4 RESULTS
4.3 Role of apoptosis inducing factor (AIF) in apoptosis under caspase arrest in HepG2 cells
4.4.1 Effect of 5-azacytidine on death receptor agonist-induced cytotoxicity
The influence of 5-azacytidine on death receptor agonist-induced apoptosis was examined in primary human hepatocyte cultures. The hepatocytes were isolated from pathological inconspicuous specimens obtained from patientsundergoing hepatic resections for the therapy of liver tumors. As seen in figure 4.15, 5-azacytidine rendered hepatocytes sensitive to the action of the death receptor agonists TNF-a; CD95L and TRAIL in a concentration-dependent manner.
ctrl.
0 20 40 60 80 100
1 2
ctrl + CD95L
+ TNF
5- Azacytidine [log µM]
cell death [%]
Fig. 4.15: Effect of increasing concentrations of 5-azacytidine on primary human hepatocytes treated with either 100 ng/ml TNF-a; 10 % v/v CD95L (a) or 100 ng/ml TRAIL (b). Cytotoxicity was determined after 18h.
Data represent mean– SD.
a b
ctrl.
0 20 40 60 80 100
1 2
ctrl.
+ TRAIL
5-Azacytidine [log µM]
cell death [%]
4. Results
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4.4.1.2 5-Azacytidine mediated sensitization in primary murine hepatocytes
The results shown above were confirmed in analogous experiments utilizing hepatocytes isolated from murine liver. Since murine liver cells were insensitive against TRAIL-induced apoptosis, the assays were conducted with TNF-a and CD95L.
The first set of experiments was done to define the optimal concentration for sensitization.
Increasing concentrations of 5-Azacytidine were added on primary murine hepatocytes alone or in combination with invariable concentrations of the cytokines TNF-a and CD95L (data not shown). From these data 75 µM 5-azacytidine was derived as the most efficient concentration for sensitization. In counter-check experiments the sensitizing effects were confirmed by treatment of primary murine hepatocytes with increasing concentrations of the cytokines at a fixed concentration of 75 µM 5-azacytidine. As seen in figure 4.16, increasing concentrations of TNF-a and CD95L resulted in enhanced cytotoxicity and effector caspase-3/-7 activity.
Fig. 4.16: Concentration-response curves of death receptor ligands with determination of cytotoxicity (a,b) and their respective effector caspase activity (c,d). Primary murine hepatocytes were treated with 75 µM 5-azacytidine prior addition of increasing concentrations of CD95L and TNF-a. Cytotoxicity was determined after 18h.
Data represent mean– SD.
4. Results
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As the methyltransferase inhibitor 5-azacytidine proved to sensitize hepatocytes towards apoptosis, we tried to work out following topics:
• Examination of DNA methyltransferase inhibitors in terms of sensitization of hepatocytes
• Mechanistic studies for clarification of the action point(s) of 5-azacytidine.
For this purpose the human hepatoma cell line HepG2 was chosen.
4.4.1.3 Capability of various DNA methyltransferase inhibitors to sensitize the human hepatoma cell line HepG2 cells against death receptor agonist-induced apoptosis To elucidate potential augmentation of death receptor-triggered cytotoxicity with different kinds of DNA methyltransferase inhibitors, HepG2 cells were incubated with increasing concentrations of 5-azacytidine, the analogue 5-aza-2’-deoxycytidine, S-(5’-adenosyl)-L-homocysteine and RG 108. To define the optimal concentration for sensitization versus a minimal basal toxicity, the indicated substances were incubated with HepG2 cell in increasing concentration alone or in combination with the cytokines TNF-a, CD95L or TRAIL (Figure 4.17).
Besides the positive controls, in terms of ActD and CHX, only the combination of 5-azacytidine combined with TRAIL or CD95L showed a significant concentration-dependent increase of cytotoxicity. Unlike in primary hepatocytes, 5-azacytidine was not capable of promoting TNF-ainduced cytotoxicity. The no-effect data for the analogue 5-aza-2’-deoxycytidine and for positive controls see figure 4.16, data for RG 108 and S-(5’-adenosyl)-L-homocysteine are not shown.
ctrl.
0 20 40 60 80 100
1 2
5-Azacytidine [log µM]
CD95L TRAIL
TNF-a a) ctrl
cell death [%]
ctrl.
0 20 40 60 80 100
1 2
b)
5-Aza-2`-deoxycytidine [log µM]
cell death [%]
4. Results
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4.4.1.4 Kinetics of caspase activity and cytotoxicity in HepG2 cells after sensitization with 5-azacytidine
After death receptor trimerisation and assembly of the death inducing signaling complex (DISC) upon binding of a death ligand, a hierarchical sequence of death signaling leads to subsequent activation of the effector caspases -3/-7, detectable by DEVD cleavage, which in turn activate other enzymes responsible for cellular degradation.
In the used cellular system an increase in cytotoxicity occurred over time after a peak of maximal caspase activity. Data in figure 4.18 demonstrated that the overall series of events was similar with CD95L and TRAIL, even though the induction was more rapid in case of TRAIL. The peak of caspase activity occurred around 8-10h, while the toxicity reached the plateau phase after 18h to 21h. It is concluded from these kinetic observations that the emerging cytotoxicity is likely to be the consequence of the activation of caspases.
Neither caspase -3/-7 like activity nor any cytotoxicity was detected in HepG2 cells treated with 5-azacytidine alone or in combination with TNF-a.
Fig. 4.17: Screening for maximal non-toxic, sensitizing concentrations of MTI and ActD/CHX toward cytokines in HepG2 cells. HepG2 cells were incubated with increasing concentrations of 5-azacytidine (a), 5-aza-2'-deoxycytidine (b), cycloheximide (c) and actinomycine D (d) 3h prior to addition TRAIL (100 ng/ml), TNFa(100 ng/ml) and CD95L (10 % v/v). Cytotoxicity was determined after 18h.
Data represent mean ± SD.
ctrl.
0 20 40 60 80 100
1 2 3
c)
Cycloheximide [log µM]
cell death [%]
ctrl.
0 20 40 60 80 100
-1 0 1
d)
Actinomycine D [log µg/ml]
cell death [%]
4. Results
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4.4.1.5 Correlation of caspase activity and cytotoxicity HepG2 cells after sensitization with 5-azacytidine
In the next series of experiments a reversed set-up was chosen in order to correlate the death receptor-agonist concentration with cytotoxicity and caspase activity. HepG2 cells, pre-treated with a constant concentration of 75 µM 5-azacytidine were incubated with increasing concentrations of either CD95L or TRAIL. As seen in fig. 4.19 increasing concentrations of death receptor agonist caused increasing caspase activity and cytotoxicity.
Also in this set of experiments the non-sensitization effect of 5-aza-2’deoxycytidine, S-(5’-adenosyl)-L-homocysteine and RG 108 were affirmed. The positive control ActD enhanced the cytotoxicity as well as caspase activity with either cytokine utilized (data not shown).
a) CD95L
Fig. 4.18: Time course of caspase activity (•) and resulting cytotoxicity () after treatment of HepG2 cells with either 75 µM 5-azacytidine alone (open symbols) or in combination with 10 % v/v CD95L (a) and TRAIL 100 ng/ml (b). Cytotoxicity and caspase activity was determined at the indicated time points. Data represent mean ± SD.
b) TRAIL
DEVD-cleavage [µU] cell death [%]
b) TRAIL
4. Results
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In conclusion, the results described in the two previous chapters showed a time- and concentration-dependent activation of caspases and a subsequent cytotoxicity.
4.4.1.6 Morphology of cell death in HepG2 cells
In order to verify the nature of the 5-azacytidine-mediated sensitization towards death receptor agonist-induced apoptosis the morphology of the dying cells was assessed by light- and fluorescence microscopy. Unlike untreated controls, the cells treated with CD95L and 5-azacytidine showed a rounding up and membrane blebbing (zeiosis), the typical nuclear morphology of condensed chromatin stained with Hoechst and exposure of phosphatidylserine on the outer membrane. The cells treated with 5-azacytidine alone showed no morphological changes in all parameters analyzed. In the case of CD95L, the cells showed only slight signs of apoptotic morphology. This could be tentatively explained with the fact that CD95L without sensitization is able to induce apoptosis to a certain degree. Similar results are observed in the center of morphological examinations of primary murine hepatocytes treated with 5-azacytidine and CD95L alone or in combination (data not shown).
Fig. 4.19: Concentration curve for caspase activation () and cytotoxicity () after treatment of HepG2 cells with increasing concentrations of following stimuli after pretreatment with 75 µM 5-azacytidine:
a) 0-10% v/v CD95L.
b) 0-100 ng/ml TRAIL.
c) 0-100 ng/ml TNF-a.
Caspase activity was determined after 8h, cytotoxicity was determined after 18h.
Data represents mean– SD.
Open symbols represent control without 5-azacytidine pretreatment.
c) TNF-a
Ctrl 0 50 100 150 200 250 300
0 1 2
0 20 40 60 80 100
TNF-aaaa [log ng/ml]
DEVD-cleavage [µU] cell death [%]
4. Results
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Fig. 4.20: Morphology (upper row); chromatin condensation (middle row) and exposure of phosphatidylserine on the outer membrane of HepG2 cells treated with either 75 µM 5-azacytidine; 10% v/v CD95L or in combination with both compounds. Pictures were taken 6h for PS exposure, 8h for chromatin condensation and 18h for morphology after stimuli.