Combination of TRAIL with Bortezomib Shifted Apoptotic Signaling from DR4 to DR5 Death Receptor by Selective Internalization and Degradation of DR4

Abstract

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) mediates apoptosis in cancer cells through death receptors DR4 and DR5 preferring often one receptor over another in the cells expressing both receptors. Receptor selective mutant variants of TRAIL and agonistic antibodies against DR4 and DR5 are highly promising anticancer agents. Here using DR5 specific mutant variant of TRAIL - DR5-B we have demonstrated for the first time that the sensitivity of cancer cells can be shifted from one TRAIL death receptor to another during co-treatment with anticancer drugs. First we have studied the contribution of DR4 and DR5 in HCT116 p53+/+ and HCT116 p53−/− cells and demonstrated that in HCT116 p53+/+ cells the both death receptors are involved in TRAIL-induced cell death while in HCT116 p53−/− cells prevailed DR4 signaling. The expression of death (DR4 and DR5) as well as decoy (DcR1 and DcR2) receptors was upregulated in the both cell lines either by TRAIL or by bortezomib. However, combined treatment of cells with two drugs induced strong time-dependent and p53-independent internalization and further lysosomal degradation of DR4 receptor. Interestingly DR5-B variant of TRAIL which do not bind with DR4 receptor also induced elimination of DR4 from cell surface in combination with bortezomib indicating the ligand-independent mechanism of the receptor internalization. Eliminatory internalization of DR4 resulted in activation of DR5 receptor thus DR4-dependent HCT116 p53−/− cells became highly sensitive to DR5-B in time-dependent manner. Internalization and degradation of DR4 receptor depended on activation of caspases as well as of lysosomal activity as it was completely inhibited by Z-VAD-FMK, E-64 and Baf-A1. In light of our findings, it is important to explore carefully which of the death receptors is active, when sensitizing drugs are combined with agonistic antibodies to the death receptors or receptor selective variants of TRAIL to enhance cancer treatment efficiency.

Mutations enhancing selectivity of antitumor cytokine TRAIL to DR5 receptor increase its cytotoxicity against tumor cells

“…However, later it was shown that in combined treatment by TRAIL and proteasome inhibitors (MG 132 or borte zomib) of primary chronic lymphocytic leukemia cells (CLL), DR4 and DR5 receptors transmitted apoptotic signal equally effectively [28]. It was shown that the chemotherapy not only regulates the expression of death receptors, but also can switch the sensitivity of cells from one death receptor to another [29].…”

“…Interestingly, the efficacy of DR5 B and TRAIL in com bination with doxorubicin and paclitaxel in MDA MB 231 cells did not differ (Tables 1 and 3), whereas in com bination with bortezomib the efficiency of DR5 B was much better both for maximal cell death and for EC 50 comparing to wild type TRAIL. Recently, it was shown that the combined action of TRAIL and bortezomib caused specific internalization and degradation of death receptor DR4 in MDA MB 231 and HCT116 cells, resulting the activation of the DR5 receptor [29].…”

“…In con trast to death receptors, the modulation of decoy recep tors by chemotherapeutic agents is practically unstudied. Perhaps under the combined action of death receptor agonists and chemotherapeutics, all TRAIL receptors are modulated, which changes the sensitivity of the cells [29]. If we consider the fact of the existence of the soluble TRAIL decoy receptor OPG in human blood, we can assume that in vivo the differences of efficacy impact of DR5 B and TRAIL will be expressed stronger as the affinity of DR5 B to OPG is reduced almost 220 fold [11].…”

Chemically modified liposomes carrying TRAIL target activated hepatic stellate cells and ameliorate hepatic fibrosis in vitro and in vivo

“…When the cells encountered the liposome particles containing rhTRAIL, cell membrane fluidity causes the release of many protein molecules, forming a higher concentration of rhTRAIL around the cells than free rhTRAIL molecules do. Besides, we also found the enriched intracellular rhTRAIL in 3T3 cells after incubation with pPB‐SSL‐TRAIL, which is probably caused by internalization of TRAIL receptors …”

Inhibition of N‐myc expression sensitizes human neuroblastoma IMR‐32 cells expressing caspase‐8 to TRAIL

“…16,17 Studies have shown that anti-cancer drugs such as bortezomib, 18,19 etoposide 20 and doxorubicin 21 sensitized cancer cells to TRAIL-mediated death through the upregulation of DR expression. 16,17 Studies have shown that anti-cancer drugs such as bortezomib, 18,19 etoposide 20 and doxorubicin 21 sensitized cancer cells to TRAIL-mediated death through the upregulation of DR expression.…”