The Influence Of Histone Deacetylase Inhibitor With Proteasome Inhibitor On The Proliferation Of Human Burkitt Lymphoma Cell Line Raji

Purpose:Burkitt lymphoma （Burkitt lymphoma, BL） is a special subtype of B-cellnon-Hodgkin’s lymphoma, which originally derived from highly malignantfollicular germinal center cells. BL cells have high proliferation rate andinvasion activity. Although recent year new clinical applications such ashigh-dose chemotherapy, rituximab, autologous hematopoietic stem celltransplantation were applied to the treatment of Burkitt’s lymphoma, whichimproved the prognosis, however, there are still some patients who aredifficult to cure. More effective treatment methods are still required. Histonedeacetylase inhibitors （histone deacetylase inhibitor, HDACi） are focusing onepigenetic targets. HDACi can inhibit tumor cell proliferation and enhance thesensitivity of tumor cells to some chemotherapy drugs through regulating theexpression of genes, influence cell histone and non-histone acetylation levels,produces cell cycle arrest, induction of cells apoptosis in a variety ofbiological effects. Proteasome inhibitors are specifically inhibiting theubiquitin-proteasome pathway, and blocking the proteasome proteolysis,thereby blocking the growth of tumor cells and angiogenesis, and thus play itsanti-tumor effects. Proteasome inhibitors have less side effects, and nocross-resistance to conventional chemotherapy drugs, therefore can inhibit theproliferation of a variety of solid tumors and hematological malignancies.The experiment combined histone deacetylase inhibitor LBH589withproteasome inhibitor boron bortezomib （bortezomib） to treat human Burkittlymphoma cell line Raji cells and examine cells growth regulation and exploreits mechanisms of action, in order to provide the theoretical basis andexperimental data for the clinical treatment and searching the new treatment method to Burkitt’s lymphomas.Methods:1Cell culture of Raji cellsThe human BL cell line Raji cells were maintained in RPMI1640,containing10%fetal bovine serum,100units/ml penicillin and100μg/mlstreptomycin. Raji cells were cultured at37℃in a humidified of5%CO₂atmosphere. The cells were passaged every two or three days. All experimentswere using logarithmically growing cells, and cells’ viability≥95%tested bytrypan blue staining.2The effect of LBH589and/or bortezomib on Raji cells tested by MTT2.1Experiment of LBH589inhibited the growth of RajiRaji cells, which in exponential growth state, were seeded in96-wellplates, at a density of2×10⁵cells/ml. The control groups were added100μl thecell nutrient fluid, and the experimental groups were added differentconcentrations of LBH589, made the final concentrations were1nM,10nM,102nM,10³nM,10⁴nM, respectively. Keep LBH589in wells for1,2, or3days before MTT assay. Four hours before the ending point, added20μl MTT（5mg/ml） in each well. After centrifuging of the plate, discarding thesupernatants, adding150μl DMSO to each well, shaking the plate until theprecipitate dissolved. Lastly, measure each well’s absorbance at570nm withelisa reader.2.2Bortezomib inhibited the proliferation of Raji cellsTaking Raji cells in logarithm vegetal period, divided into the controlgroup and the experimental groups randomly. The control group were added100μl cell culture, while the experimental groups were exposed to2,4,8,16,32nM bortezomib respectively, then using the above method to measure theabsorbance of each well.2.3LBH589and bortezomib synergistically inhibited the growth of Raji cellsThe Raji cells were seeded in96-well plates at2×10⁵/ml concentration,then divided these cells into four groups: the control group, LBH589group,bortezomib group, and LBH589plus bortezomib group. Measure the absorbance of each well with MTT test.3Assessment of apoptosis and cell cycle with FCMThe apoptosis levels were evaluated by propidium iodide （PI） stainingand Annexin V staining. Raji cells were cultured in the presence or absence ofLBH589and/or bortezomib for36h. After washing the cells with PBS, staincells with PI （50mg/ml） or Annexin Ⅴ/FITC. The samples were analyzed byFCM to detect cell apoptosis ratio and cell cycles.4Test the expression of Fas protein with FCMRaji cells were divided into control group and experimental groups. Theexperimental groups were exposed to LBH589and/or bortezomib for36h,while the control groups were added only cell culture medium. After washingthe cells with PBS at4℃, add Fas antibody to each group. Keep reaction forone hour at dark space, then add second-antibody into each group, use FCM totest the expression of Fas protein.5Test the expression of bcl-2protein with FCMRaji cells were divided into the control group, LBH589group,bortezomib group and LBH589+bortezomib group. Cells were exposed toLBH589and/or bortezomib for36h. After washing the cells with PBS at4℃,add bcl-2antibody to each group. Keep reaction for one hour at dark space,then add second-antibody into each group, use FCM to test the expression ofbcl-2protein.6Test the mRNA expression level of NF-κB with Realtime-PCRRaji cells, which in exponential growth state, were divided into controlgroup and experimental groups. The control group cells were added cellculture medium, while the experimental groups were added differentconcentrations of LBH589and/or bortezomib for36hours. Total RNA werecollected from each group, then reverse transcript into cDNA. Real-time PCRwere performed to detect expression level of NF-κB relative to GAPDH.7Test the protein levels of NF-κB with western blotRaji cells, which in exponential growth state, were divided into controlgroup and experimental groups. The control group cells were added cell culture medium, while the experimental groups were added differentconcentrations of LBH589and/or bortezomib for36hours. Total proteinwere collected from each group,100μg protein from each group were loadedinto SDS gel and electrophoresis. After bloting into film, NF-κB（p65）proteinwere detected with antibodies.Results:1LBH589inhibited the proliferation of Raji cells with time andconcentration dependency under a certain dose scope. Bortezomib inhibitedthe proliferation of Raji cells with time and concentration dependency under acertain dose scope. The combination of LBH589and bortezomib inhibitedRaji cells proliferation at synergistic effect.2LBH589and bortezomib along can both induce Raji cells apoptosiswith doeses dependency. Compare with monotherapy, the combination ofLBH589and bortezomib induced more cell apoptosis.3LBH589along treatment up-regulated Fas expression level in Raji cells.However, bortezomib along did not influence Fas expression significantly.4Both LBH589and bortezomib can down-regulate bcl-2expression inRaji cells. The combination of LBH589and bortezomib induced synergisticeffect decreased bcl-2expr ession significantly.5Both LBH589and bortezomib can down-regulate NF-κB mRNAexpression in Raji cells. The combination of LBH589and bortezomib inducedsynergistic effect decreased NF-κB mRNA expression significantly.6Both LBH589and bortezomib can down-regulate NF-κB proteinexpression in Raji cells. The combination of LBH589and bortezomib inducedsynergistic effect decreased NF-κB protein expression significantly.Conclusions:1Under a certain concentration scope, LBH589can inhibit theproliferation of human BL cell line Raji and induce cell apoptosis, with timeand concentration dependency. The possible mechanisms are up-regulate Fasexpression and downregulate the expression of NF-κB （p65） and bcl-2protein.2Under a certain concentration scope, bortezomib can inhibit the proliferation of human BL cell line Raji and induce cell apoptosis, with timeand concentration dependency. The possible mechanisms are downregulate theexpression of NF-κB （p65） and bcl-2protein.3Under a certain concentration scope, combination of LBH589andbortezomib can synergistically inhibit the cell proliferation; induce cellapoptosis of Raji cells. The possible mechanism is synergisticallydownregulate the expression of NF-κB and bcl-2protein.