towards improved death receptor targeted therapy for ... - TI Pharma

Combination study: TRAIL and TFT
cathepsin B inhibitor, CA074‐me, on TRAIL and TFT‐induced cell death was examined.
CA074‐me had no effect on the level of cell death induction of any treatment, suggesting
that cathepsin B was not directly involved in mediating TFT and/ or TRAIL‐induced cell
death (data not shown).
Effects of TFT on expression of TRAIL receptors and apoptosis regulatory proteins
The molecular mechanisms underlying the synergistic interaction between TFT and TRAIL
were investigated. First, western blotting showed that TFT increased the levels of p53
within 24 h, which was also associated with p21/ WAF1 up‐regulation in A549 and H460
cells (Fig. 5A). TFT reduced the expression of cFLIPL, FLIPs was not detectable, and XIAP in
both cell lines. Moreover, TFT increased the expression of pro‐apoptotic Bax, whereas the
expression of Bcl‐2 and Bcl‐XL did not alter (not shown). Second, the effect of TFT on the
level of expression of the TRAIL receptors was determined by flow cytometry. As depicted
in Fig. 5B, 24 h incubation of TFT increased the cell surface expression of mainly TRAIL‐R2
and to a lesser extent TRAIL‐R1 in both cell lines. As TFT increased the expression of p53,
and it is known that p53 can upregulate the death receptors [23], we examined p53‐
dependancy of this up‐regulation. A siRNA specifically directed against p53 strongly
reduced p53 expression in H460 and A549 cells, whereas control siRNA had no effect
when compared to untransfected cells (Fig. 5B). Down‐regulation of p53 expression
followed by TFT exposure resulted predominantly in reduced TRAIL‐R2 up‐regulation and
mainly
in A549 cells (Fig. 5C).
A
Figure 5. Mechanisms of TFT‐dependent TRAIL sensitization. (A) Western blots showing the
expression of FLIP L, p53, p21, XIAP, and Bax after 24, 48 and 72 h exposure to TFT, TRAIL, and the
combination. Representative blots are shown.
‐ 111 ‐