The Mechanism Of Tumor-derived Exosome-induced Gastric Cancer Cell Proliferation And T Cell Apoptosis

ObjectiveMalignant tumors seriously threaten human health,which have been one of major leading cause of death.Nowadays,traditional treatment methods such as surgery, radiotherapy and chemotherapy are effective,however,the efficacy is very limited, which is difficult to eliminate tumor cells thoroughly.Immunotherapy,in particular tumor vaccines,have attracted increasing attention and brought about good prospects for tumor treatments.The research on exosome subcellular vaccine has recently been one of the focuses of tumor immnotherapy.Exosomes are nanometer-sized membrane vesicles that are released extracellularly after fusion of multivesicular endosomes with the cell membrane.Though the discovery of exosomes could be traced back to 1987,only did recent years the exosomes attract much more attention by the immunologist and oncologist since Zitvogel L et al found that dendritic cell-derived exosomes can eradicate established murine tumors in 1998. Up to now,the research about exosomes has entered a new stage,extensive studies have been conducted about the detrimental effects on the immune system of exosomes, including inducing apoptosis of T lymphocytes and promoting proliferation of tumor cells.Therefore,a better understanding of molecular mechanisms involved in exosome-inducing gastric cancer cell proliferation and T-cell apoptosis is likely to provide theoretical support of tumor immunotherapy.Many studies showed that phosphatidylinositol 3-kinase(PI3K)/Akt and MAPK/ERK signaling pathways played a critical role in controlling the balance between cell survival and apoptosis,and correlated closely to tumor development. However,the potential role of PI3K/Akt and MAPK/ERK signaling pathways in the exosome-inducing gastric cancer cell proliferation and T-cell apoptosis has not been identified.The Cbl family proteins,including Cbl-b and c-Cbl,are members of a superfamily of RING finger E3 ubiquitin ligases.The ubiquitination of proteins by E3 ligases has recently been recognized as an important regulatory mechanism for a variety of immune functions,such as maintenance of T-cell homeostasis and self tolerance.Cbl-b and c-Cbl not only play an essential role in setting a signaling threshold for T-cell activation,but also target tyrosine kinase receptors and growth signaling proteins resulting in their ubiquitination and down-regulation.While emerging evidence suggested that the E3 ubiquitin ligases,Cbl-b was responsible for activation-induced apoptosis in murine T helper 1(Th1) cells following CD3 ligation,it is uncertain whether Cbl proteins possess the ability to regulate exosome-induced apoptosis of T cells.In the present study,we isolated and purified the exosomes from human gastric cancer cell line SGC7901 cells.We investigated the effect of exosomes on tumor cell proliferation and T-cell apoptosis and evaluated the role of PI3K/Akt,MAPK/ERK and Cbl family of ubiquitin ligases during the process.Materials and Methods1.Exosomes were purified from the supernatants of SGC7901 cells,using a modification of the serial centrifugation and sucrose gradient ultracentrifugation procedure.2.Morphological analysis of exosomes was used by transmission electron microscopy.3.The effect of exosomes on proliferation of gastric cancer cells and Jurkat T cells was measured using MTT assay and trypan blue exclusion test,respectively.4.Cell apoptosis was determined by flow cytometry with PI. 5.Morphological analysis was performed by cytospin preparation with Wright-Giemsa stain and the slides were observed under a light microscope.6.Protein expression was determined by Western blot.7.Statistical analysis:All values are expressed as means±SEM.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.Exosomes were isolated and purified from gastric cancer SGC7901 cells,then observed them by transmission electron microscopy.The exosomes had a characteristic saucer-like shape that was limited by a lipid bilayer,and their diameter ranged from 30 to 100 nm.Comparing whole-cell lysates with exosome preparations revealed that the multivesicular body marker TSG101,tetraspanin molecule CD9 and HLA-A antigens were abundant in exosomes.2.Assessment of proliferation by MTT assay revealed that gastric cancer exosomes significantly increased SGC7901 and BGC823 cell proliferation in a classical time- and dose-dependent manner.The percentages of SGC7901cell viability were 142%and 145%of control cells at 200μg/ml and 400μg/ml exosomes treatment, respectively and the percentages of BGC823 cell viability were 129%and 138%of control cells at 200μg/ml and 400μg/ml exosomes treatment,respectively.3.The results of Western blot analysis indicated that exosomes up-regulated the expressioin of phosphorylated Akt and ERK1/2 in a time- and dose-dependent manner. Treatment of 200μg/ml exosomes increased the expression of phosphorylated Akt and ERK1/2 by 3.52 and 3.16 times over the untreated control cells,respectively. Pretreatment with 10μM LY294002 for 1 h before exposure to 200μg/ml exosomes reduced expression of exosome-induced phosphorylated Akt to baseline.Similarly, combined treatment with 10μM PD98059 and exosomes reversed expression of exosome-induced phosphorylated ERK1/2.The analysis of cell proliferation showed that LY294002 and PD98059 strikingly reversed SGC7901 cell proliferation stimulated by exosomes.4.SGC7901 cells were incubated with exosomes at 200μg/ml for 24,48 and 72 h. Western blot analysis showed decreased expression of Cbl-b and c-Cbl upon exosome administration.The expression of Cbl-b and c-Cbl was gradually down-regulated at 24 h(0.78 and 0.85 versus controls,respectively),and decreased sharply at 72 h(0.35 and 0.09 versus controls,respectively).Further investigation showed that the suppression of Cbl-b and c-Cbl proteins by exosomes was dose-dependent.5.Viable Jurkat T cells were incubated in a medium supplemented with exosomes at the indicated concentrations for 24,48 and 72 h,respectively.Assessment of proliferation by trypan blue exclusion test revealed that gastric cancer exosomes significantly inhibited Jurkat T cell proliferation in a classical dose- and time-dependent manner.Treatment Jurkat T cells with 400μg/ml exosome for 48 h, 31.76%of cells underwent apoptosis,while only 2.47%cells underwent apoptosis in control group(p＜0.01).The morphological analysis demonstrated that the apoptotic cells became rounded in shape and their nuclei exhibited a fragmented morphology, forming apoptotic bodies.Treatment with 400μg/ml exosomes led to the appearance of cleaved caspase-3,caspase-8 and caspase-9.6.Jutakt T cells were treated with exosomes at 400μg/ml for 24 and 48 h,total cell lysates showed increased expression of Cbl-b and c-Cbl upon exosome administration.The expression of Cbl-b and c-Cbl was gradually up-regulated at 24 h (1.10 and 1.23 versus controls,respectively),and increased sharply at 48 h(2.04 and 1.86 versus controls,respectively).Further investigation of Akt activity,the results indicated that the activity of Akt was down-regulated in a time-dependent manner, which coincided with the increased expression of Cbl-b and c-Cbl.PS-341,the proteasome inhibitor and functional inhibitor of the Cbl family of ubiquitin ligases,was exploited to block the function of Cbl proteins and cell apoptosis was determined. Pretreatment with PS-341 partially reversed exosome-induced cell apoptosis.At the meanwhile,the up-regulation of phosphorylated Akt was also partially restored by pretreatment with PS-341.Conclusion1.Serial centrifugation and sucrose gradient ultracentrifugation procedure was feasible to purified exosomes.Gastric cancer SGC7901 cell-derived exosomes had a characteristic saucer-like shape,and their diameter ranged from 30 to 100 nm.TSG101, CD9 and HLA-A were expressed on exosomes.2.Gastric cancer exosomes significantly increased SGC7901 and BGC823 cell proliferation in a classical time- and dose-dependent manner.3.Exosomes up-regulated the expressioin of phosphorylated Akt and ERK1/2 in a time- and dose-dependent manner.PI3K or ERK inhibitor partially reversed the proliferative effect of exosomes.The results indicated that gastric cancer exosomes promoted tumor cell proliferation,at least in part,by activation of PI3K/Akt and MAPK/ERK pathways.4.SGC7901 cells responded to exosome administration with reduced expression of Cbl-b and c-Cbl in a time- and dose-dependent manner.5.Gastric cancer exosomes could inhibit proliferation and induce apoptosis of Jurkat T cells.And activation of caspase-3,8 and 9 was involed in exosome-induced apoptosis.6.The expression of Cbl-b and c-Cbl was increased and the activity of Akt was inhibited during exosome-induced apoptosis.Functional inhibitor of the Cbl proteins partially restored Akt activity as well as reversed cell apoptosis.