Bortezomib Sensitizes Gastric Cancer Cells To TRAIL-induced Apoptosis

ObjectiveGastric cancer is one of the most common malignancies worldwide,particularly in eastern Asian countries such as China,Japan,and Korea.Most patients are diagnosed at an advanced stage,and the outcome of combinational chemotherapy is very poor. The most effective regimen for the treatment of advanced disease is not yet clearly established.Recently,the combination of a biological agent,such as a monoclonal antibody, with chemotherapy has exhibited significant therapeutic benefits in the treatment of breast,colon,and lung cancer.These results confirm that a biological agent in combination with a chemotherapeutic agent is more powerful than chemotherapy alone, and further attempts to combine other biological agents,like cytokines and chemicals, has been promising.Tumor necrosis factor(TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily,which can induce apoptosis through the engagement of death receptors.Unlike other members of the TNF family,TRAIL preferentially induces apoptosis in a variety of tumor cells,whereas it has only slight toxicity towards most normal cells.More importantly,TRAIL induces a milder inflammatory response than do other TNFs.Its selective toxicity for cancer cells and its induction of low-level inflammation make TRAIL a potential tumor-specific cancer therapy.Clinical trials using agonistic monoclonal antibodies specific for TRAIL receptors(DR4 or DR5) have achieved promising results in patients with advanced solid malignancies.Despite the powerful activity of TRAIL towards most cancer cells,some tumors display intrinsic or acquired resistance to TRAIL-induced apoptosis.A thorough understanding of the mechanisms involved in TRAIL resistance is very important if we are to overcome this resistance and facilitate the clinical use of TRAIL.It has been reported that the binding of TRAIL to its receptors activates nuclear factor kappa-B (NF-κB),which in turn initiates the transcription of several antiapoptotic genes and protects cells from TRAIL-induced apoptosis.The inhibition of NF-κB activity increases the sensitivity of a variety of tumor cells towards TRAIL-induced apoptosis. However,in some cases,NF-κB inactivation fails to enhance the apoptosis triggered by TRAIL treatment.These controversial results suggest that NF-κB activation does not protect all cells from TRAIL-mediated apoptosis.The role of NF-κB in TRAIL-induced apoptosis in different tumors must be further studied.Several signaling pathways,other than NF-κB,also contribute to TRAIL resistance.Phosphatidylinositol 3-kinase(PI3K)/Akt is an important signaling pathway that regulates cell survival,growth,and apoptosis.It can be induced by a wide range of stimuli,and often acts as an antiapoptotic signal.It has been reported that highly active PI3K/Akt signaling is responsible for TRAIL resistance.Blockage of the PI3K/Akt pathway by either specific inhibitors or small interfering RNAs can reverse the resistance to TRAIL-induced apoptosis in some primarily resistant tumor cells.Attempts to increase TRAIL efficacy have had encouraging results in some tumors, such as lung cancer and colon cancer.Recently,a few chemical agents have been shown to increase the sensitivity of gastric cancer cells to TRAIL-induced apoptosis. However,none of these agents is available for clinical use.Bortezomib is a newly identified proteasomal inhibitor,which has been approved by the U.S.Food and Drug Administration(FDA) for the treatment of human multiple myeloma and mantle cell lymphoma.The main antitumor function of bortezomib acts by blocking NF-κB activation,by preventing the proteasomal degradation of its inhibitor,IκB.It has been reported that bortezomib can synergistically enhance TRAIL-mediated apoptosis in some primarily resistant tumor cells,such as non-small-cell lung cancer,ovarian cancer, and pancreatic cancer cells.This encouraged us to use a combined treatment with bortezomib and TRAIL for gastric cancer.In the present study,we performed a combined treatment with TRAIL and bortezomib for the first time in gastric cancer cells,and demonstrate that bortezomib can increase the sensitivity of gastric cancer cells to TRAIL-mediated apoptosis.The molecular mechanism involves the inhibition of the NF-κB and PI3K/Akt signaling pathways activated in response to TRAIL treatment.Materials and Methods1.The effect of TRAIL on MGC803,BGC823,and SGC7901 cell viability was measured using a 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.2.Apoptotic analysis was performed by flow cytometry following propidium iodide(PI) staining.3.Mitochondrial membrane potential(ΔΨm) was determined by flow cytometry following DiOC6 staining.4.Expressions of caspase 3,caspase 8,IκBα,phospho-Akt and Akt,et al.were analyzed by western blot assay.5.Transfection was performed using Lipofectamine 2000 reagent.6.Statistical analysis:Data are expressed as means±SD.Differences in the results for two groups were evaluated with Student's t test.P＜0.05 was considered to be statistically significant.Results1.Bortezomib sensitized gastric cancer cells to TRAIL-induced apoptosis.(1) MGC803,BGC823,and SGC7901 cells were exposed to 10-1000 ng/mL TRAIL for 24 h,and cell viability and apoptosis were measured.50 ng/mL TRAIL only reduced cell viability to 72.67±4.93%,92.33±5.86%,and 98.33±2.31%%in the MGC803,BGC823,and SGC7901 cells,respectively.Cell-cycle analysis showed that TRAIL induced less than 8%apoptosis in all three cell lines.When the dose of TRAIL was increased to 1000 ng/mL or the exposure prolonged to 48 h,there was no further increase in cytotoxicity.(2) A combined treatment with TRAIL and the proteasomal inhibitor,bortezomib, to MGC803,BGC823,and SGC7901 cells showed only slight cell death was induced in the three cell lines by bortezomib alone.In the MGC803 cells,pretreatment with 50 nM bortezomib followed by 50 ng/mL TRAIL markedly reduced cell viability from 70.42±1.14%to 34.85±2.83%and enhanced the rate of apoptosis from 8.22±0.83%to 31.27±2.02%.A similar synergistic effect of 50 nM bortezomib with TRAIL was also observed in the other two cell lines.(3) Single treatment with 50 nM bortezomib or 50 ng/mL TRAIL for 24 h both reduced the mitochondrial membrane potential(ΔΨm) in the three cell lines. Pretreatment with bortezomib followed by TRAIL exposure could further reduceΔΨm. in MGC803 cells,TRAIL alone induced 10.97±4.66%ofΔΨm reduction;combined treatment with bortezomib could further induce 54.27±7.71%ΔΨm reduction.(4) Pretreatment with 50 nM bortezomib for 2 h followed by TRAILexposure for 12 h,cell cycle was arrested at G2/M phase.Combined treatment with TRAIL could not further increase the population of G2/M phase.(5) In SGC7901 cells TRAIL or bortezomib alone did not produce the active fragments of caspase 8,but pretreatment with 50 nM bortezomib followed by TRAIL treatment strongly cleaved caspase 8,and subsequently produced the active form of caspase 3.At the same time the levels ofpro-caspase 8 and 3 were reduced significantly. Similar caspase changes were also observed in the other two cell lines.Bortezomib combined with TRAIL had no effects on the expression of death receptor 5,Bcl-2 or Bax,but slightly decreased the level of cIAP-1.Bortezomib up-regulated the expression of Survivin,and combined treatment with TRAIL did not augment the up-regulation effect.2.TRAIL activated NF-κB signal pathway and protected cells from TRAIL-induced apoptosis(1) SGC7901 cells were exposed to either 50 ng/mL TRAIL or 20 ng/mL TNF for different time and the degradation of IκB was examined by western blotting.TRAIL activated NF-κB as early as 30 min after treatment,as shown by the clear degradation of IκB.TNFαinduced activation of NF-κB after 15 min,which was earlier and more obvious than TRAIL treatment.(2) The cells were transiently transfected with either an empty vector or a cDNA encoding a phosphorylation-defective mutant IκB,and then exposed again to 50 ng/mL TRAIL for 24 h.In the cells transfected with empty vector,TRAIL induced only 7.09a±0.84%apoptosis,whereas17.80±2.34%of the cells overexpressing mutant IκB underwent apoptosis with TRAIL treatment.3.Bortezomib inhibited PI3K/Akt signaling pathway and reversed the resistance of cells to TRAIL-induced apoptosis(1) In the SGC7901 cells,TRAIL treatment induced the phosphorylation of Akt at 1 h,which increased to a peak at 8 h,after which it declined again,remaining at the basal level thereafter.(2) Bortezomib alone did not alter the basal level of phosphor-Akt,but efficiently blocked the further phosphorylation of Akt when the cells were treated with TRAIL. Pretreatment with LY294002,a specific inhibitor of PI3K,not only reduced the basal level of phosphor-Akt,but also blocked the phosphorylation of Akt triggered by TRAIL.(3) In the SGC7901 cells,pretreatment with LY294002 followed by exposure to TRAIL significantly enhanced apoptosis compared with that induced by TRAIL treatment alone.Conclusions1.Human gastric cancer cells are resistant to TRAIL-induced apoptosis.2.Bortezomib at subtoxic doses sensitizes gastric cancer cells to TRAIL-induced apoptosis.3.Bortezomib facilitates caspases cleavage,reducesΔΨm,and down-regulates cIAP-1,but has no effects on the levels of Bax,Bcl-2.4.Bortezomib causes G2/M phase arrest at gastric cancer cells.4.NF-κB is activated by TRAIL engagement.Specific inhibition of NF-κB signaling pathway partly reverses the resistance to TRAIL-induced apoptosis.This indicated in gastric cancer calls NF-κB serves as an anti-apoptotic factor and protects gastric cancer cells from TRAIL-mediated apoptosis.5.PI3K/Akt signaling pathway is also activated upon TRAIL engagement. Bortezomib interrupts PI3K/Akt signaling pathway and partly overcomes TRAIL resistance in gastric cancer cells.