RhEPO-Fc Fusion Protein Anti-anemia And Its Action Mechanism

Recombinant human erythropoietin (rhEPO) has been extensively used in the treatment of anemia associated with chronic renal failure, cancer chemotherapy and radiotherapy. However, rhEPO has a shorter serum half-life and the treatment often requires frequent injections, which results in limitation of this treatment. The long-acting recombinant human rhEPO-Fc fusion proteins consist of rhEPO and the Fc part of a human IgG molecule, which prolonged serum half-life time and increased biological activity, allowing for reduced dosing frequency in the treatment of anemia.In present studies, we first investigated the long-acting effect and dose-effect relationship of rhEPO-Fc on anemia of myelosuppression induced byγirradiation in C57 mice, anemia in partially nephrectomized rats, and anemia of myelosuppression induced by cyclophosphamide in rhesus monkeys, and Elisa assay was performed to detect the rhEPO-Fc antibody in rhesus monkey serum. Furthermore, we employed UT-7/EPO cell line which was derived from a patient's bone marrow with leukemia to observe the inhibitive effect of rhEPO-Fc on apoptosis induced byγirradiation with CCK-8 assay, flow cytometry, light microscopy and transmission electron microscopy. Immunofluorescence and western blotting were used to detect the changes of expression of apoptosis-related proteins Bcl-2, Bax and caspase 3 to investigate the molecular mechanisms of rhEPO-Fc inhibiting apoptosis induced byγirradiation in human UT-7/EPO cells.PartⅠEffect of rhEPO-Fc on anemia in different animal models1. Effect of rhEPO-Fc on anemia of myelosuppression induced byγirradiation in C57 mice.The anemia was induced by total body irradiation with a single 4Gy dose using a 137Csγ-rays source in C57 mice. The blood samples were collected before irradiation and different indicated periods after irradiation. The RBCs, hemoglobin hematocrit and reticulocyte were determined. C57 mice were randomly divided into five groups: solvent control group, rhEPO-Fc three dose groups (7.5,15,30μg·kg-1 once weekly sc.), and rhEPO group(7.5μg·kg-1 three times weekly sc.). All drugs were successively given for 25 days, and the experiment was continued until the 5th day of post-treatment. It was observed the increase in RBCs, hemoglobin, and hematocrit, they were significantly higher (p<0.01) in rhEPO, rhEPO-Fc 7.5,15,30μg·kg-1 groups than that in control group on the 11th～25th day of treatment, and reticulocyte were significantly higher (p<0.01) in rhEPO-Fc 30μg·kg-1 group from the 7th day of treatment to the end of experiment, and rhEPO group on the 18th～25th day of treatment than that in control group. There was no significant difference in spleen weight and spleen index among rhEPO-Fc 7.5,15,30μg·kg-1 groups and control group (p>0.05). Furthermore, rhEPO-Fc 7.5,15,30μg·kg-1 promote the increase in RBCs, hemoglobin, hematocrit and reticulocyte in dosing-dependent manner. Our results indicated that rhEPO-Fc could effectively correct the anemia of myelosuppression induced byγirradiation in C57 mice. The effects of rhEPO-Fc 15μg·kg-1 and 30μg·kg-1 once weekly sc.were the same as the effect of 7.5μg·kg-1 three times weekly sc. There is a dose-dependent relationship among rhEPO-Fc 7.5,15,30μg·kg-1.2. Effect of rhEPO-Fc on anemia caused by partially nephrectomy in ratsAnemia was caused by a two-step 5/6 nephrectomy in rats. The blood samples were collected before nephrectomy and different indicated periods after nephrectomy. The RBCs, hemoglobin, hematocrit, reticulocyte and BUN were determined. SD rats were randomly divided into six groups:model group, sham operated group, rhEPO-Fc three dose groups (5.4,10.7,21.4μg·kg-1 once weekly sc.), and rhEPO group(5.4μg·kg-1 three times weekly sc.). All drugs were successively given for 4 weeks, and the experiment was continued until the 2nd week of post-treatment. It was observed the increase in RBCs, hemoglobin and hematocrit, they were significantly higher (p<0.01) in rhEPO group (on the 1st～4th week of treatment), rhEPO-Fc 5.4μg·kg-1 group (on the 1st and 2nd week of treatment), rhEPO-Fc 10.7μg·kg-1 group(on the 1st～3rd week of treatment) and rhEPO-Fc 21.4μg·kg-1 group(on the 1st～4th week of treatment) than that in model group, and reticulocyte were significantly higher (p<0.01) in rhEPO-Fc three dose groups and rhEPO group (on the 1st week of treatment) than that in model group. The levels of BUN were significantly higher in model group, rhEPO-Fc 5.4,10.7,21.4μg·kg-1 groups, and rhEPO group than that in sham operated group. Furthermore, rhEPO-Fc 5.4,10.7,21.4μg·kg-1 promote the increase in RBCs, hemoglobin, hematocrit and reticulocyte in dosing-dependent manner. These results indicated that rhEPO-Fc could effectively correct the anemia caused by partially nephrectomy in rats. The effects of rhEPO-Fc 10.7 and 21.4μg·kg-1 once weekly sc.were the same as the effect of rhEPO 5.4μg·kg-1 three times weekly sc. There is a dose-dependent relationship among rhEPO-Fc 5.4,10.7,21.4μg·kg-1.3. Effect of rhEPO-Fc on anemia of myelosuppression induced by cyclophosphamide in rhesus monkeys.Anemia of myelosuppression was induced by administration of 50mg·kg-1 cyclophosphamide iv. for successive 2 days in rhesus monkeys. The blood samples were collected before administration of cyclophosphamide and different indicated periods after administration of cyclophosphamide. The RBCs, hemoglobin, hematocrit, reticulocyte, platelets and WBCs were determined. Rhesus monkeys were randomly divided into four groups:solvent control group, rhEPO-Fc two dose groups (5,10μg·kg-1 once weekly sc.), and rhEPO group(2.5μg·kg-1 three times weekly sc.). All drugs were successively given for 49 days, the experiment was continued until the 35th day of post-treatment. It was observed the increase in RBCs, hemoglobin, and hematocrit, they were significantly higher (p<0.01) in rhEPO group and rhEPO-Fc 10μg·kg-1 group (on the 35th-49th day of treatment) than that in control group, and reticulocyte were significantly higher (p<0.01) in rhEPO-Fc 10μg·kg-1 group and rhEPO group (on the 49th day of treatment) than that in model group. Furthermore, an increase in RBCs (from the 28th day of treatment to the 21th day of post-treatment), hemoglobin (from the 28th day of treatment to the 14th day of post-treatment), hematocrit (on the 28th～49th day of treatment and the 49th day of treatment) were significantly increased (p<0.01) in rhEPO group and rhEPO-Fc 10μg·kg-1 group, comparing with baseline. The increase in platelet counts were significantly higher (p<0.05) in rhEPO-Fc 5μg·kg-1 group (on the 12th and the 14th day of treatment) and rhEPO-Fc 10μg·kg-1 group (on the 14th,16th,42th day of treatment). There was no significant difference in WBCs among rhEPO-Fc 5, 10μg·kg-1, rhEPO and group. These results showed that rhEPO-Fc could effectively correct the anemia of myelosuppression induced by cyclophosphamide in rhesus monkeys. The effects of rhEPO-Fc 10μg·kg-1 once weekly sc. were the same as the effect of rhEPO 2.5μg·kg-1 three times weekly sc.4 The detection of rhEPO-Fc antibody by Elisa assay in serum of rhesus monkeys.It was observed that no rhEPO-Fc antibodies in serum of rhesus monkeys could be detected by Elisa assay before treatment, and in rhEPO-Fc 5 and 10μg·kg-1 group, and there were no rhEPO-Fc antibodies in serum of rhesus monkeys to be detected by Elisa assay throughout the study period. However, rhEPO antibody could be detected by Elisa assay in serum of one rhesus monkey in rhEPO group throughout the study period.PartⅡThe inhibitive effect and mechanism of rhEPO-Fc on apoptosis induced byγirradiation in human UT-7/EPO cell line1. The effect of rhEPO-Fc on apoptosis induced byγirradiation in UT-7/EPO cellsUT-7/EPO cells were irradiated with a single 8Gy dose using a 137Csγ-rays source. Irradiated UT-7/EPO cells were treated with or without cytokines and randomly divided into five groups:irradiation control group, rhEPO-Fc 0.7U/ml group, rhEPO-Fc 7U/ml group, rhEPO-Fc 70U/ml group and rhEPO group. The effect of rhEPO-Fc on apoptosis induced byγirradiation in UT-7/EPO cells were observed by CCK-8 assay, flow cytometry, light microscopy and transmission electron microscopy. Results showed that 24h,48h and 72h afterγirradiation, the cell viabilities were significantly increased (p<0.01) and apoptotic rates of irradiated UT-7/EPO cells were significantly decreased (p<0.01) in rhEPO-Fc 0.7,7,70U/ml and rhEPO 7 U/ml group, compared with irradiation control group, and 24h afterγirradiation, the percent of UT-7/EPO cells in G0/G1 phase were significantly decreased (p<0.01) and the percent of UT-7/EPO cells in G2/M and S phase were significantly increased (p<0.01) in rhEPO-Fc 0.7,7,70U/ml and rhEPO 7 U/ml group compared to irradiation control group. Furthermore, the irradiated UT-7/EPO cells exhibited characteristic morphology of apoptosis, including cell shrunk, nucleus fragmented, chromatin condensation and margination forming the shape of crescents or lump, cytoplasmic vacuolization, on the contrary, the irradiated UT-7/EPO cells appeared morphologically normal and showed no sign of apoptosis in rhEPO-Fc 0.7,7,70U/ml and rhEPO 7 U/ml group. These results indicated that rhEPO-Fc 0.7,7,70U/ml could inhibit the apoptosis induced byγirradiation in UT-7/EPO cells.2. The mechanisms of rhEPO-Fc inhibiting apoptosis induced byγirradiation in human UT-7/EPO cells.Immunofluorescence and western blotting were used to detected the changes of expression of apoptosis-related proteins Bcl-2, Bax and caspase 3 in different treated UT-7/EPO cells. The results of immunofluorescence showed that the expression level of anti-apoptotic protein Bcl-2 was lower at 24h followingγirradiation, however, the expression level of Bcl-2 was higher in rhEPO-Fc 0.7,7,70U/ml and rhEPO 7 U/ml group, the expression of the Bax showed no difference among variable. The results of western blotting showed that the expression level of Bcl-2 protein and the relative Bcl-2/Bax protein ratio significantly decreased, the expression levels of pro-caspase 3 significantly reduced and the cleavage of caspase 3 to catalytically active fragments were also induced at 24h and 48h followingγirradiation, however, the expression of the Bcl-2 and caspase 3 in rhEPO-Fc 0.7,7,70U/ml and rhEPO 7 U/ml group showed no significant difference compared with the normal cultured UT-7/EPO cells, the relative Bcl-2/Bax protein ratio significantly increased compared with irradiation control group. But the expression of the Bax protein showed no difference among variable. These results indicate that rhEPO-Fc could exerts anti-apoptotic effect on irradiated human UT-7/EPO cells via increase the expression of Bcl-2 and the relative Bcl-2/Bax ratio, and inhibition the activation of caspase 3.Summary1. The effects of rhEPO-Fc once weekly sc. exert the same effect as the rhEPO three times weekly sc. on anemia of myelosuppression induced byγirradiation in C57 mice, anemia caused by partially nephrectomy in rats and anemia of myelosuppression induced by cyclophosphamide in rhesus monkeys. 2. No rhEPO-Fc antibodies in serum of rhesus monkeys could be detected by Elisa assay throughout the study period in rhEPO-Fc 5 and 10μg·kg-1 group.3. rhEPO-Fc exerts anti-apoptotic effects on irradiated human UT-7/EPO cells via increase the expression of Bcl-2 and the relative Bcl-2/Bax ratio, and inhibition the activation of caspase 3.