towards improved death receptor targeted therapy for ... - TI Pharma
towards improved death receptor targeted therapy for ... - TI Pharma towards improved death receptor targeted therapy for ... - TI Pharma
RESULTS Combination study: TRAIL and TFT TRAIL, TFT and combined treatment The sensitivities of the NSCLC cell lines to TFT and TRAIL are summarized in Table 1. IC50 values between 2.9 µM and 6.3 µM were observed for TFT. H292 and H460 cells were TRAIL‐sensitive, H322 cells were moderately sensitive and A549 cells were resistant to TRAIL up to the highest tested concentration of 1500 ng/ml. The IC50 values of TRAIL for a particular cell line were used in the combination experiments, except for resistant A549 cells where we used a TRAIL concentration of 150 ng/ml. Table 1 | Growth inhibition and synergy analyses for different treatment schedules of TFT combined with TRAIL Cell line Growth inhibition (IC50) Combination index (CI) TFT (µM) TRAIL (ng/ml) 24 h Combination + 48 h TFT (schedule A) 48 h TFT + 24 h Combination (schedule B) A549 6.3 ± 1.5 >1500 0.6 ± 0.2 3.7 ± 1.3 H292 3.3 ± 0.8 25 ± 7 0.6 + 0.2 1.6 ± 0.3 H322 2.9 ± 0.2 80 ± 10 0.7 ± 0.3 1.3 ± 0.4 H460 4.2 ± 0.9 10 ± 2 0.6 ± 0.1 2.8 ± 0.9 Combination index (CI) values were calculated from the fraction affected data points from 0.5 to 0.9. CI values lower than 0.9 specify synergism; CI between 0.9-1.0 indicate an additive effect; CI values greater than 1.1 denote antagonism. Synergistic effects are illustrated as fraction affected (FA)‐combination index (CI) plots obtained in H460 and A549 cells (Fig. 1). The combination schedule of 24 h TFT and TRAIL followed by 48 h of TFT alone (Schedule A) was synergistic in all tested cell lines (CI < 0.9), including resistant A549 cells. In contrast, a schedule of 48 h TFT treatment followed by 24 h TFT and TRAIL (Schedule B) appeared to have antagonistic activity in all cell lines examined (Table 1). As TRAIL exerts its apoptotic function quite rapidly, the cells were incubated for just 24 h. TFT, on the other hand, has a long term effect; an incubation time of 72 h of this compound was therefore chosen. In the experiments described below the synergistic schedule, schedule A (24 h combination + 48 h TFT) was selected to further explore the mechanism underlying synergism of TRAIL and TFT. A Schedule A Schedule B ‐ 105 ‐
Chapter 6 B H460 A549 Figure 1. Representative FA‐CI plot of combined TFT and TRAIL treatment in H460 and A549 cells using two different exposure schedules as indicated. (A) Cells were exposed to increasing concentrations of TFT for 72 h including 24 h exposure to a fixed IC50 concentration of TRAIL. TRAIL was added either the first 24 h, or the last 24 h in the presence of TFT and cytotoxicity was determined by MTT assays. (B) An average CI was calculated for data‐points with FA higher than 0.5. Effects on cell cycle progression The effects on cell cycle distribution were analyzed in time‐course experiments using flowcytometry on PI‐stained cells (Fig. 2A). In both H460 and A549 cells, 24 h exposure to TFT alone induced the accumulation of cells in the S‐phase while longer treatment, up to 72 h, was accompanied by an increase of cells in G2/M and a decrease in G1. This effect was most prominent in A549 cells. At the same time a strong rise in the percentage (up to around 35%) of death (sub‐G1) cells was found for both cell lines. TRAIL alone mildly affected the cell cycle distribution of H460 cells with a small increase of cells in G1. This pattern was more pronounced in A549 cells. When TRAIL was combined with TFT, in H460 cells an increase in G2/M cells was seen although lower than found in TFT‐treated cells. In A549 cells, the cell cycle profile after combined treatment resembled more that of TFT‐ treated cells, with increased S‐phase after 24 h followed by a pronounced accumulation of G2/M cells. In addition, only a very small percentage of A549 cells was detected in the S‐ phase after 48 and 72 h of combined treatment. Next, the treatments were examined for affecting the cell cycle regulatory proteins Chk1, Chk2, and Cdc25c. Chk1 and Chk2 are known to become phosphorylated after DNA damage resulting amongst others in inactivation of Cdc25c phosphatase by stimulating its proteasome‐dependent degradation leading to G2/M and G1/S phase arrest [22]. TFT treatment induced strong Chk2 phosphorylation in a time‐dependent manner, which correlated with a decrease in Cdc25c levels and G2/M arrest (Fig. 2A). Total Cdc25c levels decreased after 48 and 72 h treatment with TFT and even more rapidly after combination treatment reflecting its Chk‐induced phosphorylation‐dependent degradation. In H460 ‐ 106 ‐
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Chapter 6<br />
B H460 A549<br />
Figure 1. Representative FA‐CI plot of combined TFT and TRAIL treatment in H460 and A549 cells<br />
using two different exposure schedules as indicated. (A) Cells were exposed to increasing<br />
concentrations of TFT <strong>for</strong> 72 h including 24 h exposure to a fixed IC50 concentration of TRAIL. TRAIL<br />
was added either the first 24 h, or the last 24 h in the presence of TFT and cytotoxicity was<br />
determined by MTT assays. (B) An average CI was calculated <strong>for</strong> data‐points with FA higher than 0.5.<br />
Effects on cell cycle progression<br />
The effects on cell cycle distribution were analyzed in time‐course experiments using<br />
flowcytometry on PI‐stained cells (Fig. 2A). In both H460 and A549 cells, 24 h exposure to<br />
TFT alone induced the accumulation of cells in the S‐phase while longer treatment, up to<br />
72 h, was accompanied by an increase of cells in G2/M and a decrease in G1. This effect<br />
was most prominent in A549 cells. At the same time a strong rise in the percentage (up to<br />
around 35%) of <strong>death</strong> (sub‐G1) cells was found <strong>for</strong> both cell lines. TRAIL alone mildly<br />
affected the cell cycle distribution of H460 cells with a small increase of cells in G1. This<br />
pattern was more pronounced in A549 cells. When TRAIL was combined with TFT, in H460<br />
cells an increase in G2/M cells was seen although lower than found in TFT‐treated cells. In<br />
A549 cells, the cell cycle profile after combined treatment resembled more that of TFT‐<br />
treated cells, with increased S‐phase after 24 h followed by a pronounced accumulation of<br />
G2/M cells. In addition, only a very small percentage of A549 cells was detected in the S‐<br />
phase after 48 and 72 h of combined treatment.<br />
Next, the treatments were examined <strong>for</strong> affecting the cell cycle regulatory proteins Chk1,<br />
Chk2, and Cdc25c. Chk1 and Chk2 are known to become phosphorylated after DNA<br />
damage resulting amongst others in inactivation of Cdc25c phosphatase by stimulating its<br />
proteasome‐dependent degradation leading to G2/M and G1/S phase arrest [22]. TFT<br />
treatment induced strong Chk2 phosphorylation in a time‐dependent manner, which<br />
correlated with a decrease in Cdc25c levels and G2/M arrest (Fig. 2A). Total Cdc25c levels<br />
decreased after 48 and 72 h treatment with TFT and even more rapidly after combination<br />
treatment reflecting its Chk‐induced phosphorylation‐dependent degradation. In H460<br />
‐ 106 ‐
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