Cell viability was quantified by Wst-1 assay. The same results were obtained with agonistic anti-DR5 antibodies. Thus, the effectiveness of TRAIL was rather limited due to changes in the ratio of death and decoy receptors and DR5-specific agonists may be favored in combination antitumor therapy regimens. = 4). The asterisks indicate significance (* < 0.05) and (** < 0.001) relative to cells treated with chemotherapy without ligands. TRAILtumor necrosis factor related apoptosis-inducing ligand. 2.2. The Modulation of Surface Expression of TRAIL Receptors and Decoy Receptors by Chemotherapeutic Brokers Determines the Effectiveness of Sensitization of Malignancy Cells to Ligands Next, we evaluated the effect of bortezomib, doxorubicin and panobinostat on the surface expression of the TRAIL death and decoy receptors in HT-29 and A549 cells by circulation cytometry (Physique 2A,B). Treatment of cells with these brokers for 24 h strongly enhanced DR5 expression (5C7 fold) in both cell lines. Bortezomib and doxorubicin also caused an increase in the DR4 receptor (2C2.5 occasions), while treatment with panobinostat reduced the amount of this receptor around the cell surface in both lines. Chemotherapeutic brokers enhanced the surface expression of DcR1 and DcR2 decoy receptors to varying degrees depending on the type of cells, except that panobinostat slightly reduced the expression of DcR2 in A549 cells. Open in a separate window Physique 2 Effect of modulation of surface expression of death and decoy paederosidic acid receptors by chemotherapeutic brokers on malignancy cell sensitization to TRAIL and DR5-B. Surface expression of death and decoy receptors in HT-29 (A) and A549 (B) cells before and after treatment with the chemotherapeutic brokers was determined by flow cytometry. Values of Mean Fluorescence Intensity (MFI) are offered as percent relative to control cells. Representative histograms from three impartial experiments with comparable results are shown. HT-29 (C) and A549 (D) cells were co-treated with doxorubicin (4000 paederosidic acid nM), bortezomib (200 nM) or panobinostat (400 nM) and TRAIL or DR5-B for 24 h. Cell viability was determined by WST-1 colorimetric assay. Mean Standard Deviation (= 3). The asterisks indicate significance (* < 0.05) and (** < 0.001) relative to control cells (A,B) or relative to cells treated with chemotherapy without ligands (C,D). We then compared the efficiency of TRAIL or DR5-B cytotoxicity in combination with chemotherapeutic brokers. In both cell lines, DR5-B was highly effective at concentrations of 1C10 ng/mL, while TRAIL killed the cells at concentrations one to two orders of magnitude higher depending on the type of chemotherapy (Physique 2C,D). paederosidic acid The affinity BMP2 of DR5-B to DR5 is not different from TRAIL, as previously demonstrated [18]. Therefore, it can be assumed that this large difference between the effectiveness of TRAIL and DR5-B is due to the expression of decoy receptors DcR1 and DcR2 around the cell surface. 2.3. DR5-B Induces Internalization of the DR5 Receptor More Efficiently Than TRAIL To analyze in more detail the difference in the effects of TRAIL and DR5-B in combination with chemotherapeutic brokers, we examined ligand-induced internalization of DR4 and DR5. For this, A549 and HT-29 cells were incubated with chemotherapeutic brokers for 24 h, then with ligands for 1 h, and surface expression of receptors was measured by circulation cytometry. At a higher concentration (1000 ng/mL), both ligands induced DR5 internalization at almost the same level (Physique 3A). After pretreatment of the cells with chemotherapy, a strong internalization of the DR5 receptor.