【Background and objective】Tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)can induce apoptosis in a wider range of cancer cells.The selective toxicity for cancer cells is the basis for the current enthusiasm for TRAIL as a potential novel anticancer therapy.But the intrinsic and acquired resistance to TRAIL therapy restrict its clinical application. Fortunately,a growing body of evidence has shown that combining TRAIL with conventional chemotherapy,gene therapy and radiotherapy can lead to enhance apoptosis induction in TRAIL-resistant cancer cells.Moreover,some recent studies have demonstrated that proteasome inhibitors,such as calpain inhibitor I, can overcome resistance to TRAIL in different cancer cells.However,the underlying mechanisms of this effect remain unclear.Because bortezomib (PS-341,Velcade)was recently approved by the United States Food and Drug Administration for cancer therapy,we therefore evaluate the effects of combined treatment with bortezomib and TRAIL protein on TRAIL-resistant human colon cancer cell lines,DLD1-TRAIL/R,then investigate the involved mechanisms.  【Methods】Cell viability was determined by MTT assay after treated with bortezomib and TRAIL protein either in combination or alone;flow cytometry assay was then performed for cell cycle analysis;western blot analysis was used for determining the expression of various proteins,including several molecular markers of TRAIL-induced apoptotic signaling,DRs and Bcl-2 family members. Bik protein stability was determined by western blot analysis,and band densities were qualified using Optimas softwar,the mean half-life of Bik was also calculated.After knockdown of Bik by Bik siRNA,cell apoptosis was then determined by flow cytometry assay.Differences between treatment groups were assessed using an unpaired Student's t test.  【Results】1)We pretreated DLD1-TRAIL/R cells with various concentrations of bortezomib(0.5-5.0μM)for 2h,followed by 20ng/ml of TRAIL protein for another 4h.Cell viability was then determined by using a MTT assay.We found that combining TRAIL protein and bortezomib significantly decreased cell viability,whereas TRAIL protein or bortezomib alone had minimal effect at that same time interval(p＜0.05).The same result was found 24h after the addition of TRAIL protein.These results showed that bortezomib resensitized TRAIL-resistant DLD1-TRAIL/R cells to TRAIL protein.Furthermore,flow cytometry assay found that the combination of TRAIL protein and bortezomib dramatically increased the proportion of cells in the Sub-G1 phase.2)Western blot analysis showed that the combination of TRAIL protein(20ng/ml)and bortezomib(1μM)dramatically enhanced the cleavage of molecular markers of TRAIL-induced apoptotic signaling,including caspases-8,9,3,Bid,and PARP. Moreover,western blot analysis also showed that the combination treatment increased the release of cytochrome C and Smac from mitochondria,suggesting that bortezomib initiate mainly the mitochondrial apoptotic pathway.3)To characterize the mechanisms of interaction between bortezomib and TRAIL protein,we analyzed changes in the DRs including DR4 and DR5,and in Bcl-2 family members such as Bax,Bak,Bik,Bcl-2,and Bcl-XL,which are important for initiation of the mitochondrial pathway.Western blot analysis showed that bortezomib upregulated DR5 and Bik protein,however,it had no detectable effects on DR4,Bax,Bak,Bcl-2,Bcl-XL,XIAP or survivin.4)To determine the effect of bortezomib on Bik protein degradation,we treated DLD1-TRAIL/R cells with PBS,bortezomib(1μM)for 6h and then add cycloheximide to block protein synthesis.Cells were then harvested over time and Bik levels assessed by western blot analysis.Bik protein was rapidly degraded in cells treated with PBS and had a mean half-life of about 1h.In contrast,in cells treated with bortezomib,the Bik protein level and mean half-life were stable even after 6h of cycloheximide treatment,indicating that Bik accumulation was due mainly to stabilization of Bik protein by bortezomib.5)To testing the effects of proteasome inhibitor-mediated Bik accumulation on TRAIL sensitization,we used Bik siRNA to knockdown Bik accumulation.In comparison with control siRNA,treatment with Bik siRNA significantly attenuated bortezomib-mediated TRAIL sensitization(p＜0.05).This result suggested that bortezomib-mediated Bik accumulation plays an important role in resensitization of TRAIL protein.  【Conclusions】1)Proteasome inhibitor bortezomib can overcome acquired TRAIL resistance in DLD1-TRAIL/R cells,which might be mediated by proteasome inhibitor-induced accumulation of Bik.2)Bik accumulation was caused mainly by stabilization of the protein from degradation by bortezomib.3) Combination treatment of proteasome inhibitors and TRAIL protein might be a useful strategy to simultaneously trigger both death receptor-and mitochondrion-mediated apoptosis pathways and to enhance the anti-tumor activity of these agents.