 2.5 mM CQ in combination with GSH (5 mM) or with 2.5 mM CQ irradiated for 10 s with visible blue light using a dental curing light (800 mW/cm 2 ; Elipar II, Espe, Seefeld, Germany). In addition, experiments were performed to analyze the effect of blue-light irradiation. For this, black 96 well plates with transparent bottom were used to avoid an irradiation of adjacent wells. 0.25% EtOH in HBSS (c1) and HBSS (c2) served as controls. Fluorescence was read for 90 min, directly after adding the substances and then every 15 min at Ex/Em = 485 nm/528 nm. Maximum rate of fluorescence increase ( v max ) was calculated.
2.3.3
Evaluation of apoptosis
Apoptosis was analyzed using flow cytometry after staining of the plasma membrane with Annexin V, measurement of caspase-3/7 activity, as well as by evaluating DNA ‘laddering’ on 1% agarose gels. Cells treated with staurosporine, a well known inducer of apoptosis in a wide range of cell lines, served as positive control.
2.3.4
Annexin V-FLUOS/PI assay
For flow cytometry analysis, 2 × 10 5 cells/well were seeded into 6 well tissue plates (Cellstar, Greiner bio-one, Frickenhausen, Germany) in 5 mL of HD-ker medium. After 24 h of incubation in a humidified 5% CO 2 atmosphere at 37 °C, the exponentially growing cells were exposed to various concentrations of CQ in the vicinity of the EC 50 (0.5–2.5 mM). Control cultures were grown in HD-ker with (c1) and without 0.25% EtOH (c2). After incubation, adherent OKF6/TERT2-cells were collected by trypsination and pooled together with non-attached cells in tubes on ice for FACS analysis. Cells were washed twice with phosphate buffered saline (PBS) and centrifugation cycles of 10 min at 200 × g . Cell pellets were re-suspended in the Annexin V-FLUOS and PI staining solution and stained for 15 min at room temperature in the dark.
After staining, cells were analyzed by flow cytometry using a 488 nm laser line for excitation. Green fluorescence (FLUOS) was collected between 505 nm and 545 nm and red fluorescence (PI) between 605 nm and 635 nm. Cells with lost integrity of the plasma membrane (necrotic and ‘apoptotic/necrotic’ cells) were detected with propidium iodide (PI) (annexin V +/− /PI + ). Cells that stained negative for Annexin-V-fluorescein and propidium iodide (annexin V − /PI − ) were considered viable. Cells that stained positive for Annexin-V-fluorescein and negative for PI (annexin V + /PI − ) were considered to be in early stage of apoptosis. At least 20,000 cells were analyzed per sample. All experiments were repeated at least three times. Data analysis was performed with Cell Quest software version 3.1 (Becton Dickinson, Heidelberg, Germany) and Summit software version 5.1.3.6886 (Beckman Coulter, Fullerton, USA).
2.3.5
Measurement of caspase-3/7 activity
The activity of caspase-3/7 in OKF6/TERT2 cells exposed to CQ ± DMT was assessed by a luminescence assay according to the manufacturer’s instructions (ApoToxGlo Triplex Assay, Promega, Madison, USA). In brief, OKF6/TERT2 cells were cultured on 96-well flat-bottom microtiter plates at a density of 2 × 10 4 cells/well. Semiconfluent cells were exposed to CQ (0.5–2.5 mM) ± DMT (1.0 mM, 2.5 mM) in 100 μL HD-ker medium per well for 3–10 h. After incubation, 20 μL per well Viability/Cytotoxicity Reagent was added, mixed, and incubated for 30 min at 37 °C and 5% CO 2 . Viability (intracellular protease activity) and cytotoxicity (extracellular protease activity = loss of cell membrane integrity) were estimated from fluorescence of sample at Ex/Em = 360 nm/528 nm and Ex/Em = 485 nm/528 nm, respectively, using a fluorescence microplate reader (FLx800, BioTec, Neufahrn, Germany). After that, 100 μL Caspase-Glo 3/7 Reagent was added and cells were incubated at room temperature for 30 min. Caspase-3/7 activation was determined by luminescence of samples using a luminescence microplate reader (Centro XS LB 960, Berthold Technologies, Bad Wildbad, Germany).
2.3.6
Oligonucleosomal DNA degradation analysis
OKF6/TERT2 cells were cultured on 6-well plates at 2 × 10 5 cells/well in HD-ker medium. After 48 h, cells were exposed to CQ (0.5–2.5 mM) ± DMT (1.0 mM, 2.5 mM) in 3 mL HD-ker medium per well for 24 h. After incubation, the medium was transferred into 15-mL centrifugation tubes (Sarstedt, Nuembrecht, Germany), the plates were rinsed twice with 2 mL cold PBS without Ca 2+ or Mg 2+ and then cells were scraped off the plates . Cells and the rinsing buffer were transferred to the tubes and centrifuged at 115 × g for 5 min. The pellet was re-suspended in 275 μL TE-buffer (Tris + EDTA, pH 7.5, both from Sigma, Deisenhofen, Germany) containing 1 mg/mL RNase A (Quiagen, Hilden, Germany). Cells were lysed by the addition of an equal volume of 1.2% SDS and gently mixed by inversion (six to eight times). After 5 min, 350 μl of a CsCl containing precipitation solution (3 M CsCl, 1 M potassium acetate, 0.67 M acetic acid, all from Sigma, Deisenhofen, Germany) was added, vials were carefully mixed (8–10 times) and placed on ice for 15 min. Thereafter, the mixture was spun for 15 min at 14,000 × g at ambient temperature and the clear supernatant (700 μl) either transferred into new 2-mL vials (when samples were to be stored at 4 °C for later analysis) or immediately put on a miniprep spin column (QIAprep Spin Miniprep Kit, Qiagen, Hilden, Germany). The columns were spun for 1 min at 14,000 × g , washed with 700 μl wash buffer (80 mM potassium acetate, 10 mM Tris ± HCl, pH 7.5, 40 mM EDTA, 60% ethanol), spun again and DNA was eluted with 50 μl TE buffer (pH 8.0). Extracted DNA was analyzed in 1% agarose gel in 1 mM ethylene diamine tetraacetic acid (EDTA), 40 mM Tris acetate, pH 7.6, separated for 50 min at 80 V, stained in 0.5 μg/mL of GelRed (Biotium Inc., Hayward, CA, USA) and then visualized using a TI 1 UV transilluminator (Biometra, Göttingen, Germany) equipped with a photographic camera (EDAS 290, Kodak, Lollar, Germany). Fig. 6 a and b are representative gels reproduced in three independent experiments with identical results.
2.4
Statistics
Assays were run at least three times with each six replicates (cytotoxicity, ROS formation, caspase-3/7 activity) or without replicates (FACS, DNA laddering). In experiments using microtiter plates, measured fluorescence and luminescence values were converted into % of untreated control cells (c2 = 100%). Results were expressed as means ± standard deviation (SD). Statistical analysis was performed by unpaired t -test (cytotoxicity) and one way ANOVA followed by a Bonferroni post hoc test (Prism 5.04, GraphPad Software, La Jolla, USA). p Values < 0.05 were considered significant.
2
Materials and methods
2.1
Cell cultures
The immortalized human oral keratinocyte cell line OKF6/TERT2 was provided by Dr. J. Rheinwald (Harvard University) and cultured in accordance with the protocols described by Dickson et al. . Briefly, cells were cultured in a keratinocyte serum-free medium (ker-sfm #17005-042) containing 25 μg/mL bovine pituitary extract (BPE), 0.2 ng/mL epidermal growth factor (EGF) (all from GIBCO/Invitrogen, Carlsbad, CA, USA), 0.3 mM CaCl 2 , and penicillin (100 U/mL)/streptomycin (100 μg/mL) (all from Biochrom KG, Berlin, Germany).
For passaging, a small amount of 0.125% trypsin/0.01% EDTA (T/E) solution (Sigma, Deisenhofen, Germany) in PBS and Dulbecco’s modified Eagle medium/F-12 medium (DMEM/F-12, Biochrom KG, Berlin, Germany) including 10% fetal bovine serum (FCS, Lonza, Verviers, Belgium) was used. Exponentially growing cultures (4–8 days old) were used as source of cells for the next passage.
For both experimental and control groups, cells were grown in a medium containing higher concentrations of nutrients (HD-ker medium), mixed (1:1) of GIBCO ker-sfm and a DMEM/F-12 medium containing calcium-free, glutamine-free DMEM (GIBCO/Invitrogen, #21068-028) with Ham’s F-12 supplemented with 0.2 ng/mL EGF, 25 μg/mL BPE (all GIBCO/Invitrogen, #11765-054), 1.5 mM l -glutamine, and penicillin (100 U/mL)/streptomycin (100 mg/mL). Cell viability (95–98%) was analyzed before plating for experiments using trypan blue dye (Sigma, Taufkirchen, Germany) exclusion tests. All cultures were routinely tested for mycoplasma contamination by means of the mycoplasma detection kit Venor GeM (Minerva Biolabs, Berlin, Germany).
2.2
Preparation of materials
Stock solutions (200-fold) of CQ and DMT (VOCO, Cuxhaven, Germany) were prepared in ethanol (Baker, Griesheim, Germany) and were freshly diluted in keratinocyte medium (HD-ker) prior to each experiment. GSH (Sigma, Taufkirchen, Germany) was prepared in distilled water. The final concentration of ethanol did not exceed 0.25% (v/v). In previous experiments it was found that an ethanol concentration of 0.25% (v/v) is nontoxic for OKF6/TERT2 cells and has no effect on redox balance (data not shown). Cells incubated with fresh growth medium containing 0.25% ethanol (c1) and fresh growth medium (HD-ker) without ethanol (c2) served as solvent controls (c1) and negative controls (c2). The complete preparation of the used CQ solutions was done under dim green safe light, prepared solutions were wrapped in aluminum foil and the cells were treated under dim room light to avoid the photoactivation of CQ.
2.3
Cytotoxicity
2.3.1
H33342 assay
The chromatin staining and membrane-permeable dye H33342 (Riedel de Haen, Seelze, Germany) was used to determine the effective concentration for a 50% (EC 50 ) growth reduction due to CQ and DMT (both VOCO, Cuxhaven, Germany). For this, CQ and DMT were dissolved and pre-diluted in ethanol (EtOH, 1 M stock solutions), then finally diluted at least 1:400 in culture medium (HD-ker) and used in concentrations ranging between 0.125 and 2.5 mM. Cells were seeded in 96-well microplates at a density of 2 × 10 4 cells/well and grown for 24 h. Subsequently, cultures were treated with CQ and DMT, respectively, for 24 h and 48 h. The materials were added by medium change. Untreated cells grown in HD-ker served as growth medium control (c2). After treatment, medium was removed and H33342 (working solution: 1 mg/mL in growth medium) was added to determine the total cell number. The fluorescence intensity of the cultures at 460 nm (excitation wavelength of 360 nm) (Ex/Em = 360 nm/460 nm) was evaluated using a FLx800 microplate reader (BioTec, Neufahrn, Germany). Each assay with each 6 replicates was repeated at least three times to ensure reproducibility.
2.3.2
ROS detection
Formation of reactive oxygen species (ROS) was evaluated using the oxidation-sensitive dye 2 1 ,7 1 -dichlorofluorescin diacetate (DCFH-DA, Sigma, Deisenhofen, Germany). Cells were seeded in transparent 96-well microplates at a density of 2 × 10 4 cells/well. After growing for 48 h, cells were loaded with DCFH-DA in HBSS for 20 min in the dark and then washed with HBSS. Cells were treated with CQ (0. 5–2.5 mM) in HBSS alone, with 1.0 mM CQ in combination with DMT (1.0 mM, 2.5 mM), with 2.5 mM CQ in combination with GSH (5 mM) or with 2.5 mM CQ irradiated for 10 s with visible blue light using a dental curing light (800 mW/cm 2 ; Elipar II, Espe, Seefeld, Germany). In addition, experiments were performed to analyze the effect of blue-light irradiation. For this, black 96 well plates with transparent bottom were used to avoid an irradiation of adjacent wells. 0.25% EtOH in HBSS (c1) and HBSS (c2) served as controls. Fluorescence was read for 90 min, directly after adding the substances and then every 15 min at Ex/Em = 485 nm/528 nm. Maximum rate of fluorescence increase ( v max ) was calculated.
2.3.3
Evaluation of apoptosis
Apoptosis was analyzed using flow cytometry after staining of the plasma membrane with Annexin V, measurement of caspase-3/7 activity, as well as by evaluating DNA ‘laddering’ on 1% agarose gels. Cells treated with staurosporine, a well known inducer of apoptosis in a wide range of cell lines, served as positive control.
2.3.4
Annexin V-FLUOS/PI assay
For flow cytometry analysis, 2 × 10 5 cells/well were seeded into 6 well tissue plates (Cellstar, Greiner bio-one, Frickenhausen, Germany) in 5 mL of HD-ker medium. After 24 h of incubation in a humidified 5% CO 2 atmosphere at 37 °C, the exponentially growing cells were exposed to various concentrations of CQ in the vicinity of the EC 50 (0.5–2.5 mM). Control cultures were grown in HD-ker with (c1) and without 0.25% EtOH (c2). After incubation, adherent OKF6/TERT2-cells were collected by trypsination and pooled together with non-attached cells in tubes on ice for FACS analysis. Cells were washed twice with phosphate buffered saline (PBS) and centrifugation cycles of 10 min at 200 × g . Cell pellets were re-suspended in the Annexin V-FLUOS and PI staining solution and stained for 15 min at room temperature in the dark.
After staining, cells were analyzed by flow cytometry using a 488 nm laser line for excitation. Green fluorescence (FLUOS) was collected between 505 nm and 545 nm and red fluorescence (PI) between 605 nm and 635 nm. Cells with lost integrity of the plasma membrane (necrotic and ‘apoptotic/necrotic’ cells) were detected with propidium iodide (PI) (annexin V +/− /PI + ). Cells that stained negative for Annexin-V-fluorescein and propidium iodide (annexin V − /PI − ) were considered viable. Cells that stained positive for Annexin-V-fluorescein and negative for PI (annexin V + /PI − ) were considered to be in early stage of apoptosis. At least 20,000 cells were analyzed per sample. All experiments were repeated at least three times. Data analysis was performed with Cell Quest software version 3.1 (Becton Dickinson, Heidelberg, Germany) and Summit software version 5.1.3.6886 (Beckman Coulter, Fullerton, USA).
2.3.5
Measurement of caspase-3/7 activity
The activity of caspase-3/7 in OKF6/TERT2 cells exposed to CQ ± DMT was assessed by a luminescence assay according to the manufacturer’s instructions (ApoToxGlo Triplex Assay, Promega, Madison, USA). In brief, OKF6/TERT2 cells were cultured on 96-well flat-bottom microtiter plates at a density of 2 × 10 4 cells/well. Semiconfluent cells were exposed to CQ (0.5–2.5 mM) ± DMT (1.0 mM, 2.5 mM) in 100 μL HD-ker medium per well for 3–10 h. After incubation, 20 μL per well Viability/Cytotoxicity Reagent was added, mixed, and incubated for 30 min at 37 °C and 5% CO 2 . Viability (intracellular protease activity) and cytotoxicity (extracellular protease activity = loss of cell membrane integrity) were estimated from fluorescence of sample at Ex/Em = 360 nm/528 nm and Ex/Em = 485 nm/528 nm, respectively, using a fluorescence microplate reader (FLx800, BioTec, Neufahrn, Germany). After that, 100 μL Caspase-Glo 3/7 Reagent was added and cells were incubated at room temperature for 30 min. Caspase-3/7 activation was determined by luminescence of samples using a luminescence microplate reader (Centro XS LB 960, Berthold Technologies, Bad Wildbad, Germany).
2.3.6
Oligonucleosomal DNA degradation analysis
OKF6/TERT2 cells were cultured on 6-well plates at 2 × 10 5 cells/well in HD-ker medium. After 48 h, cells were exposed to CQ (0.5–2.5 mM) ± DMT (1.0 mM, 2.5 mM) in 3 mL HD-ker medium per well for 24 h. After incubation, the medium was transferred into 15-mL centrifugation tubes (Sarstedt, Nuembrecht, Germany), the plates were rinsed twice with 2 mL cold PBS without Ca 2+ or Mg 2+ and then cells were scraped off the plates . Cells and the rinsing buffer were transferred to the tubes and centrifuged at 115 × g for 5 min. The pellet was re-suspended in 275 μL TE-buffer (Tris + EDTA, pH 7.5, both from Sigma, Deisenhofen, Germany) containing 1 mg/mL RNase A (Quiagen, Hilden, Germany). Cells were lysed by the addition of an equal volume of 1.2% SDS and gently mixed by inversion (six to eight times). After 5 min, 350 μl of a CsCl containing precipitation solution (3 M CsCl, 1 M potassium acetate, 0.67 M acetic acid, all from Sigma, Deisenhofen, Germany) was added, vials were carefully mixed (8–10 times) and placed on ice for 15 min. Thereafter, the mixture was spun for 15 min at 14,000 × g at ambient temperature and the clear supernatant (700 μl) either transferred into new 2-mL vials (when samples were to be stored at 4 °C for later analysis) or immediately put on a miniprep spin column (QIAprep Spin Miniprep Kit, Qiagen, Hilden, Germany). The columns were spun for 1 min at 14,000 × g , washed with 700 μl wash buffer (80 mM potassium acetate, 10 mM Tris ± HCl, pH 7.5, 40 mM EDTA, 60% ethanol), spun again and DNA was eluted with 50 μl TE buffer (pH 8.0). Extracted DNA was analyzed in 1% agarose gel in 1 mM ethylene diamine tetraacetic acid (EDTA), 40 mM Tris acetate, pH 7.6, separated for 50 min at 80 V, stained in 0.5 μg/mL of GelRed (Biotium Inc., Hayward, CA, USA) and then visualized using a TI 1 UV transilluminator (Biometra, Göttingen, Germany) equipped with a photographic camera (EDAS 290, Kodak, Lollar, Germany). Fig. 6 a and b are representative gels reproduced in three independent experiments with identical results.
2.4
Statistics
Assays were run at least three times with each six replicates (cytotoxicity, ROS formation, caspase-3/7 activity) or without replicates (FACS, DNA laddering). In experiments using microtiter plates, measured fluorescence and luminescence values were converted into % of untreated control cells (c2 = 100%). Results were expressed as means ± standard deviation (SD). Statistical analysis was performed by unpaired t -test (cytotoxicity) and one way ANOVA followed by a Bonferroni post hoc test (Prism 5.04, GraphPad Software, La Jolla, USA). p Values < 0.05 were considered significant.
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