Role Of α7nAChR In The Angiogenesis Induced By Multiple Myeloma Cell

Background and purposes:Multiple myeloma(MM)is a malignant neoplasm with a clonal proliferation of vicious plasm cells in bone marrow,which is characterized by a close connection between tumor cells and bone marrow microenvironment in order to support MM cells growth and proliferation.Its incidence is about 10% in hematological malignancy.Due to its high invasion,drug resistance and significant angiogenesis,most patients are vulnerable to recurrent and refractory MM.Therefore,it is a challenge for MM therapy which attracts more attention in hematological cancer.α7 nicotinic acetylcholine receptor(α7nAChR),which is widely present in peripheral and central nervous system,is one of nicotinic acetylcholine receptors(n ACh R).Recently it has been reported that α7nAChR are expressed in epithelial cells,immune cells and lung cancer cells et al.The biological function of α7nAChR involves in cognition,inflammation and cancer et al.The research showed that acetylcholinesterase inhibitor donepezil could promote angiogenesis in ischemic tissue and an inhibitor of α7nAChR,MG624 could suppress angiogenesis in lung cancer.It has been shown that,angiogenesis plays an important role in the development of MM.Angiogenesis was close to prognosis in MM patients,which was reported in Blood,2013.Patients with markedly increased angiogenesis,usually had a poor prognosis.Vascular endothelial growth factor(VEGF)is known to be one of angiogenic growth factors and is overexpressed in a series of cancer,such as gastric cancer,cervical cancer and MM.A study has indicated that the overexpression of VEGF might be related to angiogenesis and poor prognosis in MM.Basic fibroblast growth factor(b FGF),a well-known angiogenic growth factor,is also overexpressed in many cancers cells and can act synergistically with VEGF in the process of cancer development.Thrombospond1n-1(TSP-1),an endogenous angiogenesis inhibitor,is secreted by platelets,endothelium,cancer cells et al.It can directly affect proliferation and apoptosis of endothelium or antagonize VEGF to inhibit angiogenesis.Studies have shown that,TSP-1 could be used as a diagnostic indicator for prostate cancer,thyroid papillary carcinoma and non-small cell lung cancer.There is no report about α7nAChR and MM at present.Thus,we investigated the role of α7nAChR in the angiogenesis induced by multiple myeloma cells and its possible mechanism.Methods and results:MM cell line U266 and human umbilical vein endothelial cells(HUVECs)have been chosen as the research objects,PNU-282987(a selective agonist of α7nAChR)as a main drug in this study.The transwell chambers were used to co-culture U266 cells and HUVECs out of touch in vitro.1.α7nAChR is expressed in U266 cellsWe chose RAW264.7 and HUVECs,which had been reported to express α7nAChR,as positive controls in the measurement of Western blot.The result showed that U266 cells could express α7nAChR.Also,we verified the result through immunofluoresent assay and found that α7nAChR was mainly expressed in U266 cell membrane.2.Cell viability of U266 treated by different concentrations of PNU-282987CCK8 was used to assess the cell viability of U266 by PNU-282987 of 1,10 or 50μM.Results indicated that,compared to control group,PNU-282987(50μM)decreased cell viability to about 85%.The other concentrations had few influences on cell viability of U266.We chose PNU-282987(10μM)for the following experiments.3.Cell viability of U266 treated by different concentrations of MLACCK8 was used to assess the cell viability of U266 by different concentrations of MLA(100,200,300,400 or 500μM).Results showed that MLA(300,400,500μM)decreased cell viability of U266 to 80% by comparing with control group.MLA(100,200μM)had few influences on cell viability of U266.We chose MLA(200μM)for the following experiments.4.Cell viability and tube formation of HUVECs was not obviously altered by PNU-282987(10μM)HUVECs were treated PNU-282987(10μM)or vehicle for 24 h,and then cell viability and tube formation were determined.The results showed that PNU-282987(10μM)hardly altered cell viability and tube formation of HUVECs in comparison with control.5.Tube formation of HUVECs was assessed after co-culture with U266U266 cells were treated with PNU-282987(10μM)or not for 6h,and then drug was taken out.U266 cells went on culturing with HUVECs from now on.After 24 h,tube formation of HUVECs by different treatments was assessed.We found that,tube formation of HUVECs was increased by co-culture with U266 cells comparing with control(HUVECs alone).Tube formation of HUVECs was decreased by culturing with U266 which was treated by PNU-282987(10μM)for 6h,and the phenomenon could be blocked by MLA(200μM)plus PNU-282987(10μM).Meanwhile,compared to tube formation of HUVECs co-cultured with U266 cells,there were few differences in tube formation of HUVECs which were co-culturing with U266 cells pre-treated by MLA(200μM)or(200μM)plus PNU-282987(10μM).6.The possible mechanismIn order to investigate the above results of HUVECs after 24 h co-culture with U266 cells by pretreatment of PNU-282987(10μM)or not for 6h.ELISA kits were used to test whether the levels of VEGF and TSP-1 were changed or not in U266 cell culture supernatants treated by PNU-282987(0,10μM)for 6h,or cell culture supernatants in which U266 were taken out of PNU-282987 and went on culturing for 24 h.The results displayed that,PNU-282987 had few influences on VEGF and TSP-1 protein levels in cell culture supernatants of U266.Next,angiogenesis-related cytokines antibody array,which was consisted of 20 cell cytokines measurement,was used to analyze cell culture supernatants in which U266 were taken out of PNU-282987 and went on culturing for 24 h.We found that b FGF was down-regulated in cell culture supernatants of U266 after pretreatment of PNU-282987.Conclusion:α7nAChR is involved in the angiogenesis of HUVECs co-cultured with U266 cells in vitro.Pretreatment of U266 cells with PNU-282987,could decrease tube formation of HUVECs,which might be related to down-regulation of b FGF protein secreted by U266 cells.Background and purposes:Multiple myeloma(MM)is a malignant neoplasm,which is characterized by a clonal proliferation of plasm cells in bone marrow and secretion of immunoglobulin.The susceptible population of MM are the aged.The incidence of MM is rising year by year,with more and more the aged in the world.Chemotherapy and radiotherapy are the main therapies for MM at present.As one of the glucocorticoids derivatives,dexamethasone(DEX)has anti-inflammatory,anti-shock and immunosuppressive effects,et al.DEX acts as a basic drug clinically in the chemotherapy regiments recently,according to Chinese guidelines of diagnosis and treatment of multiple myeloma.It is generally known that chloroquine(CQ)is a traditional antimalarial drug.Conventional drugs in new use is a new direction for research of CQ.It has been reported that CQ has an anti-cancer effect.Tang et al found that CQ could augment cytotoxicity of gefitinib in non-small cell lung cancer and the research of Han team showed that CQ was able to increase the sensitivity of breast cancer cells to radiation.Hypoxia induced factor 1(HIF-1)which is composed of α and β subunits and α subunit determines its activity,is a ubiquitous heterologous dimer nuclear transcription factor in hypoxia.MM cells are in the relatively closed and anoxic microenvironment of bone marrow,unlike other tumors.Therefore,it is meaningful to do some research on HIF-1α in order to know more about development of MM.Bcellymphoma-2(Bcl-2)is a member of antiapoptotic family.It is also a key mediator in mitochondrial pathway of cell apoptosis,mainly acts on the outer membrane of mitochodrial.Chromosomal translocation could induce overexpression of Bcl-2,which might result in tumor.Thus,the study mainly focused on the CQ and DEX or radiation in MM cell line U266 and investigated the possible mechanism.Methods and results:MM cell line U266 has been chosen as the object,CQ and DEX as main drugs of the study.In addition,60Co-γwas a source of radiation.1.Dose dependent cytotoxic effects of CQ and DEX in U266 cells.A series of final drug concentrations of CQ(1000,500,250,125,62.5,31.3,15.6,7.8,3.9,2.0,1.0μM)and DEX(2000,1000,500,250,125,62.5,31.3,15.6,7.8,3.9,2.0μM)were prepared for use.Logarithmic growth phase U266 cells were chose in experiments,and they were cultured in 96-well plates for measuring cell proliferation or inhibition.10μl CCK8 was added into every well 24 h after drug treatment and absorbance was determined in a wave length of 450 nm.The results showed that: cytotoxic effects of both CQ and DEX were dose-dependent.The medium lethal concentration(IC50)of CQ was about 77.9μM,and the IC50 of DEX was about 237.1μM.3.Cytotoxic effect of DEX in U266 cells was enhanced by CQDifferent final concentrations of CQ(125,62.5,31.3,15.6,7.8,3.9μM)and DEX(250,125μM)were combined in U266 cells.The single drug of CQ or DEX alone were also treated to U266 cells.CCK8 was added 24 h after drug treatment and absorbance was measured.The Compusyn analysis software was used to gain combination index(CI)of CQ and DEX.When DEX was 125μM and the ratio of CQ:DEX(c/c)was between 1:32~1:4,CI≦1,it meant that combined effect of CQ and DEX in U266 cells was synergetic or additive effect.But When DEX was 125μM and the ratio of CQ:DEX(c/c)was between 1:2~1:1,CI>1,antagonism was the result of CQ and DEX in U266 cells.When DEX was 250μM and the ratio of CQ:DEX(c/c)was between 1:64~1:8,CI<1,it showed that CQ and DEX in U266 cells was synergistic effect.However,When DEX was 250μM and the ratio of CQ:DEX(c/c)was between 1:4~1:2,CI≧1,antagonistic or additive effect occurred.Interestingly,cell inhibition of DEX(125μM)was approximately 30%,and cell inhibition of CQ(3.9μM or 7.8μM)was about 8%.But when DEX(125μM)combined with CQ(3.9μM or 7.8μM)in U266 cells,the cell inhibition rose up to 45% or 36% respectively.In consideration of synergistic effect of CQ(3.9μM or 7.8μM)and DEX(125μM),DEX played a major cytotoxic effect in the combination.Thus we called the phenomenon was that cytotoxic effect of DEX in U266 cells was enhanced by CQ.4.Cytotoxic effects of various radiation doses in U266 cells had few differencesIn order to investigate cytotoxic effect of radiation in U266 cells,various doses(5,10,15,20,25Gy)of radiation were studied.Cell proliferations of U266 were decreased after various radiation doses compared to Control(0Gy).But they were about 80% and there were few differences in the treatment of various radiation doses.5.CQ sensitized U266 to radiationCells were divided into four groups: control,CQ(1μM),radiation(5Gy),and radiation + CQ.CCK8 and flow cytometry were used to assess cell proliferation and apoptosis respectively.The results showed that: in comparing to control,CQ hardly had effects on cell proliferation and apoptosis of U266;cell proliferate ability descended and cell apoptosis rose after radiation;the decline of cell proliferation and increase of cell apoptosis had significant changes in radiation pretreatment with CQ.6.The possible mechanism in the sensitization of CQ to DEX or radiationThe protein levels of HIF-1α and Bcl-2 in different groups of U266 cells were measured by Western blot.There were few changes of HIF-1α protein level in CQ combined with DEX or radiation.Besides,compared with control,CQ(3.9μM)nearly had no alteration in expression level of Bcl-2 in U266 cells and DEX(125μM)decreased expression level of Bcl-2.Combination of CQ(3.9μM)and DEX(125μM)in U266 cells,the protein level of Bcl-2 notably declined.However,there were no statistical differences of Bcl-2 protein levels in radiation pretreatment with CQ or not.Conclusion:Traditional antimalarial CQ could sensitize cytotoxic effect of DEX or radiation in MM cell line U266.The former might be related to the down-regulation of Bcl-2.