| Background and ObjectCatecholamine accelerates the proliferation of different solid tumors. In contrast, growth of tumor cells can be inhibited by blocking the activity of adrenergic receptor. To some extent, it explains the correlation between overstress and tumorigenesis. Whether catecholamine affects multiple myeloma cells is still unknown. This study aims to explore the effect of adrenoreceptor signaling pathway on myeloma cells and bone marrow mesechymal stem cells. The mechanism of affecting cell proliferation by blocking0adrenoreceptor on multiple myeloma cells is explored.Materials and MethodsHuman myeloma cell line U266and RPMI8226were cultured in vitro. mRNA of key enzymes including Tyrosine Hydroxylase and Dopamineβ Hydroxylase in synthesizing catecholamine were detected using reverse transcriptase PCR. MTS cell proliferation assay were used to detect the growth of myeloma cells and control lymphoblast cell line NCI-BL2009in three following conditions.1. Different dose concentrations of adrenaline were tested.2. Adrenaline and velcade were treated together.3. Agonist and antagonist of α, β receptors were used to explore the functional subtype.Next, we studied the function of subtype of β adrenoreceptor. The expression of mRNA of β1β2β3subtypes were tested. Cell proliferation was evaluated after treating cells with specific β receptor agonist. To explore the mechanism how β adrenoreceptor affects myeloma cells, following experiments were done.1. VEGF in cell supernant was detected using ELISA after treatment of adrenaline and β adrenoreceptor agonist isoprenaline.2. cytotoxic effects of beta blocker propranolol was evaluated by MTS cell proliferation assay. Trypan Blue staining and cell counting were used to detect cell number changes after lower concentration (0-50umol/1) propranolol treating for a long time (16d). Apoptotic effect of propranolol were evaluated by DAPI staining of cell nucleus, Annexin V-PI expression assessment by flow cytometry in myeloma cells and control group. Apoptotic related proteins of caspase pathway was tested by Caspase-Glo assay and western blotting. Bone marrow mononuclear cells from myeloma patients were harvested and mesenchymal stem cells (MSC) were cultured in vitro. Cell proliferation assay was performed to compare changes after treated with adrenaline and propronolol. Primary CD138positive plasma cells were isolated and treated with propranolol, FDA staining and assessment by flow cytometry were performed to detect propranolol effect on primary myeloma cells.ResultsMyeloma cell line RPMI8226expressed TH, Dβ H mRNA. Adrenaline (>5uM,24h) can stimulat myeloma cell line U266, RPMI8226proliferation in a dose-dependent manner. The maximum effect of cell proliferation percentage changes is415%±34%、408%±34%(p<0.05) after treating with50umol/1adrenaline for24h. Adrenaline can diminish the inhibition effect of velcade on myeloma cells. The promotion effect of adrenaline involves α，βreceptors. All these two cell line expressβ2,β3mRNA. Myeloma cell line U266, RPMI8226proliferation can be inhibited by β receptor antagonist propranolol in a dose dependent manner (>50umol/1,24/48h).400umol/1propranolol can induce U266, RPMI826cell proliferation to29.2%±4.6%,18.3%±2.0%(p<0.05) after treating48hours. Low concentration propranolol (10-50umol/1) treating for16d can also inhibit these two myeloma cell line proliferation for29.4%±2.3%-95.2%±1.7%(U266cell,10umol/1-50umol/1, p<0.05)、84.4%±2.5%-93.0%±5.2%(RPMI8226cell,20umol/1-50umol/l, p<0.05). Propranolol (200umol/1) can induce RPMI8226cell nucleus to break into pieces. Propranolol (50-200umol/1) can upregulate the percentage of late apoptotic cell (Annexin V+PI+). Propranolol can promote the expression of caspase3/7,8,9. The changes of luminimeter of caspase3/7,8,9for U266cell line after200umol/1propranolol treating6h are26743±7113RLU,7978±1805RLU、27705±2335RLU (p<0.05); whereas for RPMI8226cell line are21751±3760RLU、12070±1317RLU、53627±6882RLU (p<0.05). Westernblotting results show that50-100umol/1propranolol can promote the expression of promoting-apoptotic protein Bax, whereas decrease the expression of anti-apoptotic protein Bcl-2. Adrenaline and β agonist can not promote the expression of VEGF.Adrenaline (100umol/1) can promote the proliferation of MM-MSC, the changes of cell proliferation were235%±85.5%(p<0.05). Propranolol can inhibit the proliferation of MM-MSC, the changes of cell proliferation were76.3%±11.2%(p<0.05) Propranolol can decrease FDA positive percentage of primary CD138positive cells in flow cytometry for71.4%±30.5%(p<0.05)Conclusion:Myeloma cell express the key enzymes of synthesizing catecholamine. The activation of adrenoreceptor translated into promotion of cell proliferation in myeloma cells and BM-MSCs. Long-term stress probably has unfavorable effect to chemotherapy. Both α and βadrenoreceptor are involved.β2and β3subtypes were dominant rather than β1. Propranolol inhibited myeloma cell proliferation by inducing apoptosis, indicating its potential use as an anti-myeloma drug. |
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