Oxaliplatin Enhances TRAIL-induced Apoptosis In Gastric Cancer Cells By CBL-regulated Death Receptor Redistribution In Lipid Rafts

ObjectiveGastric cancer is one of the most common malignancies worldwide and the major cause of cancer death in Asian countries.Most patients present with advanced or metastatic disease at diagnosis and five-year survival rates are only 10%-15%. Chemotherapy is main therapeutic method for advanced gastric cancer.Although chemotherapy for advanced disease improves survival,the median overall survival is no more than 12 months.Biotherapy holds great promise for its potential to treat cancer selectively.Tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) is a member of the tumor necrosis factor(TNF) family.TRAIL is known to induce apoptosis in many cancer cells in vitro but spares most normal cells.However,gastric cancer cells are relatively insensitive to TRAIL.The apoptotic signal induced by TRAIL is transduced by its binding to death receptor 4(DR4) and death receptor 5(DR5).Binding to DR4 and DR5 results in receptors aggregation and recruitment of FADD and procaspase-8/procaspase-10 to the intracellular domain of receptors to form the death-inducing signaling complex(DISC). The formation of DISC triggers downstream effector caspases and leads to apoptosis. Lipid rafts provide a membrane platform for death receptors aggregation.Resent studies have shown that lipid rafts play an important role in initiating death signaling transmission.Whether the dysfunction of lipid raft is one reason that gastric cancer cells are insensitive to TRAIL is unknown.Some chemotherapeutic drugs,such as doxorubicin,paclitaxel and cisplatin,have been shown to sensitize cancer cells to TRAIL.Oxaliplatin is a third-generation platinum-containing drug,and oxaliplatin-based regimens have significantly improved efficacy compared with cisplatin-based regimens in patients with advanced gastric cancer.Cisplatin has been shown to have the ability to sensitize colon cancer cells to Fas ligand by triggering lipid raft aggregation.Whether oxaliplatin can enhance the sensitivity of gastric cancer cells to TRAIL by regulating lipid rafts is unknown.The function of lipid rafts is regulated by several factors.Casitas B-lineage Lymphoma(Cbl) family of ubiquitin ligases is an important regulator.The Cbl family of proteins consists of three homologues known as c-Cbl,Cbl-b and Cbl-3.c-Cbl and Cbl-b share an abundance of similar motifs and have equal capacity.It has been demonstrated that c-Cbl and Cbl-b can sequester signaling molecules from lipid rafts in T cells and mast cells,which results in ineffective lipid raft aggregation.Moreover,the loss of Cbl-b in T cells triggers antigen-induced receptor clustering and lipid raft aggregation.However,whether c-Cbl and Cbl-b regulate lipid rafts and therefore influence the sensitivity of gastric cancer cells to TRAIL is not yet clear.To sensitize gastric cancer cells to TRAIL-induced apoptosis,we treated them with a combination of oxaliplatin and TRAIL.In this study,we investigated the function of lipid raft in oxaliplatin+TRAIL-induced gastric cancer MGC803,BGC823 and SGC7901 cell apoptosis.We also investigated the regulatory mechanism of ubiquitin ligase c-Cbl and Cbl-b,a regulator of lipid rafts aggregation,in oxaliplatin+TRAIL-induced apoptosis.Materials and Methods1.Cell proliferation was measured using MTT assay.2.Cell apoptosis was determined by flow cytometry with Annexin V and PI.3.Mitochondrial membrane potential was determined by means of DiOC6 staining.4.Expressionofc-Cbl,Cbl-b,caspase-3,caspase-8,Bax,Bcl-2 proteins was analyzed by Western blot.5.The distribution of lipid raft and death receptors was analyzed by immunofluorescence microscopy.6.Transient transfection was performed using Lipofectamine 2000 reagent.7.Statistical analysis.All values are expressed as means±SD.The differences of the results between two groups were evaluated by Student's t-test.P＜0.05 was considered to be statistically significant.Results1.100 ng/mL TRAIL resulted in a slight reduction in cell viability in MGC803, BGC823,and SGC7901 cells.Treatment with TRAIL alone caused no more than 6% cell apoptosis.The IC₅₀ doses for oxaliplatin were 22.56±6.55,37.47±7.67,and 33.92±8.01μg/mL in the MGC803,BGC823,and SGC7901 cells,respectively. Treatment with oxaliplatin(with the respective IC₅₀ doses) plus TRAIL(100 ng/mL) for 24 h resulted in a significant reduction in cell viability and a dramatic increase in cell apoptosis compared with treatment with oxaliplatin or TRAIL alone.When two drugs were used in combination,a higher degree of mitochondrial depolarization, cleavage of procaspase-3 and procaspase-8 and decreased expression of Bcl-2 were detected.The synergistic activity of 5-FU and TRAIL on gastric cancer cells has been demonstrated,so 5-FU was regarded as a positive control.The IC50 dose for 5-FU at 48h was 2.07±1.14μg/mL in the MGC803 cells.Treatment with 5-FU(with the IC₅₀ dose) plus TRAIL(100 ng/mL) for 48 h resulted in a significant reduction in cell viability and a dramatic increase in cell apoptosis compared with treatment with 5-FU or TRAIL alone.When two drugs were used in combination,a higher degree of mitochondrial depolarization,cleavage ofprocaspase-3 and procaspase-8 and decreased expression of Bcl-2 were detected.2.The exposure of MGC803 cells to 100 ng/mL TRAIL alone for 16 h did not induce any obvious lipid raft aggregation or DR4 or DR5 clustering compared with those in the untreated control cells.However,22.56μg/mL oxaliplatin significantly promoted the localization of DR4 and DR5 in aggregated lipid rafts.The combined treatment with oxaliplatin and TRAIL showed similar results to the treatment with oxaliplatin alone.3.Nystatin is a cholesterol-sequestering agent that disrupts lipid rafts. Pretreatment for 1 h with 50μ/mL nystatin partially prevented the lipid raft aggregation and DR4 and DR5 clustering induced by oxaliplatin in MGC803 cells. Although nystatin alone triggered a small amount of cell apoptosis after 24 h, pretreatment with nystatin for 1 h before the addition of oxaliplatin caused a decreased tendency to cell apoptosis compared with that caused by treatment with oxaliplatin alone.Moreover,pretreatment with nystatin for 1 h significantly suppressed oxaliplatin+TRAIL-induced apoptosis,and the proportion of apoptotic cells decreased from 41.79±5.48%to 29.52±3.99%.4.Oxaliplatin(22.56μg/mL) alone slightly suppressed the expression of c-Cbl and Cbl-b after 8 h,and strongly reduced c-Cbl and Cbl-b protein levels after 16 h and 24 h compared with those of the untreated control group.However,100 ng/mL TRAIL alone did not change the expression of c-Cbl,but slightly upregulated the expression of Cbl-b at 16 h.Exposure to oxaliplatin+TRAIL induced the downregulation of c-Cbl and Cbl-b,similar to exposure to oxaliplatin alone at 16 h.5.MGC803 cells were transiently transfected with wild-type c-Cbl and Cbl-b expression constructs using Lipofectamine 2000 reagent.After 48 h transfection,the expression of c-Cbl and Cbl-b was upregulated by western blot method.Lipid raft aggregation was partially reversed in c-Cbl-transfected,Cbl-b-transfected,and c-Cbl-and Cbl-b-cotransfected cells induced by oxaliplatin and oxaliplatin+TRAIL compared with the negative control.Conclusion1.Oxaliplatin enhanced TRAIL-induced apoptosis of MGC803,BGC823,and SGC7901 cells.In the synergistic action,a higher degree of mitochondrial depolarization, cleavage of procaspase-3 and procaspase-8 and decreased expression of Bcl-2 were detected.5-FU and TRAIL induced the similar results in gastric cancer cells.2.Oxaliplatin promoted DR4 and DR5 clustering into aggregated lipid rafts in MGC803 cells.3.Nystatin partially prevented oxaliplatin-induced lipid raft aggregation and DR4 and DR5 clustering,and decreased oxaliplatin+TRAIL-induced MGC803 cell apoptosis.4.In MGC803 cells,oxaliplatin downregulated the expression of c-Cbl and Cbl-b.5.In MGC803 cells,overexpression of c-Cbl and/or Cbl-b partially reversed oxaliplatin-induced lipid raft aggregation.