Cancer Therapy Vol 4, page 61 cancer cell lines and, through this mechanism, sensitize these cells to the pro-apoptotic effects of TRAIL. Experiments on a battery of renal carcinoma cell lines have shown that Bortezomib may either increase TRAILR1 and/or TRAIL-R2 expression or decrease c-FLIP levels, but in all istances it sensitize these cells to the apoptogenic effects of TRAIL (Sayers and Murphy, 2005). The increased TRAIL-R2 expression induced by proteasome inhibitors is related to a transcriptional mechanism dependent upon an enhanced promoter activity, mediated by the binding of the CHOP transcription factor (Yoshida et al, 2005). Since TRAIL binding to its receptors induces NF-#B activation, that can induce several anti-apoptotic genes, it was suggested that proteasome inhibitors may sensitize the cells to TRAIL-mediated apoptosis by blocking NF-#B activity. However, several studies have shown that NF-#B blocking by proteasome inhibition is not essential for the sensitization of several tumor cell types to TRAIL (Sayers et al, 2003). In some tumor cells, NF-#B inhibition may represent an important event contributing to the sensitization to TRAIL (Luo et al, 2004). Interestingly, the combined addition of proteasome inhibitors and TRAIL resulted in a cytotoxic effect in chemoresistant Bcl-2-overexpressing cells that are otherwise resistant to TRAIL or cytotoxic drugs or proteasome inhibitors alone (Nencioni et al, 2005). The combination of TRAIL and antiblastic drugs was unable to induce the apoptosis of these cells (Nencioni et al, 2005). These observations indicate that proteasome inhibitors plus TRAIL induce mitochondrial dysfunction irrespective of upregulated Bcl-2. The molecular mechanisms through which proteasome inhibitors induce a decrease of c-FLIP levels and upmodulate TRAIL-R1 and TRAIL-R2 are largely unknown. In this context, it is important to note that several components of the apoptotic machinery, including some members of the Bcl-2 family, IAP family proteins, I#B and p53, are ubiquitinated (Zhang et al, 2004). The decrease in c-FLIP observed in cells treated with proteasome inhibitors is surprising in that c-FLIP was reported to be degraded by proteasome in some cells (Sayers et al, 2003). One explanation could be related to the effect of the proteasome inhibitor on cell cycle: cell cycle arrest in S-G2/M phase could result in c-FLIP decrease because during the normal cell cycle peak levels of this protein are observed in G1, with a marked decrease in G2/S phase. In contrast, the increase of c-FLIP levels induced by proteasome inhibitors observed in other tumor cells may be related to a reduced degradation of c-FLIP protein (Ganten et al, 2005). Recent studies carried out in bladder and prostate cancer cells have shown that the cell cycle regulatory protein p21, whose levels are greatly enhanced by Bortezomib+TRAIL, plays a key role in the mechanism through which the proteasome inhibitor allows to bypass TRAIL resistance (Lashinger et al, 2005). The increased p21 levels are required for optimal caspase-8 processing (Lashinger et al, 2005).
IV. Strategies to overcome TRAIL resistance As outlined in the previous chapters, very frequently primary cancer cells are resistant to TRAIL-mediated apoptosis. This observation had stimulated many studies focused to develop strategies to circumvent TRAIL resistance by cancer cells. The philosophy of these various strategies is based on the combination of TRAIL with another drug: the role of the other drug consists in sensitizing tumor cells to the apoptotic effects of TRAIL.
A. TRAIL and proteasome inhibitors The 26S is a multicatalytic enzyme present in the cytoplasm and in the nucleus of virtually all eukaryotic cells, involved in the degradation of proteins targeted by ubiquitin conjugation. The proteasome plays a key role in the control of cell homeostasis in that it regulates the halflife of cellular proteins essential for the life of the cell, such as transcription factors, tumor suppressors, oncogenes and proteins involved in cell cycle control. These observations have suggested that the proteasome may represent an important therapeutic target in cancer. Several proteasome inhibitors have been synthesized and one of them, Bortezomib (known also as VELCADE or PS-341), was approved by the Food and Drug Aministration for cancer treatment (Rajikumar et al, 2005). Bortezomib, a peptide boronate, selectively inhibits the chymotrypsic-like activity of the proteasome at nanomolar concentrations (Rajikumar et al, 2005). In vitro studies have shown that the effect of Bortezomib on cell lines mainly consists on cell cycle inhibition and induction of apoptosis (Rajikumar et al, 2005). Treatment of tumor cells with Bortezomib results in multiple biological effects, including inhibition of cell cycling, inhibition of NF-#B acivity, changes in cell adherence and increased apoptosis. Among these various effects it was initially observed that Bortezomib treatment may considerably increase the sensitivity of myeloma cells to TRAIL (Mitsiades et al, 2001). In the absence of the proteasome inhibitor, myeloma cells are resistant to TRAIL-mediated apoptosis. Subsequent studies have confirmed these initial observations in other tumor cell types and have explored the potential molecular mechanisms responsible for this phenomenon. Thus, Bortezomib was found to sensitize mouse myeloid leukaemia C1498 cells to TRAIL-mediated apoptosis through a mechanism related to a decreased expression on the anti-apoptotic protein c-FLIP, without significant decrease of Bcl-2 anti-apoptotic members or of the various IAP members (Sayers et al, 2003). In contrast, other studies carried out on different tumor cells, like hepatocellular carcinoma cells, have shown that the proteasome inhibitor MG-132 clearly increased c-FLIP levels; in spite the c-FLIP increase, MG-132-pretreated cells became more sensitive to TRAIL-mediated apoptosis (Ganten et al, 2005). Other studies have shown that Bortezomib (Johnson et al, 2003) or MG-132, another proteasome inhibitor, (He et al, 2004) induce a marked increase of TRAIL-R1 and TRAIL-R2 expression in prostate, colon and bladder
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