| Objective: Erythropoietin receptor (EPOR) expression and the role of erythropoietin (EPO) in the melanoma cell line B16 proliferation, the anticancer activity of dacarbazine (DTIC), cisplatin (DDP) and its relation to the expression of Bcl-2 family proteins in vitro and in vivo were studied. In addition, The effects of erythropoietin on immunoregulation in vitro and in vivo was examined. The goal is to provide some theoretical evidence for reasonable use of EPO for the patients with cancer-related anemia and estimating prognosis.Methods: 1 Expression of EPOR mRNA and protein was detected by RT-PCR, immunofluorescence and laser scanning microscope (LSM) and western blot.2 Effects of EPO and combinding with dacarbazine(1, 10, 50, 100μg/ml)and cisplatin(0.5, 1, 10, 100μg/ml)on B16 cells proliferation was measured by MTT methods after treatment with different doses EPO (5, 10, 100U/ml) for 24h.3 Cell-cycle analysis was performed by flow cytometry after treatment with EPO (5, 10, 100U/ml) for 24h.4 Expressions of Bcl-2, Bcl-xL, Bax proteins were analyzed by Western blot after treated with different doses EPO (5, 10, 100U/ml) for 24h.5 Construct murine melanoma B16 cell xenografts in C57BL/6 mice and treated with EPO alone, the designated chemotherapeutic drug (DTIC or DDP) alone, or EPO combined with the drug, mice injected with saline was designated control. Tumor growth and tumor appearance were assessed daily. The following day after stoping administration of drugs, part of mice were removed eye balls and collected whole blood in EDTA-containing tubes, and the blood was used to analyze the levels of red blood cell (RBC), hematocrit (Hct), hemoglobin (Hb) and white blood cell (WBC). Subsequently, mice were sacrificed, the tumors were separated from the surrounding muscles and dermis, and weighed. The rest mice were analyzed used in observation of survival time.6 The part of mice in normal control group, saline-treated group and EPO-treated group were sacrificed after treated with drugs for two weeks. The changes of spleen index, CD4+ and CD8+ T-cell subpopulations of mice spleen, the concentrations of interleukins (IL)-2, tumor necrosis factor (TNF)-αin serum of 3 group mice were analyzed.7 The effects of EPO on ConA-mediated proliferation ability of normal spleen cell and the concentrations of the cytokines (IL-2, TNF-α) in the supernatants from ConA -stimulated normal spleen cell was examined in vitro.Results: 1 EPOR mRNA and protein expresse in B16 cells.2 EPO can significantly enhance B16 cells proliferation (P<0.05). Pretreatment of B16 cells with EPO can significantly increase resistance to dacarbazine treatment (P<0.05), while EPO did not affect the antineoplastic activity of cisplatin (P>0.05).3 The results of FCM analysis showed that after treatment with EPO, the percentage of B16 cells in S phase is increase, whereas cell numbers in G0/G1 phase were significantly reduced (P<0.05).4 The results by Western blot analysis reveals that expression of both Bcl-2 and Bcl-xL proteins in B16 cell is increase (P<0.05), while Bax protein remained unchanged (P>0.05).5 Tumor volume and weight of mice injected with EPO alone was not different compared to tumor-bearing animals injected with saline (P>0.05). Injections of DTIC alone or EPO combined with DTIC induced a great decrease in tumor volume compared to tumor-bearing animals injected with Saline (P<0.01), but the difference was not observed between the two groups (P>0.05). Cisplatin alone induced a decrease in tumor volume and weight compared to tumor-bearing animals injected with saline (P<0.05). Furthermore, a significantly greater reduction in tumor mass was observed in Epo and cisplatin group compared to the group treated with cisplatin alone (P<0.05). Blood analysis indicated that a significant increase in RBC, Hct, and Hb was found in animals injected with EPO compared to animals that did not inject with EPO (P<0.05), and a significant increase in WBC was found in animals injected with cisplatin and EPO compared to cisplatin -injected mice.6 Spleen index in tumor-bearing mice was significantly increased in the EPO treatment mice (P<0.05). CD4+T cell percentage and CD4+/CD8+T ratio of spleen mononuclearcell in saline-treated or EPO-treated mice was significantly decreased compared to the normal control group (P<0.05). CD4+T cell percentage of EPO-treated mice did not changed (P>0.05). EPO treatment induced a deep decrease of CD8+T cell percentage leading to increase of the CD4+/CD8+T ratio compared to the saline-treated group(P<0.05); The level of IL-2 and TNF–αin serum from saline-treated or EPO-treated mice was significantly decreased compared to the normal control group (P<0.05) . The concentrations of IL-2 in EPO-treated mice was significantly increased compared to the saline-treated group (P<0.05), while that of TNF-αwas not defferent in both study groups (P>0.05).7 EPO treatment did not affects ConA-mediated proliferation ability of spleen cell from normal mice and the concentrations of the cytokines (IL-2, TNF–α) in the supernatants from ConA-stimulated spleen cell of normal mice in vitro (P>0.05).Conclusion: 1 B16 cells expressed EPOR.2 EPO can significantly enhance the proliferation of murine melanoma cell line B16 in vitro, its effect may be related to its affecting the cell-cycle distribution. In addition, EPO can regulate antineoplastic activity of chemotherapeutic agents and the effect is drug related.3 The proliferation and regulating antineoplastic activity of B16 cells treatment with chemotherapeutic agents modulated by EPO was probably related to regulating expression of Bcl-2 family proteins.4 EPO alone could not effect melanoma growth engrafted in mice. EPO could modulate the antitumor activity of various chemotherapeutic agents in melanoma -bearing mice, and the effect is drug related.5 EPO-induced immunoregulation is mediated by an indirect stimulatory effect on the immune system in tumor-bearing mice, this stimulation being accompanied by an improvement in anemia condition.
|
PDF Full Text Request